普鲁兰酶的产生菌筛选及其表达与分泌调控
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摘要
普鲁兰酶（pullulanase, EC3.2.1.41）是一种可以特异地水解普鲁兰多糖、淀粉及相关分支多糖中α-1,6糖苷键的淀粉脱支酶。由于它对α-1,6糖苷键的特异水解功能，普鲁兰酶被广泛地应用于淀粉糖加工工业、医药领域、饲料、啤酒及白酒酿造等工业中。但是，工业用普鲁兰酶由于异源表达菌株的分泌性能较差，产酶效率过低等因素，在我国极大地限制了普鲁兰酶的规模化工业生产，长期依赖进口。
为此，本研究针对普鲁兰酶工业化生产为目标，从普鲁兰酶生产菌株的筛选入手，对采自于全国多地的数十个土壤样品进行了普鲁兰酶生产菌株的筛选。之后，利用分子生物学技术对其进行鉴定并克隆其中的普鲁兰酶编码基因，成功地构建了高效表达分泌重组普鲁兰酶的工程菌株。最后，通过自诱导培养、分泌调控等策略将胞外普鲁兰酶的产量提高了270多倍，达到97U mL-1。
（1）筛选得到了一株生产胞外普鲁兰酶的微生物菌株。为克服普通筛选方法工作量大、目标不明确、操作繁杂的缺点，本研究采用平板显色法和普鲁兰沉淀法相结合的策略，对32个土壤样品进行了筛选。通过平板显色的方法缩小了筛选目标，共筛选得到60多株具有分解红色普鲁兰能力的微生物菌株。根据透明圈大小对这些菌株进行分类之后，选择透明圈与菌落大小之比大于2的菌株进行验证和液体培养基中的复筛。最终得到一株产酶能力较高的微生物菌株。经过生理生化及分子遗传学鉴定，将其鉴定为克雷伯氏菌SHN-1（Klebsiella sp. SHN-1）。向NCBI提交其16S rDNA序列，获得数据库登记号为：HM037179。通过对野生菌株产酶条件的初步探索，最终将该普鲁兰酶生产菌株的产酶能力提升到10.98U mL-1。
（2）构建了一株可以高效生产胞外普鲁兰酶的大肠杆菌的工程菌株。虽然胞外分泌对于普鲁兰酶的规模化生产意义重大，但是，众多研究者构建的普鲁兰酶生产菌株都不具备分泌能力。考虑到大肠杆菌与克雷伯氏菌的亲缘关系及野生菌株生产胞外普鲁兰酶的能力，本研究根据NCBI上公布的克雷伯氏菌属的普鲁兰酶编码基因设计引物，克隆了Klebsiella sp. SHN-1的普鲁兰酶及信号肽的编码序列（NCBI登陆号为JX087429），以pET-28a（+）为载体构建了重组表达质粒pET-28a（+）-pulA，转化大肠杆菌后获得了重组大肠杆菌E. coli BL21（DE3）/pET-28a(+)-pulA。当该重组大肠杆菌在LB培养基中生长时，在IPTG的诱导下，重组普鲁兰酶可以得到有效表达并分泌到细胞外部。该重组菌的这些生产特性展现出了它作为工业用生产菌株的潜力。
（3）构建了信号肽编码序列删除的表达质粒pET-28a(+)-pulA-sig-。有研究表明，为了实现重组普鲁兰酶在大肠杆菌中的高效分泌表达，必须同时克隆野生克雷伯氏菌中与普鲁兰酶分泌相关的分泌基因（包括信号肽编码序列）。然而本研究在仅克隆了普鲁兰酶的编码基因及其信号序列的情况下就实现了重组普鲁兰酶在大肠杆菌中的高效分泌表达。为了考察普鲁兰酶编码基因内部与分泌相关的信号序列，本研究构建了信号肽编码基因删除的表达质粒pET-28a(+)-pulA-sig-。当在LB液体培养基中培养该工程菌时，加入诱导物IPTG，重组普鲁兰酶仍然能够得到表达并分泌到发酵液中。随后对重组普鲁兰酶在胞外、胞膜、周质空间和胞内部分分布的考察表明，有高达65.49%重组普鲁兰酶被释放到细胞外部，这说明重组普鲁兰酶在大肠杆菌中的分泌与信号肽的存在关系不大。通过对普鲁兰酶成熟肽序列的分析发现，普鲁兰酶编码基因的内部存在着两个与蛋白分泌相关的序列，这可能是重组普鲁兰酶在没有信号肽的情况下仍然能够被释放到细胞外部的原因。随后，以该工程菌为研究对象，对其在LB培养基中以IPTG为诱导物生产胞外普鲁兰酶的条件进行了研究。最终，本研究将胞外重组普鲁兰酶的产量提高到了10U mL-1左右的水平。接下来，本研究又通过镍柱对重组普鲁兰酶进行纯化并考察了该重组普鲁兰酶的部分酶学性质，确定其最适作用温度和pH分别为55℃和5.0。当以纯酶作用于1%的普鲁兰多糖的溶液时，得到的主要产物为麦芽三糖，再次证明该酶是I型普鲁兰酶。
（4）采用涉及可视化筛选、自诱导培养和温度调控的组合策略生产高水平的胞外重组普鲁兰酶。重组蛋白表达水平的下降会导致最终目标蛋白产量的下降，为此，本研究提出了可视化筛选策略来考察各个克隆子的产酶能力。在此基础之上，针对发酵过程中细胞浓度过低和普鲁兰酶胞内积累严重的问题，提出了自诱导——双阶段温度调控策略，使胞外普鲁兰酶产量提高到50U mL-1。另外，细胞渗透物的使用也有效地提升了胞外重组普鲁兰酶的产量，达到68.23U mL-1。最后，在7L NBS发酵罐上进行了重组普鲁兰酶的分批发酵和分批-补料发酵实验，在进一步提高细胞密度的基础上，又将胞外普鲁兰酶的产量提高到97U mL-1。
本课题通过开展系统的研究工作，极大地提高胞外普鲁兰酶的产量。为了得到一株高效生产胞外普鲁兰酶的微生物菌株，本研究提出了平板显色与普鲁兰多糖沉淀相结合的策略。通过构建高效生产胞外普鲁兰酶的工程菌株发现了普鲁兰酶在大肠杆菌中的信号肽非依赖型分泌现象，为研究蛋白的高效分泌提供了参考和理论依据。根据重组蛋白的表达与分泌的特点，本研究又提出了高效生产胞外普鲁兰酶的自诱导——双阶段温度调控策略，结合细胞渗透物的使用，极大地提高了工程大肠杆菌生产胞外重组普鲁兰酶的能力。因此，本研究的开展将会为今后重组普鲁兰酶的规模化生产提供重要的理论指导。
Pullulanase (EC3.2.1.41) is a debranching enzyme, which specifcally cleaves α-1,6glycosidic bonds in pullulan, starch and other related amylaceous polysaccharides. Hence,pullulanase can be employed to break down starch to produce glucose, fructose, maltosesyrups, and amylose, in conjunction with or without α-amylase, β-amylase, glucoamylase. Inaddition, pullulanase could also be used in the industry of pharmaceuticals, feed, brewing etc.to enhance the utilization rate of starch. However, low yield and productivity have been thebottleneck for the large-scale production and widespread application of pullulanase.
In order to obtain pullulanase-producing microbes of high efficiency, this study beganwith the screening of pullulanase producers from the soil samples taken from severalprovinces in China. Subsequently, an engineered Escherichia coli was constructed, whichcould produce extracellular pullulanase with high efficiency. For the sake of improved yield, acombined strategy involving auto-induction, temperature regulation, and osmolyte additionwas developed. As a result, its application greatly enhanced the yield of extracellularpullulanase.
(1) A pullulanase-producing strain was obtained. In order to overcome the problem oftraditional screening method, such as heavy workload and complex operation, a plate assay,based on red-pullulan, was employed to reduce the scope of screening. Subsequent abilityconfirmation of the isolates was carried out with the traditional screening method.Consequently, more than60isolates were obtained, which presented the ability to de-color thered-pullulan. In order to achieve an excellent strain, the isolates with haloes2times thecolonies were subjected to the second round of screening. Fortunately, an isolate with thehighest capability was obtained, which was identified as Klebsiella sp. SHN-1(GenBankAccession Number: HM037179). Under the optimized fermentation condition, the Klebsiellasp. SHN-1could produce about10.98U mL-1extracellular pullulanase.
(2) An engineered E. coli BL21(DE3) with the ability to produce extracellularpullulanase was constructed. Although extracellular production is important for the large-scaleproduction of pullulanase, most constructed engineered strains produced pullulanaseintracellularly. Considering the close relationship between E. coli and Klebsiella sp. SHN-1, aprimer pair was designed based on the published pullulanase sequence to clone thepullulanase encoding gene. As a result, a DNA fragment of3000bp was obtained, whichshared99%homogeneity with that from K. variicola At-2and K. pneumoniae342.Subsequently, the sequence was subjected to GenBank (Accession Number: JX087429).When the DNA fragment was cloned into E. coli BL21(DE3) with the plasmid of pET-28a(+)as expression vector, recombinant pullulanase was detected in the supernatant of culture brothin the presence of IPTG.
(3) An expression vector without signal peptide sequence was constructed. Numerousstudies suggested that the secretion of pullulanase from Klebsiella in E. coli required theexistence of secretion-related genes, including the signal peptide sequence. In order to explorethe reasons of pullulanase secretion in E. coli, a signal peptide-deleted plasmid was constructed. However, the engineered E. coli BL21(DE) carrying the expression vector couldstill express the gene encoding pullulanase and release the product into surroundingenvironment. The distribution of recombinant pullulanase in subcellular fractions, such as cellmembrane, cytoplasm, petriplasmic space, cell-associated fraction was also investigated. Theresult showed that up to65.49%recombinant pullulanase was secreted, indicating the releaseof recombinant pullulanase was not close to the existence of the signal peptide sequence.Analysis on the sequence of mature pullulanase suggested two inner regions might beresponsible for the secretion of pullulanase. Subsequently, the fermentation conditions of thisstrain in LB medium were optimized to enhance the production of extracellular pullulanaseand10U mL-1extracellular pullulanase was obtained. The recombinant pullulanase was alsopurified by one-step affinity chromatography. Investigation on the purified pullulanaseshowed its optimum reaction pH and temperature were5.0and55oC, respectively. When itwas employed to hydrolyze1%pullulan, maltotriose as the main end products was obtained,demonstrating the property of type I pullulanase.
(4) The yield of extracellular pullulanase was enhanced by using a combined strategyinvolving the visible screening method, auto-induction, temperature control strategy. In orderto ensure the stability of the engineered strain, colonies used to carry out the production ofpullulanase were evaluated with the visible screening method. Subsequently, auto-inductionmethod was employed to simplify the production of pullulanase and increase the cell densityof culture. Results showed that the yield of extracellular pullulanase was improved to50U mL-1. Additionally, the ability of the engineered strain was further improved to68.23U mL-1by applying various natural osmolytes. Finally, pullulanase production was performedin a7L fermentor with batch and fed-batch strategy. As a result, the yield of extracellularpullulanase was elevated to97U mL-1.
The yield of extracellular pullulanase was greatly improved by performing systematicresearch work. An isolate producing extracellular pullulanase was obtained with color platemethod and pullulan precipitation method. During the construction of an engineered E. coliproducing extracellular pullulanase, the signal peptide-independent secretion of pullulanase inE. coli was discovered. This observation provided the study of effective proteins secretionwith reference and theoretical basis. According to the property of recombinant proteinexpression and secretion, auto-induction-two stage temperature control strategy wasdeveloped. By combination with osmolytes, the yield of extracellular pullulanase was greatlyincreased. Therefore, the study presented theoretical guideline for the large-scale productionof pullulanase in the future.
为此，本研究针对普鲁兰酶工业化生产为目标，从普鲁兰酶生产菌株的筛选入手，对采自于全国多地的数十个土壤样品进行了普鲁兰酶生产菌株的筛选。之后，利用分子生物学技术对其进行鉴定并克隆其中的普鲁兰酶编码基因，成功地构建了高效表达分泌重组普鲁兰酶的工程菌株。最后，通过自诱导培养、分泌调控等策略将胞外普鲁兰酶的产量提高了270多倍，达到97U mL-1。
（1）筛选得到了一株生产胞外普鲁兰酶的微生物菌株。为克服普通筛选方法工作量大、目标不明确、操作繁杂的缺点，本研究采用平板显色法和普鲁兰沉淀法相结合的策略，对32个土壤样品进行了筛选。通过平板显色的方法缩小了筛选目标，共筛选得到60多株具有分解红色普鲁兰能力的微生物菌株。根据透明圈大小对这些菌株进行分类之后，选择透明圈与菌落大小之比大于2的菌株进行验证和液体培养基中的复筛。最终得到一株产酶能力较高的微生物菌株。经过生理生化及分子遗传学鉴定，将其鉴定为克雷伯氏菌SHN-1（Klebsiella sp. SHN-1）。向NCBI提交其16S rDNA序列，获得数据库登记号为：HM037179。通过对野生菌株产酶条件的初步探索，最终将该普鲁兰酶生产菌株的产酶能力提升到10.98U mL-1。
（2）构建了一株可以高效生产胞外普鲁兰酶的大肠杆菌的工程菌株。虽然胞外分泌对于普鲁兰酶的规模化生产意义重大，但是，众多研究者构建的普鲁兰酶生产菌株都不具备分泌能力。考虑到大肠杆菌与克雷伯氏菌的亲缘关系及野生菌株生产胞外普鲁兰酶的能力，本研究根据NCBI上公布的克雷伯氏菌属的普鲁兰酶编码基因设计引物，克隆了Klebsiella sp. SHN-1的普鲁兰酶及信号肽的编码序列（NCBI登陆号为JX087429），以pET-28a（+）为载体构建了重组表达质粒pET-28a（+）-pulA，转化大肠杆菌后获得了重组大肠杆菌E. coli BL21（DE3）/pET-28a(+)-pulA。当该重组大肠杆菌在LB培养基中生长时，在IPTG的诱导下，重组普鲁兰酶可以得到有效表达并分泌到细胞外部。该重组菌的这些生产特性展现出了它作为工业用生产菌株的潜力。
（3）构建了信号肽编码序列删除的表达质粒pET-28a(+)-pulA-sig-。有研究表明，为了实现重组普鲁兰酶在大肠杆菌中的高效分泌表达，必须同时克隆野生克雷伯氏菌中与普鲁兰酶分泌相关的分泌基因（包括信号肽编码序列）。然而本研究在仅克隆了普鲁兰酶的编码基因及其信号序列的情况下就实现了重组普鲁兰酶在大肠杆菌中的高效分泌表达。为了考察普鲁兰酶编码基因内部与分泌相关的信号序列，本研究构建了信号肽编码基因删除的表达质粒pET-28a(+)-pulA-sig-。当在LB液体培养基中培养该工程菌时，加入诱导物IPTG，重组普鲁兰酶仍然能够得到表达并分泌到发酵液中。随后对重组普鲁兰酶在胞外、胞膜、周质空间和胞内部分分布的考察表明，有高达65.49%重组普鲁兰酶被释放到细胞外部，这说明重组普鲁兰酶在大肠杆菌中的分泌与信号肽的存在关系不大。通过对普鲁兰酶成熟肽序列的分析发现，普鲁兰酶编码基因的内部存在着两个与蛋白分泌相关的序列，这可能是重组普鲁兰酶在没有信号肽的情况下仍然能够被释放到细胞外部的原因。随后，以该工程菌为研究对象，对其在LB培养基中以IPTG为诱导物生产胞外普鲁兰酶的条件进行了研究。最终，本研究将胞外重组普鲁兰酶的产量提高到了10U mL-1左右的水平。接下来，本研究又通过镍柱对重组普鲁兰酶进行纯化并考察了该重组普鲁兰酶的部分酶学性质，确定其最适作用温度和pH分别为55℃和5.0。当以纯酶作用于1%的普鲁兰多糖的溶液时，得到的主要产物为麦芽三糖，再次证明该酶是I型普鲁兰酶。
（4）采用涉及可视化筛选、自诱导培养和温度调控的组合策略生产高水平的胞外重组普鲁兰酶。重组蛋白表达水平的下降会导致最终目标蛋白产量的下降，为此，本研究提出了可视化筛选策略来考察各个克隆子的产酶能力。在此基础之上，针对发酵过程中细胞浓度过低和普鲁兰酶胞内积累严重的问题，提出了自诱导——双阶段温度调控策略，使胞外普鲁兰酶产量提高到50U mL-1。另外，细胞渗透物的使用也有效地提升了胞外重组普鲁兰酶的产量，达到68.23U mL-1。最后，在7L NBS发酵罐上进行了重组普鲁兰酶的分批发酵和分批-补料发酵实验，在进一步提高细胞密度的基础上，又将胞外普鲁兰酶的产量提高到97U mL-1。
本课题通过开展系统的研究工作，极大地提高胞外普鲁兰酶的产量。为了得到一株高效生产胞外普鲁兰酶的微生物菌株，本研究提出了平板显色与普鲁兰多糖沉淀相结合的策略。通过构建高效生产胞外普鲁兰酶的工程菌株发现了普鲁兰酶在大肠杆菌中的信号肽非依赖型分泌现象，为研究蛋白的高效分泌提供了参考和理论依据。根据重组蛋白的表达与分泌的特点，本研究又提出了高效生产胞外普鲁兰酶的自诱导——双阶段温度调控策略，结合细胞渗透物的使用，极大地提高了工程大肠杆菌生产胞外重组普鲁兰酶的能力。因此，本研究的开展将会为今后重组普鲁兰酶的规模化生产提供重要的理论指导。
Pullulanase (EC3.2.1.41) is a debranching enzyme, which specifcally cleaves α-1,6glycosidic bonds in pullulan, starch and other related amylaceous polysaccharides. Hence,pullulanase can be employed to break down starch to produce glucose, fructose, maltosesyrups, and amylose, in conjunction with or without α-amylase, β-amylase, glucoamylase. Inaddition, pullulanase could also be used in the industry of pharmaceuticals, feed, brewing etc.to enhance the utilization rate of starch. However, low yield and productivity have been thebottleneck for the large-scale production and widespread application of pullulanase.
In order to obtain pullulanase-producing microbes of high efficiency, this study beganwith the screening of pullulanase producers from the soil samples taken from severalprovinces in China. Subsequently, an engineered Escherichia coli was constructed, whichcould produce extracellular pullulanase with high efficiency. For the sake of improved yield, acombined strategy involving auto-induction, temperature regulation, and osmolyte additionwas developed. As a result, its application greatly enhanced the yield of extracellularpullulanase.
(1) A pullulanase-producing strain was obtained. In order to overcome the problem oftraditional screening method, such as heavy workload and complex operation, a plate assay,based on red-pullulan, was employed to reduce the scope of screening. Subsequent abilityconfirmation of the isolates was carried out with the traditional screening method.Consequently, more than60isolates were obtained, which presented the ability to de-color thered-pullulan. In order to achieve an excellent strain, the isolates with haloes2times thecolonies were subjected to the second round of screening. Fortunately, an isolate with thehighest capability was obtained, which was identified as Klebsiella sp. SHN-1(GenBankAccession Number: HM037179). Under the optimized fermentation condition, the Klebsiellasp. SHN-1could produce about10.98U mL-1extracellular pullulanase.
(2) An engineered E. coli BL21(DE3) with the ability to produce extracellularpullulanase was constructed. Although extracellular production is important for the large-scaleproduction of pullulanase, most constructed engineered strains produced pullulanaseintracellularly. Considering the close relationship between E. coli and Klebsiella sp. SHN-1, aprimer pair was designed based on the published pullulanase sequence to clone thepullulanase encoding gene. As a result, a DNA fragment of3000bp was obtained, whichshared99%homogeneity with that from K. variicola At-2and K. pneumoniae342.Subsequently, the sequence was subjected to GenBank (Accession Number: JX087429).When the DNA fragment was cloned into E. coli BL21(DE3) with the plasmid of pET-28a(+)as expression vector, recombinant pullulanase was detected in the supernatant of culture brothin the presence of IPTG.
(3) An expression vector without signal peptide sequence was constructed. Numerousstudies suggested that the secretion of pullulanase from Klebsiella in E. coli required theexistence of secretion-related genes, including the signal peptide sequence. In order to explorethe reasons of pullulanase secretion in E. coli, a signal peptide-deleted plasmid was constructed. However, the engineered E. coli BL21(DE) carrying the expression vector couldstill express the gene encoding pullulanase and release the product into surroundingenvironment. The distribution of recombinant pullulanase in subcellular fractions, such as cellmembrane, cytoplasm, petriplasmic space, cell-associated fraction was also investigated. Theresult showed that up to65.49%recombinant pullulanase was secreted, indicating the releaseof recombinant pullulanase was not close to the existence of the signal peptide sequence.Analysis on the sequence of mature pullulanase suggested two inner regions might beresponsible for the secretion of pullulanase. Subsequently, the fermentation conditions of thisstrain in LB medium were optimized to enhance the production of extracellular pullulanaseand10U mL-1extracellular pullulanase was obtained. The recombinant pullulanase was alsopurified by one-step affinity chromatography. Investigation on the purified pullulanaseshowed its optimum reaction pH and temperature were5.0and55oC, respectively. When itwas employed to hydrolyze1%pullulan, maltotriose as the main end products was obtained,demonstrating the property of type I pullulanase.
(4) The yield of extracellular pullulanase was enhanced by using a combined strategyinvolving the visible screening method, auto-induction, temperature control strategy. In orderto ensure the stability of the engineered strain, colonies used to carry out the production ofpullulanase were evaluated with the visible screening method. Subsequently, auto-inductionmethod was employed to simplify the production of pullulanase and increase the cell densityof culture. Results showed that the yield of extracellular pullulanase was improved to50U mL-1. Additionally, the ability of the engineered strain was further improved to68.23U mL-1by applying various natural osmolytes. Finally, pullulanase production was performedin a7L fermentor with batch and fed-batch strategy. As a result, the yield of extracellularpullulanase was elevated to97U mL-1.
The yield of extracellular pullulanase was greatly improved by performing systematicresearch work. An isolate producing extracellular pullulanase was obtained with color platemethod and pullulan precipitation method. During the construction of an engineered E. coliproducing extracellular pullulanase, the signal peptide-independent secretion of pullulanase inE. coli was discovered. This observation provided the study of effective proteins secretionwith reference and theoretical basis. According to the property of recombinant proteinexpression and secretion, auto-induction-two stage temperature control strategy wasdeveloped. By combination with osmolytes, the yield of extracellular pullulanase was greatlyincreased. Therefore, the study presented theoretical guideline for the large-scale productionof pullulanase in the future.
引文
1. Singh R，Saini G, Kennedy J. Pullulan: microbial sources, production and applications [J]. CarbohydPolym，2008，73(4):515-531
2. Bender H，Lehmann J, Wallenfels K. Pullulan, an extracellular glucan from Pullularia pullulans [J].Biochimica Et Biophysica Acta，1959，36:309-316
3. Leathers T D. Biotechnological production and applications of pullulan [J]. Appl MicrobiolBiotechnol，2003，62(5):468-473
4. Kulicke W M, Heinze T. Improvements in polysaccharides for use as blood plasma expanders [J].Macromol Symp，2005，231(1):47-59
5. Doman-Pytka M, Bardowski J. Pullulan degrading enzymes of bacterial origin [J]. Crit Rev Microbiol，2004，30(2):107-121
6. Suginoshita Y，Tabata Y，Matsumura T， et al. Liver targeting of human interferon-β with pullulanbased on metal coordination [J]. J Control Release，2002，83(1):75-88
7. Kumar P, Satyanarayana T. Microbial glucoamylases: characteristics and applications [J]. Crit RevBiotechnol，2009，29(3):225-255
8. Feng P H，Berensmeier S，Buchholz K， et al. Production, purification, and characterization ofThermoanaerobacterium thermosaccharolyticum glucoamylase [J]. Starch-starke，2002，54(8):328-337
9. Bertoldo C，Duffner F，Jorgensen P L， et al. Pullulanase type I from Fervidobacterium pennavoransVen5: cloning, sequencing, and expression of the gene and biochemical characterization of the recombinantenzyme [J]. Appl Environ Microbiol，1999，65(5):2084-2091
10. Bertoldo C，Armbrecht M，Becker F， et al. Cloning, sequencing, and characterization of a heat-andalkali-stable type I pullulanase from Anaerobranca gottschalkii [J]. Appl Environ Microbiol，2004，70(6):3407-3416
11. Kim H J，Park J N，Kim H O， et al. Cloning and expression of a Paenibacillus sp neopullulanasegene in Saccharomyces cerevisiae producing Schwanniomyces occidentalis glucoamylase [J]. J MicrobiolBiotechn，2002，12(2):340-344
12. Matzke J，Herrmann A，Schneider E， et al. Gene cloning, nucleotide sequence and biochemicalproperties of a cytoplasmic cyclomaltodextrinase (neopullulanase) from Alicyclobacillus acidocaldarius,reclassification of a group of enzymes [J]. FEMS Microbiol Lett，2000，183(1):55-61
13. Imanaka T, Kuriki T. Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan [J].J Bacteriol，1989，171(1):369-374
14. Aoki H，Yopi, Sakano Y. Molecular cloning and heterologous expression of the isopullulanase genefrom Aspergillus niger A.T.C.C.9642[J]. Biochem J，1997，323(3):757-764
15. Mizuno M，Koide A，Yamamura A， et al. Crystal structure of Aspergillus niger isopullulanase, amember of glycoside hydrolase family49[J]. J Mol Biol，2008，376(1):210-220
16. Akeboshi H，Kashiwagi Y，Aoki H， et al. Construction of an efficient expression system forAspergillus isopullulanase in Pichia pastoris, and a simple purification method [J]. Biosci Biotech Bioch，2003，67(5):1149-1153
17. Bertoldo C, Antranikian G. Starch-hydrolyzing enzymes from thermophilic archaea and bacteria [J].Curr Opin Chem Biol，2002，6(2):151-160
18. Niehaus F，Peters A，Groudieva T， et al. Cloning, expression and biochemical characterisation of aunique thermostable pullulan-hydrolysing enzyme from the hyperthermophilic archaeon Thermococcusaggregans [J]. FEMS Microbiol Lett，2000，190(2):223-229
19. Zhang Y，Liu Y-h，Li Y， et al. Extracellular expression of pullulanase from Bacillus naganoensis inEscherichia coli [J]. Ann Microbiol，2012，63(1):289-294
20. Kunamneni A, Singh S. Improved high thermal stability of pullulanase from a newly isolatedthermophilic Bacillus sp AN-7[J]. Enzyme Microb Tech，2006，39(7):1399-1404
21. Saha B C, Zeikus J G. Novel highly thermostable pullulanase from thermophiles [J]. TrendsBiotechnol，1989，7(9):234-239
22. Abdullah M, French D. Reversible action of pulluanase [J]. Nature，1966，210:200
23. Abdullah M，Catley B. J，Lee E. Y. C， et al. The mechanism of carbohydrase action. II. pullulanase,an enzyme specific forthe hydrolysis of alpha-1,6-bonds in amylous oligosaccharides.[J]. Cereal Chem，1966，43:111-118
24. Bender H, Wallenfels K. Untersuchungen an pullulan. II. spezifischer abbau durch ein bakteriellesEnzym.[J]. Biochemische Zeitschrift，1961，334:79-95
25. Pugsley A P，Francetic O，Hardie K， et al. Pullulanase: model protein substrate for the generalsecretory pathway of gram-negative bacteria [J]. Folia Microbiol，1997，42(3):184-192
26. Mathupala S P，Lowe S E，Podkovyrov S M， et al. Sequencing of the amylopullulanase(APU) geneof Thermoanaerobacter ethanlicus39E, and identification of the active-site by site-directed mutagenesis [J].J Biol Chem，1993，268(22):16332-16344
27. Ganhofner D， Kellermann J， Staudenbauer W， et al. Purification and properties of anamylopullulanase, a glucoamylase, and an α-glucosidase in the amylolytic enzyme system ofThermoanaerobacterium thermosaccharolyticum [J]. Biosci Biotech Bioch，1998，62(2):302-308
28. Hii S L，Ling T C，Rosfarizan M， et al. Characterization of pullulanase type II from Bacillus cereusH1.5[J]. Am J Biochem Biotechnol，2009，5(4):170-179
29. Erra-Pujada M，Debeire P，Duchiron F， et al. The type II pullulanase of Thermococcushydrothermalis: molecular characterization of the gene and expression of the catalytic domain [J]. JBacteriol，1999，181(10):3284-3287
30. Duffner F，Bertoldo C，Andersen J T， et al. A new thermoactive pullulanase from Desulfurococcusmucosus: cloning, sequencing, purification, and characterization of the recombinant enzyme afterexpression in Bacillus subtilis [J]. J Bacteriol，2000，182(22):6331-6338
31. Abdullah M, French D. Substrate specificity of pullulanase [J]. Arch Biochem Biophys，1970，137(2):483-493
32. Drummond G S，Smith E E，Whelan W J， et al. Mechanism of action of pullulanase [J]. FEMSLetters，1969，5(1):85-88
33. Wallenfels K，Bender H, Rached J R. Pullulanase from Aerobacter aerogenes production in acell-bound state. Purification and properties of enzyme [J]. Biochem Bioph Res Co，1966，22(3):254-261
34. Bender H, Wallenfels K. Pullulanase (an amylopectin and glycogen debranching enzyme) fromAerobacter aerogenes [J]. Method Enzymol，1966，8:555-559
35. Hurst T L. Process for producing dextrose [P]. United States.1975.
36. Dessein A, Schwartz M. Is there a pullulanase in Escherichia coli?[J]. Eur J Biochem，1974，45(2):363-366
37. W ber G. Pullulanase is a characteristic of many Klebsiella species and functions in the degradationof starch [J]. Appl Microbiol Biotechnol，1976，3(1b):71-80
38. Adams K R, Priest F G. Extracellular pullulanase synthesis in Bacillus macerans [J]. FEMSMicrobiol Lett，1977，1(5):269-273
39. Morgan F J，Adams K R, Priest F G. A culture method for the detection of pullulan-degrading enzymein bacteria and its application to the Genus Bacillus [J]. J Appl Bacteriol，1979，46:291-294
40. Ram K A, Venkatasubramanian K. Enhancement of starch conversion efficiency with free andimmobilized pullulanase and α-1,4-glucosidase [J]. Biotechnol Bioeng，1982，24(2):355-369
41. Norman B E. A novel debranching enzyme for application in the glucose syrup industry [J]. Starch-St rke，1982，34(10):340-346
42. Jensen B, Norman B. Bacillus acidopullulyticus pullulanase: application and regulatory aspects foruse in the food industry [J]. Process Biochem，1984，19(4):129-134
43. Takasaki Y. Purification and enzymatic properties of β-amylase and pullulanase from Bacillus cereusvar. mycoides [J]. Agric Biol Chem，1976，40:1523-1530
44. Takasaki Y. Pullulanase-amylase complex enzyme from Bacillus subtilis [J]. Agric Biol Chem，1987，51(1):9-16
45. Tomimura E，Zeman N W，Frankiewicz J R， et al. Description of Bacillus naganoensis sp. nov [J].Int J Syst Bacteriol，1990，40(2):123-125
46. Tomimura E. Thermoduric and aciduric pullulanase enzyme and method for its production [P]. UnitedStates19911.
47. Hii S L，Ling T C，Mohamad R， et al. Enhancement of extracellular pullulanase production byRaoultella planticola DSMZ4617using optimized medium based on sago starch [J]. Open Biotechnol J，2009，3:1-8
48. Nielsen G C T, DK), Diers, Ivan V.(Vaerlose, DK), Outtrup, Helle (Vaerlose, DK), Norman, Barrie E.(Farum, DK). Debranching enzyme product, preparation and use thereof [P]. United States.1985.
49. Deweer P A, BE), Amory, Antoine (Rixensart, BE). Pullulanase producing microrganisms [P]. UnitedStates.1998.
50. Kambourova M S, Emanuilova E I. Purification and general biochemical properties of thermostablepullulanase from Bacillus stearothermoplilus G-82[J]. Appl Biochem Biotech，1992，33(3):193-203
51. Kang J，Park K-M，Choi K-H， et al. Molecular cloning and biochemical characterization of aheat-stable type I pullulanase from Thermotoga neapolitana [J]. Enzyme Microb Tech，2011，48(3):260-266
52. Lin F P, Leu K L. Cloning, expression, and characterization of thermostable region ofamylopullulanase gene from Thermoanaerobacter ethanolicus39E [J]. Appl Biochem Biotech，2002，97(1):33-44
53. Spreinat A, Antranikian G. Purification and properties of a thermostable pullulanase from Clostridiumthermosulfurogenes EM1which hydrolyses both α-1,6and α-1,4-glycosidic linkages [J]. Appl MicrobiolBiotechnol，1990，33(5):511-518
54. Kang S，Vieille C, Zeikus J G. Identification of Pyrococcus furiosus amylopullulanase catalyticresidues [J]. Appl Microbiol Biotechnol，2005，66:408-413
55. Knapp S，Rudiger A，Antranikian G， et al. Crystallization and preliminary crystallographic analysisof an amylopullulanase from the hyperthermophilic archaeon Pyrococcus woesei [J]. Proteins，1995，23(4):595-597
56. Rudiger A，Jorgensen P L, Antranikian G. Isolation and characterization of a heat-stable pullulanasefrom the hyperthermophilic archaeon Pyrococcus woesei after cloning and expression of its gene inEscherichia coli [J]. Appl Environ Microbiol，1995，61(2):567-575
57. Nair S U，Singhal R S, Kamat M Y. Induction of pullulanase production in Bacillus cereus FDTA-13[J]. Bioresource Technol，2007，98(4):856-859
58. Kim J-H，Sunako M，Ono H， et al. Characterization of the C-terminal truncated form ofamylopullulanase from Lactobacillus plantarum L137[J]. J Biosci Bioeng，2009，107(2):124-129
59. Wu S J，Kim J M，Zhou C A， et al. Estimation of pullulan by hydrolysis with pullulanase [J].Biotechnol Lett，2010，32(8):1143-1145
60. Zouari Ayadi D，Ben Ali M，Jemli S， et al. Heterologous expression, secretion and characterizationof the Geobacillus thermoleovorans US105type I pullulanase [J]. Appl Microbiol Biotechnol，2008，78(3):473-481
61. Yamashita M，Nakagawa A，Katsuragi N， et al. Role of lipid modification on a starch-debranchingenzyme, Klebsiella pullulanase: comparison of properties of lipid-modified and unmodified pullulanases [J].Mol Microbiol，1992，6(3):389-394
62. Francetic O, Pugsley A P. Towards the identification of type II secretion signals in a nonacylatedvariant of pullulanase from Klebsiella oxytoca [J]. J Bacteriol，2005，187(20):7045-7055
63. Buddelmeijer N，Francetic O, Pugsley A P. Green fluorescent chimeras indicate nonpolar localizationof pullulanase secreton components PulL and PulM [J]. J Bacteriol，2006，188(8):2928-2935
64. Mikami B，Iwamoto H，Malle D，et al. Crystal structure of pullulanase: evidence for parallel bindingof oligosaccharides in the active site [J]. J Mol Biol，2006，359(3):690-707
65. Hytonen J，Haataja S, Finne J. Use of flow cytometry for the adhesion analysis of Streptococcuspyogenes mutant strains to epithelial cells: investigation of the possible role of surface pullulanase andcysteine protease, and the transcriptional regulator Rgg [J]. BMC Microbiology，2006，6(1):18
66. Gourlay L J，Santi I，Pezzicoli A， et al. Group B Streptococcus pullulanase crystal structures in thecontext of a novel strategy for vaccine development [J]. J Bacteriol，2009，191(11):3544-3552
67. Smith K A, Salyers A A. Cell-associated pullulanase from Bacteroides thetaiotaomicron: cloning,characterization, and insertional mutagenesis to determine role in pullulan utilization [J]. J Bacteriol，1989，171(4):2116-2123
68. Kitahata S，Tanimoto T，Ikuta A， et al. Synthesis of novel heterobranched β-cyclodextrins from42-O-β-D-galactosy l-maltose and β-cyclodextrin by the reverse action of pullulanase, and isolation andcharacterization of the products [J]. Biosci Biotech Bioch，2000，64(6):1223-1229
69. Moubasher H，Wahsh S, El-Kassem N. Purification of pullulanase from Aureobasidium pullulans [J].Microbiology，2010，79(6):759-766
70.徐金利，吕明生，王淑军，等.嗜热古菌Thermococcus sp. HJ21产高温普鲁兰酶条件和酶学性质[J].食品与生物技术学报，2009，28(2):243-249
71. Gomes I，Gomes J, Steiner W. Highly thermostable amylase and pullulanase of the extremethermophilic eubacterium Rhodothermus marinus: production and partial characterization [J]. BioresourceTechnol，2003，90(2):207-214
72. Jiao Y L，Wang S J，Lv M S， et al. A GH57family amylopullulanase from deep-sea Thermococcussiculi: expression of the gene and characterization of the recombinant enzyme [J]. Curr Microbiol，2011，62(1):222-228
73. Plant A，Morgan H, Daniel R. A highly stable pullulanase from Thermus aquaticus YT-1[J]. EnzymeMicrob Tech，1986，8(11):668-672
74. Kim C H，Nashiru O, Ko J H. Purification and biochemical characterization of pullulanase type I fromThermus caldophilus GK-24[J]. FEMS Microbiol Lett，1996，138(2-3):147-152
75. Tomiyasu K，Yato K，Yasuda M， et al. Cloning and nucleotide sequence of the pullulanase gene ofThermus thermophilus HB8and production of the enzyme in Escherichia coli [J]. Biosci Biotech Bioch，2001，65(9):2090-2094
76. Masuda H，Takahashi T, Sugawara S. Purification and properties of starch hydrolyzing enzymes inmature root of sugar beets [J]. Plant Physiol，1987，84(2):361-365
77. McDougall G J，Ross H A，Swanston J S， et al. Limit dextrinase from germinating barley hasendotransglycosylase activity, which explains its activation by maltodextrins [J]. Planta，2004，218(4):542-551
78. Yamasaki Y，Nakashima S, Konno H. Pullulanase from rice endosperm [J]. Acta Biochim Pol，2008，55(3):507-510
79. Fujita N，Toyosawa Y，Utsumi Y， et al. Characterization of pullulanase (PUL)-deficient mutants ofrice (Oryza sativa L.) and the function of PUL on starch biosynthesis in the developing rice endosperm [J].J Exp Bot，2009，60(3):1009-1023
80. Wu C Y，Colleoni C，Myers A M， et al. Enzymatic properties and regulation of ZPU1, the maizepullulanase-type starch debranching enzyme [J]. Arch Biochem Biophys，2002，406(1):21-32
81. Dinges J R，Colleoni C，James M G， et al. Mutational analysis of the pullulanase-type debranchingenzyme of maize indicates multiple functions in starch metabolism [J]. Plant Cell，2003，15(3):666-680
82. Beatty M K，Rahman A，Cao H P，et al. Purification and molecular genetic characterization of ZPU1,a pullulanase-type starch-debranching enzyme from maize [J]. Plant Physiol，1999，119(1):255-266
83. Schindler I，Renz A，Schmid F X， et al. Activation of spinach pullulanase by reduction results in adecrease in the number of isomeric forms [J]. Bba-protein Struct M，2001，1548(2):175-186
84. Dumbrepatil A B，Choi J-H，Park J T， et al. Structural features of the Nostoc punctiformedebranching enzyme reveal the basis of its mechanism and substrate specificity [J]. Proteins: Struct, Funct,Bioinf，2010，78(2):348-356
85. Tabata S, Hizukuri S. Properties of yeast debranching enzyme and its specificity toward branchedcyclodextrins [J]. Eur J Biochem，1992，206(2):345-348
86. Stenholm K, Home S. A new approach to limit dextrinase and its role in mashing [J]. J Inst Brewing，1999，105(4):205-210
87. Nakamura Y，Umemoto T，Ogata N， et al. Starch debranching enzyme (R-enzyme or pullulanase)from developing rice endosperm: Purification, cDNA and chromosomal localization of the gene [J]. Planta，1996，199(2):209-218
88. MacGregor A W，Bazin S L，Macri L J， et al. Modelling the contribution of alpha-amylase,beta-amylase and limit dextrinase to starch degrading during mashing [J]. J Cereal Sci，1999，29:161-169
89. Harada T，Misaki A，Akai H， et al. Characterization of Pseudomonas isoamylase by its actions onamylopectin and glycogen: comparison with Aerobacter pullulanase [J]. Biochim Biophys Acta，1972，268(2):497-505
90. Kainuma K，Kobayashi S, Harada T. Action of Pseudomonas isoamylase on various branched oligoand poly-saccharides [J]. Carbohyd Res，1978，61:345-357
91. Slomińska L, M czyński M. Studies on the application of pullulanase in starch saccharificationprocess [J]. Starch-St rke，1985，37(11):386-390
92. Takizawa N, Murooka Y. Cloning of the pullulanase gene and overproduction of pullulanase inEscherichia coli and Klebsiella aerogenes [J]. Appl Environ Microbiol，1985，49(2):294-298
93. Bisgard-frantzen H (Bagsverd, DK), Svendsen, Allan (Birkerod, DK). Starch debranching enzymes[P]. United States.2008.
94. Crabb W D, Shetty J K. Commodity scale production of sugars from starches [J]. Curr OpinMicrobiol，1999，2(3):252-256
95. van der Maarel M J E C. Starch-processing enzymes [M]：2nd ed. Singapore：Wiley-Blackwell，2009.320-331
96. Jòzef S. The use of starch processing enzymes in the food industry [M]. Berlin：Springer，2007.19-34
97.张志达，张蓉真，邱宏端，等.高麦芽糖浆的工艺研究[J].中国粮油学报，1995，10(1):26-33
98.寿泉洪，杨国军,徐大新.应用普鲁兰酶提高黄酒残糟出酒率研究[J].酿酒，2006，33(1):53-55
99. Yim D K，Park Y H, Park Y H. Production of branched cyclodextrins by reverse reaction of microbialdebranching enzymes [J]. Starch-St rke，1997，49(2):75-78
100.张永伟，徐学明，赵建伟，等.普鲁兰酶逆向合成麦芽糖基-α-环糊精[J].食品工业与发酵，2009，35:46-49
101.王少杰,金征宇.普鲁兰酶逆向合成麦芽糖基-CD的工艺研究[J].食品工业科技，2005，26(9):105-107
102.崔波,金征宇.普鲁蓝酶逆向合成麦芽糖基β-环状糊精[J].食品科学，2005，26(12):128-131
103.唐宝英，朱晓慧,刘佳.耐酸耐热普鲁兰酶菌株的筛选及发酵条件的研究[J].微生物学通报，2001，(01):39-43
104. Asp N-G, Bj rck I. Resistant starch [J]. Trends Food Sci Tech，1992，3:111-114
105. Te Wierik G H，Eissens A C，Besemer A C，et al. Preparation, characterization, and pharmaceuticalapplication of linear dextrins. I. Preparation and characterization of amylodextrin, metastable amylodextrins,and metastable amylose [J]. Pharmaceut Res，1993，10(9):1274-1279
106. Katsuragi N，Takizawa N, Murooka Y. Entire nucleotide sequence of the pullulanase gene ofKlebsiella aerogenes W70[J]. J Bacteriol，1987，169(5):2301-2306
107. Lebbink J H G， Bertoldo C， Tibbelin G， et al. Crystallization and preliminary X-raycrystallographic studies of the thermoactive pullulanase type I, hydrolyzing alpha-1,6glycosidic linkages,from Fervidobacterium pennivorans Ven5[J]. Acta Crystallogr D Biol Crytstallogr，2000，56:1470-1472
108. Bibel M，Brettl C，Gosslar U， et al. Isolation and analysis of genes for amylolytic enzymes of thehyperthermophilic bacterium Thermotoga maritima [J]. FEMS Microbiol Lett，1998，158(1):9-15
109. Stefanova M E，Schwerdtfeger R，Antranikian G， et al. Heat-stable pullulanase from Bacillusacidopullulyticus: characterization and refolding after guanidinium chloride-induced unfolding [J].Extremophiles，1999，3(2):147-152
110. Albertson G D，McHale R H，Gibbs M D， et al. Cloning and sequence of a type I pullulanase froman extremely thermophilic anaerobic bacterium, Caldicellulosiruptor saccharolyticus [J]. Bba-gene StructExpr，1997，1354(1):35-39
111. Kornacker M G, Pugsley A P. The normally periplasmic enzyme β-lactamase is specifically andefficiently translocated through the Escherichia coli outer membrane when it is fused to the cell-surfaceenzyme pullulanase [J]. Mol Microbiol，1990，4(7):1101-1109
112. Kuriki T， Park J, Imanaka T. Characteristics of thermostable pullulanase from Bacillusstearothermophilus and the nucleotide sequence of the gene [J]. J Ferment Bioeng，1990，69(4):204-210
113. Kashiwabara S-i，Ogawa S，Miyoshi N， et al. Three domains comprised in thermostable molecularweight54,000pullulanase of type I from Bacillus flavocaldarius KP1228[J]. Biosci Biotechnol Biochem，1999，62(10):1737-1748
114. Murooka Y, Ikeda R. Biosynthesis and secretion of pullulanase, a lipoprotein from Klebsiellaaerogenes [J]. J Biol Chem，1989，264(29):17524-17531
115. Ohdan K，Kuriki T，Kaneko H， et al. Characteristics of two forms of α-amylases and structuralimplication [J]. Appl Environ Microbiol，1999，65(10):4652-4658
116. Renz A，Schikora S，Schmid R， et al. cDNA sequence and heterologous expression of monomericspinach pullulanase: multiple isomeric forms arise from the same polypeptide [J]. Biochem J，1998，331:937-945
117. Kuriki T, Imanaka T. The concept of the α-amylase family: structural similarity and commoncatalytic mechanism [J]. J Biosci Bioeng，1999，87(5):557-565
118. Kuriki T, Imanaka T. Nucleotide sequence of the neopullulanase gene from Bacillusstearothermophilus [J]. J Gen Microbiol，1989，135(6):1521-1528
119. Yamashita M，Matsumoto D, Murooka Y. Amino acid residues specific for the catalytic actiontowards α-1,6-glucosidic linkages in Klebsiella pullulanase [J]. J Ferment Bioeng，1997，84(4):283-290
120. Takizawa N, Murooka Y. Intergeneric transfer of the pullulanase gene between Klebsiella aerogenesand Escherichia coli by in vivo genetic manipulation [J]. Agric Biol Chem，1984，48(6):1451-1458
121. Takizawa N，Shiro H，Hatta T， et al. Extracellular production of Klebsiella pullulanase byEscherichia coli that carries the pullulanase secretion genes [J]. Agric Biol Chem，1991，55(6):1467-1473
122. Janse B J H, Pretorius I S. Expression of the Klebsiella pneumoniae pullulanase-encoding gene inSaccharomyces cerevisiae [J]. Curr Genet，1993，24(1):32-37
123. Kuriki T，Park J H，Okada S， et al. Purification and characterization of thermostable pullulanasefrom Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis [J].Appl Environ Microbiol，1988，54(11):2881-2883
124. Xu B，Yang Y J, Huang Z X. Cloning and overexpression of gene encoding the pullulanase fromBacillus naganoensis in Pichia pastoris [J]. J Microbiol Biotechn，2006，16(8):1185-1191
125. Studier F W. Protein production by auto-induction in high-density shaking cultures [J]. Protein ExprPurif，2005，41(1):207-234
126. Sivashanmugam A，Murray V，Cui C， et al. Practical protocols for production of very high yieldsof recombinant proteins using Escherichia coli [J]. Protein Sci，2009，18(5):936-948
127. Blommel P G，Becker K J，Duvnjak P， et al. Enhanced bacterial protein expression duringauto-induction obtained by alteration of lac repressor dosage and medium composition [J]. BiotechnolProg，2007，23:585-598
128. Li Z，Kessler W，van den Heuvel J， et al. Simple defined autoinduction medium for high-levelrecombinant protein production using T7-based Escherichia coli expression systems [J]. Appl MicrobiolBiotechnol，2011，91(4):1203-1213
129. Placido D，Fernandes C G，Isidro A， et al. Auto-induction and purification of a Bacillus subtilistransglutaminase (Tgl) and its preliminary crystallographic characterization [J]. Protein Expr Purif，2008，59(1):1-8
130. Tyler R C，Sreenath H K，Singh S，et al. Auto-induction medium for the production of [U-15N]-and[U-13C, U-15N]-labeled proteins for NMR screening and structure determination [J]. Protein Expr Purif，2005，40(2):268-278
131. Sang Yup L. High cell-density culture of Escherichia coli [J]. Trends Biotechnol，1996，14(3):98-105
132.黎鸿平，黄海婵,钟卫鸿.大肠杆菌高密度培养研究进展[J].化学与生物工程，2012，29(8):1-5
133. Riesenberg D, Guthke R. High-cell-density cultivation of microorganisms [J]. Appl MicrobiolBiotechnol，1999，51(4):422-430
134.刘子宇，李平兰，郑海涛，等.微生物高密度培养的研究进展[J].中国乳业，2005，12:47-51
135. Carneiro S，Ferreira E C, Rocha I. Metabolic responses to recombinant bioprocesses in Escherichiacoli [J]. J Biotechnol，2013，164(3):396-408
136. Choi J H，Keum K C, Lee S Y. Production of recombinant proteins by high cell density culture ofEscherichia coli [J]. Chem Eng Sci，2006，61(3):876-885
137. Shiloach J, Fass R. Growing E. coli to high cell density-A historical perspective on methoddevelopment [J]. Biotechnol Adv，2005，23(5):345-357
138. Nakano K，Rischke M，Sato S，et al. Influence of acetic acid on the growth of Escherichia coli K12during high-cell-density cultivation in a dialysis reactor [J]. Appl Microbiol Biotechnol，1997，48(5):597-601
139. Gallup D M, Gerhardt P. Dialysis fermentor systems for concentrated culture of microorganisms [J].Appl Microbiol，1963，11(6):506-512
140. P rtner R, M rkl H. Dialysis cultures [J]. Appl Microbiol Biotechnol，1998，50(4):403-414
141. Hestrin S，Avineri-Shapiro S, Aschner M. The enzymic production of levan [J]. Biochem J，1943，37(4):450-456
142. Castro G，Santopietro L, Si eriz F. Acid pullulanase from Bacillus polymyxa MIR-23[J]. ApplBiochem Biotech，1992，37(3):227-233
143. Nair S U，Singhal R S, Kamat M Y. Enhanced production of thermostable pullulanase type I usingBacillus cereus FDTA13and its mutant [J]. Food Technol Biotech，2006，44(2):275-282
144.杨华第，沈微,王正祥.普鲁兰酶产生菌的分离与鉴定[J].食品研究与开发，2007，(09):4-7
145. Yoon S H，Kim S K, Kim J F. Secretory production of recombinant protein in Escherichia coli [J].Recent Pat Biotechnol，2010，4:23-29
146. Choi J H, Lee S Y. Secretory and extracellular production of recombinant proteins using Escherichiacoli [J]. Appl Microbiol Biotechnol，2004，64(5):625-635
147. Makrides S C. Strategies for achieving high-level expression of genes in Escherichia coli [J].Microbiol Mol Biol R，1996，60(3):512-538
148. Chou C. Engineering cell physiology to enhance recombinant protein production in Escherichia coli[J]. Appl Microbiol Biotechnol，2007，76(3):521-532
149. Duan X，Chen J, Wu J. Improving the thermostability and catalytic efficiency of Bacillusderamificans pullulanase by site-directed mutagenesis [J]. Appl Environ Microbiol，2013，79(13):4072-4077
150. Duan X，Chen J, Wu J. Optimization of pullulanase production in E. coli by regulation of processconditions and supplement with natural osmolytes [J]. Bioresource Technol，2013，(0):
151. Li Y，Zhang L，Niu D， et al. Cloning, expression, characterization, and biocatalytic investigationof a novel bacilli thermostable type I pullulanase from Bacillus sp. CICIM263[J]. J Agr Food Chem，2012，60(44):11164-11172
152. R. A，N. N F, M. S S. Purification and properties of pullulanase from Bacillus halodurans [J]. IntRes J Biol Sci，2013，2(3):35-43
153. Han T，Zeng F，Li Z， et al. Biochemical characterization of a recombinant pullulanase fromThermococcus kodakarensis KOD1[J]. Lett Appl Microbiol，2013，: n/a-n/a
154. Abraham B， Orban C K， Dima R， et al. Heterologous expression of pullulanase fromFervidobacterium pennivorans in E. coli [J]. UPB Sci Bull，2013，75(2):75-80
155. Silva F，Queiroz J A, Domingues F C. Evaluating metabolic stress and plasmid stability in plasmidDNA production by Escherichia coli [J]. Biotechnol Adv，2012，30(3):691-708
156. Kesik-Brodacka M，Romanik A，Mikiewicz-Sygula D， et al. A novel system for stable, high-levelexpression from the T7promoter [J]. Microb Cell Fact，2012，11(1):109
157. Spehr V，Frahm D, Meyer T F. Improvement of the T7expression system by the use of T7lysozyme[J]. Gene，2000，257(2):259-267
158. Vethanayagam J G, Flower A M. Decreased gene expression from T7promoters may be due toimpaired production of active T7RNA polymerase [J]. Microb Cell Fact，2005，4(1):3
159. Striedner G，Pfaffenzeller I，Markus L， et al. Plasmid-free T7-based Escherichia coli expressionsystems [J]. Biotechnol Bioeng，2010，105(4):786-794
160. Walia R，Deb J K, Mukherjee K J. Stability studies with different vector backbones utilizing the T7expression system in Escherichia coli [J]. J Chem Technol Biot，2008，83(8):1120-1125
161. Fran ois B. Recombinant protein expression in Escherichia coli [J]. Curr Opin Biotech，1999，10(5):411-421
162. Corchero J L, Villaverde A. Plasmid maintenance in Escherichia coli recombinant cultures isdramatically, steadily, and specifically influenced by features of the encoded proteins [J]. BiotechnolBioeng，1998，58(6):625-632
163. Mertens N，Remaut E, Fiers W. Tight transcriptional control mechanism ensures stable high-levelexpression from T7promoter-based expression plasmids [J]. Nat Biotechnol，1995，13(2):175-179
164. Chen R. Bacterial expression systems for recombinant protein production: E. coli and beyond [J].Biotechnol Adv，2012，30(5):1102-1107
165. Petrova P，Petrov K, Stoyancheva G. Starch-modifying enzymes of lactic acid bacteria–structures,properties, and applications [J]. Starch-St rke，2013，65(1-2):34-47
166. Pérez S, Bertoft E. The molecular structures of starch components and their contribution to thearchitecture of starch granules: a comprehensive review [J]. Starch-St rke，2010，62(8):389-420
167. Hii S L，Tan J S，Ling T C， et al. Pullulanase: role in starch hydrolysis and potential industrialapplications [J]. Enzyme Res，2012，2012:921362-921362
168. van der Maarel M J E C，van der Veen B，Uitdehaag J C M， et al. Properties and applications ofstarch-converting enzymes of the α-amylase family [J]. J Biotechnol，2002，94(2):137-155
169. Haki G D, Rakshit S K. Developments in industrially important thermostable enzymes: a review [J].Bioresource Technol，2003，89(1):17-34
170.谢银珠，沈微,王正祥.酸性普鲁兰酶基因在地衣芽孢杆菌中的表达[J].食品与发酵工业，2011，37(2):7-10
171.朱梦，孙海彦,彭明.普鲁兰酶产生菌的筛选鉴定与发酵条件的研究[J].食品研究与开发，2011，32(7):136-140
172.周念波，魏丙卓，孙杰，等.普鲁兰酶产生菌的筛选及部分酶学性质研究[J].现代农业科技，2008，(13):7-8
173. Ben Messaoud E，Ben Ammar Y，Mellouli L， et al. Thermostable pullulanase type I from newisolated Bacillus thermoleovorans US105: cloning, sequencing and expression of the gene in E. coli [J].Enzyme Microb Tech，2002，31(6):827-832
174. Roy A，Messaoud E B, Bejar S. Isolation and purification of an acidic pullulanase type II fromnewly isolated Bacillus sp. US149[J]. Enzyme Microb Tech，2003，33(5):720-724
175.魏丙卓，孙杰，王晶，等.普鲁兰酶产生菌的筛选、鉴定及其发酵条件优化[J].化学与生物工程，2008，25(10):35-38
176.郭宏文，江成英，江洁，等.普鲁兰酶产生菌的筛选[J].食品研究与开发，2008，29(9):52-55
177. Antranikian G，Vorgias C E, Bertoldo C. Extreme environments as a resource for microorganismsand novel biocatalysts [J]. Adv Biochem Eng Biotechnol，2005，96:219-262
178. Vandenburg B. Extremophiles as a source for novel enzymes [J]. Curr Opin Microbiol，2003，6(3):213-218
179. Tomimura E H, JP). Thermoduric and aciduric pullulanase enzyme and method for its production [P].United States19911.
180.程池.普鲁兰酶Promozyme200L及其生产菌种[J].食品与发酵工业，1992，(6):72-76
181. Tomimura M K E. A plate culture method for the simultaneous detection of bacterial producingpulluan-and or starch-hydrolyzing enzymes [J]. Agric Biol Chem，1985，49(5):1529-1530
182. Ruijssenaars H J, Hartmans S. Plate screening methods for the detection ofpolysaccharase-producing microorganisms [J]. Appl Environ Microbiol，2001，55:143-149
183.陈均辉，李俊，张冬梅，等.生物化学实验[M]：第4版.北京：科学出版社，2008.
184.萧能赓，余瑞元，袁明秀，等.生物化学实验原理和方法[M]：第2版.北京：北京大学出版社，2005.
185.周德庆.微生物学实验教程[M]：第2版.北京：高等教育出版社，2006.
186. Israilides C J，Smith A，Harthill J E， et al. Pullulan content of the ethanol prfecipitate fromfermented agro-industrial wastes [J]. Appl Microbiol Biotechnol，1998，49:613-617
187.沈萍,陈向东.微生物学实验[M]：第4版.北京：高等教育出版社，2007.
188.东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京：科学出版社，2001.
189. Riichiro O, Seinosuke U. Optimum medium components and culture conditions for the production ofintra-and extra-cellular pullulanase by Aerobacter aerogenes [J]. J Fac Agr Kyushu U，1980，23:135-151
190. Zitouni M，Fortin M，Thibeault J-S， et al. A dye-labelled soluble substrate for the assay ofendo-chitosanase activity [J]. Carbohyd Polym，2010，80(2):521-524
191. Santoshkumar M，Nayak A，Anjaneya O， et al. A plate method for screening of bacteria capable ofdegrading aliphatic nitriles [J]. J Ind Microbiol Biot，2010，37(1):111-115
192. Ten L N，Im W T，Kim M K，et al. A plate assay for simultaneous screening of polysaccharide-andprotein-degrading micro-organisms [J]. Lett Appl Microbiol，2005，40(2):92-98
193. Rinderknecht H，Wilding P, Haverback B J. A new method for the determination of α-amylase [J].Experientia，1967，23(10):805
194. Gaidhani H K，McNeil B, Ni X. Fermentation of pullulan using an oscillatory baffled fermenter [J].Chem Eng Res Des，2005，83(6):640-645
195. Peiris J S M，Lai S T，Poon L L M， et al. Coronavirus as a possible cause of severe acuterespiratory syndrome [J]. The Lancet，2003，361(9366):1319-1325
196. Ksiazek T G，Erdman D，Goldsmith C S， et al. A novel coronavirus associated with severe acuterespiratory syndrome [J]. New Engl J Med，2003，348(20):1953-1966
197. Nisha M, Satyanarayana T. Thermophilic microbes in environmental and industrial biotechnology[M].：Springer，2013.535-587
198. d'Enfert C，Chapon C, Pugsley A P. Export and secretion of the lipoprotein pullulanase by Klebsiellapneumoniae [J]. Mol Microbiol，1987，1(3):107-116
199. Wang L，Ridgway D，Gu T，et al. Effects of process parameters on heterologous protein productionin Aspergillus niger fermentation [J]. J Chem Technol Biot，2003，78(12):1259-1266
200. Cheng J，Wu D，Chen S， et al. High-level extracellular production of alpha-cyclodextringlycosyltransferase with recombinant Escherichia coli BL21(DE3)[J]. J Agr Food Chem，2011，59(8):3797-3802
201. Terpe K. Overview of bacterial expression systems for heterologous protein production: frommolecular and biochemical fundamentals to commercial systems [J]. Appl Microbiol Biotechnol，2006，72(2):211-222
202. Aehle W. Enzymes in Industry [M]：3rd ed. Germany：Wiley，2007.
203.华子春.蛋白质高效表达技术[M].北京：化学工业出版社，2011.
204. Demain A L, Vaishnav P. Production of recombinant proteins by microbes and higher organisms [J].Biotechnol Adv，2009，27(3):297-306
205. Tolia N H, Joshua-Tor L. Strategies for protein coexpression in Escherichia coli [J]. Naturemethods，2006，3(1):55-64
206. Vincentelli R, Romier C. Expression in Escherichia coli: becoming faster and more complex [J].Curr Opin Struc Biol，2013，23(3):326-334
207.奥斯伯F M，布伦特R，金斯顿R E，等.精编分子生物学实验指南[M]：第5版.金由辛等译.北京：科学出版社，2008.
208.陈德富,陈喜文.现代分子生物学实验原理与技术[M].北京：科学出版社，2006.
209. Lee K S，Kim J S，Heo P， et al. Characterization of Saccharomyces cerevisiae promoters forheterologous gene expression in Kluyveromyces marxianus [J]. Appl Microbiol Biotechnol，2012，:
210. Pinto-Tomas A A，Anderson M A，Suen G，et al. Symbiotic nitrogen fixation in the fungus gardensof leaf-cutter ants [J]. Science，2009，326(5956):1120-1123
211. Fouts D E，Tyler H L，DeBoy R T， et al. Complete genome sequence of the N2-fixing broad hostrange endophyte Klebsiella pneumoniae342and virulence predictions verified in mice [J]. PLoS Genetics，2008，4(7): e1000141
212. E S G, J D. A strategy for in vivo screening of subtilisin E reaction specificity in E. coli perplasm [J].Biotechnol Bioeng，2002，78:761-169
213. Nouwen N，Ranson N，Saibil H， et al. Secretin PulD: Association with pilot PulS, structure, andion-conducting channel formation [J]. Proc Natl Acad Sci USA，1999，96(14):8173-8177
214. d'Enfert C, Pugsley A P. Klebsiella pneumoniae pulS gene encodes an outer membrane lipoproteinrequired for pullulanase secretion [J]. J Bacteriol，1989，171(7):3673-3679
215. Kornacker M G, Pugsley A P. Molecular characterization of pulA and its product, pullulanase, asecreted enzyme of Klebsiella pneumoniae UNF5023[J]. Mol Microbiol，1989，4(1):73-85
216. Reyss I, Pugsley A P. Five additional genes in the pulC-O operon of the gram-negative bacteriumKlebsiella oxytoca UNF5023which are required for pullulanase secretion.[J]. Mol Gen Genet，1990，222(2-3):176-184
217. Pugsley A P, Reyss I. Five genes at the3' end of the Klebsiella pneumoniae pulC operon arerequired for pullulanase secretion [J]. Mol Microbiol，1990，4(3):365-379
218. Dupuy B，Taha M K，Possot O， et al. PulO, a component of the pullulanase secretion pathway ofKlebsiella oxytoca, correctly and efficiently processes gonococcal type IV prepilin in Escherichia coli [J].Mol Microbiol，1992，6(14):1887-1894
219. Michaelis S，Chapon C，d'Enfert C， et al. Characterization and expression of the structural gene forpullulanase, a maltose-inducible secreted protein of Klebsiella pneumoniae [J]. J Bacteriol，1985，164(2):633-638
220. Kuroiwa T，Shoda H，Ichikawa S， et al. Immobilization and stabilization of pullulanase fromKlebsiella pneumoniae by a multipoint attachment method using activated agar gel supports [J]. ProcessBiochem，2005，40(8):2637-2642
221. Lee M-S，Hseu Y-C，Lai G-H， et al. High yield expression in a recombinant E. coli of a codonoptimized chicken anemia virus capsid protein VP1useful for vaccine development [J]. Microb Cell Fact，2011，10:56
222. Li Z， Nimtz M, Rinas U. Optimized procedure to generate heavy isotope andselenomethionine-labeled proteins for structure determination using Escherichia coli-based expressionsystems [J]. Appl Microbiol Biotechnol，2011，92(4):823-833
223. Paracuellos P， hman A，Sauer-Eriksson A E， et al. Expression and purification of SfaXII, aprotein involved in regulating adhesion and motility genes in extraintestinal pathogenic Escherichia coli [J].Protein Expr Purif，2012，86(2):127-134
224. Mergulh o F J M，Summers D K, Monteiro G A. Recombinant protein secretion in Escherichia coli[J]. Biotechnol Adv，2005，23(3):177-202
225. Sauvonnet N, Pugsley A P. Identification of two regions of Klebsiella oxytoca pullulanase thattogether are capable of promoting β-lactamase secretion by the general secretory pathway [J]. MolMicrobiol，1996，22(1):1-7
226. Rodríguez-Carmona E，Cano-Garrido O，Dragosits M， et al. Recombinant Fab expression andsecretion in Escherichia coli continuous culture at medium cell densities: influence of temperature [J].Process Biochem，2012，47(3):446-452
227. Yamabhai M，Emrat S，Sukasem S， et al. Secretion of recombinant Bacillus hydrolytic enzymesusing Escherichia coli expression systems [J]. J Biotechnol，2008，133(1):50-57
228. Donovan R S，Robinson C W, Glick B R. Review: Optimizing inducer and culture conditions forexpression of foreign proteins under the control of the lac promoter [J]. J Ind Microbiol Biot，1996，16(3):145-154
229. Glick B R. Metabolic load and heterologous gene expression [J]. Biotechnol Adv，1995，13(2):247-261
230. Bentley W E，Davis R H, Kompala D S. Dynamics of induced CAT expression in E. coli [J].Biotechnol Bioeng，1991，38(7):749-760
231. Ohba R, Ueda S. Purification, crystallization and some properties of intracellular pullulanase fromAerobacter aerogenes [J]. Agric Biol Chem，1973，37(12):2821-2826
232. Ueda S, Ohba R. Purification, crystallization and some properties of extracellular pullulanase fromAerobacter aerogenes [J]. Agric Biol Chem，1972，36(13):2381-2391
233. Sarduy E S，Mu oz A C，Trejo S A， et al. High-level expression of Falcipain-2in Escherichia coliby codon optimization and auto-induction [J]. Protein Expr Purif，2012，83(1):59-69
234. Xu J， Li W， Wu J， et al. Stability of plasmid and expression of a recombinantgonadotropin-releasing hormone (GnRH) vaccine in Escherichia coli [J]. Appl Microbiol Biotechnol，2006，73(4):780-788
235. Ding R，Li Z，Chen S， et al. Enhanced secretion of recombinant α-cyclodextrin glucosyltransferasefrom E. coli by medium additives [J]. Process Biochem，2010，45(6):880-886
236. Wong M S，Wu S，Causey T B， et al. Reduction of acetate accumulation in Escherichia colicultures for increased recombinant protein production [J]. Metab Eng，2008，10(2):97-108
237. Jana S, Deb J K. Strategies for efficient production of heterologous proteins in Escherichia coli [J].Appl Microbiol Biotechnol，2005，67(3):289-298
238. S rensen H P, Mortensen K K. Advanced genetic strategies for recombinant protein expression inEscherichia coli [J]. J Biotechnol，2005，115(2):113-128
239. Li Z，Gu Z，Wang M， et al. Delayed supplementation of glycine enhances extracellular secretionof the recombinant α-cyclodextrin glycosyltransferase in Escherichia coli [J]. Appl Microbiol Biotechnol，2010，85(3):553-561
240. Arnesen S，Havn Eriksen S，Olsen J，et al. Increased production of α-amylase from Thermomyceslanuginosus by the addition of Tween80[J]. Enzyme Microb Tech，1998，23(3–4):249-252
241. Yang J，Moyana T，MacKenzie S， et al. One hundred seventy-fold increase in excretion of an FVfragment-tumor necrosis factor alpha fusion protein (sFV/TNF-α) from Escherichia coli caused by thesynergistic effects of glycine and Triton X-100[J]. Appl Environ Microbiol，1998，64(8):2869-2874
242. Hammes W，Schleifer K H, Kandler O. Mode of action of glycine on the biosynthesis ofpeptidoglycan [J]. J Bacteriol，1973，116(2):1029-1053
243. Thies E，Jenkins T, Stutzenberger F. Effects of the detergent Tween80on Thermomonosporacurvata [J]. World J Microb Biot，1994，10(6):657-663
244.陈坚,李寅.发酵过程优化原理与实践[M].北京：化学工业出版社，2002.
245.乔宇，丁宏标，闫俊艳，等.重组大肠杆菌产普鲁兰酶的高密度发酵工艺研究[J].生物技术进展，2012，2(3):195-200
2. Bender H，Lehmann J, Wallenfels K. Pullulan, an extracellular glucan from Pullularia pullulans [J].Biochimica Et Biophysica Acta，1959，36:309-316
3. Leathers T D. Biotechnological production and applications of pullulan [J]. Appl MicrobiolBiotechnol，2003，62(5):468-473
4. Kulicke W M, Heinze T. Improvements in polysaccharides for use as blood plasma expanders [J].Macromol Symp，2005，231(1):47-59
5. Doman-Pytka M, Bardowski J. Pullulan degrading enzymes of bacterial origin [J]. Crit Rev Microbiol，2004，30(2):107-121
6. Suginoshita Y，Tabata Y，Matsumura T， et al. Liver targeting of human interferon-β with pullulanbased on metal coordination [J]. J Control Release，2002，83(1):75-88
7. Kumar P, Satyanarayana T. Microbial glucoamylases: characteristics and applications [J]. Crit RevBiotechnol，2009，29(3):225-255
8. Feng P H，Berensmeier S，Buchholz K， et al. Production, purification, and characterization ofThermoanaerobacterium thermosaccharolyticum glucoamylase [J]. Starch-starke，2002，54(8):328-337
9. Bertoldo C，Duffner F，Jorgensen P L， et al. Pullulanase type I from Fervidobacterium pennavoransVen5: cloning, sequencing, and expression of the gene and biochemical characterization of the recombinantenzyme [J]. Appl Environ Microbiol，1999，65(5):2084-2091
10. Bertoldo C，Armbrecht M，Becker F， et al. Cloning, sequencing, and characterization of a heat-andalkali-stable type I pullulanase from Anaerobranca gottschalkii [J]. Appl Environ Microbiol，2004，70(6):3407-3416
11. Kim H J，Park J N，Kim H O， et al. Cloning and expression of a Paenibacillus sp neopullulanasegene in Saccharomyces cerevisiae producing Schwanniomyces occidentalis glucoamylase [J]. J MicrobiolBiotechn，2002，12(2):340-344
12. Matzke J，Herrmann A，Schneider E， et al. Gene cloning, nucleotide sequence and biochemicalproperties of a cytoplasmic cyclomaltodextrinase (neopullulanase) from Alicyclobacillus acidocaldarius,reclassification of a group of enzymes [J]. FEMS Microbiol Lett，2000，183(1):55-61
13. Imanaka T, Kuriki T. Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan [J].J Bacteriol，1989，171(1):369-374
14. Aoki H，Yopi, Sakano Y. Molecular cloning and heterologous expression of the isopullulanase genefrom Aspergillus niger A.T.C.C.9642[J]. Biochem J，1997，323(3):757-764
15. Mizuno M，Koide A，Yamamura A， et al. Crystal structure of Aspergillus niger isopullulanase, amember of glycoside hydrolase family49[J]. J Mol Biol，2008，376(1):210-220
16. Akeboshi H，Kashiwagi Y，Aoki H， et al. Construction of an efficient expression system forAspergillus isopullulanase in Pichia pastoris, and a simple purification method [J]. Biosci Biotech Bioch，2003，67(5):1149-1153
17. Bertoldo C, Antranikian G. Starch-hydrolyzing enzymes from thermophilic archaea and bacteria [J].Curr Opin Chem Biol，2002，6(2):151-160
18. Niehaus F，Peters A，Groudieva T， et al. Cloning, expression and biochemical characterisation of aunique thermostable pullulan-hydrolysing enzyme from the hyperthermophilic archaeon Thermococcusaggregans [J]. FEMS Microbiol Lett，2000，190(2):223-229
19. Zhang Y，Liu Y-h，Li Y， et al. Extracellular expression of pullulanase from Bacillus naganoensis inEscherichia coli [J]. Ann Microbiol，2012，63(1):289-294
20. Kunamneni A, Singh S. Improved high thermal stability of pullulanase from a newly isolatedthermophilic Bacillus sp AN-7[J]. Enzyme Microb Tech，2006，39(7):1399-1404
21. Saha B C, Zeikus J G. Novel highly thermostable pullulanase from thermophiles [J]. TrendsBiotechnol，1989，7(9):234-239
22. Abdullah M, French D. Reversible action of pulluanase [J]. Nature，1966，210:200
23. Abdullah M，Catley B. J，Lee E. Y. C， et al. The mechanism of carbohydrase action. II. pullulanase,an enzyme specific forthe hydrolysis of alpha-1,6-bonds in amylous oligosaccharides.[J]. Cereal Chem，1966，43:111-118
24. Bender H, Wallenfels K. Untersuchungen an pullulan. II. spezifischer abbau durch ein bakteriellesEnzym.[J]. Biochemische Zeitschrift，1961，334:79-95
25. Pugsley A P，Francetic O，Hardie K， et al. Pullulanase: model protein substrate for the generalsecretory pathway of gram-negative bacteria [J]. Folia Microbiol，1997，42(3):184-192
26. Mathupala S P，Lowe S E，Podkovyrov S M， et al. Sequencing of the amylopullulanase(APU) geneof Thermoanaerobacter ethanlicus39E, and identification of the active-site by site-directed mutagenesis [J].J Biol Chem，1993，268(22):16332-16344
27. Ganhofner D， Kellermann J， Staudenbauer W， et al. Purification and properties of anamylopullulanase, a glucoamylase, and an α-glucosidase in the amylolytic enzyme system ofThermoanaerobacterium thermosaccharolyticum [J]. Biosci Biotech Bioch，1998，62(2):302-308
28. Hii S L，Ling T C，Rosfarizan M， et al. Characterization of pullulanase type II from Bacillus cereusH1.5[J]. Am J Biochem Biotechnol，2009，5(4):170-179
29. Erra-Pujada M，Debeire P，Duchiron F， et al. The type II pullulanase of Thermococcushydrothermalis: molecular characterization of the gene and expression of the catalytic domain [J]. JBacteriol，1999，181(10):3284-3287
30. Duffner F，Bertoldo C，Andersen J T， et al. A new thermoactive pullulanase from Desulfurococcusmucosus: cloning, sequencing, purification, and characterization of the recombinant enzyme afterexpression in Bacillus subtilis [J]. J Bacteriol，2000，182(22):6331-6338
31. Abdullah M, French D. Substrate specificity of pullulanase [J]. Arch Biochem Biophys，1970，137(2):483-493
32. Drummond G S，Smith E E，Whelan W J， et al. Mechanism of action of pullulanase [J]. FEMSLetters，1969，5(1):85-88
33. Wallenfels K，Bender H, Rached J R. Pullulanase from Aerobacter aerogenes production in acell-bound state. Purification and properties of enzyme [J]. Biochem Bioph Res Co，1966，22(3):254-261
34. Bender H, Wallenfels K. Pullulanase (an amylopectin and glycogen debranching enzyme) fromAerobacter aerogenes [J]. Method Enzymol，1966，8:555-559
35. Hurst T L. Process for producing dextrose [P]. United States.1975.
36. Dessein A, Schwartz M. Is there a pullulanase in Escherichia coli?[J]. Eur J Biochem，1974，45(2):363-366
37. W ber G. Pullulanase is a characteristic of many Klebsiella species and functions in the degradationof starch [J]. Appl Microbiol Biotechnol，1976，3(1b):71-80
38. Adams K R, Priest F G. Extracellular pullulanase synthesis in Bacillus macerans [J]. FEMSMicrobiol Lett，1977，1(5):269-273
39. Morgan F J，Adams K R, Priest F G. A culture method for the detection of pullulan-degrading enzymein bacteria and its application to the Genus Bacillus [J]. J Appl Bacteriol，1979，46:291-294
40. Ram K A, Venkatasubramanian K. Enhancement of starch conversion efficiency with free andimmobilized pullulanase and α-1,4-glucosidase [J]. Biotechnol Bioeng，1982，24(2):355-369
41. Norman B E. A novel debranching enzyme for application in the glucose syrup industry [J]. Starch-St rke，1982，34(10):340-346
42. Jensen B, Norman B. Bacillus acidopullulyticus pullulanase: application and regulatory aspects foruse in the food industry [J]. Process Biochem，1984，19(4):129-134
43. Takasaki Y. Purification and enzymatic properties of β-amylase and pullulanase from Bacillus cereusvar. mycoides [J]. Agric Biol Chem，1976，40:1523-1530
44. Takasaki Y. Pullulanase-amylase complex enzyme from Bacillus subtilis [J]. Agric Biol Chem，1987，51(1):9-16
45. Tomimura E，Zeman N W，Frankiewicz J R， et al. Description of Bacillus naganoensis sp. nov [J].Int J Syst Bacteriol，1990，40(2):123-125
46. Tomimura E. Thermoduric and aciduric pullulanase enzyme and method for its production [P]. UnitedStates19911.
47. Hii S L，Ling T C，Mohamad R， et al. Enhancement of extracellular pullulanase production byRaoultella planticola DSMZ4617using optimized medium based on sago starch [J]. Open Biotechnol J，2009，3:1-8
48. Nielsen G C T, DK), Diers, Ivan V.(Vaerlose, DK), Outtrup, Helle (Vaerlose, DK), Norman, Barrie E.(Farum, DK). Debranching enzyme product, preparation and use thereof [P]. United States.1985.
49. Deweer P A, BE), Amory, Antoine (Rixensart, BE). Pullulanase producing microrganisms [P]. UnitedStates.1998.
50. Kambourova M S, Emanuilova E I. Purification and general biochemical properties of thermostablepullulanase from Bacillus stearothermoplilus G-82[J]. Appl Biochem Biotech，1992，33(3):193-203
51. Kang J，Park K-M，Choi K-H， et al. Molecular cloning and biochemical characterization of aheat-stable type I pullulanase from Thermotoga neapolitana [J]. Enzyme Microb Tech，2011，48(3):260-266
52. Lin F P, Leu K L. Cloning, expression, and characterization of thermostable region ofamylopullulanase gene from Thermoanaerobacter ethanolicus39E [J]. Appl Biochem Biotech，2002，97(1):33-44
53. Spreinat A, Antranikian G. Purification and properties of a thermostable pullulanase from Clostridiumthermosulfurogenes EM1which hydrolyses both α-1,6and α-1,4-glycosidic linkages [J]. Appl MicrobiolBiotechnol，1990，33(5):511-518
54. Kang S，Vieille C, Zeikus J G. Identification of Pyrococcus furiosus amylopullulanase catalyticresidues [J]. Appl Microbiol Biotechnol，2005，66:408-413
55. Knapp S，Rudiger A，Antranikian G， et al. Crystallization and preliminary crystallographic analysisof an amylopullulanase from the hyperthermophilic archaeon Pyrococcus woesei [J]. Proteins，1995，23(4):595-597
56. Rudiger A，Jorgensen P L, Antranikian G. Isolation and characterization of a heat-stable pullulanasefrom the hyperthermophilic archaeon Pyrococcus woesei after cloning and expression of its gene inEscherichia coli [J]. Appl Environ Microbiol，1995，61(2):567-575
57. Nair S U，Singhal R S, Kamat M Y. Induction of pullulanase production in Bacillus cereus FDTA-13[J]. Bioresource Technol，2007，98(4):856-859
58. Kim J-H，Sunako M，Ono H， et al. Characterization of the C-terminal truncated form ofamylopullulanase from Lactobacillus plantarum L137[J]. J Biosci Bioeng，2009，107(2):124-129
59. Wu S J，Kim J M，Zhou C A， et al. Estimation of pullulan by hydrolysis with pullulanase [J].Biotechnol Lett，2010，32(8):1143-1145
60. Zouari Ayadi D，Ben Ali M，Jemli S， et al. Heterologous expression, secretion and characterizationof the Geobacillus thermoleovorans US105type I pullulanase [J]. Appl Microbiol Biotechnol，2008，78(3):473-481
61. Yamashita M，Nakagawa A，Katsuragi N， et al. Role of lipid modification on a starch-debranchingenzyme, Klebsiella pullulanase: comparison of properties of lipid-modified and unmodified pullulanases [J].Mol Microbiol，1992，6(3):389-394
62. Francetic O, Pugsley A P. Towards the identification of type II secretion signals in a nonacylatedvariant of pullulanase from Klebsiella oxytoca [J]. J Bacteriol，2005，187(20):7045-7055
63. Buddelmeijer N，Francetic O, Pugsley A P. Green fluorescent chimeras indicate nonpolar localizationof pullulanase secreton components PulL and PulM [J]. J Bacteriol，2006，188(8):2928-2935
64. Mikami B，Iwamoto H，Malle D，et al. Crystal structure of pullulanase: evidence for parallel bindingof oligosaccharides in the active site [J]. J Mol Biol，2006，359(3):690-707
65. Hytonen J，Haataja S, Finne J. Use of flow cytometry for the adhesion analysis of Streptococcuspyogenes mutant strains to epithelial cells: investigation of the possible role of surface pullulanase andcysteine protease, and the transcriptional regulator Rgg [J]. BMC Microbiology，2006，6(1):18
66. Gourlay L J，Santi I，Pezzicoli A， et al. Group B Streptococcus pullulanase crystal structures in thecontext of a novel strategy for vaccine development [J]. J Bacteriol，2009，191(11):3544-3552
67. Smith K A, Salyers A A. Cell-associated pullulanase from Bacteroides thetaiotaomicron: cloning,characterization, and insertional mutagenesis to determine role in pullulan utilization [J]. J Bacteriol，1989，171(4):2116-2123
68. Kitahata S，Tanimoto T，Ikuta A， et al. Synthesis of novel heterobranched β-cyclodextrins from42-O-β-D-galactosy l-maltose and β-cyclodextrin by the reverse action of pullulanase, and isolation andcharacterization of the products [J]. Biosci Biotech Bioch，2000，64(6):1223-1229
69. Moubasher H，Wahsh S, El-Kassem N. Purification of pullulanase from Aureobasidium pullulans [J].Microbiology，2010，79(6):759-766
70.徐金利，吕明生，王淑军，等.嗜热古菌Thermococcus sp. HJ21产高温普鲁兰酶条件和酶学性质[J].食品与生物技术学报，2009，28(2):243-249
71. Gomes I，Gomes J, Steiner W. Highly thermostable amylase and pullulanase of the extremethermophilic eubacterium Rhodothermus marinus: production and partial characterization [J]. BioresourceTechnol，2003，90(2):207-214
72. Jiao Y L，Wang S J，Lv M S， et al. A GH57family amylopullulanase from deep-sea Thermococcussiculi: expression of the gene and characterization of the recombinant enzyme [J]. Curr Microbiol，2011，62(1):222-228
73. Plant A，Morgan H, Daniel R. A highly stable pullulanase from Thermus aquaticus YT-1[J]. EnzymeMicrob Tech，1986，8(11):668-672
74. Kim C H，Nashiru O, Ko J H. Purification and biochemical characterization of pullulanase type I fromThermus caldophilus GK-24[J]. FEMS Microbiol Lett，1996，138(2-3):147-152
75. Tomiyasu K，Yato K，Yasuda M， et al. Cloning and nucleotide sequence of the pullulanase gene ofThermus thermophilus HB8and production of the enzyme in Escherichia coli [J]. Biosci Biotech Bioch，2001，65(9):2090-2094
76. Masuda H，Takahashi T, Sugawara S. Purification and properties of starch hydrolyzing enzymes inmature root of sugar beets [J]. Plant Physiol，1987，84(2):361-365
77. McDougall G J，Ross H A，Swanston J S， et al. Limit dextrinase from germinating barley hasendotransglycosylase activity, which explains its activation by maltodextrins [J]. Planta，2004，218(4):542-551
78. Yamasaki Y，Nakashima S, Konno H. Pullulanase from rice endosperm [J]. Acta Biochim Pol，2008，55(3):507-510
79. Fujita N，Toyosawa Y，Utsumi Y， et al. Characterization of pullulanase (PUL)-deficient mutants ofrice (Oryza sativa L.) and the function of PUL on starch biosynthesis in the developing rice endosperm [J].J Exp Bot，2009，60(3):1009-1023
80. Wu C Y，Colleoni C，Myers A M， et al. Enzymatic properties and regulation of ZPU1, the maizepullulanase-type starch debranching enzyme [J]. Arch Biochem Biophys，2002，406(1):21-32
81. Dinges J R，Colleoni C，James M G， et al. Mutational analysis of the pullulanase-type debranchingenzyme of maize indicates multiple functions in starch metabolism [J]. Plant Cell，2003，15(3):666-680
82. Beatty M K，Rahman A，Cao H P，et al. Purification and molecular genetic characterization of ZPU1,a pullulanase-type starch-debranching enzyme from maize [J]. Plant Physiol，1999，119(1):255-266
83. Schindler I，Renz A，Schmid F X， et al. Activation of spinach pullulanase by reduction results in adecrease in the number of isomeric forms [J]. Bba-protein Struct M，2001，1548(2):175-186
84. Dumbrepatil A B，Choi J-H，Park J T， et al. Structural features of the Nostoc punctiformedebranching enzyme reveal the basis of its mechanism and substrate specificity [J]. Proteins: Struct, Funct,Bioinf，2010，78(2):348-356
85. Tabata S, Hizukuri S. Properties of yeast debranching enzyme and its specificity toward branchedcyclodextrins [J]. Eur J Biochem，1992，206(2):345-348
86. Stenholm K, Home S. A new approach to limit dextrinase and its role in mashing [J]. J Inst Brewing，1999，105(4):205-210
87. Nakamura Y，Umemoto T，Ogata N， et al. Starch debranching enzyme (R-enzyme or pullulanase)from developing rice endosperm: Purification, cDNA and chromosomal localization of the gene [J]. Planta，1996，199(2):209-218
88. MacGregor A W，Bazin S L，Macri L J， et al. Modelling the contribution of alpha-amylase,beta-amylase and limit dextrinase to starch degrading during mashing [J]. J Cereal Sci，1999，29:161-169
89. Harada T，Misaki A，Akai H， et al. Characterization of Pseudomonas isoamylase by its actions onamylopectin and glycogen: comparison with Aerobacter pullulanase [J]. Biochim Biophys Acta，1972，268(2):497-505
90. Kainuma K，Kobayashi S, Harada T. Action of Pseudomonas isoamylase on various branched oligoand poly-saccharides [J]. Carbohyd Res，1978，61:345-357
91. Slomińska L, M czyński M. Studies on the application of pullulanase in starch saccharificationprocess [J]. Starch-St rke，1985，37(11):386-390
92. Takizawa N, Murooka Y. Cloning of the pullulanase gene and overproduction of pullulanase inEscherichia coli and Klebsiella aerogenes [J]. Appl Environ Microbiol，1985，49(2):294-298
93. Bisgard-frantzen H (Bagsverd, DK), Svendsen, Allan (Birkerod, DK). Starch debranching enzymes[P]. United States.2008.
94. Crabb W D, Shetty J K. Commodity scale production of sugars from starches [J]. Curr OpinMicrobiol，1999，2(3):252-256
95. van der Maarel M J E C. Starch-processing enzymes [M]：2nd ed. Singapore：Wiley-Blackwell，2009.320-331
96. Jòzef S. The use of starch processing enzymes in the food industry [M]. Berlin：Springer，2007.19-34
97.张志达，张蓉真，邱宏端，等.高麦芽糖浆的工艺研究[J].中国粮油学报，1995，10(1):26-33
98.寿泉洪，杨国军,徐大新.应用普鲁兰酶提高黄酒残糟出酒率研究[J].酿酒，2006，33(1):53-55
99. Yim D K，Park Y H, Park Y H. Production of branched cyclodextrins by reverse reaction of microbialdebranching enzymes [J]. Starch-St rke，1997，49(2):75-78
100.张永伟，徐学明，赵建伟，等.普鲁兰酶逆向合成麦芽糖基-α-环糊精[J].食品工业与发酵，2009，35:46-49
101.王少杰,金征宇.普鲁兰酶逆向合成麦芽糖基-CD的工艺研究[J].食品工业科技，2005，26(9):105-107
102.崔波,金征宇.普鲁蓝酶逆向合成麦芽糖基β-环状糊精[J].食品科学，2005，26(12):128-131
103.唐宝英，朱晓慧,刘佳.耐酸耐热普鲁兰酶菌株的筛选及发酵条件的研究[J].微生物学通报，2001，(01):39-43
104. Asp N-G, Bj rck I. Resistant starch [J]. Trends Food Sci Tech，1992，3:111-114
105. Te Wierik G H，Eissens A C，Besemer A C，et al. Preparation, characterization, and pharmaceuticalapplication of linear dextrins. I. Preparation and characterization of amylodextrin, metastable amylodextrins,and metastable amylose [J]. Pharmaceut Res，1993，10(9):1274-1279
106. Katsuragi N，Takizawa N, Murooka Y. Entire nucleotide sequence of the pullulanase gene ofKlebsiella aerogenes W70[J]. J Bacteriol，1987，169(5):2301-2306
107. Lebbink J H G， Bertoldo C， Tibbelin G， et al. Crystallization and preliminary X-raycrystallographic studies of the thermoactive pullulanase type I, hydrolyzing alpha-1,6glycosidic linkages,from Fervidobacterium pennivorans Ven5[J]. Acta Crystallogr D Biol Crytstallogr，2000，56:1470-1472
108. Bibel M，Brettl C，Gosslar U， et al. Isolation and analysis of genes for amylolytic enzymes of thehyperthermophilic bacterium Thermotoga maritima [J]. FEMS Microbiol Lett，1998，158(1):9-15
109. Stefanova M E，Schwerdtfeger R，Antranikian G， et al. Heat-stable pullulanase from Bacillusacidopullulyticus: characterization and refolding after guanidinium chloride-induced unfolding [J].Extremophiles，1999，3(2):147-152
110. Albertson G D，McHale R H，Gibbs M D， et al. Cloning and sequence of a type I pullulanase froman extremely thermophilic anaerobic bacterium, Caldicellulosiruptor saccharolyticus [J]. Bba-gene StructExpr，1997，1354(1):35-39
111. Kornacker M G, Pugsley A P. The normally periplasmic enzyme β-lactamase is specifically andefficiently translocated through the Escherichia coli outer membrane when it is fused to the cell-surfaceenzyme pullulanase [J]. Mol Microbiol，1990，4(7):1101-1109
112. Kuriki T， Park J, Imanaka T. Characteristics of thermostable pullulanase from Bacillusstearothermophilus and the nucleotide sequence of the gene [J]. J Ferment Bioeng，1990，69(4):204-210
113. Kashiwabara S-i，Ogawa S，Miyoshi N， et al. Three domains comprised in thermostable molecularweight54,000pullulanase of type I from Bacillus flavocaldarius KP1228[J]. Biosci Biotechnol Biochem，1999，62(10):1737-1748
114. Murooka Y, Ikeda R. Biosynthesis and secretion of pullulanase, a lipoprotein from Klebsiellaaerogenes [J]. J Biol Chem，1989，264(29):17524-17531
115. Ohdan K，Kuriki T，Kaneko H， et al. Characteristics of two forms of α-amylases and structuralimplication [J]. Appl Environ Microbiol，1999，65(10):4652-4658
116. Renz A，Schikora S，Schmid R， et al. cDNA sequence and heterologous expression of monomericspinach pullulanase: multiple isomeric forms arise from the same polypeptide [J]. Biochem J，1998，331:937-945
117. Kuriki T, Imanaka T. The concept of the α-amylase family: structural similarity and commoncatalytic mechanism [J]. J Biosci Bioeng，1999，87(5):557-565
118. Kuriki T, Imanaka T. Nucleotide sequence of the neopullulanase gene from Bacillusstearothermophilus [J]. J Gen Microbiol，1989，135(6):1521-1528
119. Yamashita M，Matsumoto D, Murooka Y. Amino acid residues specific for the catalytic actiontowards α-1,6-glucosidic linkages in Klebsiella pullulanase [J]. J Ferment Bioeng，1997，84(4):283-290
120. Takizawa N, Murooka Y. Intergeneric transfer of the pullulanase gene between Klebsiella aerogenesand Escherichia coli by in vivo genetic manipulation [J]. Agric Biol Chem，1984，48(6):1451-1458
121. Takizawa N，Shiro H，Hatta T， et al. Extracellular production of Klebsiella pullulanase byEscherichia coli that carries the pullulanase secretion genes [J]. Agric Biol Chem，1991，55(6):1467-1473
122. Janse B J H, Pretorius I S. Expression of the Klebsiella pneumoniae pullulanase-encoding gene inSaccharomyces cerevisiae [J]. Curr Genet，1993，24(1):32-37
123. Kuriki T，Park J H，Okada S， et al. Purification and characterization of thermostable pullulanasefrom Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis [J].Appl Environ Microbiol，1988，54(11):2881-2883
124. Xu B，Yang Y J, Huang Z X. Cloning and overexpression of gene encoding the pullulanase fromBacillus naganoensis in Pichia pastoris [J]. J Microbiol Biotechn，2006，16(8):1185-1191
125. Studier F W. Protein production by auto-induction in high-density shaking cultures [J]. Protein ExprPurif，2005，41(1):207-234
126. Sivashanmugam A，Murray V，Cui C， et al. Practical protocols for production of very high yieldsof recombinant proteins using Escherichia coli [J]. Protein Sci，2009，18(5):936-948
127. Blommel P G，Becker K J，Duvnjak P， et al. Enhanced bacterial protein expression duringauto-induction obtained by alteration of lac repressor dosage and medium composition [J]. BiotechnolProg，2007，23:585-598
128. Li Z，Kessler W，van den Heuvel J， et al. Simple defined autoinduction medium for high-levelrecombinant protein production using T7-based Escherichia coli expression systems [J]. Appl MicrobiolBiotechnol，2011，91(4):1203-1213
129. Placido D，Fernandes C G，Isidro A， et al. Auto-induction and purification of a Bacillus subtilistransglutaminase (Tgl) and its preliminary crystallographic characterization [J]. Protein Expr Purif，2008，59(1):1-8
130. Tyler R C，Sreenath H K，Singh S，et al. Auto-induction medium for the production of [U-15N]-and[U-13C, U-15N]-labeled proteins for NMR screening and structure determination [J]. Protein Expr Purif，2005，40(2):268-278
131. Sang Yup L. High cell-density culture of Escherichia coli [J]. Trends Biotechnol，1996，14(3):98-105
132.黎鸿平，黄海婵,钟卫鸿.大肠杆菌高密度培养研究进展[J].化学与生物工程，2012，29(8):1-5
133. Riesenberg D, Guthke R. High-cell-density cultivation of microorganisms [J]. Appl MicrobiolBiotechnol，1999，51(4):422-430
134.刘子宇，李平兰，郑海涛，等.微生物高密度培养的研究进展[J].中国乳业，2005，12:47-51
135. Carneiro S，Ferreira E C, Rocha I. Metabolic responses to recombinant bioprocesses in Escherichiacoli [J]. J Biotechnol，2013，164(3):396-408
136. Choi J H，Keum K C, Lee S Y. Production of recombinant proteins by high cell density culture ofEscherichia coli [J]. Chem Eng Sci，2006，61(3):876-885
137. Shiloach J, Fass R. Growing E. coli to high cell density-A historical perspective on methoddevelopment [J]. Biotechnol Adv，2005，23(5):345-357
138. Nakano K，Rischke M，Sato S，et al. Influence of acetic acid on the growth of Escherichia coli K12during high-cell-density cultivation in a dialysis reactor [J]. Appl Microbiol Biotechnol，1997，48(5):597-601
139. Gallup D M, Gerhardt P. Dialysis fermentor systems for concentrated culture of microorganisms [J].Appl Microbiol，1963，11(6):506-512
140. P rtner R, M rkl H. Dialysis cultures [J]. Appl Microbiol Biotechnol，1998，50(4):403-414
141. Hestrin S，Avineri-Shapiro S, Aschner M. The enzymic production of levan [J]. Biochem J，1943，37(4):450-456
142. Castro G，Santopietro L, Si eriz F. Acid pullulanase from Bacillus polymyxa MIR-23[J]. ApplBiochem Biotech，1992，37(3):227-233
143. Nair S U，Singhal R S, Kamat M Y. Enhanced production of thermostable pullulanase type I usingBacillus cereus FDTA13and its mutant [J]. Food Technol Biotech，2006，44(2):275-282
144.杨华第，沈微,王正祥.普鲁兰酶产生菌的分离与鉴定[J].食品研究与开发，2007，(09):4-7
145. Yoon S H，Kim S K, Kim J F. Secretory production of recombinant protein in Escherichia coli [J].Recent Pat Biotechnol，2010，4:23-29
146. Choi J H, Lee S Y. Secretory and extracellular production of recombinant proteins using Escherichiacoli [J]. Appl Microbiol Biotechnol，2004，64(5):625-635
147. Makrides S C. Strategies for achieving high-level expression of genes in Escherichia coli [J].Microbiol Mol Biol R，1996，60(3):512-538
148. Chou C. Engineering cell physiology to enhance recombinant protein production in Escherichia coli[J]. Appl Microbiol Biotechnol，2007，76(3):521-532
149. Duan X，Chen J, Wu J. Improving the thermostability and catalytic efficiency of Bacillusderamificans pullulanase by site-directed mutagenesis [J]. Appl Environ Microbiol，2013，79(13):4072-4077
150. Duan X，Chen J, Wu J. Optimization of pullulanase production in E. coli by regulation of processconditions and supplement with natural osmolytes [J]. Bioresource Technol，2013，(0):
151. Li Y，Zhang L，Niu D， et al. Cloning, expression, characterization, and biocatalytic investigationof a novel bacilli thermostable type I pullulanase from Bacillus sp. CICIM263[J]. J Agr Food Chem，2012，60(44):11164-11172
152. R. A，N. N F, M. S S. Purification and properties of pullulanase from Bacillus halodurans [J]. IntRes J Biol Sci，2013，2(3):35-43
153. Han T，Zeng F，Li Z， et al. Biochemical characterization of a recombinant pullulanase fromThermococcus kodakarensis KOD1[J]. Lett Appl Microbiol，2013，: n/a-n/a
154. Abraham B， Orban C K， Dima R， et al. Heterologous expression of pullulanase fromFervidobacterium pennivorans in E. coli [J]. UPB Sci Bull，2013，75(2):75-80
155. Silva F，Queiroz J A, Domingues F C. Evaluating metabolic stress and plasmid stability in plasmidDNA production by Escherichia coli [J]. Biotechnol Adv，2012，30(3):691-708
156. Kesik-Brodacka M，Romanik A，Mikiewicz-Sygula D， et al. A novel system for stable, high-levelexpression from the T7promoter [J]. Microb Cell Fact，2012，11(1):109
157. Spehr V，Frahm D, Meyer T F. Improvement of the T7expression system by the use of T7lysozyme[J]. Gene，2000，257(2):259-267
158. Vethanayagam J G, Flower A M. Decreased gene expression from T7promoters may be due toimpaired production of active T7RNA polymerase [J]. Microb Cell Fact，2005，4(1):3
159. Striedner G，Pfaffenzeller I，Markus L， et al. Plasmid-free T7-based Escherichia coli expressionsystems [J]. Biotechnol Bioeng，2010，105(4):786-794
160. Walia R，Deb J K, Mukherjee K J. Stability studies with different vector backbones utilizing the T7expression system in Escherichia coli [J]. J Chem Technol Biot，2008，83(8):1120-1125
161. Fran ois B. Recombinant protein expression in Escherichia coli [J]. Curr Opin Biotech，1999，10(5):411-421
162. Corchero J L, Villaverde A. Plasmid maintenance in Escherichia coli recombinant cultures isdramatically, steadily, and specifically influenced by features of the encoded proteins [J]. BiotechnolBioeng，1998，58(6):625-632
163. Mertens N，Remaut E, Fiers W. Tight transcriptional control mechanism ensures stable high-levelexpression from T7promoter-based expression plasmids [J]. Nat Biotechnol，1995，13(2):175-179
164. Chen R. Bacterial expression systems for recombinant protein production: E. coli and beyond [J].Biotechnol Adv，2012，30(5):1102-1107
165. Petrova P，Petrov K, Stoyancheva G. Starch-modifying enzymes of lactic acid bacteria–structures,properties, and applications [J]. Starch-St rke，2013，65(1-2):34-47
166. Pérez S, Bertoft E. The molecular structures of starch components and their contribution to thearchitecture of starch granules: a comprehensive review [J]. Starch-St rke，2010，62(8):389-420
167. Hii S L，Tan J S，Ling T C， et al. Pullulanase: role in starch hydrolysis and potential industrialapplications [J]. Enzyme Res，2012，2012:921362-921362
168. van der Maarel M J E C，van der Veen B，Uitdehaag J C M， et al. Properties and applications ofstarch-converting enzymes of the α-amylase family [J]. J Biotechnol，2002，94(2):137-155
169. Haki G D, Rakshit S K. Developments in industrially important thermostable enzymes: a review [J].Bioresource Technol，2003，89(1):17-34
170.谢银珠，沈微,王正祥.酸性普鲁兰酶基因在地衣芽孢杆菌中的表达[J].食品与发酵工业，2011，37(2):7-10
171.朱梦，孙海彦,彭明.普鲁兰酶产生菌的筛选鉴定与发酵条件的研究[J].食品研究与开发，2011，32(7):136-140
172.周念波，魏丙卓，孙杰，等.普鲁兰酶产生菌的筛选及部分酶学性质研究[J].现代农业科技，2008，(13):7-8
173. Ben Messaoud E，Ben Ammar Y，Mellouli L， et al. Thermostable pullulanase type I from newisolated Bacillus thermoleovorans US105: cloning, sequencing and expression of the gene in E. coli [J].Enzyme Microb Tech，2002，31(6):827-832
174. Roy A，Messaoud E B, Bejar S. Isolation and purification of an acidic pullulanase type II fromnewly isolated Bacillus sp. US149[J]. Enzyme Microb Tech，2003，33(5):720-724
175.魏丙卓，孙杰，王晶，等.普鲁兰酶产生菌的筛选、鉴定及其发酵条件优化[J].化学与生物工程，2008，25(10):35-38
176.郭宏文，江成英，江洁，等.普鲁兰酶产生菌的筛选[J].食品研究与开发，2008，29(9):52-55
177. Antranikian G，Vorgias C E, Bertoldo C. Extreme environments as a resource for microorganismsand novel biocatalysts [J]. Adv Biochem Eng Biotechnol，2005，96:219-262
178. Vandenburg B. Extremophiles as a source for novel enzymes [J]. Curr Opin Microbiol，2003，6(3):213-218
179. Tomimura E H, JP). Thermoduric and aciduric pullulanase enzyme and method for its production [P].United States19911.
180.程池.普鲁兰酶Promozyme200L及其生产菌种[J].食品与发酵工业，1992，(6):72-76
181. Tomimura M K E. A plate culture method for the simultaneous detection of bacterial producingpulluan-and or starch-hydrolyzing enzymes [J]. Agric Biol Chem，1985，49(5):1529-1530
182. Ruijssenaars H J, Hartmans S. Plate screening methods for the detection ofpolysaccharase-producing microorganisms [J]. Appl Environ Microbiol，2001，55:143-149
183.陈均辉，李俊，张冬梅，等.生物化学实验[M]：第4版.北京：科学出版社，2008.
184.萧能赓，余瑞元，袁明秀，等.生物化学实验原理和方法[M]：第2版.北京：北京大学出版社，2005.
185.周德庆.微生物学实验教程[M]：第2版.北京：高等教育出版社，2006.
186. Israilides C J，Smith A，Harthill J E， et al. Pullulan content of the ethanol prfecipitate fromfermented agro-industrial wastes [J]. Appl Microbiol Biotechnol，1998，49:613-617
187.沈萍,陈向东.微生物学实验[M]：第4版.北京：高等教育出版社，2007.
188.东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京：科学出版社，2001.
189. Riichiro O, Seinosuke U. Optimum medium components and culture conditions for the production ofintra-and extra-cellular pullulanase by Aerobacter aerogenes [J]. J Fac Agr Kyushu U，1980，23:135-151
190. Zitouni M，Fortin M，Thibeault J-S， et al. A dye-labelled soluble substrate for the assay ofendo-chitosanase activity [J]. Carbohyd Polym，2010，80(2):521-524
191. Santoshkumar M，Nayak A，Anjaneya O， et al. A plate method for screening of bacteria capable ofdegrading aliphatic nitriles [J]. J Ind Microbiol Biot，2010，37(1):111-115
192. Ten L N，Im W T，Kim M K，et al. A plate assay for simultaneous screening of polysaccharide-andprotein-degrading micro-organisms [J]. Lett Appl Microbiol，2005，40(2):92-98
193. Rinderknecht H，Wilding P, Haverback B J. A new method for the determination of α-amylase [J].Experientia，1967，23(10):805
194. Gaidhani H K，McNeil B, Ni X. Fermentation of pullulan using an oscillatory baffled fermenter [J].Chem Eng Res Des，2005，83(6):640-645
195. Peiris J S M，Lai S T，Poon L L M， et al. Coronavirus as a possible cause of severe acuterespiratory syndrome [J]. The Lancet，2003，361(9366):1319-1325
196. Ksiazek T G，Erdman D，Goldsmith C S， et al. A novel coronavirus associated with severe acuterespiratory syndrome [J]. New Engl J Med，2003，348(20):1953-1966
197. Nisha M, Satyanarayana T. Thermophilic microbes in environmental and industrial biotechnology[M].：Springer，2013.535-587
198. d'Enfert C，Chapon C, Pugsley A P. Export and secretion of the lipoprotein pullulanase by Klebsiellapneumoniae [J]. Mol Microbiol，1987，1(3):107-116
199. Wang L，Ridgway D，Gu T，et al. Effects of process parameters on heterologous protein productionin Aspergillus niger fermentation [J]. J Chem Technol Biot，2003，78(12):1259-1266
200. Cheng J，Wu D，Chen S， et al. High-level extracellular production of alpha-cyclodextringlycosyltransferase with recombinant Escherichia coli BL21(DE3)[J]. J Agr Food Chem，2011，59(8):3797-3802
201. Terpe K. Overview of bacterial expression systems for heterologous protein production: frommolecular and biochemical fundamentals to commercial systems [J]. Appl Microbiol Biotechnol，2006，72(2):211-222
202. Aehle W. Enzymes in Industry [M]：3rd ed. Germany：Wiley，2007.
203.华子春.蛋白质高效表达技术[M].北京：化学工业出版社，2011.
204. Demain A L, Vaishnav P. Production of recombinant proteins by microbes and higher organisms [J].Biotechnol Adv，2009，27(3):297-306
205. Tolia N H, Joshua-Tor L. Strategies for protein coexpression in Escherichia coli [J]. Naturemethods，2006，3(1):55-64
206. Vincentelli R, Romier C. Expression in Escherichia coli: becoming faster and more complex [J].Curr Opin Struc Biol，2013，23(3):326-334
207.奥斯伯F M，布伦特R，金斯顿R E，等.精编分子生物学实验指南[M]：第5版.金由辛等译.北京：科学出版社，2008.
208.陈德富,陈喜文.现代分子生物学实验原理与技术[M].北京：科学出版社，2006.
209. Lee K S，Kim J S，Heo P， et al. Characterization of Saccharomyces cerevisiae promoters forheterologous gene expression in Kluyveromyces marxianus [J]. Appl Microbiol Biotechnol，2012，:
210. Pinto-Tomas A A，Anderson M A，Suen G，et al. Symbiotic nitrogen fixation in the fungus gardensof leaf-cutter ants [J]. Science，2009，326(5956):1120-1123
211. Fouts D E，Tyler H L，DeBoy R T， et al. Complete genome sequence of the N2-fixing broad hostrange endophyte Klebsiella pneumoniae342and virulence predictions verified in mice [J]. PLoS Genetics，2008，4(7): e1000141
212. E S G, J D. A strategy for in vivo screening of subtilisin E reaction specificity in E. coli perplasm [J].Biotechnol Bioeng，2002，78:761-169
213. Nouwen N，Ranson N，Saibil H， et al. Secretin PulD: Association with pilot PulS, structure, andion-conducting channel formation [J]. Proc Natl Acad Sci USA，1999，96(14):8173-8177
214. d'Enfert C, Pugsley A P. Klebsiella pneumoniae pulS gene encodes an outer membrane lipoproteinrequired for pullulanase secretion [J]. J Bacteriol，1989，171(7):3673-3679
215. Kornacker M G, Pugsley A P. Molecular characterization of pulA and its product, pullulanase, asecreted enzyme of Klebsiella pneumoniae UNF5023[J]. Mol Microbiol，1989，4(1):73-85
216. Reyss I, Pugsley A P. Five additional genes in the pulC-O operon of the gram-negative bacteriumKlebsiella oxytoca UNF5023which are required for pullulanase secretion.[J]. Mol Gen Genet，1990，222(2-3):176-184
217. Pugsley A P, Reyss I. Five genes at the3' end of the Klebsiella pneumoniae pulC operon arerequired for pullulanase secretion [J]. Mol Microbiol，1990，4(3):365-379
218. Dupuy B，Taha M K，Possot O， et al. PulO, a component of the pullulanase secretion pathway ofKlebsiella oxytoca, correctly and efficiently processes gonococcal type IV prepilin in Escherichia coli [J].Mol Microbiol，1992，6(14):1887-1894
219. Michaelis S，Chapon C，d'Enfert C， et al. Characterization and expression of the structural gene forpullulanase, a maltose-inducible secreted protein of Klebsiella pneumoniae [J]. J Bacteriol，1985，164(2):633-638
220. Kuroiwa T，Shoda H，Ichikawa S， et al. Immobilization and stabilization of pullulanase fromKlebsiella pneumoniae by a multipoint attachment method using activated agar gel supports [J]. ProcessBiochem，2005，40(8):2637-2642
221. Lee M-S，Hseu Y-C，Lai G-H， et al. High yield expression in a recombinant E. coli of a codonoptimized chicken anemia virus capsid protein VP1useful for vaccine development [J]. Microb Cell Fact，2011，10:56
222. Li Z， Nimtz M, Rinas U. Optimized procedure to generate heavy isotope andselenomethionine-labeled proteins for structure determination using Escherichia coli-based expressionsystems [J]. Appl Microbiol Biotechnol，2011，92(4):823-833
223. Paracuellos P， hman A，Sauer-Eriksson A E， et al. Expression and purification of SfaXII, aprotein involved in regulating adhesion and motility genes in extraintestinal pathogenic Escherichia coli [J].Protein Expr Purif，2012，86(2):127-134
224. Mergulh o F J M，Summers D K, Monteiro G A. Recombinant protein secretion in Escherichia coli[J]. Biotechnol Adv，2005，23(3):177-202
225. Sauvonnet N, Pugsley A P. Identification of two regions of Klebsiella oxytoca pullulanase thattogether are capable of promoting β-lactamase secretion by the general secretory pathway [J]. MolMicrobiol，1996，22(1):1-7
226. Rodríguez-Carmona E，Cano-Garrido O，Dragosits M， et al. Recombinant Fab expression andsecretion in Escherichia coli continuous culture at medium cell densities: influence of temperature [J].Process Biochem，2012，47(3):446-452
227. Yamabhai M，Emrat S，Sukasem S， et al. Secretion of recombinant Bacillus hydrolytic enzymesusing Escherichia coli expression systems [J]. J Biotechnol，2008，133(1):50-57
228. Donovan R S，Robinson C W, Glick B R. Review: Optimizing inducer and culture conditions forexpression of foreign proteins under the control of the lac promoter [J]. J Ind Microbiol Biot，1996，16(3):145-154
229. Glick B R. Metabolic load and heterologous gene expression [J]. Biotechnol Adv，1995，13(2):247-261
230. Bentley W E，Davis R H, Kompala D S. Dynamics of induced CAT expression in E. coli [J].Biotechnol Bioeng，1991，38(7):749-760
231. Ohba R, Ueda S. Purification, crystallization and some properties of intracellular pullulanase fromAerobacter aerogenes [J]. Agric Biol Chem，1973，37(12):2821-2826
232. Ueda S, Ohba R. Purification, crystallization and some properties of extracellular pullulanase fromAerobacter aerogenes [J]. Agric Biol Chem，1972，36(13):2381-2391
233. Sarduy E S，Mu oz A C，Trejo S A， et al. High-level expression of Falcipain-2in Escherichia coliby codon optimization and auto-induction [J]. Protein Expr Purif，2012，83(1):59-69
234. Xu J， Li W， Wu J， et al. Stability of plasmid and expression of a recombinantgonadotropin-releasing hormone (GnRH) vaccine in Escherichia coli [J]. Appl Microbiol Biotechnol，2006，73(4):780-788
235. Ding R，Li Z，Chen S， et al. Enhanced secretion of recombinant α-cyclodextrin glucosyltransferasefrom E. coli by medium additives [J]. Process Biochem，2010，45(6):880-886
236. Wong M S，Wu S，Causey T B， et al. Reduction of acetate accumulation in Escherichia colicultures for increased recombinant protein production [J]. Metab Eng，2008，10(2):97-108
237. Jana S, Deb J K. Strategies for efficient production of heterologous proteins in Escherichia coli [J].Appl Microbiol Biotechnol，2005，67(3):289-298
238. S rensen H P, Mortensen K K. Advanced genetic strategies for recombinant protein expression inEscherichia coli [J]. J Biotechnol，2005，115(2):113-128
239. Li Z，Gu Z，Wang M， et al. Delayed supplementation of glycine enhances extracellular secretionof the recombinant α-cyclodextrin glycosyltransferase in Escherichia coli [J]. Appl Microbiol Biotechnol，2010，85(3):553-561
240. Arnesen S，Havn Eriksen S，Olsen J，et al. Increased production of α-amylase from Thermomyceslanuginosus by the addition of Tween80[J]. Enzyme Microb Tech，1998，23(3–4):249-252
241. Yang J，Moyana T，MacKenzie S， et al. One hundred seventy-fold increase in excretion of an FVfragment-tumor necrosis factor alpha fusion protein (sFV/TNF-α) from Escherichia coli caused by thesynergistic effects of glycine and Triton X-100[J]. Appl Environ Microbiol，1998，64(8):2869-2874
242. Hammes W，Schleifer K H, Kandler O. Mode of action of glycine on the biosynthesis ofpeptidoglycan [J]. J Bacteriol，1973，116(2):1029-1053
243. Thies E，Jenkins T, Stutzenberger F. Effects of the detergent Tween80on Thermomonosporacurvata [J]. World J Microb Biot，1994，10(6):657-663
244.陈坚,李寅.发酵过程优化原理与实践[M].北京：化学工业出版社，2002.
245.乔宇，丁宏标，闫俊艳，等.重组大肠杆菌产普鲁兰酶的高密度发酵工艺研究[J].生物技术进展，2012，2(3):195-200