β-甘露聚糖酶的基因克隆、分子改造及低聚甘露糖的酶法制备
|
摘要
β-甘露聚糖酶(EC3.2.1.78)是β-1,4-甘露聚糖甘露糖苷水解酶的简称，它可以从甘露聚糖分子主链的内部随机水解β-1,4甘露糖苷键，产生不同长度的低聚甘露糖。低聚甘露糖具有重要的生理活性，可当作膳食纤维和益生元。传统的借助高温、酸或碱水解魔芋精粉生产低聚甘露糖的方法需要用到大量的酸或碱，后期处理较为复杂、生产成本较高且对环境的污染较大，而酶法生产具有反应条件温和、环境友好的优势。低聚甘露糖的酶法生产中最关键的生物催化剂主要是β-甘露聚糖酶，这也使得β-甘露聚糖酶越来越受到广大研究者的关注和重视。目前国内外已有大量关于β-甘露聚糖酶的研究，然而它的应用仍然受限于其较低的催化活性和在极端环境中较差的稳定性。
为了获得热稳定性好和/或催化活性高的β-甘露聚糖酶工程酶，并实现低聚甘露糖的酶法制备，本论文克隆了宇佐美曲霉(Aspergillus usamii) YL-01-78β-甘露聚糖酶的完整cDNA和部分DNA序列，将该酶成熟肽的编码基因实现了在毕赤酵母(Pichia pastoris)GS115中的异源表达，并在此基础上对该酶进行了理性改造和应用的研究，主要研究结果如下：
(1)通过3′-RACE、5′-RACE及T载体介导的PCR技术获得了AuMan5A基因完整的cDNA和部分DNA序列，获得的DNA长度为2168bp，包括275bp的5′-端侧翼调控序列、1427bp的完整cDNA序列、两个分别为63和60bp的内含子，以及一个343bp的3′-端侧翼调控序列。其中完整的cDNA包括51bp的5′-端非翻译区、224bp的3′-端非翻译区和1152bp的开放读码框，共编码383个氨基酸残基。AuMan5A推测的氨基酸序列包括21个氨基酸残基的信号肽、17个氨基酸残基的前导肽和345个氨基酸残基的成熟肽，在成熟肽的序列中存在2个潜在的N-连接糖基化位点。AuMan5A的二级结构和三级结构预测的结果表明，该酶是一个由8个α-螺旋和8个β-折叠组成的蛋白，其三级结构符合典型的(α/β)8TIM-桶状结构，在β4和β7折叠片的C-末端分别存在1个Glu催化残基。
(2)借助pPIC9K质粒将AuMan5A成熟肽的编码基因整合入了P. pastoris GS115基因组中，从第一批转化子中筛选到了一株高拷贝重组子GSKM4-8，其产酶水平最高可达54.6U mL-1。而后，借助pPICZαA质粒构建出了双重重组子GSKZαM4-2，其产酶水平最高可达78.1U mL-1，且具有良好的遗传稳定性。对GSKZαM4-2的表达条件进行了优化，最佳的表达条件为：起始pH值6.5、诱导时间120h、甲醇浓度1.5%、诱导温度32℃，在此条件下GSKZαM4-2的产酶水平可达162.8U mL-1，较优化前的表达水平提高了1.08倍，较GSKM4-8的表达水平提高了1.98倍。此外，对Glu168和Glu276残基进行了突变分析，证实了Glu168和Glu276残基对AuMan5A保持其催化活性起着重要的作用。reAuMan5A是一个经糖基化修饰的蛋白质，其含糖量为21.3%。酶学特性分析的结果表明，reAuMan5A的最适反应pH值为3.0、最适反应温度为70℃、在60℃以下及pH值3.0~7.0之间具有较好的稳定性；除Ag+和Hg2+外，其它所测的金属离子及EDTA对reAuMan5A的酶活性没有明显的影响；reAuMan5A对角豆胶有较好的催化活性，对魔芋粉和瓜尔豆胶的催化活性较低，而对桦木木聚糖、可溶性淀粉和羧甲基纤维素钠无催化活性；reAuMan5A对角豆胶的Km和Vmax值分别为1.36mg mL-1和415.8U mg-1。
(3)以结合自由能为参考标准设计出了一种C-端带有纤维素结合结构域(CBM)的AuMan5A-CBM融合酶，利用重叠PCR技术构建出了AuMan5A-CBM的编码基因并在P. pastoris GS115中实现了异源表达。P. pastoris GSAuMC4-5的产酶活性最高可以达到40.6U mL-1，且具有良好的遗传稳定性。reAuMan5A-CBM的最适反应温度为75℃，较reAuMan5A提高了5℃，reAuMan5A-CBM耐受的温度较reAuMan5A也提高了8℃；另外，reAuMan5A-CBM对角豆胶的Km和Vmax值分别为0.66mg mL-1和389.1U mg-1，Km值较reAuMan5A的1.36mg mL-1有明显的降低；另外，reAuMan5A-CBM具有较好的纤维素结合能力。
(4)基于AuMan5A的3-D结构并借助理性设计的手段设计出了一个RMSD值和结合自由能均较低的截短突变体AuMan5AN3C3，借助pPIC9KM质粒将其编码基因整合入了P. pastoris GS115中并实现了表达，P. pastoris GSNC4-7菌株的产酶活性最高可以达到73.4U mL-1，较GS115/Auman5A的52.8U mL-1提高了39.0%。reAuMan5AN3C3的比酶活性较reAuMan5A有显著的提高，其它酶学特性与reAuMan5A的基本一致。借助二硫键设计、分子对接和分子动力学模拟的软件在AuMan5A中理性地引入了一个二硫键，设计了一个引入二硫键的突变体AuMan5Ads324330，将其编码基因在P. pastoris GS115中实现了表达。遗憾的是，实验获得的结果与预期的结果存在一定的差异，这也表明理性设计的条件及参数仍有改进的空间。
(5)经单因素实验确定的β-甘露聚糖酶酶法水解魔芋精粉制备低聚甘露糖的条件为：用去离子水配制的30g L-1魔芋胶溶液，酶液添加量为60U g-1魔芋精粉，水解温度为60℃，水解时间为6h，在此条件下魔芋精粉的水解率可达36.6%。β-内切葡聚糖酶对β-甘露聚糖酶水解魔芋精粉具有一定的协同作用，水解率最高可达65.4%。薄层层析分析结果显示魔芋精粉经酶水解后的产物主要是二糖以上的寡糖，且主要介于二糖与六糖之间，无单糖的产生。这些研究为生物催化制备低聚甘露糖的工业化生产奠定了基础。
β-Mannanases (EC3.2.1.78), abbreviated from β-1,4-D-mannan mannohydrolases, canrandomly hydrolyze the internal β-1,4-D-mannosidic linkages of mannans to form varyinglength mannooligosaccharides, which have important physiological activities and can be usedas dietary fiber and prebiotics. The traditional production method that high temperature andacid or alkaline were used to hydrolyze konjac gum to mannooligosaccharides needs a largeamount of acid or alkaline. That can cause complex post-processing, high cost and severeenvironmental pollution, while enzymatic production has the advantages of ambienttemperature and environmentally friendliness. The key biocatalyst is β-mannanase in theenzymatic production of mannooligosaccharides, which was paid more concern and attentionby the researchers. Although there are a lot of reports about the-mannanases, theirapplications are still limited by their low activities and poor stability in an extremeenvironment.
To obtain excellent engineered β-mannanases that have good thermostability and/or highcatalytic activity, and realize the biosynthesis of glucomannan oligosaccharides, we cloned thefull-length cDNA and partial DNA sequences of A. usamii YL-01-78-mannanase(abbreviated as AuMan5A) and made its mature peptide-encoding gene (Auman5A) expressedin P. pastoris GS115. On the basis of that, it was done that the preliminary research on therational modification and application of this-mannanase. The main results are listed asfollows:
(1) The full-length cDNA and partial DNA sequences of AuMan5A gene were amplifiedby3,5rapid amplification of cDNA ends (RACE) and T vector-mediated PCR techniques.The DNA sequence is2168bp in length, harboring275bp5′flanking regulatory regions,343bp3′flanking regulatory regions and the1427bp full-length cDNA sequence in which twoshort introns with63and60bp are inserted, respectively. Among which, the cDNA includes51bp5′non-coding region,224bp3′non-coding region and an1152bp open reading frame(ORF). The ORF encodes a21-aa signal peptide, a17-aa propeptide, and a345-aa maturepeptide (AuMan5A) with two putative N-glycosylation sites. Then, its secondary andthree-dimensional (3-D) structures were predicted. The modelled3-D structure of AuMan5Aconsists principally of the (/)8barrel fold. One Glu catalytic-residue is located at theC-terminus of4and7, respectively.
(2) The Auman5A was integrated into P. pastoris GS115genome by pPIC9K vecotor,and one strain labeled as GSKM4-8having the highest recombinant-mannanase activity of54.6U mL-1was chosen from the first-batch P. pastoris transformants. Then, the double P.pastoris transformant GSKZαM4-2with the highest-mannanase activity of78.1U mL-1andfavorable genetic stability was obtained by pPICZαAvecotor, and used to optimize expressionconditions. As GSKZαM4-2was induced under the optimized conditions (initial pH value6.5,induction period120h, methanol concentration1.5%and induction temperature32℃),
-mannanase activity reached162.8U mL-1, being2.08times as high as that expressed using the standard protocol (Invitrogen, USA), and1.98-fold higher than that expressed byGSKM4-8. In addition, mutation analyses were carried out on the Glu168and Glu276residuesof AuMan5A, these results confirmed that the Glu168and Glu276residues of AuMan5A playedimportant roles in keeping its catalytic activity. The reAuMan5A is an N-glycosylated protein,and its carbohydrate content was determined to be21.3%. It displayed the maximum activityat pH3.0and70℃, and was stable at a pH range of3.0~7.0and at60℃or below. Its activitywas not significantly affected by metal ions tested and EDTA, but inhibited by Ag+and Hg2+.The reAuMan5A showed the highest activity towards locust bean gum, but lower activitiestowards konjac gum and guar gum. No activity was detected towards birchwood xylan,soluble starch or carboxyl methyl cellulose. The kinetic parameters, Kmand Vmax, of thereAuMan5A towards locust bean gum were1.36mg mL-1and415.8U mg-1, respectively.
(3) The AuMan5A-CBM was designed based on the binding free energy, then theAuMan5A-CBM encoding gene was constructed by overlapping PCR and expressed in P.pastoris GS115. The GSAuMC4-5with the highest-mannanase activity of40.6U mL-1possesses favorable genetic stability. The temperature optimum of the reAuMan5A-CBM was75℃, being5℃higher than that of the reAuMan5A. And it was stable at68℃, being8℃higher than that of the reAuMan5A. The kinetic parameters, Kmand Vmax, of thereAuMan5A-CBM towards locust bean gum were0.66mg mL-1and389.1U mg-1, showingan obvious decrease in Km. In addition, the reAuMan5A-CBM possesses favorablecellulose-binding capacity.
(4) Based on the3-D structure of AuMan5A, a truncated mutant AuMan5AN3C3with thelowest RMSD value and binding free energy was designed by in silico design. TheAuMan5AN3C3encoding gene was integrated into P. pastoris GS115genome by pPIC9KMvecotor and expressed, the β-mannanase activity of P. pastoris GSNC4-7expressionsupernatant was73.4U mL-1, which increased by39.0%as compared with that (52.8U mL-1)of reAuMan5A. Compared with the reAuMan5A, the reAuMan5AN3C3showed an obviousincrease in the specific activity and no significant change in the other enzymatic properties. Adisulfide bond was rationally introduced into AuMan5A forming a mutant AuMan5Ads324330by disulfide bond design, molecular docking and molecular dynamics simulation softwares,and its encoding gene was expressed in P. pastoris GS115. Unfortunately, there were somedifferences between the expected result and experimental result, which indicated that therewere still some room for improvement in the conditions and parameters of the in silico design.
(5) The optimal hydrolytic condition of konjak gum for producing glucomannanoligosaccharides were as follows:30g L-1konjak gum solution (with deionized water),60U g-1konjak gum β-mannanase dosage,60℃hydrolytic temperature and6h hydrolytictime. Under this condition, the hydrolytic rate of konjak gum can reach36.6%. In addtion, theβ-endo-glucanase can play a synergistic role in the enzymatic hydrolysis of konjak gum byβ-mannanase, and its hydrolytic rate can reach65.4%. Analysis of oligosaccharide productsobtained by enzymatic hydrolysis of konjak gum using thin layer chromatography revealedthat these enzymes yielded mixture of mannobiose to mannohexose as their main products,and no trace of mannose could be detected in these hydrolysis experiments. These studies can lay the foundation for industrialized production of glucomannan oligosaccharides bybiocatalysis.
为了获得热稳定性好和/或催化活性高的β-甘露聚糖酶工程酶，并实现低聚甘露糖的酶法制备，本论文克隆了宇佐美曲霉(Aspergillus usamii) YL-01-78β-甘露聚糖酶的完整cDNA和部分DNA序列，将该酶成熟肽的编码基因实现了在毕赤酵母(Pichia pastoris)GS115中的异源表达，并在此基础上对该酶进行了理性改造和应用的研究，主要研究结果如下：
(1)通过3′-RACE、5′-RACE及T载体介导的PCR技术获得了AuMan5A基因完整的cDNA和部分DNA序列，获得的DNA长度为2168bp，包括275bp的5′-端侧翼调控序列、1427bp的完整cDNA序列、两个分别为63和60bp的内含子，以及一个343bp的3′-端侧翼调控序列。其中完整的cDNA包括51bp的5′-端非翻译区、224bp的3′-端非翻译区和1152bp的开放读码框，共编码383个氨基酸残基。AuMan5A推测的氨基酸序列包括21个氨基酸残基的信号肽、17个氨基酸残基的前导肽和345个氨基酸残基的成熟肽，在成熟肽的序列中存在2个潜在的N-连接糖基化位点。AuMan5A的二级结构和三级结构预测的结果表明，该酶是一个由8个α-螺旋和8个β-折叠组成的蛋白，其三级结构符合典型的(α/β)8TIM-桶状结构，在β4和β7折叠片的C-末端分别存在1个Glu催化残基。
(2)借助pPIC9K质粒将AuMan5A成熟肽的编码基因整合入了P. pastoris GS115基因组中，从第一批转化子中筛选到了一株高拷贝重组子GSKM4-8，其产酶水平最高可达54.6U mL-1。而后，借助pPICZαA质粒构建出了双重重组子GSKZαM4-2，其产酶水平最高可达78.1U mL-1，且具有良好的遗传稳定性。对GSKZαM4-2的表达条件进行了优化，最佳的表达条件为：起始pH值6.5、诱导时间120h、甲醇浓度1.5%、诱导温度32℃，在此条件下GSKZαM4-2的产酶水平可达162.8U mL-1，较优化前的表达水平提高了1.08倍，较GSKM4-8的表达水平提高了1.98倍。此外，对Glu168和Glu276残基进行了突变分析，证实了Glu168和Glu276残基对AuMan5A保持其催化活性起着重要的作用。reAuMan5A是一个经糖基化修饰的蛋白质，其含糖量为21.3%。酶学特性分析的结果表明，reAuMan5A的最适反应pH值为3.0、最适反应温度为70℃、在60℃以下及pH值3.0~7.0之间具有较好的稳定性；除Ag+和Hg2+外，其它所测的金属离子及EDTA对reAuMan5A的酶活性没有明显的影响；reAuMan5A对角豆胶有较好的催化活性，对魔芋粉和瓜尔豆胶的催化活性较低，而对桦木木聚糖、可溶性淀粉和羧甲基纤维素钠无催化活性；reAuMan5A对角豆胶的Km和Vmax值分别为1.36mg mL-1和415.8U mg-1。
(3)以结合自由能为参考标准设计出了一种C-端带有纤维素结合结构域(CBM)的AuMan5A-CBM融合酶，利用重叠PCR技术构建出了AuMan5A-CBM的编码基因并在P. pastoris GS115中实现了异源表达。P. pastoris GSAuMC4-5的产酶活性最高可以达到40.6U mL-1，且具有良好的遗传稳定性。reAuMan5A-CBM的最适反应温度为75℃，较reAuMan5A提高了5℃，reAuMan5A-CBM耐受的温度较reAuMan5A也提高了8℃；另外，reAuMan5A-CBM对角豆胶的Km和Vmax值分别为0.66mg mL-1和389.1U mg-1，Km值较reAuMan5A的1.36mg mL-1有明显的降低；另外，reAuMan5A-CBM具有较好的纤维素结合能力。
(4)基于AuMan5A的3-D结构并借助理性设计的手段设计出了一个RMSD值和结合自由能均较低的截短突变体AuMan5AN3C3，借助pPIC9KM质粒将其编码基因整合入了P. pastoris GS115中并实现了表达，P. pastoris GSNC4-7菌株的产酶活性最高可以达到73.4U mL-1，较GS115/Auman5A的52.8U mL-1提高了39.0%。reAuMan5AN3C3的比酶活性较reAuMan5A有显著的提高，其它酶学特性与reAuMan5A的基本一致。借助二硫键设计、分子对接和分子动力学模拟的软件在AuMan5A中理性地引入了一个二硫键，设计了一个引入二硫键的突变体AuMan5Ads324330，将其编码基因在P. pastoris GS115中实现了表达。遗憾的是，实验获得的结果与预期的结果存在一定的差异，这也表明理性设计的条件及参数仍有改进的空间。
(5)经单因素实验确定的β-甘露聚糖酶酶法水解魔芋精粉制备低聚甘露糖的条件为：用去离子水配制的30g L-1魔芋胶溶液，酶液添加量为60U g-1魔芋精粉，水解温度为60℃，水解时间为6h，在此条件下魔芋精粉的水解率可达36.6%。β-内切葡聚糖酶对β-甘露聚糖酶水解魔芋精粉具有一定的协同作用，水解率最高可达65.4%。薄层层析分析结果显示魔芋精粉经酶水解后的产物主要是二糖以上的寡糖，且主要介于二糖与六糖之间，无单糖的产生。这些研究为生物催化制备低聚甘露糖的工业化生产奠定了基础。
β-Mannanases (EC3.2.1.78), abbreviated from β-1,4-D-mannan mannohydrolases, canrandomly hydrolyze the internal β-1,4-D-mannosidic linkages of mannans to form varyinglength mannooligosaccharides, which have important physiological activities and can be usedas dietary fiber and prebiotics. The traditional production method that high temperature andacid or alkaline were used to hydrolyze konjac gum to mannooligosaccharides needs a largeamount of acid or alkaline. That can cause complex post-processing, high cost and severeenvironmental pollution, while enzymatic production has the advantages of ambienttemperature and environmentally friendliness. The key biocatalyst is β-mannanase in theenzymatic production of mannooligosaccharides, which was paid more concern and attentionby the researchers. Although there are a lot of reports about the-mannanases, theirapplications are still limited by their low activities and poor stability in an extremeenvironment.
To obtain excellent engineered β-mannanases that have good thermostability and/or highcatalytic activity, and realize the biosynthesis of glucomannan oligosaccharides, we cloned thefull-length cDNA and partial DNA sequences of A. usamii YL-01-78-mannanase(abbreviated as AuMan5A) and made its mature peptide-encoding gene (Auman5A) expressedin P. pastoris GS115. On the basis of that, it was done that the preliminary research on therational modification and application of this-mannanase. The main results are listed asfollows:
(1) The full-length cDNA and partial DNA sequences of AuMan5A gene were amplifiedby3,5rapid amplification of cDNA ends (RACE) and T vector-mediated PCR techniques.The DNA sequence is2168bp in length, harboring275bp5′flanking regulatory regions,343bp3′flanking regulatory regions and the1427bp full-length cDNA sequence in which twoshort introns with63and60bp are inserted, respectively. Among which, the cDNA includes51bp5′non-coding region,224bp3′non-coding region and an1152bp open reading frame(ORF). The ORF encodes a21-aa signal peptide, a17-aa propeptide, and a345-aa maturepeptide (AuMan5A) with two putative N-glycosylation sites. Then, its secondary andthree-dimensional (3-D) structures were predicted. The modelled3-D structure of AuMan5Aconsists principally of the (/)8barrel fold. One Glu catalytic-residue is located at theC-terminus of4and7, respectively.
(2) The Auman5A was integrated into P. pastoris GS115genome by pPIC9K vecotor,and one strain labeled as GSKM4-8having the highest recombinant-mannanase activity of54.6U mL-1was chosen from the first-batch P. pastoris transformants. Then, the double P.pastoris transformant GSKZαM4-2with the highest-mannanase activity of78.1U mL-1andfavorable genetic stability was obtained by pPICZαAvecotor, and used to optimize expressionconditions. As GSKZαM4-2was induced under the optimized conditions (initial pH value6.5,induction period120h, methanol concentration1.5%and induction temperature32℃),
-mannanase activity reached162.8U mL-1, being2.08times as high as that expressed using the standard protocol (Invitrogen, USA), and1.98-fold higher than that expressed byGSKM4-8. In addition, mutation analyses were carried out on the Glu168and Glu276residuesof AuMan5A, these results confirmed that the Glu168and Glu276residues of AuMan5A playedimportant roles in keeping its catalytic activity. The reAuMan5A is an N-glycosylated protein,and its carbohydrate content was determined to be21.3%. It displayed the maximum activityat pH3.0and70℃, and was stable at a pH range of3.0~7.0and at60℃or below. Its activitywas not significantly affected by metal ions tested and EDTA, but inhibited by Ag+and Hg2+.The reAuMan5A showed the highest activity towards locust bean gum, but lower activitiestowards konjac gum and guar gum. No activity was detected towards birchwood xylan,soluble starch or carboxyl methyl cellulose. The kinetic parameters, Kmand Vmax, of thereAuMan5A towards locust bean gum were1.36mg mL-1and415.8U mg-1, respectively.
(3) The AuMan5A-CBM was designed based on the binding free energy, then theAuMan5A-CBM encoding gene was constructed by overlapping PCR and expressed in P.pastoris GS115. The GSAuMC4-5with the highest-mannanase activity of40.6U mL-1possesses favorable genetic stability. The temperature optimum of the reAuMan5A-CBM was75℃, being5℃higher than that of the reAuMan5A. And it was stable at68℃, being8℃higher than that of the reAuMan5A. The kinetic parameters, Kmand Vmax, of thereAuMan5A-CBM towards locust bean gum were0.66mg mL-1and389.1U mg-1, showingan obvious decrease in Km. In addition, the reAuMan5A-CBM possesses favorablecellulose-binding capacity.
(4) Based on the3-D structure of AuMan5A, a truncated mutant AuMan5AN3C3with thelowest RMSD value and binding free energy was designed by in silico design. TheAuMan5AN3C3encoding gene was integrated into P. pastoris GS115genome by pPIC9KMvecotor and expressed, the β-mannanase activity of P. pastoris GSNC4-7expressionsupernatant was73.4U mL-1, which increased by39.0%as compared with that (52.8U mL-1)of reAuMan5A. Compared with the reAuMan5A, the reAuMan5AN3C3showed an obviousincrease in the specific activity and no significant change in the other enzymatic properties. Adisulfide bond was rationally introduced into AuMan5A forming a mutant AuMan5Ads324330by disulfide bond design, molecular docking and molecular dynamics simulation softwares,and its encoding gene was expressed in P. pastoris GS115. Unfortunately, there were somedifferences between the expected result and experimental result, which indicated that therewere still some room for improvement in the conditions and parameters of the in silico design.
(5) The optimal hydrolytic condition of konjak gum for producing glucomannanoligosaccharides were as follows:30g L-1konjak gum solution (with deionized water),60U g-1konjak gum β-mannanase dosage,60℃hydrolytic temperature and6h hydrolytictime. Under this condition, the hydrolytic rate of konjak gum can reach36.6%. In addtion, theβ-endo-glucanase can play a synergistic role in the enzymatic hydrolysis of konjak gum byβ-mannanase, and its hydrolytic rate can reach65.4%. Analysis of oligosaccharide productsobtained by enzymatic hydrolysis of konjak gum using thin layer chromatography revealedthat these enzymes yielded mixture of mannobiose to mannohexose as their main products,and no trace of mannose could be detected in these hydrolysis experiments. These studies can lay the foundation for industrialized production of glucomannan oligosaccharides bybiocatalysis.
引文
1.蒙海林. β-甘露聚糖酶产生菌的选育及发酵条件的研究[D]:[硕士学位论文].南宁:广西大学,2006.
2.杨立志,喻春皓,何志敏.功能性低聚糖功效及生产概况[J].天津化工,2001,1:13-15.
3. Otieno D O, Ahring B K. The potential for oligosaccharide production from the hemicellulose fractionof biomasses through pretreatment processes: xylooligosaccharides (XOS), arabinooligosaccharides(AOS), and mannooligosaccharides (MOS)[J]. Carbohydr Res,2012,360:84-92.
4. Chung Y C, Hsu C K, Ko C Y, et al. Dietary intake of xylooligosaccharides improves the intestinalmicrobiota, fecal moisture, and pH value in the elderly [J]. Nutr Res,2007,27(12):756-761.
5. Moreira L R S, Filho E X F. An overview of mannan structure and mannan-degrading enzyme systems[J]. Appl Microbiol Biot,2008,79(2):165-178.
6. Sittikijyothin W, Torres D, Gon alves M P. Modelling the rheological behaviour of galactomannanaqueous solutions [J]. Carbohyd Polym,2005,59(3):339-350.
7. de O. Petkowicz CL, Reicher F, Chanzy H, et al. Linear mannan in the endosperm of Schizolobiumamazonicum [J]. Carbohyd Polym,2001,44(2):107-112.
8. Parvathy K S, Susheelamma N S, Tharanathan R N, et al. A simple non-aqueous method forcarboxymethylation of galactomannans [J]. Carbohyd Polym,2005,62(2):137-141.
9. Shobha M S, Vishu Kumar A B, Tharanathan R N, et al. Modification of guar galactomannan with theaid of Aspergillus niger pectinase [J]. Carbohyd Polym,2005,62(3):267-273.
10. Bresolin T M B, Sander P C, Reicher F, et al. Viscometric studies on xanthan and galactomannansystems [J]. Carbohyd Polym,1997,33(2/3):131-138.
11. Popa V I, Spiridon J. Polysaccharides: structural diversity and functional versatility. In:Hemicelluloses:structure and properties. Edited by Dumitriu S, New York: Marcel Dekker,1998.297-311.
12. Hongshu Z, Jinggan Y, Yan Z. The glucomannan from ramie [J]. Carbohyd Polym,2002,47(1):83-86.
13. Ishurd O, Kermagi A, Elghazoun M, et al. Structural of a glucomannan from Lupinus varius seed [J].Carbohyd Polym,2006,65(4):410-413.
14. Aspinall G O. Structural chemistry of the hemicelluloses. In: Advances in Carbohydrate Chemistry.Edited by Melville LW: Academic Press,1959.429-468.
15. Timell T E. Wood Hemicelluloses: Part II. In: Advances in Carbohydrate Chemistry. Edited byMelville LW: Academic Press,1965.409-483.
16.尤新.功能性低聚糖发展动向及前景[J].中国食品添加剂,2008,3:45-49.
17. Mussatto SI, Mancilha IM. Non-digestible oligosaccharides: A review [J]. Carbohyd Polym,2007,68(3):587-597.
18. Gibson G R, Roberfroid M B. Dietary modulation of the human colonic microbiota: introducing theconcept of prebiotics [J]. J Nutr,1995,125(6):1401-1412.
19. Bornscheuer U T, Huisman G W, Kazlauskas R J, et al. Engineering the third wave of biocatalysis [J].Nature,2012,485(7397):185-194.
20. Zhang M, Chen X L, Zhang Z H, et al. Purification and functional characterization ofendo-β-mannanase MAN5and its application in oligosaccharide production from konjac flour [J].Appl Microbiol Biot,2009,83(5):865-873.
21. Blibech M, Chaari F, Bhiri F, et al. Production of manno-oligosaccharides from locust bean gum usingimmobilized Penicillium occitanis mannanase [J]. J Mol Catal B-Enzym,2011,111-115.
22.陶兴无.酸酶结合法水解魔芋葡甘露聚糖工艺研究[J].武汉工业学院学报,2005,24(3):1-4.
23.徐春梅,邬敏辰,李剑芳等.魔芋葡甘露聚糖的酶水解工艺条件[J].食品与生物技术学报,2008,27(3):120-124.
24. Kurakake M, Sumida T, Masuda D, et al. Production of galacto-manno-oligosaccharides from guargum by β-mannanase from Penicillium oxalicum SO [J]. JAgr Food Chem,2006,54(20):7885-7889.
25. Albrecht S, van Muiswinkel G C, Xu J, et al. Enzymatic production and characterization of konjacglucomannan oligosaccharides [J]. J Agr Food Chem,2011,59(23):12658-12666.
26.李剑芳,邬敏辰,程科等. β-甘露聚糖酶制备魔芋葡甘露低聚糖的研究[J].食品与发酵工业2007,33(1):21-24.
27. Pan X, Zhou J, Tian A, et al. High level expression of a truncated β-mannanase from alkaliphilicBacillus sp. N16-5in Kluyveromyces cicerisporus [J]. Biotechnol Lett,2010,33(3):565-570.
28. Li J F, Zhao S G, Tang C D, et al. Cloning and functional expression of an acidophilic β-mannanasegene (Anman5A) from Aspergillus niger LW-1in Pichia pastoris [J]. J Agr Food Chem,2012,60(3):765-773.
29. van Zyl W H, Rose S H, Trollope K, et al. Fungal β-mannanases: Mannan hydrolysis, heterologousproduction and biotechnological applications [J]. Process Biochem,2010,45(8):1203-1213.
30. Duruksu G, Ozturk B, Biely P, et al. Cloning, expression and characterization ofendo-β-1,4-mannanase from Aspergillus fumigates in Aspergillus sojae and Pichia pastoris [J].Biotechnol Progr,2009,25:271-276.
31.李剑芳.黑曲霉酸性β-甘露聚糖酶及制备魔芋低聚糖研究[D]:[博士学位论文].江南大学,
2007.
32. Meenakshi, Singh G, Bhalla A, et al. Solid state fermentation and characterization of partially purifiedthermostable mannanase from Bacillus sp. MG-33[J]. Bioresources,2010,5(3):1689-1701.
33. Titapoka S, Keawsompong S, Haltrich D, et al. Selection and characterization ofmannanase-producing bacteria useful for the formation of prebiotic manno-oligosaccharides fromcopra meal [J]. World J Microb Biot,2008,24(8):1425-1433.
34. Kote N, Patil A, Mulimani V H. Optimization of the production of thermostable endo-β-1,4mannanases from a newly isolated Aspergillus niger gr and Aspergillus flavus gr [J]. Appl BiochemBiotechnol,2009,152(2):213-223.
35. Blibech M, Ghorbel R, Fakhfakh I, et al. Purification and characterization of a low molecular weightof β-mannanase from Penicillium occitanis Pol6[J].Appl Biochem Biotech,2010,160(4):1227-1240.
36. Malheirosferreira H. Purification and characterization of a β-mannanase from Trichoderma harzianumstrain T4[J]. Carbohyd Polym,2004,57(1):23-29.
37. Parker K N, Chhabra S R, Lam D, et al. Galactomannanases Man2and Man5from Thermotogaspecies:Growth physiology on galactomannans, gene sequence analysis, and biochemical properties ofrecombinant enzymes [J]. Biotechnol Bioeng,2001,75(3):322-333.
38. Zhao W, Zheng J, Zhou H B. A thermotolerant and cold-active mannan endo-1,4-β-mannosidase fromAspergillus niger CBS513.88: Constitutive overexpression and high-density fermentation in Pichiapastoris [J]. Bioresource Technol,2011,102(16):7538-7547.
39. Zhao J, Shi P, Luo H, et al. An acidophilic and acid-stable β-mannanase from Phialophora sp. P13with high mannan hydrolysis activity under simulated gastric gonditions [J]. J Agr Food Chem,2010,58(5):3184-3190.
40. Zahura U A, Rahman M M, Inoue A, et al. An endo-β-1,4-mannanase, AkMan, from the common seahare Aplysia kurodai [J]. Comp Biochem Phys B,2010,157(1):137-143.
41. Tanaka M, Umemoto Y, Okamura H, et al. Cloning and characterization of a β-1,4-mannanase5Cpossessing a family27carbohydrate-binding module from a Marine Bacterium, Vibrio sp. strainMA-138[J]. Biosci Biotech Bioch,2009,73(1):109-116.
42. Bien-Cuong D, Thi-Thu D, Berrin J-G, et al. Cloning, expression in Pichia pastoris, andcharacterization of a thermostable GH5mannan endo-1,4-β-mannosidase from Aspergillus nigerBK01[J]. Microb Cell Fact,2009,8(1):59.
43. Chen X, Cao Y, Ding Y, et al. Cloning, functional expression and characterization of Aspergillussulphureus β-mannanase in Pichia pastoris [J]. J Biotechnol,2007,128(3):452-461.
44. Songsiriritthigul C, Buranabanyat B, Haltrich D, et al. Efficient recombinant expression and secretionof a thermostable GH26mannan endo-1,4-β-mannosidase from Bacillus licheniformis in Escherichiacoli [J]. Microb Cell Fact,2010,9(1):20.
45. Jiang Z, Wei Y, Li D, et al. High-level production, purification and characterization of a thermostableβ-mannanase from the newly isolated Bacillus subtilis WY34[J]. Carbohyd Polym,2006,66(1):88-96.
46. Luo H, Wang K, Huang H, et al. Gene cloning, expression, and biochemical characterization of analkali-tolerant β-mannanase from Humicola insolens Y1[J]. J Ind Microbiol Biot,2012,39(4):547-555.
47. Katrolia P, Zhou P, Zhang P, et al. High level expression of a novel β-mannanase from Chaetomium sp.exhibiting efficient mannan hydrolysis [J]. Carbohyd Polym,2012,87(1):480-490.
48. Luo H, Wang Y, Wang H, et al. A novel highly acidic β-mannanase from the acidophilic fungusBispora sp. MEY-1: gene cloning and overexpression in Pichia pastoris [J]. Appl Microbiol Biot,2009,82(3):453-461.
49. Chauhan P S, Puri N, Sharma P, et al. Mannanases: microbial sources, production, properties andpotential biotechnological applications [J]. Appl Microbiol Biot,2012,93(5):1817-1830.
50. Oosterveld A, Voragen A G J, Schols H A. Effect of roasting on the carbohydrate composition ofCoffea arabica beans [J]. Carbohyd Polym,2003,54(2):183-192.
51. Comfort D A, Chhabra S R, Conners S B, et al. Strategic biocatalysis with hyperthermophilic enzymes[J]. Green Chem,2004,6(9):459-465.
52. Varnai A, Huikko L, Pere J, et al. Synergistic action of xylanase and mannanase improves the totalhydrolysis of softwood [J]. Bioresource technol,2011,102(19):9096-9104.
53. Dhawan S, Kaur J. Microbial mannanases: an overview of production and applications [J]. Crit RevBiotechnol,2007,27(4):197-216.
54. El-Helow E, Sabry S, Khattab A. Production of α-mannanase by B. subtilis from agro-industrialby-products: screening and optimization [J]. Antonie Van Leeuwenhoek,1997,71(3):189-193.
55. Politz O, Krah M, Thomsen K K, et al. A highly thermostable endo-(1,4)-beta-mannanase from themarine bacterium Rhodothermus marinus [J]. Appl Microbiol Biot,2000,53(6):715-721.
56. Yin L J, Tai H M, Jiang S T. Characterization of mannanase from a novel mannanase-producingBacterium [J]. J Agr Food Chem,2012,60(25):6425-6431.
57. Wu M C, Tang C D, Li J F, et al. Bimutation breeding of Aspergillus niger strain for enhancingβ-mannanase production by solid-state fermentation [J]. Carbohyd Res,2011,346(14):2149-2155.
58. Yang P, Li Y, Wang Y et al. A novel β-mannanase with high specific activity from Bacillus circulansCGMCC1554: gene cloning, expression and enzymatic characterization [J]. Appl Biochem Biotech,2009,159(1):85-94.
59. Summpunn P, Chaijan S, Isarangkul D, et al. Characterization, gene cloning, and heterologousexpression of β-mannanase from a thermophilic Bacillus subtilis [J]. J Microbiol,2011,49(1):86-93.
60. Zhou H Y, Pan H Y, Rao L Q, et al. Redesign the alpha/beta fold to enhance the stability of mannanaseman23from Bacillus subtilis [J]. Appl Biochem Biotech,2011,163(1):186-194.
61. Qiao J, Rao Z, Dong B, et al. Expression of Bacillus subtilis MA139β-mannanase in Pichia pastorisand the enzyme characterization [J]. Appl Biochem Biotech,2010,160(5):1362-1370.
62. Zyl P, Moodley V, Rose S H, et al. Production of the Aspergillus aculeatus endo-1,4-β-mannanase in A.niger [J]. J Ind Microbiol Biot,2009,36(4):611-617.
63. Yang X S, Jiang Z B, Song H T, et al. Cell-surface display of the active mannanase in Yarrowialipolytica with a novel surface-display system [J]. Biotechnol Appl Bioc,2009,54(3):171-176.
64. Pham T A, Berrin J G, Record E, et al. Hydrolysis of softwood by Aspergillus mannanase: role of acarbohydrate-binding module [J]. J Biotechnol,2010,148(4):163-170.
65.郜赵伟,张宇宏,张伟.微生物酶分子改造研究进展[J].中国生物工程杂志2010,30(1):98-103.
66.孔祥禄.计算机辅助耐热性研究及分子设计[D]:[硕士学位论文].广州:华南理工大学,2010.
67. Kim M-S, Lei X. Enhancing thermostability of Escherichia coli phytase AppA2by error-prone PCR[J]. Appl Microbiol Biot,2008,79(1):69-75.
68.孔荣,钮利喜,袁静明.易错PCR法定向进化D-海因酶的初步研究[J].山西大学学报(自然科学版),2006,29:425-427.
69. Ang E L, Obbard J P, Zhao H. Probing the molecular determinants of aniline dioxygenase substratespecificity by saturation mutagenesis [J]. FEBS J,2007,274(4):928-939.
70. Gabor E M, Janssen D B. Increasing the synthetic performance of penicillin acylase PAS2bystructure-inspired semi-random mutagenesis [J]. Protein Eng Des Sel,2004,17(7):571-579.
71. Cho C M, Mulchandani A, Chen W. Altering the substrate specificity of organophosphorus hydrolasefor enhanced hydrolysis of chlorpyrifos [J]. Appl Environ Microbiol,2004,70(8):4681-4685.
72. Shi C, Lu X, Ma C, et al. Enhancing the thermostability of a novel β-agarase AgaB through directedevolution [J]. Appl Biochemi Biotech,2008,151(1):51-59.
73. Davoodi J, Wakarchuk W W, Carey P R, et al. Mechanism of stabilization of Bacillus circulansxylanase upon the introduction of disulfide bonds [J]. Biophys Chem,2007,125(2–3):453-461.
74. Fenel F, Leisola M, J nis J, et al. A de novo designed N-terminal disulphide bridge stabilizes theTrichoderma reesei endo-1,4-β-xylanase II [J]. J Biotechnol,2004,108(2):137-143.
75. Huang Y H, Huang C T, Hseu R S. Effects of dockerin domains on Neocallimastix frontalis xylanases[J]. FEMS Microbiol Lett,2005,243(2):455-460.
76. Sokkar P, Mohandass S, Ramachandran M. Multiple templates-based homology modeling enhancesstructure quality of AT1receptor: validation by molecular dynamics and antagonist docking [J]. J MolModel,2011,17(7):1565-1577.
77. Gao S J, Wang J Q, Wu M C, et al. Engineering hyperthermostability into a mesophilic family11xylanase from Aspergillus oryzae by in silico design of N-terminus substitution [J]. Biotechnol Bioeng,2013,110(4):1028-1038.
78.田健.计算机辅助分子设计提高蛋白质热稳定性的研究[D]:[博士学位论文].北京:中国农业科学院,2011.
79. Le QA, Joo JC, Yoo YJ et al. Development of thermostable Candida antarctica lipase B through novelin silico design of disulfide bridge [J]. Biotechnol Bioeng,2012,109(4):867-876.
80.史红玲.米曲霉木聚糖酶基因的克隆表达及耐热性改造[D]:[硕士学位论文].无锡:江南大学,
2012.
81. Timmers L F, Ducati R G, Sanchez-Quitian Z A, et al. Combining molecular dynamics and dockingsimulations of the cytidine deaminase from Mycobacterium tuberculosis H37Rv [J]. J Mol Model,2012,18(2):467-479.
82. Christelle B, Eduardo Bde O, Latifa C, et al. Combined docking and molecular dynamics simulationsto enlighten the capacity of Pseudomonas cepacia and Candida antarctica lipases to catalyzequercetin acetylation [J]. J Biotechnol,2011,156(3):203-210.
83. Zhang Y, Ju J, Peng H, et al. Biochemical and structural characterization of the intracellularmannanase AaManA of Alicyclobacillus acidocaldarius reveals a novel glycoside hydrolase familybelonging to clan GH-A [J]. J Biol Chem,2008,283(46):31551-31558.
84. Le Nours J, Anderson L, Stoll D, et al. The structure and characterization of a modularendo-β-1,4-mannanase from Cellulomonas fimi [J]. Biochemistry,2005,44(38):12700-12708.
85. Roth R, Moodley V, Zyl P. Heterologous expression and optimized production of an Aspergillusaculeatus endo-1,4-β-mannanase in Yarrowia lipolytica [J]. Mol Biotechnol,2009,43(2):112-120.
86.李剑芳,马丽萍,邬敏辰等.宇佐美曲霉酸性β-甘露聚糖酶的纯化及性质研究[J].食品与发酵工业,2006,32(9):5-9.
87. Sambrook J, Russell D W. Molecular cloning: a laboratory manual, Cold Spring Harbor, NY, USA:Cold Spring Harbor Laboratory Press,2001.1595-1604.
88. Wang J Q, Zhang H M, Wu M C. et al. Cloning and sequence analysis of a novel xylanase gene,Auxyn10A, from Aspergillus usamii [J]. Biotechnol Lett,2011,33(5):1029-1038.
89. Wenkel S, Turck F, Singer K, et al. CONSTANS and the CCAAT box binding complex share afunctionally important domain and interact to regulate flowering of Arabidopsis [J]. Plant Cell,2006,18(11):2971-2984.
90. Petersen T N, Brunak S, von Heijne G, et al. SignalP4.0: discriminating signal peptides fromtransmembrane regions [J]. Nat Meth,2011,8(10):785-786.
91.赵月菊,薛燕芬,马延和. β-甘露聚糖酶的结构生物学研究现状和展望[J].微生物学报,2009,49(9):1131-1137.
92. Zhang J H, Wu D, Chen J, et al. Enhancing functional expression of β-glucosidase in Pichia pastorisby co-expressing protein disulfide isomerase [J]. Biotechnol Bioproc E,2011,16(6):1196-1200.
93. Sreekrishna K, Brankamp R G, Kropp K E, et al. Strategies for optimal synthesis and secretion ofheterologous proteins in the methylotrophic yeast Pichia pastoris [J]. Gene,1997,190:55-62.
94. Chen X, Qiao J, Yu H, et al. Overexpression of an optimized Aspergillus sulphureus β-mannanasegene in Pichia pastoris [J]. Biologia,2009,64(2):235-238.
95. Akcapinar G B, Gul O, Sezerman U. Effect of codon optimization on the expression of Trichodermareesei endo-glucanase1in Pichia pastoris [J]. Biotechnol Progr,2011,27(5):1257-1263.
96. Tan Z, Li J, Wu M, et al. High-level heterologous expression of an alkaline lipase gene fromPenicillium cyclopium PG37in Pichia pastoris [J]. World J Microb Biot,2011,27(12):2767-2774.
97.李剑芳,赵顺阁,邬敏辰等. β-甘露聚糖酶基因和木聚糖酶基因在毕赤酵母中的共表达[J].食品与生物技术学报2012,31(11):1136-1141.
98. Lin S S, Dou W F, Xu H Y, et al. Optimization of medium composition for the production of alkalineβ-mannanase by alkaliphilic Bacillus sp. N16-5using response surface methodology [J]. ApplMicrobiol Biot,2007,75(5):1015-1022.
99. Ma S, Wang H, Wang Y, et al. Bio-hydrogen production from cornstalk wastes by orthogonal designmethod [J]. Renew Energ,2011,36(2):709-713.
100. Li J F, Tang C D, Shi H L, et al. Cloning and optimized expression of a neutral endoglucanase gene(ncel5A) from Volvariella volvacea WX32in Pichia pastoris [J]. J Biosci Bioeng,2011,111(5):537-540.
101.Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J].Nature,1970,227:680-685.
102. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of proteinutilizing the principle of protein-dye binding [J]. Anal Biochem,1976,72:248-254.
103. Dubois M, Gilles K A, Hamilton J K, et al. Colorimetric method for determination of sugars andrelated substances [J]. Anal Chem,1956,28:350-356.
104. Katakura Y, Zhang W, Zhuang G, et al. Effect of methanol concentration on the production of humanβ2-glycoproteinⅠ domain V by a recombinant Pichia pastoris: A simple system for the control ofmethanol concentration using a semiconductor gas sensor [J]. Ferment Bioeng,1998,86:482-487.
105. Cregg J M, Higgins D R. Production of foreign proteins in the yeast Pichia pastoris [J]. Can J Bot,1995,73(S1):891-897.
106. Srimathi S, Jayaraman G. Effect of glycosylation on the catalytic and conformational stability ofhomologous α-amylases [J]. Protein J,2005,24(2):79-88.
107. Shental-Bechor D, Levy Y. Effect of glycosylation on protein folding: a close look at thermodynamicstabilization [J]. Proc Natl Acad Sci U S A,2008,105(24):8256-8261.
108. Guillen D, Sanchez S, Rodriguez-Sanoja R. Carbohydrate-binding domains: multiplicity of biologicalroles [J]. Appl Microbiol Biot,2010,85(5):1241-1249.
109. Thongekkaew J, Ikeda H, Iefuji H. Increases thermal stability and cellulose-binding capacity ofCryptococcus sp. S-2lipase by fusion of cellulose binding domain derived from Trichoderma reesei[J]. Biochem Bioph Res Co,2012,420(1):183-187.
110. Ravalason H, Herpoel-Gimbert I, Record E, et al. Fusion of a family1carbohydrate binding moduleof Aspergillus niger to the Pycnoporus cinnabarinus laccase for efficient softwood kraft pulpbiobleaching [J]. J Biotechnol,2009,142(3-4):220-226.
111. Phosrithong N, Ungwitayatorn J. Molecular docking study on anticancer activity of plant-derivednatural products [J]. Med Chem Res,2009,19(8):817-835.
112. Poole D M, Hazlewood G P, Huskisson N S, et al. The role of conserved tryptophan residues in theinteraction of a bacterial cellulose binding domain with its ligand [J]. FEMS Microbiol Lett,1993,106(1):77-83.
113. Shoseyov O, Shani Z, Levy I. Carbohydrate binding modules: biochemical properties and novelapplications [J]. Microbiol Mol Biol R,2006,70(2):283-295.
114. Neeraja C, Moerschbacher B, Podile A R. Fusion of cellulose binding domain to the catalytic domainimproves the activity and conformational stability of chitinase in Bacillus licheniformis DSM13[J].Bioresource Technol,2010,101(10):3635-3641.
115. Zhang Y, Chen S, Xu M, et al. Characterization of Thermobifida fusca cutinase-carbohydrate-bindingmodule fusion proteins and their potential application in bioscouring [J]. Appl Environ Microbiol,2010,76(20):6870-6876.
116. Ye X, Zhu Z, Zhang C, et al. Fusion of a family9cellulose-binding module improves catalyticpotential of Clostridium thermocellum cellodextrin phosphorylase on insoluble cellulose [J]. ApplMicrobiol Biot,2011,92(3):551-560.
117. Wan W, Wang D, Gao X, et al. Expression of family3cellulose-binding module (CBM3) as an affinitytag for recombinant proteins in yeast [J]. Appl Microbiol Biot,2011,91(3):789-798.
118. Sriprang R, Asano K, Gobsuk J, et al. Improvement of thermostability of fungal xylanase by usingsite-directed mutagenesis [J]. J Biotechnol,2006,126(4):454-462.
119. Liu L, Zhang G, Zhang Z, et al. Terminal amino acids disturb xylanase thermostability and activity [J].J Biol Chem,2011,286(52):44710-44715.
120. Wen T, Chen J, Lee S, et al. A truncated Fibrobacter succinogenes1,3-1,4-β-D-glucanase withimproved enzymatic activity and thermotolerance [J]. Biochemistry,2005,44(25):9197-9205.
121. Dombkowski A A. Disulfide by DesignTM: a computational method for the rational design of disulfidebonds in proteins [J]. Bioinformatics,2003,19(14):1852-1853.
122. Albrecht S, van Muiswinkel G C, Schols H A, et al. Introducing capillary electrophoresis withlaser-induced fluorescence detection (CE-LIF) for the characterization of konjac glucomannanoligosaccharides and their in vitro fermentation behavior [J]. J Agr Food Chem,2009,57(9):3867-3876.
123. Shi H L, Yin X, Wu M C, et al. Cloning and bioinformatics analysis of an endo-glucanase gene(Aucel12A) from Aspergillus usamii and its functional expression in Pichia pastoris [J]. J IndMicrobiol Biot,2012,39(2):347-357.
2.杨立志,喻春皓,何志敏.功能性低聚糖功效及生产概况[J].天津化工,2001,1:13-15.
3. Otieno D O, Ahring B K. The potential for oligosaccharide production from the hemicellulose fractionof biomasses through pretreatment processes: xylooligosaccharides (XOS), arabinooligosaccharides(AOS), and mannooligosaccharides (MOS)[J]. Carbohydr Res,2012,360:84-92.
4. Chung Y C, Hsu C K, Ko C Y, et al. Dietary intake of xylooligosaccharides improves the intestinalmicrobiota, fecal moisture, and pH value in the elderly [J]. Nutr Res,2007,27(12):756-761.
5. Moreira L R S, Filho E X F. An overview of mannan structure and mannan-degrading enzyme systems[J]. Appl Microbiol Biot,2008,79(2):165-178.
6. Sittikijyothin W, Torres D, Gon alves M P. Modelling the rheological behaviour of galactomannanaqueous solutions [J]. Carbohyd Polym,2005,59(3):339-350.
7. de O. Petkowicz CL, Reicher F, Chanzy H, et al. Linear mannan in the endosperm of Schizolobiumamazonicum [J]. Carbohyd Polym,2001,44(2):107-112.
8. Parvathy K S, Susheelamma N S, Tharanathan R N, et al. A simple non-aqueous method forcarboxymethylation of galactomannans [J]. Carbohyd Polym,2005,62(2):137-141.
9. Shobha M S, Vishu Kumar A B, Tharanathan R N, et al. Modification of guar galactomannan with theaid of Aspergillus niger pectinase [J]. Carbohyd Polym,2005,62(3):267-273.
10. Bresolin T M B, Sander P C, Reicher F, et al. Viscometric studies on xanthan and galactomannansystems [J]. Carbohyd Polym,1997,33(2/3):131-138.
11. Popa V I, Spiridon J. Polysaccharides: structural diversity and functional versatility. In:Hemicelluloses:structure and properties. Edited by Dumitriu S, New York: Marcel Dekker,1998.297-311.
12. Hongshu Z, Jinggan Y, Yan Z. The glucomannan from ramie [J]. Carbohyd Polym,2002,47(1):83-86.
13. Ishurd O, Kermagi A, Elghazoun M, et al. Structural of a glucomannan from Lupinus varius seed [J].Carbohyd Polym,2006,65(4):410-413.
14. Aspinall G O. Structural chemistry of the hemicelluloses. In: Advances in Carbohydrate Chemistry.Edited by Melville LW: Academic Press,1959.429-468.
15. Timell T E. Wood Hemicelluloses: Part II. In: Advances in Carbohydrate Chemistry. Edited byMelville LW: Academic Press,1965.409-483.
16.尤新.功能性低聚糖发展动向及前景[J].中国食品添加剂,2008,3:45-49.
17. Mussatto SI, Mancilha IM. Non-digestible oligosaccharides: A review [J]. Carbohyd Polym,2007,68(3):587-597.
18. Gibson G R, Roberfroid M B. Dietary modulation of the human colonic microbiota: introducing theconcept of prebiotics [J]. J Nutr,1995,125(6):1401-1412.
19. Bornscheuer U T, Huisman G W, Kazlauskas R J, et al. Engineering the third wave of biocatalysis [J].Nature,2012,485(7397):185-194.
20. Zhang M, Chen X L, Zhang Z H, et al. Purification and functional characterization ofendo-β-mannanase MAN5and its application in oligosaccharide production from konjac flour [J].Appl Microbiol Biot,2009,83(5):865-873.
21. Blibech M, Chaari F, Bhiri F, et al. Production of manno-oligosaccharides from locust bean gum usingimmobilized Penicillium occitanis mannanase [J]. J Mol Catal B-Enzym,2011,111-115.
22.陶兴无.酸酶结合法水解魔芋葡甘露聚糖工艺研究[J].武汉工业学院学报,2005,24(3):1-4.
23.徐春梅,邬敏辰,李剑芳等.魔芋葡甘露聚糖的酶水解工艺条件[J].食品与生物技术学报,2008,27(3):120-124.
24. Kurakake M, Sumida T, Masuda D, et al. Production of galacto-manno-oligosaccharides from guargum by β-mannanase from Penicillium oxalicum SO [J]. JAgr Food Chem,2006,54(20):7885-7889.
25. Albrecht S, van Muiswinkel G C, Xu J, et al. Enzymatic production and characterization of konjacglucomannan oligosaccharides [J]. J Agr Food Chem,2011,59(23):12658-12666.
26.李剑芳,邬敏辰,程科等. β-甘露聚糖酶制备魔芋葡甘露低聚糖的研究[J].食品与发酵工业2007,33(1):21-24.
27. Pan X, Zhou J, Tian A, et al. High level expression of a truncated β-mannanase from alkaliphilicBacillus sp. N16-5in Kluyveromyces cicerisporus [J]. Biotechnol Lett,2010,33(3):565-570.
28. Li J F, Zhao S G, Tang C D, et al. Cloning and functional expression of an acidophilic β-mannanasegene (Anman5A) from Aspergillus niger LW-1in Pichia pastoris [J]. J Agr Food Chem,2012,60(3):765-773.
29. van Zyl W H, Rose S H, Trollope K, et al. Fungal β-mannanases: Mannan hydrolysis, heterologousproduction and biotechnological applications [J]. Process Biochem,2010,45(8):1203-1213.
30. Duruksu G, Ozturk B, Biely P, et al. Cloning, expression and characterization ofendo-β-1,4-mannanase from Aspergillus fumigates in Aspergillus sojae and Pichia pastoris [J].Biotechnol Progr,2009,25:271-276.
31.李剑芳.黑曲霉酸性β-甘露聚糖酶及制备魔芋低聚糖研究[D]:[博士学位论文].江南大学,
2007.
32. Meenakshi, Singh G, Bhalla A, et al. Solid state fermentation and characterization of partially purifiedthermostable mannanase from Bacillus sp. MG-33[J]. Bioresources,2010,5(3):1689-1701.
33. Titapoka S, Keawsompong S, Haltrich D, et al. Selection and characterization ofmannanase-producing bacteria useful for the formation of prebiotic manno-oligosaccharides fromcopra meal [J]. World J Microb Biot,2008,24(8):1425-1433.
34. Kote N, Patil A, Mulimani V H. Optimization of the production of thermostable endo-β-1,4mannanases from a newly isolated Aspergillus niger gr and Aspergillus flavus gr [J]. Appl BiochemBiotechnol,2009,152(2):213-223.
35. Blibech M, Ghorbel R, Fakhfakh I, et al. Purification and characterization of a low molecular weightof β-mannanase from Penicillium occitanis Pol6[J].Appl Biochem Biotech,2010,160(4):1227-1240.
36. Malheirosferreira H. Purification and characterization of a β-mannanase from Trichoderma harzianumstrain T4[J]. Carbohyd Polym,2004,57(1):23-29.
37. Parker K N, Chhabra S R, Lam D, et al. Galactomannanases Man2and Man5from Thermotogaspecies:Growth physiology on galactomannans, gene sequence analysis, and biochemical properties ofrecombinant enzymes [J]. Biotechnol Bioeng,2001,75(3):322-333.
38. Zhao W, Zheng J, Zhou H B. A thermotolerant and cold-active mannan endo-1,4-β-mannosidase fromAspergillus niger CBS513.88: Constitutive overexpression and high-density fermentation in Pichiapastoris [J]. Bioresource Technol,2011,102(16):7538-7547.
39. Zhao J, Shi P, Luo H, et al. An acidophilic and acid-stable β-mannanase from Phialophora sp. P13with high mannan hydrolysis activity under simulated gastric gonditions [J]. J Agr Food Chem,2010,58(5):3184-3190.
40. Zahura U A, Rahman M M, Inoue A, et al. An endo-β-1,4-mannanase, AkMan, from the common seahare Aplysia kurodai [J]. Comp Biochem Phys B,2010,157(1):137-143.
41. Tanaka M, Umemoto Y, Okamura H, et al. Cloning and characterization of a β-1,4-mannanase5Cpossessing a family27carbohydrate-binding module from a Marine Bacterium, Vibrio sp. strainMA-138[J]. Biosci Biotech Bioch,2009,73(1):109-116.
42. Bien-Cuong D, Thi-Thu D, Berrin J-G, et al. Cloning, expression in Pichia pastoris, andcharacterization of a thermostable GH5mannan endo-1,4-β-mannosidase from Aspergillus nigerBK01[J]. Microb Cell Fact,2009,8(1):59.
43. Chen X, Cao Y, Ding Y, et al. Cloning, functional expression and characterization of Aspergillussulphureus β-mannanase in Pichia pastoris [J]. J Biotechnol,2007,128(3):452-461.
44. Songsiriritthigul C, Buranabanyat B, Haltrich D, et al. Efficient recombinant expression and secretionof a thermostable GH26mannan endo-1,4-β-mannosidase from Bacillus licheniformis in Escherichiacoli [J]. Microb Cell Fact,2010,9(1):20.
45. Jiang Z, Wei Y, Li D, et al. High-level production, purification and characterization of a thermostableβ-mannanase from the newly isolated Bacillus subtilis WY34[J]. Carbohyd Polym,2006,66(1):88-96.
46. Luo H, Wang K, Huang H, et al. Gene cloning, expression, and biochemical characterization of analkali-tolerant β-mannanase from Humicola insolens Y1[J]. J Ind Microbiol Biot,2012,39(4):547-555.
47. Katrolia P, Zhou P, Zhang P, et al. High level expression of a novel β-mannanase from Chaetomium sp.exhibiting efficient mannan hydrolysis [J]. Carbohyd Polym,2012,87(1):480-490.
48. Luo H, Wang Y, Wang H, et al. A novel highly acidic β-mannanase from the acidophilic fungusBispora sp. MEY-1: gene cloning and overexpression in Pichia pastoris [J]. Appl Microbiol Biot,2009,82(3):453-461.
49. Chauhan P S, Puri N, Sharma P, et al. Mannanases: microbial sources, production, properties andpotential biotechnological applications [J]. Appl Microbiol Biot,2012,93(5):1817-1830.
50. Oosterveld A, Voragen A G J, Schols H A. Effect of roasting on the carbohydrate composition ofCoffea arabica beans [J]. Carbohyd Polym,2003,54(2):183-192.
51. Comfort D A, Chhabra S R, Conners S B, et al. Strategic biocatalysis with hyperthermophilic enzymes[J]. Green Chem,2004,6(9):459-465.
52. Varnai A, Huikko L, Pere J, et al. Synergistic action of xylanase and mannanase improves the totalhydrolysis of softwood [J]. Bioresource technol,2011,102(19):9096-9104.
53. Dhawan S, Kaur J. Microbial mannanases: an overview of production and applications [J]. Crit RevBiotechnol,2007,27(4):197-216.
54. El-Helow E, Sabry S, Khattab A. Production of α-mannanase by B. subtilis from agro-industrialby-products: screening and optimization [J]. Antonie Van Leeuwenhoek,1997,71(3):189-193.
55. Politz O, Krah M, Thomsen K K, et al. A highly thermostable endo-(1,4)-beta-mannanase from themarine bacterium Rhodothermus marinus [J]. Appl Microbiol Biot,2000,53(6):715-721.
56. Yin L J, Tai H M, Jiang S T. Characterization of mannanase from a novel mannanase-producingBacterium [J]. J Agr Food Chem,2012,60(25):6425-6431.
57. Wu M C, Tang C D, Li J F, et al. Bimutation breeding of Aspergillus niger strain for enhancingβ-mannanase production by solid-state fermentation [J]. Carbohyd Res,2011,346(14):2149-2155.
58. Yang P, Li Y, Wang Y et al. A novel β-mannanase with high specific activity from Bacillus circulansCGMCC1554: gene cloning, expression and enzymatic characterization [J]. Appl Biochem Biotech,2009,159(1):85-94.
59. Summpunn P, Chaijan S, Isarangkul D, et al. Characterization, gene cloning, and heterologousexpression of β-mannanase from a thermophilic Bacillus subtilis [J]. J Microbiol,2011,49(1):86-93.
60. Zhou H Y, Pan H Y, Rao L Q, et al. Redesign the alpha/beta fold to enhance the stability of mannanaseman23from Bacillus subtilis [J]. Appl Biochem Biotech,2011,163(1):186-194.
61. Qiao J, Rao Z, Dong B, et al. Expression of Bacillus subtilis MA139β-mannanase in Pichia pastorisand the enzyme characterization [J]. Appl Biochem Biotech,2010,160(5):1362-1370.
62. Zyl P, Moodley V, Rose S H, et al. Production of the Aspergillus aculeatus endo-1,4-β-mannanase in A.niger [J]. J Ind Microbiol Biot,2009,36(4):611-617.
63. Yang X S, Jiang Z B, Song H T, et al. Cell-surface display of the active mannanase in Yarrowialipolytica with a novel surface-display system [J]. Biotechnol Appl Bioc,2009,54(3):171-176.
64. Pham T A, Berrin J G, Record E, et al. Hydrolysis of softwood by Aspergillus mannanase: role of acarbohydrate-binding module [J]. J Biotechnol,2010,148(4):163-170.
65.郜赵伟,张宇宏,张伟.微生物酶分子改造研究进展[J].中国生物工程杂志2010,30(1):98-103.
66.孔祥禄.计算机辅助耐热性研究及分子设计[D]:[硕士学位论文].广州:华南理工大学,2010.
67. Kim M-S, Lei X. Enhancing thermostability of Escherichia coli phytase AppA2by error-prone PCR[J]. Appl Microbiol Biot,2008,79(1):69-75.
68.孔荣,钮利喜,袁静明.易错PCR法定向进化D-海因酶的初步研究[J].山西大学学报(自然科学版),2006,29:425-427.
69. Ang E L, Obbard J P, Zhao H. Probing the molecular determinants of aniline dioxygenase substratespecificity by saturation mutagenesis [J]. FEBS J,2007,274(4):928-939.
70. Gabor E M, Janssen D B. Increasing the synthetic performance of penicillin acylase PAS2bystructure-inspired semi-random mutagenesis [J]. Protein Eng Des Sel,2004,17(7):571-579.
71. Cho C M, Mulchandani A, Chen W. Altering the substrate specificity of organophosphorus hydrolasefor enhanced hydrolysis of chlorpyrifos [J]. Appl Environ Microbiol,2004,70(8):4681-4685.
72. Shi C, Lu X, Ma C, et al. Enhancing the thermostability of a novel β-agarase AgaB through directedevolution [J]. Appl Biochemi Biotech,2008,151(1):51-59.
73. Davoodi J, Wakarchuk W W, Carey P R, et al. Mechanism of stabilization of Bacillus circulansxylanase upon the introduction of disulfide bonds [J]. Biophys Chem,2007,125(2–3):453-461.
74. Fenel F, Leisola M, J nis J, et al. A de novo designed N-terminal disulphide bridge stabilizes theTrichoderma reesei endo-1,4-β-xylanase II [J]. J Biotechnol,2004,108(2):137-143.
75. Huang Y H, Huang C T, Hseu R S. Effects of dockerin domains on Neocallimastix frontalis xylanases[J]. FEMS Microbiol Lett,2005,243(2):455-460.
76. Sokkar P, Mohandass S, Ramachandran M. Multiple templates-based homology modeling enhancesstructure quality of AT1receptor: validation by molecular dynamics and antagonist docking [J]. J MolModel,2011,17(7):1565-1577.
77. Gao S J, Wang J Q, Wu M C, et al. Engineering hyperthermostability into a mesophilic family11xylanase from Aspergillus oryzae by in silico design of N-terminus substitution [J]. Biotechnol Bioeng,2013,110(4):1028-1038.
78.田健.计算机辅助分子设计提高蛋白质热稳定性的研究[D]:[博士学位论文].北京:中国农业科学院,2011.
79. Le QA, Joo JC, Yoo YJ et al. Development of thermostable Candida antarctica lipase B through novelin silico design of disulfide bridge [J]. Biotechnol Bioeng,2012,109(4):867-876.
80.史红玲.米曲霉木聚糖酶基因的克隆表达及耐热性改造[D]:[硕士学位论文].无锡:江南大学,
2012.
81. Timmers L F, Ducati R G, Sanchez-Quitian Z A, et al. Combining molecular dynamics and dockingsimulations of the cytidine deaminase from Mycobacterium tuberculosis H37Rv [J]. J Mol Model,2012,18(2):467-479.
82. Christelle B, Eduardo Bde O, Latifa C, et al. Combined docking and molecular dynamics simulationsto enlighten the capacity of Pseudomonas cepacia and Candida antarctica lipases to catalyzequercetin acetylation [J]. J Biotechnol,2011,156(3):203-210.
83. Zhang Y, Ju J, Peng H, et al. Biochemical and structural characterization of the intracellularmannanase AaManA of Alicyclobacillus acidocaldarius reveals a novel glycoside hydrolase familybelonging to clan GH-A [J]. J Biol Chem,2008,283(46):31551-31558.
84. Le Nours J, Anderson L, Stoll D, et al. The structure and characterization of a modularendo-β-1,4-mannanase from Cellulomonas fimi [J]. Biochemistry,2005,44(38):12700-12708.
85. Roth R, Moodley V, Zyl P. Heterologous expression and optimized production of an Aspergillusaculeatus endo-1,4-β-mannanase in Yarrowia lipolytica [J]. Mol Biotechnol,2009,43(2):112-120.
86.李剑芳,马丽萍,邬敏辰等.宇佐美曲霉酸性β-甘露聚糖酶的纯化及性质研究[J].食品与发酵工业,2006,32(9):5-9.
87. Sambrook J, Russell D W. Molecular cloning: a laboratory manual, Cold Spring Harbor, NY, USA:Cold Spring Harbor Laboratory Press,2001.1595-1604.
88. Wang J Q, Zhang H M, Wu M C. et al. Cloning and sequence analysis of a novel xylanase gene,Auxyn10A, from Aspergillus usamii [J]. Biotechnol Lett,2011,33(5):1029-1038.
89. Wenkel S, Turck F, Singer K, et al. CONSTANS and the CCAAT box binding complex share afunctionally important domain and interact to regulate flowering of Arabidopsis [J]. Plant Cell,2006,18(11):2971-2984.
90. Petersen T N, Brunak S, von Heijne G, et al. SignalP4.0: discriminating signal peptides fromtransmembrane regions [J]. Nat Meth,2011,8(10):785-786.
91.赵月菊,薛燕芬,马延和. β-甘露聚糖酶的结构生物学研究现状和展望[J].微生物学报,2009,49(9):1131-1137.
92. Zhang J H, Wu D, Chen J, et al. Enhancing functional expression of β-glucosidase in Pichia pastorisby co-expressing protein disulfide isomerase [J]. Biotechnol Bioproc E,2011,16(6):1196-1200.
93. Sreekrishna K, Brankamp R G, Kropp K E, et al. Strategies for optimal synthesis and secretion ofheterologous proteins in the methylotrophic yeast Pichia pastoris [J]. Gene,1997,190:55-62.
94. Chen X, Qiao J, Yu H, et al. Overexpression of an optimized Aspergillus sulphureus β-mannanasegene in Pichia pastoris [J]. Biologia,2009,64(2):235-238.
95. Akcapinar G B, Gul O, Sezerman U. Effect of codon optimization on the expression of Trichodermareesei endo-glucanase1in Pichia pastoris [J]. Biotechnol Progr,2011,27(5):1257-1263.
96. Tan Z, Li J, Wu M, et al. High-level heterologous expression of an alkaline lipase gene fromPenicillium cyclopium PG37in Pichia pastoris [J]. World J Microb Biot,2011,27(12):2767-2774.
97.李剑芳,赵顺阁,邬敏辰等. β-甘露聚糖酶基因和木聚糖酶基因在毕赤酵母中的共表达[J].食品与生物技术学报2012,31(11):1136-1141.
98. Lin S S, Dou W F, Xu H Y, et al. Optimization of medium composition for the production of alkalineβ-mannanase by alkaliphilic Bacillus sp. N16-5using response surface methodology [J]. ApplMicrobiol Biot,2007,75(5):1015-1022.
99. Ma S, Wang H, Wang Y, et al. Bio-hydrogen production from cornstalk wastes by orthogonal designmethod [J]. Renew Energ,2011,36(2):709-713.
100. Li J F, Tang C D, Shi H L, et al. Cloning and optimized expression of a neutral endoglucanase gene(ncel5A) from Volvariella volvacea WX32in Pichia pastoris [J]. J Biosci Bioeng,2011,111(5):537-540.
101.Laemmli U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J].Nature,1970,227:680-685.
102. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of proteinutilizing the principle of protein-dye binding [J]. Anal Biochem,1976,72:248-254.
103. Dubois M, Gilles K A, Hamilton J K, et al. Colorimetric method for determination of sugars andrelated substances [J]. Anal Chem,1956,28:350-356.
104. Katakura Y, Zhang W, Zhuang G, et al. Effect of methanol concentration on the production of humanβ2-glycoproteinⅠ domain V by a recombinant Pichia pastoris: A simple system for the control ofmethanol concentration using a semiconductor gas sensor [J]. Ferment Bioeng,1998,86:482-487.
105. Cregg J M, Higgins D R. Production of foreign proteins in the yeast Pichia pastoris [J]. Can J Bot,1995,73(S1):891-897.
106. Srimathi S, Jayaraman G. Effect of glycosylation on the catalytic and conformational stability ofhomologous α-amylases [J]. Protein J,2005,24(2):79-88.
107. Shental-Bechor D, Levy Y. Effect of glycosylation on protein folding: a close look at thermodynamicstabilization [J]. Proc Natl Acad Sci U S A,2008,105(24):8256-8261.
108. Guillen D, Sanchez S, Rodriguez-Sanoja R. Carbohydrate-binding domains: multiplicity of biologicalroles [J]. Appl Microbiol Biot,2010,85(5):1241-1249.
109. Thongekkaew J, Ikeda H, Iefuji H. Increases thermal stability and cellulose-binding capacity ofCryptococcus sp. S-2lipase by fusion of cellulose binding domain derived from Trichoderma reesei[J]. Biochem Bioph Res Co,2012,420(1):183-187.
110. Ravalason H, Herpoel-Gimbert I, Record E, et al. Fusion of a family1carbohydrate binding moduleof Aspergillus niger to the Pycnoporus cinnabarinus laccase for efficient softwood kraft pulpbiobleaching [J]. J Biotechnol,2009,142(3-4):220-226.
111. Phosrithong N, Ungwitayatorn J. Molecular docking study on anticancer activity of plant-derivednatural products [J]. Med Chem Res,2009,19(8):817-835.
112. Poole D M, Hazlewood G P, Huskisson N S, et al. The role of conserved tryptophan residues in theinteraction of a bacterial cellulose binding domain with its ligand [J]. FEMS Microbiol Lett,1993,106(1):77-83.
113. Shoseyov O, Shani Z, Levy I. Carbohydrate binding modules: biochemical properties and novelapplications [J]. Microbiol Mol Biol R,2006,70(2):283-295.
114. Neeraja C, Moerschbacher B, Podile A R. Fusion of cellulose binding domain to the catalytic domainimproves the activity and conformational stability of chitinase in Bacillus licheniformis DSM13[J].Bioresource Technol,2010,101(10):3635-3641.
115. Zhang Y, Chen S, Xu M, et al. Characterization of Thermobifida fusca cutinase-carbohydrate-bindingmodule fusion proteins and their potential application in bioscouring [J]. Appl Environ Microbiol,2010,76(20):6870-6876.
116. Ye X, Zhu Z, Zhang C, et al. Fusion of a family9cellulose-binding module improves catalyticpotential of Clostridium thermocellum cellodextrin phosphorylase on insoluble cellulose [J]. ApplMicrobiol Biot,2011,92(3):551-560.
117. Wan W, Wang D, Gao X, et al. Expression of family3cellulose-binding module (CBM3) as an affinitytag for recombinant proteins in yeast [J]. Appl Microbiol Biot,2011,91(3):789-798.
118. Sriprang R, Asano K, Gobsuk J, et al. Improvement of thermostability of fungal xylanase by usingsite-directed mutagenesis [J]. J Biotechnol,2006,126(4):454-462.
119. Liu L, Zhang G, Zhang Z, et al. Terminal amino acids disturb xylanase thermostability and activity [J].J Biol Chem,2011,286(52):44710-44715.
120. Wen T, Chen J, Lee S, et al. A truncated Fibrobacter succinogenes1,3-1,4-β-D-glucanase withimproved enzymatic activity and thermotolerance [J]. Biochemistry,2005,44(25):9197-9205.
121. Dombkowski A A. Disulfide by DesignTM: a computational method for the rational design of disulfidebonds in proteins [J]. Bioinformatics,2003,19(14):1852-1853.
122. Albrecht S, van Muiswinkel G C, Schols H A, et al. Introducing capillary electrophoresis withlaser-induced fluorescence detection (CE-LIF) for the characterization of konjac glucomannanoligosaccharides and their in vitro fermentation behavior [J]. J Agr Food Chem,2009,57(9):3867-3876.
123. Shi H L, Yin X, Wu M C, et al. Cloning and bioinformatics analysis of an endo-glucanase gene(Aucel12A) from Aspergillus usamii and its functional expression in Pichia pastoris [J]. J IndMicrobiol Biot,2012,39(2):347-357.