乳酸片球菌素的表达纯化与高密度发酵研究
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摘要
天然防腐剂的研究与开发随着人们对食品安全性的认识和要求的提高,近年来成为食品工业的一个热点。细菌素(bacteriocin)是一些细菌产生的一类蛋白抗菌物质,可以用作防腐剂,因其安全有效性而得到人们的重视。但细菌素产生菌许多因为产量低、基因表达不稳定而未得到应用。从乳酸菌分离的细菌素由于它们的抗食源性病菌、食物腐败细菌活性已经被一些研究者研究。乳酸片球菌素(Pediocin)由于它的强抗利斯特氏菌活性而被较多关注。在本课题的前期研究中,我们从牛初乳中筛选到了一株产Pediocin的乳酸片球菌Pediococcus acidilacticii LH311。本课题研究分离纯化来自P.acidilacticiiLH31的细菌素(pediocin PA-1),鉴定其理化性质,并克隆Pediocin基因,进行异源表达,研究其表达特性,进行高密度发酵,实施碳氮营养源平衡流加策略,实现高效表达。
利用细胞吸附法初步分离乳酸片球菌素,再经半制备型反相液相色谱得到纯品。对纯化的乳酸片球菌素进行Tris-Tricine系统电泳,测定其分子量为5 000 Da左右。采用酶蛋白(来源于纤维化纤维微细菌(Cellulosimicrobium cellulans)的纤维素酶和木聚糖酶)特性鉴定的思路、经验、方法,来鉴定短肽乳酸片球菌素(pediocin PA-1)的特性,得出最适抑菌温度为25℃,最适抑菌pH为6；乳酸片球菌素在沸水浴处理一段时间后,仍有一定活性,但是随着时间的增长,其活性逐渐降低；在较宽范围pH处理后仍有较稳定的活性,在pH接近酸性时稳定性更高；较高浓度的乳酸片球菌素的稳定性较高,这可能是由于高浓度时分子间集聚反应起到了对极端温度和pH的缓冲作用。由系列梯度乳酸片球菌素对粪肠球菌实验得出其最低抑菌浓度(MIC)为50μg/mL,制定了不同剂量乳酸片球菌素的时间杀菌曲线。
为在大肠杆菌中高效表达乳酸片球菌素pediocin PA-1 (PED),按照NCBI上公布的PED基因序列设计合成引物,以乳酸片球菌(Pediococcus acidilacticii LH31)基因组DNA为模板,用PCR扩增PED结构基因特异片段,构建编码组氨酸标记硫氧还原蛋白融合蛋白PED-N的重组表达质粒pET32c-PED,并转化大肠杆菌BL21(DE3)和BL21(DE3)pLysS。在IPTG诱导后融合型乳酸片球菌素(fusion-typed pediocin PA-1, PED-N)以包涵体形式表达,经镍亲合层析纯化的PED-N用肠激酶裂解,再经超滤纯化,可得78%的收率。PED-N没有抗粪肠球菌(Enterococcus faecalis)细菌素活性,被分开的PED恢复了它的抑菌活性。研究实现了细菌素PED在大肠杆菌中的高效融合表达,并得到有生物学活性的PED,为进一步研究其应用奠定了一定的基础,同时也为研究细菌素表达提供了一种方法。
分析比较不同IPTG诱导条件(诱导温度、诱导时间、诱导剂浓度)对于PED-N表达的影响,以确定最佳IPTG诱导条件。对于BL21(DE3),PED-N的表达水平在诱导后3小时达到最大；对于BL21(DE3)pLysS,PED-N的表达水平在诱导后5小时达到最大。对于BL21(DE3),PED-N的表达水平在诱导剂浓度为0.2 mmol/L最大,对于BL21(DE3)pLysS,PED-N的表达水平在诱导剂浓度为2.0 mmol/L时最大。
BL21(DE3)pLysS的菌浓、PED-N产率均比同培养条件下BL21(DE3)要高。在37℃条件下诱导,两菌株均获得高效表达,目标蛋白以包涵体形式为主；BL21(DE3)pLysS表达水平高于BL21(DE3)；而BL21(DE3)表达的可溶性蛋白比例略高于BL21(DE3) pLysS.在30℃条件下诱导,两菌株同样获得高效表达,但表达水平均较于37℃条件下低；目标蛋白仍然均以包涵体形式为主,但是可溶性蛋白比例均比37℃条件下高；BL21(DE3)pLysS表达水平低于BL21(DE3),而BL21(DE3)pLysS表达的可溶性蛋白比例略高于BL21(DE3)。
Triton X-100对包涵体PED-N溶解性有显著影响,裂解液和洗涤液中均含有1%Triton X-100作用于包涵体后,可溶性PED-N蛋白的含量高于裂解液不含Triton X-100、洗涤液中含有1% Triton X-100的可溶性PED-N蛋白含量。尿素的浓度可对融合蛋白PED-N包涵体溶解起着重要作用,随着尿素浓度的增加,可溶性蛋白的含量呈上升之势。3 mol/L尿素即可使近一半的PED-N包涵体溶解,5 mol/L尿素时可使大部分的PED-N包涵体变为可溶性的。在尿素中加入STT可以大大增加对PED-N包涵体溶解作用。
优化表达融合型乳酸片球菌素(PED-N)工程菌的高密度发酵条件。筛选半合成培养基作为发酵培养基,其作为高密度培养基的成分甘油最适浓度为10 mL/L,氮源蛋白胨最适浓度为5 g/L。选择在37℃下,恒pH 6.5-7.0培养,在工程菌对数生长中期进行诱导。采用1g/L乳糖作为诱导剂,诱导表达的时间为5 h。用自控发酵罐进行溶氧反馈一限制性补料高密度发酵,控制补料速率以稳定比生长率在0.2 h-1上下,溶氧值反馈值设定于30%-40%。最终菌体密度A600值达到了116,PED-N的表达量为1.09 g/L。DO-stat流加发酵时间相对于恒速流加较短、相对于加速流加较长,甘油利用充分、残余量少,表达目的蛋白PED-N效率最高。
本文还对碳氮比、碳氮平衡补料流加策略对重组E.coliBL21(DE3)pLysS生长与表达PED-N的影响进行了研究。摇瓶实验结果当碳氮比为1.0时菌体浓度达到最大值,可溶性PED-N有最大比例,碳氮比过高或过低均不利于菌体生长；碳氮比较高或较低时,可溶性PED-N比例较低、包涵体PED-N所占比例较高。在重组E.coliBL21(DE3)pLysS培养的不同时间阶段补料流加不同碳氮比的营养源,实现碳氮实时流加平衡,有效地避免了乙酸的积累,获得了高密度的菌体(最大菌体密度湿重可达81.8 g/L),PED-N表达产率、可溶性PED-N比例也较高(PED-N含量可达0.463 g/L,可溶性PED-N蛋白含量可达0.157 g/L)。
Due to people's awareness of food safety and demand increasing in recent years, research and development of natural preservatives wasing a hot. Bacteriocin is a protein produced by certain bacteria antibacterial substances. Bacteriocins as a "green preservative" has been paid an attention. Bacteriocin-producing strains in the application is limited mainly because of low output and instability of gene expression. Bacteriocins isolated from lactic acid bacteria (LAB) have been studied by many researchers due to their bactericidal activities against food-borne pathogens and food-spoilage bacteria. Pediocin was paid more attention because of its strong anti-Listeria activity. We have isolated a lactic acid bacteria producing pediocin from bovine colostrum, Pediococcus acidilacticii LH31. Object of this research is about the purification of pediocin PA-1 from P. acidilacticii LH31, identifying their physical and chemical properties, and Cloning Pediocin genes for heterologous expression, studying the expression characteristics, investigating the optimal high cell-density culture procedure and feeding strategy, to obtain high-level expression.
The bacteriocin from P. acidilacticii LH31, dsignated as pediocin PA-1, was rapidly purified to homogeneity by the pH mediated cell adsorption-desorption method and semi-preparative reversed-phase HPLC. It gave a single peak on tricine-SDS-PAGE, the protein molecular weight was determined as 5000 Da. Characteristics of pediocin PA-1 was identified by use of the ideas, experience and methods for characterization of cellulases and xylanases from Cellulosimicrobium cellulans. The activity of pediocin PA-1 against Enterococcus faecalis was determined, optimum temperature at 25℃and optimum pH at 6.0. Pediocin treated in boiling water bath for some time remained active, but its activity gradually reduced as time growing. After treated in wide range of pH, pediocin remained stable activity; stability is higher in lower pH. Its stability is higher as concentration of pediocin increasing, High concentration of pediocin (>50μg/ml) showed good resistance to extremes of pH (13) and temperature (121℃). By a series of gradient pediocin against E. faecalis experiments its minimum inhibitory concentration (MIC) was 50μg/mL, formulating time bactericidal curve with different doses of pediocin.
To effectively express pediocin PA-1 (PED) in Escherichia coli, the primers specific for PED coding sequence was designed and synthesized according to the NCBI. E. coli BL21(DE3)/pET expression system was used to produce recombinant pediocin, Pediocin PA-1 fused with thioredoxin (PED-N). The gene fragment of PED was amplified from genomic DNA of Pediococcus acidilacticii LH31 by PCR. The plasmid pET32c-PED, encoding PED fused with His-tagged thioredoxin protein, was constructed and introduced into Escherichia coli BL21 (DE3) strain. The plasmid stabilities is higher in BL21(ED3)plysS than in BL21(DE3). The fusion protein was expressed in the strain after induction of isopropyl-β-D-thiogalactopyranoside (IPTG) and purified by nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography. For the recovery of biologically active PED, the purified fusion protein was cleaved by enterokinase and the liberated PED was finally purified by ultrafiltration with a 78% yield. The fusion protein, which did not have bactericidal activity against Enterococcus faecalis, was cleaved by enterokinase and the cleaved PED recovered its own bactericidal activity. The paper could be of great value for effective expression of a fusion-typed PED in E. coli, obtaining biologically active PED, and providing some basis to study application of PED, and also provides some approach to study the expression of bacteriocin.
The research was conducted to investigate the characteristics of PED-N expression in BL21 (DE3) and BL21 (DE3) pLysS. For the BL21 (DE3), PED-N expression levels was maximum 3 hours after the induction; for the BL21 (DE3) pLysS, PED-N expression levels was maximum 5 hours after the induction. For the BL21 (DE3), PED-N expression levels was maximum when IPTG concentration was 0.2 mmol/L; for the BL21 (DE3) pLysS, PED-N expression levels was maximum when IPTG concentration was 2.0 mmol/L. Biomass and PED-N yield of BL21 (DE3) pLysS, were higher than those of BL21 (DE3) in the same culture conditions. At 37℃or 30℃, PED-N are effectively expressed in both strains, mainly in form of inclusion bodies; At 37℃, expression level was higher than at 30℃; At 30℃, the proportion of soluble protein was higher than at 37℃. At 37℃, expression level in BL21 (DE3) pLys was higher than BL21 (DE3), but the proportion of soluble protein of BL21 (DE3) was slightly higher than that of BL21 (DE3) pLys. At 30℃, expression level in BL21 (DE3) pLys was lower than the BL21 (DE3), while the proportion of soluble protein of BL21 (DE3) pLysS was slightly higher than BL21 (DE3). The fusion proteins were very sensitive to 1.0% Triton X-100, most of fusion protein became soluble when inclusion bodies were washed with 1.0% Triton X-100. Approximately half of the inclusion body was redissolved in 3 moI/L urea; when increasing the concentration of the urea to 5 mol/L, most of proteins were redissolved.
Optimization of the high cell-density culture procedure of fusion-typed pediocin PA-1 (PED-N) in recombinant E. coli was carried out in 250 mL shaking flasks to investigate the effects of the composition of the culture medium, the range of pH, and induction condition on the growth of bacteria and the expression yield of PED-N and then transferred to a 5 L DO feed-back fed-batch culture system to analyze the optimal dissolved oxygen and specific growth rates. The results indicate that by keeping dissolved oxygen at 30%-40%and controlling nutrient feeding rate with DO feed back strategy, keeping pH at 6.5-7.0, culturing in optimized semi-synthetical medium and inducing for 5 h in the presence of 1 g/L lactose at 37℃during fed-batch cultivation, a high cell density (A600=16) and 1.09 g PED-N per liter of broth were obtained under constant (0.2 h-1) specific growth rates in shorter fermentation periods.
Excessive carbon source in medium may easily lead to acetic acid production, inhibit cell growth and exogenous protein expression. Cell concentration reached the maximum when C/N ratio was 1.0 in shake-flask, while soluble PED-N has the largest proportion. C/N ratio was too high or too low, not conducive to cell growth, the low percentage of soluble PED-N, a high proportion of inclusion bodies. New feeding strategies based on feeding balance between carbon source and nitrogen source were designed. Using a multistage carbon-nitrogen ratios feeding strategy to achive real-time feeding balance of carbon and nitrogen during fermentation process, a high density of biomass (maximum biomass density of wet weight of up to 81.8 g L-1) and high expression of fusion protein (fusion-typed pediocin PA-1) (content of up to 0.463 g L-1) and soluble protein (content of up to 0.157 g L-1) were obtained. The accumulation of acetate, which usually occurs during the process of high-density fermentation of recombinant Escherichia coli, was effectively avoided.
利用细胞吸附法初步分离乳酸片球菌素,再经半制备型反相液相色谱得到纯品。对纯化的乳酸片球菌素进行Tris-Tricine系统电泳,测定其分子量为5 000 Da左右。采用酶蛋白(来源于纤维化纤维微细菌(Cellulosimicrobium cellulans)的纤维素酶和木聚糖酶)特性鉴定的思路、经验、方法,来鉴定短肽乳酸片球菌素(pediocin PA-1)的特性,得出最适抑菌温度为25℃,最适抑菌pH为6；乳酸片球菌素在沸水浴处理一段时间后,仍有一定活性,但是随着时间的增长,其活性逐渐降低；在较宽范围pH处理后仍有较稳定的活性,在pH接近酸性时稳定性更高；较高浓度的乳酸片球菌素的稳定性较高,这可能是由于高浓度时分子间集聚反应起到了对极端温度和pH的缓冲作用。由系列梯度乳酸片球菌素对粪肠球菌实验得出其最低抑菌浓度(MIC)为50μg/mL,制定了不同剂量乳酸片球菌素的时间杀菌曲线。
为在大肠杆菌中高效表达乳酸片球菌素pediocin PA-1 (PED),按照NCBI上公布的PED基因序列设计合成引物,以乳酸片球菌(Pediococcus acidilacticii LH31)基因组DNA为模板,用PCR扩增PED结构基因特异片段,构建编码组氨酸标记硫氧还原蛋白融合蛋白PED-N的重组表达质粒pET32c-PED,并转化大肠杆菌BL21(DE3)和BL21(DE3)pLysS。在IPTG诱导后融合型乳酸片球菌素(fusion-typed pediocin PA-1, PED-N)以包涵体形式表达,经镍亲合层析纯化的PED-N用肠激酶裂解,再经超滤纯化,可得78%的收率。PED-N没有抗粪肠球菌(Enterococcus faecalis)细菌素活性,被分开的PED恢复了它的抑菌活性。研究实现了细菌素PED在大肠杆菌中的高效融合表达,并得到有生物学活性的PED,为进一步研究其应用奠定了一定的基础,同时也为研究细菌素表达提供了一种方法。
分析比较不同IPTG诱导条件(诱导温度、诱导时间、诱导剂浓度)对于PED-N表达的影响,以确定最佳IPTG诱导条件。对于BL21(DE3),PED-N的表达水平在诱导后3小时达到最大；对于BL21(DE3)pLysS,PED-N的表达水平在诱导后5小时达到最大。对于BL21(DE3),PED-N的表达水平在诱导剂浓度为0.2 mmol/L最大,对于BL21(DE3)pLysS,PED-N的表达水平在诱导剂浓度为2.0 mmol/L时最大。
BL21(DE3)pLysS的菌浓、PED-N产率均比同培养条件下BL21(DE3)要高。在37℃条件下诱导,两菌株均获得高效表达,目标蛋白以包涵体形式为主；BL21(DE3)pLysS表达水平高于BL21(DE3)；而BL21(DE3)表达的可溶性蛋白比例略高于BL21(DE3) pLysS.在30℃条件下诱导,两菌株同样获得高效表达,但表达水平均较于37℃条件下低；目标蛋白仍然均以包涵体形式为主,但是可溶性蛋白比例均比37℃条件下高；BL21(DE3)pLysS表达水平低于BL21(DE3),而BL21(DE3)pLysS表达的可溶性蛋白比例略高于BL21(DE3)。
Triton X-100对包涵体PED-N溶解性有显著影响,裂解液和洗涤液中均含有1%Triton X-100作用于包涵体后,可溶性PED-N蛋白的含量高于裂解液不含Triton X-100、洗涤液中含有1% Triton X-100的可溶性PED-N蛋白含量。尿素的浓度可对融合蛋白PED-N包涵体溶解起着重要作用,随着尿素浓度的增加,可溶性蛋白的含量呈上升之势。3 mol/L尿素即可使近一半的PED-N包涵体溶解,5 mol/L尿素时可使大部分的PED-N包涵体变为可溶性的。在尿素中加入STT可以大大增加对PED-N包涵体溶解作用。
优化表达融合型乳酸片球菌素(PED-N)工程菌的高密度发酵条件。筛选半合成培养基作为发酵培养基,其作为高密度培养基的成分甘油最适浓度为10 mL/L,氮源蛋白胨最适浓度为5 g/L。选择在37℃下,恒pH 6.5-7.0培养,在工程菌对数生长中期进行诱导。采用1g/L乳糖作为诱导剂,诱导表达的时间为5 h。用自控发酵罐进行溶氧反馈一限制性补料高密度发酵,控制补料速率以稳定比生长率在0.2 h-1上下,溶氧值反馈值设定于30%-40%。最终菌体密度A600值达到了116,PED-N的表达量为1.09 g/L。DO-stat流加发酵时间相对于恒速流加较短、相对于加速流加较长,甘油利用充分、残余量少,表达目的蛋白PED-N效率最高。
本文还对碳氮比、碳氮平衡补料流加策略对重组E.coliBL21(DE3)pLysS生长与表达PED-N的影响进行了研究。摇瓶实验结果当碳氮比为1.0时菌体浓度达到最大值,可溶性PED-N有最大比例,碳氮比过高或过低均不利于菌体生长；碳氮比较高或较低时,可溶性PED-N比例较低、包涵体PED-N所占比例较高。在重组E.coliBL21(DE3)pLysS培养的不同时间阶段补料流加不同碳氮比的营养源,实现碳氮实时流加平衡,有效地避免了乙酸的积累,获得了高密度的菌体(最大菌体密度湿重可达81.8 g/L),PED-N表达产率、可溶性PED-N比例也较高(PED-N含量可达0.463 g/L,可溶性PED-N蛋白含量可达0.157 g/L)。
Due to people's awareness of food safety and demand increasing in recent years, research and development of natural preservatives wasing a hot. Bacteriocin is a protein produced by certain bacteria antibacterial substances. Bacteriocins as a "green preservative" has been paid an attention. Bacteriocin-producing strains in the application is limited mainly because of low output and instability of gene expression. Bacteriocins isolated from lactic acid bacteria (LAB) have been studied by many researchers due to their bactericidal activities against food-borne pathogens and food-spoilage bacteria. Pediocin was paid more attention because of its strong anti-Listeria activity. We have isolated a lactic acid bacteria producing pediocin from bovine colostrum, Pediococcus acidilacticii LH31. Object of this research is about the purification of pediocin PA-1 from P. acidilacticii LH31, identifying their physical and chemical properties, and Cloning Pediocin genes for heterologous expression, studying the expression characteristics, investigating the optimal high cell-density culture procedure and feeding strategy, to obtain high-level expression.
The bacteriocin from P. acidilacticii LH31, dsignated as pediocin PA-1, was rapidly purified to homogeneity by the pH mediated cell adsorption-desorption method and semi-preparative reversed-phase HPLC. It gave a single peak on tricine-SDS-PAGE, the protein molecular weight was determined as 5000 Da. Characteristics of pediocin PA-1 was identified by use of the ideas, experience and methods for characterization of cellulases and xylanases from Cellulosimicrobium cellulans. The activity of pediocin PA-1 against Enterococcus faecalis was determined, optimum temperature at 25℃and optimum pH at 6.0. Pediocin treated in boiling water bath for some time remained active, but its activity gradually reduced as time growing. After treated in wide range of pH, pediocin remained stable activity; stability is higher in lower pH. Its stability is higher as concentration of pediocin increasing, High concentration of pediocin (>50μg/ml) showed good resistance to extremes of pH (13) and temperature (121℃). By a series of gradient pediocin against E. faecalis experiments its minimum inhibitory concentration (MIC) was 50μg/mL, formulating time bactericidal curve with different doses of pediocin.
To effectively express pediocin PA-1 (PED) in Escherichia coli, the primers specific for PED coding sequence was designed and synthesized according to the NCBI. E. coli BL21(DE3)/pET expression system was used to produce recombinant pediocin, Pediocin PA-1 fused with thioredoxin (PED-N). The gene fragment of PED was amplified from genomic DNA of Pediococcus acidilacticii LH31 by PCR. The plasmid pET32c-PED, encoding PED fused with His-tagged thioredoxin protein, was constructed and introduced into Escherichia coli BL21 (DE3) strain. The plasmid stabilities is higher in BL21(ED3)plysS than in BL21(DE3). The fusion protein was expressed in the strain after induction of isopropyl-β-D-thiogalactopyranoside (IPTG) and purified by nickel-nitrilotriacetic acid (Ni-NTA) metal affinity chromatography. For the recovery of biologically active PED, the purified fusion protein was cleaved by enterokinase and the liberated PED was finally purified by ultrafiltration with a 78% yield. The fusion protein, which did not have bactericidal activity against Enterococcus faecalis, was cleaved by enterokinase and the cleaved PED recovered its own bactericidal activity. The paper could be of great value for effective expression of a fusion-typed PED in E. coli, obtaining biologically active PED, and providing some basis to study application of PED, and also provides some approach to study the expression of bacteriocin.
The research was conducted to investigate the characteristics of PED-N expression in BL21 (DE3) and BL21 (DE3) pLysS. For the BL21 (DE3), PED-N expression levels was maximum 3 hours after the induction; for the BL21 (DE3) pLysS, PED-N expression levels was maximum 5 hours after the induction. For the BL21 (DE3), PED-N expression levels was maximum when IPTG concentration was 0.2 mmol/L; for the BL21 (DE3) pLysS, PED-N expression levels was maximum when IPTG concentration was 2.0 mmol/L. Biomass and PED-N yield of BL21 (DE3) pLysS, were higher than those of BL21 (DE3) in the same culture conditions. At 37℃or 30℃, PED-N are effectively expressed in both strains, mainly in form of inclusion bodies; At 37℃, expression level was higher than at 30℃; At 30℃, the proportion of soluble protein was higher than at 37℃. At 37℃, expression level in BL21 (DE3) pLys was higher than BL21 (DE3), but the proportion of soluble protein of BL21 (DE3) was slightly higher than that of BL21 (DE3) pLys. At 30℃, expression level in BL21 (DE3) pLys was lower than the BL21 (DE3), while the proportion of soluble protein of BL21 (DE3) pLysS was slightly higher than BL21 (DE3). The fusion proteins were very sensitive to 1.0% Triton X-100, most of fusion protein became soluble when inclusion bodies were washed with 1.0% Triton X-100. Approximately half of the inclusion body was redissolved in 3 moI/L urea; when increasing the concentration of the urea to 5 mol/L, most of proteins were redissolved.
Optimization of the high cell-density culture procedure of fusion-typed pediocin PA-1 (PED-N) in recombinant E. coli was carried out in 250 mL shaking flasks to investigate the effects of the composition of the culture medium, the range of pH, and induction condition on the growth of bacteria and the expression yield of PED-N and then transferred to a 5 L DO feed-back fed-batch culture system to analyze the optimal dissolved oxygen and specific growth rates. The results indicate that by keeping dissolved oxygen at 30%-40%and controlling nutrient feeding rate with DO feed back strategy, keeping pH at 6.5-7.0, culturing in optimized semi-synthetical medium and inducing for 5 h in the presence of 1 g/L lactose at 37℃during fed-batch cultivation, a high cell density (A600=16) and 1.09 g PED-N per liter of broth were obtained under constant (0.2 h-1) specific growth rates in shorter fermentation periods.
Excessive carbon source in medium may easily lead to acetic acid production, inhibit cell growth and exogenous protein expression. Cell concentration reached the maximum when C/N ratio was 1.0 in shake-flask, while soluble PED-N has the largest proportion. C/N ratio was too high or too low, not conducive to cell growth, the low percentage of soluble PED-N, a high proportion of inclusion bodies. New feeding strategies based on feeding balance between carbon source and nitrogen source were designed. Using a multistage carbon-nitrogen ratios feeding strategy to achive real-time feeding balance of carbon and nitrogen during fermentation process, a high density of biomass (maximum biomass density of wet weight of up to 81.8 g L-1) and high expression of fusion protein (fusion-typed pediocin PA-1) (content of up to 0.463 g L-1) and soluble protein (content of up to 0.157 g L-1) were obtained. The accumulation of acetate, which usually occurs during the process of high-density fermentation of recombinant Escherichia coli, was effectively avoided.
引文
[1]杜芸.天然食品防腐剂的研究进展.食品研究与开发,2006,27(3)：162-165
[2]马文山,廖富蘋,范雪云,等.细菌抗菌肽的研究进展及其应用.生物技术通报,2008,3：39-43,49
[3]甘晓玲.细菌素在食品工业中的应用价值.科学咨询,2008,17：48
[4]吴琦,胡建平.抑菌活性蛋白(肽)作为食品防腐剂的研究进展.食品研究与开发,2008,29(1)：168-172
[5]田召芳,牛钟相,常维山,等.乳酸菌细菌素的研究进展.微生物学杂志,2003,6：119-122
[6]黄现青,高晓平,韩庆功,等.乳酸菌类细菌素的研究进展.中国乳品工业,2007,35(9)：41-43,57
[7]李红,赵春燕.乳酸链球菌素的研究进展.食品科技,2006,31(1)：75-78
[8]还连栋,陈秀珠.天然食品防腐剂乳链菌肽的性质及其应用.天然产物研究与开发,1995,7(1)：55-60
[9]Kanatani K, Oshimura M. Plasmid-associated bacteriocin production by a Lactobacillus plantarum strain. Bioscience, Biotechnology and Biochemistry,1994,58(11): 2084-2086
[10]Miller KW, Schamber R, Osmanagaoglu O, et al. Isolation and characterization of pediocin AcH chimeric protein mutants with altered bactericidal activity. Applied And Environmental Microbiology,1998,64 (6):1997-2005
[11]McAuliffe O, Ross R P, Hill C. Lantibiotics:structure, biosynthesis and mode of action. FEMS Microbiology Reviews,2001,25:285-308.
[12]吕燕妮,李平兰,周伟,等.Ⅱa类细菌素的结构、生物合成与活性.微生物学通报,2007,34(5)：955-959
[13]Ennahar S, Sashihara T, Sonomoto K, et al. Class IIa bacteriocins:biosynthesis, structure and activity. FEMS Microbiology Reviews,2000,24:85-106.
[14]韩雪,周志江.乳酸片球菌细菌素的活性及特性的研究.食品研究与开发,2006,3：19-21
[15]Sablon E, Contreras B, Vandamme, E. Antimicrobial peptides of lactic acid bacteria: mode of action, genetics and biosynthesis. Advances in Biochemical Engineering/ Biotechnology,2000,68:21-59
[16]Eijsink V G H, Axelsson L, Diep D B, et al. Production of class Ⅱ bacteriocins by lactic acid bacteria; an example of biological warfare and communication. Antonie van Leeuwenhoek,2002,81:639-654
[17]Somkuti G A, Steinberg D H. Pediocin production by recombinant lactic acid bacteria. Biotechnol Lett,2003,25:473-477
[18]King W., Creek W., Ming X., Grove C., Antibacterial composition for control of gram positive bactertia in food applications. US6451365B1,2002.
[19]Ray B, Schamber R, Miller K W. The pediocin AcH precursor is biologically active. Applied and Environmental Microbiology,1999,65:2281-2286.
[20]Fimland G, Johnsen L, Axelsson L, et al. A C-terminal disulfide bridge in pediocin-like bacteriocins renders bacteriocin activity less temperature dependent and is a major determinant of the antimicrobial spectrum. Journal of Bacteriology,2000,182: 2643-2648.
[21]Johnsen L, Fimland G., Eijsink V, et al. Engineering increased stability in the antimicrobial peptide pediocin PA-1. Applied and Environmental Microbiology,2000,66: 47984802.
[22]Chen Y, Ludescher R D, Montville T J. Electrostatic interactions, but not the YGNGV consensus motif, govern the binding of pediocin PA-1 and its fragments to phospholipid vesicles. Appl Environ Microbiol,1997,63:4770-4777
[23]Moon G S, Pyun Y R, Kim W J. Characterization of pediocin operon of Pediococcus acidilactici K10 and expression of His-tagged recombinant pediocin PA-1 in Escherichia coli. Journal of Microbiology and Biotechnology,2005,15:403-411
[24]Henderson J T, Chopko A L, Van Wassenaar P D. Purification and primary structure of pediocin PA-1 produced by Pediococcus acidilactici PAC-1.0. Arch Biochem Biophys, 1992,295:5-12
[25]Jack R W, Tagg J R, Ray B. Bacteriocins of Gram-Positive bacteria. Microbiol Rev, 1995,59:171-200
[26]Lucie B., Denis G., Carlos B.M., Jean-Francois J., Hafida A., Muriel S.. Production of pediocin PA-1 in the methylotrophic yeast Pichia pastoris reveals unexpected inhibition of its biological activity due to the presence of collagen-like material. Protein Expression and Purification,2005,43:111-125
[27]Reviriego C, Fernandez A., Horn N., Rodriguez E., Marin M.L., Fernandez L. Rodriguez J.M. Production of pediocin PA-1 and coproduction of nisin A and pediocin PA-1, by wild Lactococcus lactis strains of dairy origin, international dairy journal,2005: 45-49
[28]Horn N, Fernandez A, Dodd H M, et al. Nisin-controlled production of pediocin PA-1 and colicin V in nisin-and non-nisin-producing Lactococcus lactis strains. Applied and Environmental Microbiology,2004,70,5030-5032.
[29]Miller K W, Schamber R, Chen Y, et al. Production of active chimeric pediocin AcH in Escherichia coli in the absence of processing and secretion genes from the Pediococcus pap operon. Applied and Environmental Microbiology,1998,64:14-20.
[30]Gibbs G M, Davidson B E, Hillier A J. Novel expression system for large-scale production and purification of recombinant class IIa bacteriocins and its application to piscicolin 126. Applied and Environmental Microbiology,2004,70:3292-3297.
[31]李亚玲,周志江,韩烨,等.乳酸片球菌细菌素的分离纯化及理化性质.食品工业科技,2007,28(8)：94-97
[32]丁成为,周志江,韩烨,等.产片球菌素的乳酸片球菌培养条件的优化.食品工业科技,2007,28(10)：66-69
[33]张小霞.外源蛋白质表达系统类型的研究进展.国外医学卫生学分册,2004,31(4)：203-208
[34]张惠展.基因工程.上海：华东理工大学出版社.2005
[35]Swartz JR. Advances in Escherichia coli production of therapeutic proteins. Current Opinion in Biotechnology,2001,12(2):195-201
[36]Baneyx F. Recombinant protein expression in Escherichia coli. Current Opinion in Biotechnology,1999,10:411-421
[37]Makrides SC. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiological Reviews,1996,60(3):512-538
[38]Sorensen HP, Mortensen KK. Advanced genetic strategies for recombinant protein expression in Escherichia coli. Journal of Biotechnology,2005,115:113-128
[39]Baneyx F, Mujacic M. Recombinant protein folding and misfolding in Escherichia coli. Nature Biotechnology,2004,22(11):1399-1408
[40]Georgiou G, Valax P. Expression of correctly folded protein in Escherichia coli. Current opinion in Biotechnology,1996,7:190-197
[41]Studier FW, Moffatt BA. Use of Bacteriophage T7 RNA Polymerase to Direct Selective High-level Expression of Cloned Genes. Journal of Molecular Biology,1986,189: 113-130
[42]Studier FW, Rosenberg AH, Dunn JJ, et al. Use of T7 RNA polymerase to direct expression of cloned genes. Methods in Enzymology,1990,185:60-89
[43]Dubendorff JW, Studier FW. Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. Journal of Molecular Biology,1991,219:37-44
[44]Studier FW. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. Journal of Molecular Biology,1991,219:37-44
[45]Wood WB. Host specificity of DNA produced by Escherichia coli:bacterial mutations affecting the restriction and modification of DNA. Journal of Molecular Biology,1966, 16:118-133
[46]Novy R, Morris B. Use of glucose to control basal expression in the pET system. in Novations,2001,13:8-10
[47]Neubrauer P, Wolff C, Hecker M, et al. Introduction of the tac-promoter by lactose under fermentation conditions. Acta Biotechnol,1991,11(1):23-29
[48]Glick B R, Whiteney G K. Factors affecting the expression of foreign proteins in Escherichia coli. Journ. of Indust. Microbiol,1987,1:277-282
[49]Bellara, Sanjay R, McFarlane, CM, Thomas, CR, Fryer, PJ. Growth of Escherichia coli in a food simulant during conduction cooling:Combining engineering and microbiological modeling, Chemical Engineering Sciences,2002,55(24):6085-6095
[50]Fuchs C, Koster D, Wiebusch S, et al. Scale-up of dialysis fermentation for high cell density cultivation of Escherichia coli. J Biotechnol,2002,93(3):243-251
[51]赵霞,叶勤.重组大肠杆菌W3110(pEC901)培养过程中乙酸生成规律的研究.华东理工大学学报,1995,21(6):685-689 Fuchs C, Koster D, Wiebusch S, et at. Scale-up of
[52]周宇苟,曹巍,魏东芝,等.抗菌肽Adenoregulin基因工程菌培养条件的优化及分批发酵研究.生物工程学报,2005,21(4)：615-621
[53]张彩,王郡甫,刘金生,等.重组人白细胞介素15工程菌高密度发酵条件探讨.中国生化药物杂志,2007,28(1)：1-3
[54]Markl H, Zenneck C, Dubach AC, et al. Cultivation of Escherichia coli to high cell densities in a dialysis reactor. Appl Microbiol Biotechnol,1993,39(1):48-52
[55]Fuchs C, Koster D, Wiebusch S, et al. Scale-up of dialysis fermentation for high cell density cultivation of Escherichia coli. J Biotechnol,2002,93(3):243-251
[56]Shao-YangHu, Jen-Leih Wu, Jan-Hsiung Huang. Productionof tilapia insulin-like growth factor-2 in high cell density cultures of recombinant Escherichia coli. Journal of Biotechnology,2004,107:161-171
[57]Wang F, Lee SY. Production of poly (3-hydroxybutyrate) by fed-batch culture of filamentation-suppressed recombinant Escherichia coli. Appl Environ Microbiol,1997, 34:382-385
[58]Xia XX, Han MJ, Lee SY, et al. Comparison of the extracellular proteomes of Escherichia coli B and K-12 strains during high cell density cultivation. Proteomics, 2008,8(10):2089-2103
[59]Phue JN, Noronha SB, Hattacharyya R, et al. Glucose metabolism at high density growth of E. coli B and E. coli K:differences in metabolic pathways are responsible for efficient glucose utilization in E. coli B as determined by microarrays and Northern blot analyses. Biotechnol Bioeng,2005,90(7):805-820
[60]Vidal L, Pinsach J, Striedner G, et al. Development of an antibiotic-free plasmid selection system based on glycine auxotrophy for recombinant protein overproduction in Escherichia coli. J Biotechnol,2008,134(1-2):127-136
[61]Chang CH, Winans SC. Resection and mutagenesis of the acid pH-inducible P2 promoter of the Agrobacterium tumefaciens virG gene. J Bsv rtiol,1996,178 (15):4717-4720
[62]Mattanovich D, Kramer W, Luttich C, et al. Rational design of an improved induction scheme for recombinant Escherichia coli. Biotechnol Bioeng,1998,58 (2,3):296-298
[63]Ogawa T, Uchida H. Differential induction of IL-1 beta and IL-6 production by the nontoxic lipid A from Porphyromonas gingivalis in comparison with synthetic Escherichia coli lipid A in hupman peripheral blood mononuclear cells. FEMS Immunol Med Microbiol,1996,14 (1):1-13
[64]Mogk A, Volker A, Engelmann S, et al. Normative proteins induce expression of the Bacillus subtilis CIRCE regulon. J Bacteriol,1998,180 (11):2895-2900
[65]Chen Y, Xing XH, Ye F, et al. Production of MBP-HepA fusion protein in recombinant Escherichia coli by optimization of culture medium. Biochem Engineer J,2007,34(2): 114-121
[66]Tabandeh F, Khodabandeh M, Yakhchali B, et al. Response surface methodology for optimizing the induction conditions of recombinant interferon beta during high cell density culture. Chem Engineer Sci,2008,63(9):2477-2483
[67]Maldonado LM, Hernandez VE, Rivero EM, et al. Optimization of culture conditions for a synthetic gene expression in Escherichia coli using response surface methodology:the case of human interferon beta. Biomol Eng,2007,24(2):217-222
[68]王海波,欧俊杰,耿信笃.基因工程菌生产rhG-CSF发酵培养基的研究.中国生物工程杂志,2003,23(11)：68-71
[69]Janice Lau, Carnie Tran, Peter Licari, et al. Development of a high cell-density fed-batch bioprocess for the heterologous production of 6-deoxyerythronolide B in Escherichia colo. Journal of biotechnology,2004,110:95-103
[70]Riesenberg D. High-cell-density cultivation of Escherichia coli. Curr Opin Biotechnol, 1991,2(3):380-384
[71]郑梦杰.大肠杆菌工程菌的高密度培养.国外医学(预防、诊断、治疗用生物制品分册),2001,24(4)：151-154
[72]Shiloach J, Fass R. Growing E. coli to high cell densitya historical perspective on method development. Biotechnol Adv,2005,23(5):345-357
[73]Wei XX, Chen GQ. Applications of the VHb gene vgb for improved microbial fermentation processes. Methods Enzymol,2008,436:273-287
[74]Kim DH, wang DS, Kang DG, et al. Enhancement of mussel adhesive protein production in Escherichia coli by co-expression of bacterial hemoglobin. Biotechnol Prog,2008, 24(3):663-666
[75]Nakano K, Rischke M, Sato S, et al. Influence of acetic acid on the growth of Escherichia coli K12 during high-cell-density cultivation in a dialysis reactor. Appl Microbiol Biotechnol,1997,48(5):597-601
[76]Curless C, Baclaski J and Sachdev R. Phosphate glass as a phosphate source in high cell density Escherichia coli fermentations. Biotechnol Prog,1996,12:22-25
[77]Weikert C, Sauer U and Baily JE. An Escherichia coli host strain useful for efficient overproduction of secreted recombinant protein. Biotechnol Bioeng,1998,98:386-391
[78]Yuan H, Yang X, Hua ZC. Optimization of expression of anannexin V-hirudin chimeric protein in Escherichia coli. Microbiological Research,2004,159:147-156
[79]Wang J, Chen J, Xu R, et al. Batch and fed-batch cultivation forexcretive production of human epidermal growth factor (hEGF) with recombinant E. coli K12 system. Prep Biochem Biotechnol,2008,38(3):271-281
[80]Yan Q, Du G, Chen J. Biosynthesis of polyhydroxyalkanoates (PHAs) with continuous feeding of mixed organic acids as carbon sources by Ralstonia eutropha. Process Biochem,2003,39(3):387-391
[81]Korz DJ, Rinas U, Hellmuth K, et al. Simple fed-batch technique for high cell density cultivation of Escherichia coli. J Biotechnol,1995,39(1):59-65
[82]Babaeipour V, Shojaosadati SA, Robatjazi SM, et al. Over-production of human interferon-y by HCDC of recombinant Escherichia coli. Process Biochem,2007,42(1): 112-117
[83]Babaeipour V, Shojaosadati SA, Khalilzadeh R, et al. A proposed feeding strategy for the overproduction of recombinant proteins in Escherichia coli. Biotechnol Appl Biochem, 2008,49(2):141-147
[84]Kim S, Cheung LH, Zhang W, et al. Improved expression of a soluble single chain antibody fusion protein containing tumor necrosis factor in Escherichia coli. Appl Microbiol Biotechnol,2007,77(1):99-106
[85]Ting TE, Thoma GJ, Beitle RR, et al. A simple substrate feeding strategy using a pH control trigger in fed-batch fermentation. Appl Biochem Biotechnol,2008,149(1):89-98
[86]Kleman GL, Chalmers JJ, Luli GW, et al. A predictive and feedback control algorithm maintains a constant glucose concentration in fed-batch fermentations. Appl Environ Microbiol,1991,57(4):910-917
[87]Chauhan V, Singh A, Waheed M, et al. Constitutive expression of protective antigen gene of Bacillus anthracis in Escherichia coli. Biochemical and Biophysical Research communications，2001，283:308-315
[88]Kotik M, Kocanova M, Maresova H, et al. High level expression of a fungal pyranose oxidase in high cell-density fed-batch cultivations of Escherichia coli using lactose as inducer. Protein Expression and Purification,2004,36:61-69
[89]侯松旺,李小洁,马辉文.受乳糖启动子控制的基因表达过程的优化.武汉大学学报(自然科学版),1998,44(4)：513-516
[90]吴一凡,张双全,高秀玉,等.乳糖诱导pET载体表达重组蛋白的研究.南京师大学报(自然科学版),2002,25(1)：89-93
[91]张毅,屈贤铭,杨胜利.乳糖作为诱导剂对重组目的蛋白表达的影响.生物工程学报,2000,16(4)：464-468
[92]Baheri HR, Roesler WJ, Hill GA. Modeling of recombinant bacteria fermentation for enhanced productivity. Biotechnol Techniq,1997,11(1):47-50
[93]Reiling HE, Laurila H, Fiechter A. Mass culture of Escherichia coli:medium development for low and high density cultivation of Escherichia coli B/r in minimal and complex media. J Biotechnol,1985,2:191-206
[94]Doelle HW, Ewing KN, Hollywood NW. Regulation of glucose metabolism in bacterial systems. Adv Biochem Eng,1982,23:1-35
[95]Majewski RA, Domach MM. Simple constrained optimization view of acetate overflow in E. coli. Biotechnol.Bioeng,1990,35:732-738
[96]Han K, Lim HC, and Hong J. Acetic acid formation in Escherichia coli fermentation. Biotechnol Bioeng,1992,39:663-671
[97]Wolfe AJ. The acetate switch. Microbiol Mol Biol Rev,2005,69(1):12-50
[98]Shimtiz N, Fukuzono S, Fujimori K, et al. Fed-batch cultures of recombinant Escherichia coli with inhibitory substance concentration monitoring. J Ferment Technol,1988,66: 187-191
[99]Yee L, Blanch HW. Recombinant protein expression in high cell density fed-batch cultures of Escherichia coli. Biotechnology,1992,10(12):1550-1556
[100]Luo Q, Shen YL, Wei DZ, et al. Optimization of culture on the overproduction of TRAIL in high-cell-density culture by recombinant Escherichia coli. Appl Microbiol Biotechnol,2006,71(2):184-191
[101]Fass, R, Clem, TR, Shiloach J. Use of novel air separation system in a fed-batch fermentative culture of Escherichia coli. Appl Environ Microbiol,1989,55:1305-1307
[102]杜鹏,叶勤,俞俊棠.大肠杆菌偶合乙酸分离的过滤培养.生物工程学报,2000,16(4)：528-530
[103]Lara AR, Vazquez-Limon C, Gosset G, et al. Engineering Escherichia coli to improve culture performance and reduce formation of by-products during recombinant protein production under transient intermittent anaerobic conditions. Biotechnol Bioeng,2006, 94(6):1164-1175
[104]Contiero J, Beatty C, Kumari S, et al. Effects of mutations in acetate metabolism on high-cell-density growth of Escherichia coli. J Industrial Microbiol Biotech,2000, 24(6):421-430
[105]Brown TDK. The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli. J Gen Microb,1977,102:327-336
[106]Aristidou, AA, San KY and Bennett GN. Modification of central metabolic pathway in Escherichia coli to reduce acetate accumulation by heterologous expression of the Bacillus subtilis acetolactate synthase gene. Biotechnol Bioeng,1994,44:944-951
[107]Lara AR, Caspeta L, Gosset G, et al. Utility of an Escherichia coli strain engineered in the substrate uptake system for improved culture performance at high glucose and cell concentrations:an alternative to fed-batch cultures. Biotechnol Bioeng,2008,99(4): 893-901
[108]Cho S, Shin D, Ji GE, et al. High-level recombinant protein production by overexpression of Mlc in Escherichia coli. J Biotechnol,2005,119(2):197-203
[109]Bentley WE, Kompals DS. Optimal induction of protein synthesis in recombinant bacterial culture. Ann N Y Acad Sci,1990,589 (1):121-138
[110]Khalilzadeh R, Shojaosadati SA, Bahrami A, et al. Over expression of recombinant human interferon-gamma in high cell density fermentation of Escherichia coli. Biotechnology Letters,2003,25:1989-1992
[111]Wang MY, Kwong S, Bentley WE. Effects of oxygen glucose glutamine feeding on insect cell baculovirus protein expression:a study on epoxide hydrolase production. Biotechnol Prog,1993,9(4):355-361
[112]刘晓铭,林玉.G3基因工程菌发酵中pH值对包涵体形成及目的蛋白表达的影响.生物技术,1998,8(5)：16-18
[113]吴军,于公义,冯尔玲.乙酸积累对基因工程菌培养的影响及与pH的关系.微生物学报,1996,36(6)：433-437
[114]Sitnsy KC, Mann MB, Stearns GW, et al. Use of a modified Tryptophanase promoter to direct high-level expression of foreign proteins in E. coli. Ann N Y Acad Sci,1996,782: 297
[115]Allen BR, Luli GW. A gradient-feed process for E. coli fermentation. Biopharm,1987, 1:38
[116]Fiseschko J, Ritch T. Production of human alpha consensus interferon in recombinant Escherichia coli. Chem Eng Commun,1986,45:229-240
[117]孙爱友,魏东芝,宋建民,秦鲁敏.一株乳酸片球菌和乳酸片球菌素的生产方法.中国专利,公开号：CN 101050437A
[118]De Vuyst L, Vandamme EJ. Antimicrobial potential of lactic acid bacteria. In:de Vuyst L and Vandamme EJ (Eds), Bacteriocins of Lactic Acid Bacteria:Microbiology, Genetics and Applications. London:Blackie Academic and Professional.1994,91-142
[119]Ray B, Daeschel MA. Bacteriocins of starter culture bacteria. In:Dillon VM and Board RG (Eds), Natural Antimicrobial Systems and Food Preservation. CAB International, Wallingford.1994,133-165
[120]Berry ED, Hutkins RW, Mandlgo RW. The use of bacteriocin-producing Pediococcus acidilactici to control post processing Listeria monocytogenes contamination of frankfurters. J Food Prot,1991,54,681-686
[121]Joosten HMLJ, Nunel M. Prevention of histamine formation in cheese by bacteriocin-producing lactic acid bacteria. Appl Environ Microbial,1996,62: 1178-1181
[122]谢继辉,陈晓琳,张明.有关细菌素分离纯化方法的评价.安徽农学通报,2008,14(15)：60-61,14
[123]Coventry MJ, Gordon JB, Alexander M, et al. A food-grade process for isolation and partial purification of bacteriocins of lactic acid bacteria that uses diatomite calcium silicate. Appl Environ Microbial,1996,62:1764-1769
[124]De Vos WM. Future prospects for research and applications of nisin and other bacteriocins. In:Hoover DG and Steenson LR (Eds), Bacteriocins of Lactic Acid Bactena. San Diego:Academic Press.1993,250-265
[125]Yang R, Johnson MC, Ray B. Novel method to extract large amounts of bacteriocins from lactic acid bacteria. Appl Environ Microbial,1992,58(10):3355-3359
[126]Daba H, Lacroix C, Huang J, et al. Simple method of purification and sequencing of a bacteriocin produced by Pediococcus acidilacticiUL5. J. Appl. Bacteriol.,1994,77, 682-688
[127]Francisco BE, Wang JK, Dae YK. Rapid purification, partial characterization, and antimicrobial spectrum of the bacteriocin, Pediocin AcM, from Pediococcus acidilactici M. International Journal of Food Microbiology,1997,37:1-11
[128]Kim WJ, Hong SS, Cha SK, Koo YJ. Use of bacteriocinogenic pediococcus acidilactici in sausage fermentation. J Microbial Biotechnol,1993,3:199-203
[129]Ray B, Daeschel MA. Food Biopreservatives of Microbial Origin. Boca Raton:CRC Press,1992:57-80
[130]Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry,1951,193:265-275
[131]Schagger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Analytical Biochemistry,1987,166(2):368-379
[132]王旭,何冰芳,李霜等Tricine-SDS-PAGE电泳分析小分子多肽.南京工业大学学报,2003,25(2)：79-81
[133]邢芳芳,黄瑞林,张友明,等.改进Tricine-SDS-PAGE法分析重组牛乳铁蛋白肽.饲料工业,2007,28(9)：10-13
[134]Schneider R, Fernandez FJ, Aguilar MB. Partial characterization of a class Ⅱa pediocin produced by Pediococcus parvulus 133 strain isolated from meat. Food Control,2006,17: 909-915
[135]Bauer R, Chikindas ML, Dicks LMT. Purification, partial amino acid sequence and mode of action of pediocin PD-1, a bacteriocin produced by Pediococcus damnosus NCFB 1832. International Journal of Food Microbiologe,2005,101:17-27
[136]解树涛,宋晓妍,石梅,等.康宁木霉(Trichoderma koningii) SMF2分泌的pepaibols类抗菌肽Trichokonins抑菌活性研究.山东大学学报(理学版),2006,41：140-144
[137]Biswas SR, Ray P, Johnson MC, et al. Influence of growth conditions on the production of a bacterioein, pediocin AcH by Pediocoeeus acidilactici H. Applied and Enviromental Microbiology,1991,57:1265-1267
[138]Tichaczek PS, Meyer JN, Ness IF, et al. Characterization of the bacteriocins curvacin a from Lactobacllus curvatus LTH673. Systematic Applied Microbiology,1992,15: 460-468
[139]Campos CA, Rodriguse O, Mata CP. Preliminary characterization of bacteriocins from Lactococcus lactis, Enterococcus faecium and Enterococcus mundtii strain isolated from turbot (Psetta maxima). Food Research International,2006,39:356-364
[140]MIRHOSSEINI MAHBOUBEH, NAHVI 1RAJ, EMTIAZI GITI. Incidence and antibiotic susceptibility of bacteriocin-producing lactic acid bacteria from dairy products. International Journal of Dairy Technology,2008,61(4):391-396
[141]Davey GP, Richardson BC. Purification and Some Properties of Diplococcin from Streptococcus cremoris 346. Applied and Enviromental Microbiology,1981,41(1): 84-89
[142]Bhunia AK, Johnson MC, Ray B, et al. Mode of action of Pediocin AcH from Pediococcus acidilactici H on sensitive bacterial strains. J Appl Bacterial,1941,70, 25-33
[143]Kim WJ, Ha DM, Ray B. Plasmid linkage of bacteriocin production and sucrose fermentation phenotypes m Pediococcus acidilactici M. J. Microbial. Biotechnol. J., 1991,169-175
[144]Gi-Seong Moon, Yu-Ryang Pyun, Wang June Kim. Expression and purification of a fusion-typed pediocin PA-1 in Escherichia coli and recovery of biologically active pediocin PA-1. International Journal of Food Microbiology,2006,108:136-140
[145]Sambrook J, Russell DW. Molecular cloning:A laboratory manual, (3rd ed.) New York: Cold Spring Harbor Laboratory Press.2001
[146]赵永芳.生物化学技术原理及应用.北京：科学出版社.2002
[147]汪家政,范明.蛋白质技术手册.北京：科学出版社.2000
[148][澳]查理德J,辛普.蛋白质与蛋白质组学实验指南.化学工业出版社.2006
[149]McLoughlin AJ. Plasmid stability and ecological competence in recombinant cultures. Biotechnology Advances,1994,12(2):279-324
[150]Friehs K. Plasmid copy number and plasmid stability. Advances in Biochemical Engineering/Biotechnology,2004,86:47-82
[151]Studier FW. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. Journal of Molecular Biology,1991,219:37-44
[152]Summers DK. The kinetics of plasmid loss. Trends in Biotechnology,1991,9(8): 273-278
[153]Pogliano J, Ho TQ, Zhong Z, et al. Multicopy plasmids are clustered and localized in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America,2001,98(8):4486-4491
[154]Pan SH, Malcolm BA. Reduced background expression and improved plasmid stability with pET vectors in BL21 (DE3). Biotechniques.2000,29(6):1234-1238
[155]陈卫,葛佳佳.半乳糖苷酶基因在大肠杆菌中过量表达及IPTG诱导条件.无锡轻工业大学学报,2002,21(5)：492-495
[156]Fischer B, Sumner I. Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in E. coli as inclusion bodies. Biotech Bioeng,1993, 41(1):3-13
[157]金元昌,陈春岚,苏晓艳,等GST-GnRH/TRS融合蛋白(包涵体)的分离、纯化与复性研究.西北农业学报,2006,15(4)：173-176
[158]刘永庆,陈溥言,杜念兴,等.生长抑素(SS)与硫氧还原蛋白(Thioredoxin)相融合生成的2种基因工程化融合蛋白SS-N/X和：SS-N/S的表达特性.中国兽医学报,2003,23(2)：172-176
[159]Miroux B, Walker JE. Over-production of proteins in Escherichia coli:Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. Journal of Molecular Biology,1996,260(3):289-298
[160]安平,董志伟,寿成超.可溶性VEGF受体Flt-1的基因克隆及其活性产物在原核中的表达.中国生物化学与分子生物学报,2000,16(1)：62-65
[161]Kleman GL, Strohl WR. Acetate metabolism by Escherichia coli in High-cell-density fermentation. Appl Environ Microbiol,1994,60 (11):3952-3958
[162]Corchero JL, Villaverde D. Plasmid maintenance in Escherichia coli recombinant culture is dramatically, steadily, and specifically influncened by features of the encoded proteins. Biotechnol Bioeng,1998,58(60):625-632
[163]Jacques N, Guillerez J, Dreyfus M. Culture conditions differentially affect the translation of individual Escherichia coli mRNA. J Mol Biol,1992,226:597-608
[164]Stratton J, Chiruvolu V, Meagher M. High cell-density fermentation. Methods Mol Biol, 1998,103(1):107-120
[165]Van de Walle M, Shiloach J. Proposed mechanism of acetate accumulation in two recombinant Escherichia coli strains during high density fermentation. Biotechnol Bioeng,1998,57(1):71-78
[166]Cheng CY, Huang YL, Yang ST. A novel feeding strategy for enhanced plasmid stability and protein production in recombinant yeast Fed-batch fermentation. Biotechnol Bioeng,1997,56(1):23-31
[167]Mizutani S, Mori H, Shimizu, et al. Effect of amino acid supplement on cell yield and gene product in Escherichia coli harboring plasmid. Biotechnol Bioeng,1986,28: 203-211
[168]潘春梅,孙西玉,樊耀亭.气相色谱法测定秸秆水解液中的糠醛和乙酸含量.酿酒科技,2008,5：108-110
[169]李毅,蒲勤,赵忠良,等.溶氧反馈-分批补料高密度培养人骨形成蛋白-2工程菌.生物工程学报,2002,18(6)：718-723
[170]钟根深,石炳兴,王海东,等.表达重组葡激酶-水蛭素融合蛋白的大肠杆菌工程菌的高密度培养.中国生物工程杂志,2006,26(9)：11-15
[171]Yin L, Jian C, Ying YM, et al. Effect of additives and fed-batch culture strategies on the production of glutathione by recombinant Escherichia coli. Proc Biochem,1998,33: 709-714
[172]Castan A, Enfors SO. Characteristics of a DO-controlled fed-batch culture of Escherichia coli. Bioproc Eng,2000,22:509-515
[173]Shen YL, Zhang Y, Sun AY. High-level production of soluble tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) in high-density cultivation of recombinant Escherichia coli using a combined feeding strategy. Biotechnol Lett,2004, 26:981-984
[174]Luli GW, Strohl WR. Comparison of growth, acetate production and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations. Appl Environ Microbiol,1990,56:1004-1011
[175]Suarez DC, Kilikian BV. Acetic acid accumulation in aerobic growth of recombinant Escherichia coli. Process Biochem,2000,35:1051-1055.
[176]Jana S, Deb JK. Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol,2005,67:289-298.
[177]储炬,李友荣.现代工业发酵调控学.北京：化学工业出版社.2002
[178]Schultz T, Martinez L, de Marco A. The evaluation of the factors that cause aggregation during recombinant expression in E. coli is simplified by the employment of an aggregation-sensitive reporter. Microbial Cell Factories,2006,5:28
[179]Belin P, Dassa J, Drevet P, et al. Toxicity-based selection of Escherichia coli mutants for functional recombinant protein production:Application to an antibody fragment. Protein Engineering, Design and Selection,2004,17(5):491-500
[2]马文山,廖富蘋,范雪云,等.细菌抗菌肽的研究进展及其应用.生物技术通报,2008,3：39-43,49
[3]甘晓玲.细菌素在食品工业中的应用价值.科学咨询,2008,17：48
[4]吴琦,胡建平.抑菌活性蛋白(肽)作为食品防腐剂的研究进展.食品研究与开发,2008,29(1)：168-172
[5]田召芳,牛钟相,常维山,等.乳酸菌细菌素的研究进展.微生物学杂志,2003,6：119-122
[6]黄现青,高晓平,韩庆功,等.乳酸菌类细菌素的研究进展.中国乳品工业,2007,35(9)：41-43,57
[7]李红,赵春燕.乳酸链球菌素的研究进展.食品科技,2006,31(1)：75-78
[8]还连栋,陈秀珠.天然食品防腐剂乳链菌肽的性质及其应用.天然产物研究与开发,1995,7(1)：55-60
[9]Kanatani K, Oshimura M. Plasmid-associated bacteriocin production by a Lactobacillus plantarum strain. Bioscience, Biotechnology and Biochemistry,1994,58(11): 2084-2086
[10]Miller KW, Schamber R, Osmanagaoglu O, et al. Isolation and characterization of pediocin AcH chimeric protein mutants with altered bactericidal activity. Applied And Environmental Microbiology,1998,64 (6):1997-2005
[11]McAuliffe O, Ross R P, Hill C. Lantibiotics:structure, biosynthesis and mode of action. FEMS Microbiology Reviews,2001,25:285-308.
[12]吕燕妮,李平兰,周伟,等.Ⅱa类细菌素的结构、生物合成与活性.微生物学通报,2007,34(5)：955-959
[13]Ennahar S, Sashihara T, Sonomoto K, et al. Class IIa bacteriocins:biosynthesis, structure and activity. FEMS Microbiology Reviews,2000,24:85-106.
[14]韩雪,周志江.乳酸片球菌细菌素的活性及特性的研究.食品研究与开发,2006,3：19-21
[15]Sablon E, Contreras B, Vandamme, E. Antimicrobial peptides of lactic acid bacteria: mode of action, genetics and biosynthesis. Advances in Biochemical Engineering/ Biotechnology,2000,68:21-59
[16]Eijsink V G H, Axelsson L, Diep D B, et al. Production of class Ⅱ bacteriocins by lactic acid bacteria; an example of biological warfare and communication. Antonie van Leeuwenhoek,2002,81:639-654
[17]Somkuti G A, Steinberg D H. Pediocin production by recombinant lactic acid bacteria. Biotechnol Lett,2003,25:473-477
[18]King W., Creek W., Ming X., Grove C., Antibacterial composition for control of gram positive bactertia in food applications. US6451365B1,2002.
[19]Ray B, Schamber R, Miller K W. The pediocin AcH precursor is biologically active. Applied and Environmental Microbiology,1999,65:2281-2286.
[20]Fimland G, Johnsen L, Axelsson L, et al. A C-terminal disulfide bridge in pediocin-like bacteriocins renders bacteriocin activity less temperature dependent and is a major determinant of the antimicrobial spectrum. Journal of Bacteriology,2000,182: 2643-2648.
[21]Johnsen L, Fimland G., Eijsink V, et al. Engineering increased stability in the antimicrobial peptide pediocin PA-1. Applied and Environmental Microbiology,2000,66: 47984802.
[22]Chen Y, Ludescher R D, Montville T J. Electrostatic interactions, but not the YGNGV consensus motif, govern the binding of pediocin PA-1 and its fragments to phospholipid vesicles. Appl Environ Microbiol,1997,63:4770-4777
[23]Moon G S, Pyun Y R, Kim W J. Characterization of pediocin operon of Pediococcus acidilactici K10 and expression of His-tagged recombinant pediocin PA-1 in Escherichia coli. Journal of Microbiology and Biotechnology,2005,15:403-411
[24]Henderson J T, Chopko A L, Van Wassenaar P D. Purification and primary structure of pediocin PA-1 produced by Pediococcus acidilactici PAC-1.0. Arch Biochem Biophys, 1992,295:5-12
[25]Jack R W, Tagg J R, Ray B. Bacteriocins of Gram-Positive bacteria. Microbiol Rev, 1995,59:171-200
[26]Lucie B., Denis G., Carlos B.M., Jean-Francois J., Hafida A., Muriel S.. Production of pediocin PA-1 in the methylotrophic yeast Pichia pastoris reveals unexpected inhibition of its biological activity due to the presence of collagen-like material. Protein Expression and Purification,2005,43:111-125
[27]Reviriego C, Fernandez A., Horn N., Rodriguez E., Marin M.L., Fernandez L. Rodriguez J.M. Production of pediocin PA-1 and coproduction of nisin A and pediocin PA-1, by wild Lactococcus lactis strains of dairy origin, international dairy journal,2005: 45-49
[28]Horn N, Fernandez A, Dodd H M, et al. Nisin-controlled production of pediocin PA-1 and colicin V in nisin-and non-nisin-producing Lactococcus lactis strains. Applied and Environmental Microbiology,2004,70,5030-5032.
[29]Miller K W, Schamber R, Chen Y, et al. Production of active chimeric pediocin AcH in Escherichia coli in the absence of processing and secretion genes from the Pediococcus pap operon. Applied and Environmental Microbiology,1998,64:14-20.
[30]Gibbs G M, Davidson B E, Hillier A J. Novel expression system for large-scale production and purification of recombinant class IIa bacteriocins and its application to piscicolin 126. Applied and Environmental Microbiology,2004,70:3292-3297.
[31]李亚玲,周志江,韩烨,等.乳酸片球菌细菌素的分离纯化及理化性质.食品工业科技,2007,28(8)：94-97
[32]丁成为,周志江,韩烨,等.产片球菌素的乳酸片球菌培养条件的优化.食品工业科技,2007,28(10)：66-69
[33]张小霞.外源蛋白质表达系统类型的研究进展.国外医学卫生学分册,2004,31(4)：203-208
[34]张惠展.基因工程.上海：华东理工大学出版社.2005
[35]Swartz JR. Advances in Escherichia coli production of therapeutic proteins. Current Opinion in Biotechnology,2001,12(2):195-201
[36]Baneyx F. Recombinant protein expression in Escherichia coli. Current Opinion in Biotechnology,1999,10:411-421
[37]Makrides SC. Strategies for achieving high-level expression of genes in Escherichia coli. Microbiological Reviews,1996,60(3):512-538
[38]Sorensen HP, Mortensen KK. Advanced genetic strategies for recombinant protein expression in Escherichia coli. Journal of Biotechnology,2005,115:113-128
[39]Baneyx F, Mujacic M. Recombinant protein folding and misfolding in Escherichia coli. Nature Biotechnology,2004,22(11):1399-1408
[40]Georgiou G, Valax P. Expression of correctly folded protein in Escherichia coli. Current opinion in Biotechnology,1996,7:190-197
[41]Studier FW, Moffatt BA. Use of Bacteriophage T7 RNA Polymerase to Direct Selective High-level Expression of Cloned Genes. Journal of Molecular Biology,1986,189: 113-130
[42]Studier FW, Rosenberg AH, Dunn JJ, et al. Use of T7 RNA polymerase to direct expression of cloned genes. Methods in Enzymology,1990,185:60-89
[43]Dubendorff JW, Studier FW. Controlling basal expression in an inducible T7 expression system by blocking the target T7 promoter with lac repressor. Journal of Molecular Biology,1991,219:37-44
[44]Studier FW. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. Journal of Molecular Biology,1991,219:37-44
[45]Wood WB. Host specificity of DNA produced by Escherichia coli:bacterial mutations affecting the restriction and modification of DNA. Journal of Molecular Biology,1966, 16:118-133
[46]Novy R, Morris B. Use of glucose to control basal expression in the pET system. in Novations,2001,13:8-10
[47]Neubrauer P, Wolff C, Hecker M, et al. Introduction of the tac-promoter by lactose under fermentation conditions. Acta Biotechnol,1991,11(1):23-29
[48]Glick B R, Whiteney G K. Factors affecting the expression of foreign proteins in Escherichia coli. Journ. of Indust. Microbiol,1987,1:277-282
[49]Bellara, Sanjay R, McFarlane, CM, Thomas, CR, Fryer, PJ. Growth of Escherichia coli in a food simulant during conduction cooling:Combining engineering and microbiological modeling, Chemical Engineering Sciences,2002,55(24):6085-6095
[50]Fuchs C, Koster D, Wiebusch S, et al. Scale-up of dialysis fermentation for high cell density cultivation of Escherichia coli. J Biotechnol,2002,93(3):243-251
[51]赵霞,叶勤.重组大肠杆菌W3110(pEC901)培养过程中乙酸生成规律的研究.华东理工大学学报,1995,21(6):685-689 Fuchs C, Koster D, Wiebusch S, et at. Scale-up of
[52]周宇苟,曹巍,魏东芝,等.抗菌肽Adenoregulin基因工程菌培养条件的优化及分批发酵研究.生物工程学报,2005,21(4)：615-621
[53]张彩,王郡甫,刘金生,等.重组人白细胞介素15工程菌高密度发酵条件探讨.中国生化药物杂志,2007,28(1)：1-3
[54]Markl H, Zenneck C, Dubach AC, et al. Cultivation of Escherichia coli to high cell densities in a dialysis reactor. Appl Microbiol Biotechnol,1993,39(1):48-52
[55]Fuchs C, Koster D, Wiebusch S, et al. Scale-up of dialysis fermentation for high cell density cultivation of Escherichia coli. J Biotechnol,2002,93(3):243-251
[56]Shao-YangHu, Jen-Leih Wu, Jan-Hsiung Huang. Productionof tilapia insulin-like growth factor-2 in high cell density cultures of recombinant Escherichia coli. Journal of Biotechnology,2004,107:161-171
[57]Wang F, Lee SY. Production of poly (3-hydroxybutyrate) by fed-batch culture of filamentation-suppressed recombinant Escherichia coli. Appl Environ Microbiol,1997, 34:382-385
[58]Xia XX, Han MJ, Lee SY, et al. Comparison of the extracellular proteomes of Escherichia coli B and K-12 strains during high cell density cultivation. Proteomics, 2008,8(10):2089-2103
[59]Phue JN, Noronha SB, Hattacharyya R, et al. Glucose metabolism at high density growth of E. coli B and E. coli K:differences in metabolic pathways are responsible for efficient glucose utilization in E. coli B as determined by microarrays and Northern blot analyses. Biotechnol Bioeng,2005,90(7):805-820
[60]Vidal L, Pinsach J, Striedner G, et al. Development of an antibiotic-free plasmid selection system based on glycine auxotrophy for recombinant protein overproduction in Escherichia coli. J Biotechnol,2008,134(1-2):127-136
[61]Chang CH, Winans SC. Resection and mutagenesis of the acid pH-inducible P2 promoter of the Agrobacterium tumefaciens virG gene. J Bsv rtiol,1996,178 (15):4717-4720
[62]Mattanovich D, Kramer W, Luttich C, et al. Rational design of an improved induction scheme for recombinant Escherichia coli. Biotechnol Bioeng,1998,58 (2,3):296-298
[63]Ogawa T, Uchida H. Differential induction of IL-1 beta and IL-6 production by the nontoxic lipid A from Porphyromonas gingivalis in comparison with synthetic Escherichia coli lipid A in hupman peripheral blood mononuclear cells. FEMS Immunol Med Microbiol,1996,14 (1):1-13
[64]Mogk A, Volker A, Engelmann S, et al. Normative proteins induce expression of the Bacillus subtilis CIRCE regulon. J Bacteriol,1998,180 (11):2895-2900
[65]Chen Y, Xing XH, Ye F, et al. Production of MBP-HepA fusion protein in recombinant Escherichia coli by optimization of culture medium. Biochem Engineer J,2007,34(2): 114-121
[66]Tabandeh F, Khodabandeh M, Yakhchali B, et al. Response surface methodology for optimizing the induction conditions of recombinant interferon beta during high cell density culture. Chem Engineer Sci,2008,63(9):2477-2483
[67]Maldonado LM, Hernandez VE, Rivero EM, et al. Optimization of culture conditions for a synthetic gene expression in Escherichia coli using response surface methodology:the case of human interferon beta. Biomol Eng,2007,24(2):217-222
[68]王海波,欧俊杰,耿信笃.基因工程菌生产rhG-CSF发酵培养基的研究.中国生物工程杂志,2003,23(11)：68-71
[69]Janice Lau, Carnie Tran, Peter Licari, et al. Development of a high cell-density fed-batch bioprocess for the heterologous production of 6-deoxyerythronolide B in Escherichia colo. Journal of biotechnology,2004,110:95-103
[70]Riesenberg D. High-cell-density cultivation of Escherichia coli. Curr Opin Biotechnol, 1991,2(3):380-384
[71]郑梦杰.大肠杆菌工程菌的高密度培养.国外医学(预防、诊断、治疗用生物制品分册),2001,24(4)：151-154
[72]Shiloach J, Fass R. Growing E. coli to high cell densitya historical perspective on method development. Biotechnol Adv,2005,23(5):345-357
[73]Wei XX, Chen GQ. Applications of the VHb gene vgb for improved microbial fermentation processes. Methods Enzymol,2008,436:273-287
[74]Kim DH, wang DS, Kang DG, et al. Enhancement of mussel adhesive protein production in Escherichia coli by co-expression of bacterial hemoglobin. Biotechnol Prog,2008, 24(3):663-666
[75]Nakano K, Rischke M, Sato S, et al. Influence of acetic acid on the growth of Escherichia coli K12 during high-cell-density cultivation in a dialysis reactor. Appl Microbiol Biotechnol,1997,48(5):597-601
[76]Curless C, Baclaski J and Sachdev R. Phosphate glass as a phosphate source in high cell density Escherichia coli fermentations. Biotechnol Prog,1996,12:22-25
[77]Weikert C, Sauer U and Baily JE. An Escherichia coli host strain useful for efficient overproduction of secreted recombinant protein. Biotechnol Bioeng,1998,98:386-391
[78]Yuan H, Yang X, Hua ZC. Optimization of expression of anannexin V-hirudin chimeric protein in Escherichia coli. Microbiological Research,2004,159:147-156
[79]Wang J, Chen J, Xu R, et al. Batch and fed-batch cultivation forexcretive production of human epidermal growth factor (hEGF) with recombinant E. coli K12 system. Prep Biochem Biotechnol,2008,38(3):271-281
[80]Yan Q, Du G, Chen J. Biosynthesis of polyhydroxyalkanoates (PHAs) with continuous feeding of mixed organic acids as carbon sources by Ralstonia eutropha. Process Biochem,2003,39(3):387-391
[81]Korz DJ, Rinas U, Hellmuth K, et al. Simple fed-batch technique for high cell density cultivation of Escherichia coli. J Biotechnol,1995,39(1):59-65
[82]Babaeipour V, Shojaosadati SA, Robatjazi SM, et al. Over-production of human interferon-y by HCDC of recombinant Escherichia coli. Process Biochem,2007,42(1): 112-117
[83]Babaeipour V, Shojaosadati SA, Khalilzadeh R, et al. A proposed feeding strategy for the overproduction of recombinant proteins in Escherichia coli. Biotechnol Appl Biochem, 2008,49(2):141-147
[84]Kim S, Cheung LH, Zhang W, et al. Improved expression of a soluble single chain antibody fusion protein containing tumor necrosis factor in Escherichia coli. Appl Microbiol Biotechnol,2007,77(1):99-106
[85]Ting TE, Thoma GJ, Beitle RR, et al. A simple substrate feeding strategy using a pH control trigger in fed-batch fermentation. Appl Biochem Biotechnol,2008,149(1):89-98
[86]Kleman GL, Chalmers JJ, Luli GW, et al. A predictive and feedback control algorithm maintains a constant glucose concentration in fed-batch fermentations. Appl Environ Microbiol,1991,57(4):910-917
[87]Chauhan V, Singh A, Waheed M, et al. Constitutive expression of protective antigen gene of Bacillus anthracis in Escherichia coli. Biochemical and Biophysical Research communications，2001，283:308-315
[88]Kotik M, Kocanova M, Maresova H, et al. High level expression of a fungal pyranose oxidase in high cell-density fed-batch cultivations of Escherichia coli using lactose as inducer. Protein Expression and Purification,2004,36:61-69
[89]侯松旺,李小洁,马辉文.受乳糖启动子控制的基因表达过程的优化.武汉大学学报(自然科学版),1998,44(4)：513-516
[90]吴一凡,张双全,高秀玉,等.乳糖诱导pET载体表达重组蛋白的研究.南京师大学报(自然科学版),2002,25(1)：89-93
[91]张毅,屈贤铭,杨胜利.乳糖作为诱导剂对重组目的蛋白表达的影响.生物工程学报,2000,16(4)：464-468
[92]Baheri HR, Roesler WJ, Hill GA. Modeling of recombinant bacteria fermentation for enhanced productivity. Biotechnol Techniq,1997,11(1):47-50
[93]Reiling HE, Laurila H, Fiechter A. Mass culture of Escherichia coli:medium development for low and high density cultivation of Escherichia coli B/r in minimal and complex media. J Biotechnol,1985,2:191-206
[94]Doelle HW, Ewing KN, Hollywood NW. Regulation of glucose metabolism in bacterial systems. Adv Biochem Eng,1982,23:1-35
[95]Majewski RA, Domach MM. Simple constrained optimization view of acetate overflow in E. coli. Biotechnol.Bioeng,1990,35:732-738
[96]Han K, Lim HC, and Hong J. Acetic acid formation in Escherichia coli fermentation. Biotechnol Bioeng,1992,39:663-671
[97]Wolfe AJ. The acetate switch. Microbiol Mol Biol Rev,2005,69(1):12-50
[98]Shimtiz N, Fukuzono S, Fujimori K, et al. Fed-batch cultures of recombinant Escherichia coli with inhibitory substance concentration monitoring. J Ferment Technol,1988,66: 187-191
[99]Yee L, Blanch HW. Recombinant protein expression in high cell density fed-batch cultures of Escherichia coli. Biotechnology,1992,10(12):1550-1556
[100]Luo Q, Shen YL, Wei DZ, et al. Optimization of culture on the overproduction of TRAIL in high-cell-density culture by recombinant Escherichia coli. Appl Microbiol Biotechnol,2006,71(2):184-191
[101]Fass, R, Clem, TR, Shiloach J. Use of novel air separation system in a fed-batch fermentative culture of Escherichia coli. Appl Environ Microbiol,1989,55:1305-1307
[102]杜鹏,叶勤,俞俊棠.大肠杆菌偶合乙酸分离的过滤培养.生物工程学报,2000,16(4)：528-530
[103]Lara AR, Vazquez-Limon C, Gosset G, et al. Engineering Escherichia coli to improve culture performance and reduce formation of by-products during recombinant protein production under transient intermittent anaerobic conditions. Biotechnol Bioeng,2006, 94(6):1164-1175
[104]Contiero J, Beatty C, Kumari S, et al. Effects of mutations in acetate metabolism on high-cell-density growth of Escherichia coli. J Industrial Microbiol Biotech,2000, 24(6):421-430
[105]Brown TDK. The enzymic interconversion of acetate and acetyl-coenzyme A in Escherichia coli. J Gen Microb,1977,102:327-336
[106]Aristidou, AA, San KY and Bennett GN. Modification of central metabolic pathway in Escherichia coli to reduce acetate accumulation by heterologous expression of the Bacillus subtilis acetolactate synthase gene. Biotechnol Bioeng,1994,44:944-951
[107]Lara AR, Caspeta L, Gosset G, et al. Utility of an Escherichia coli strain engineered in the substrate uptake system for improved culture performance at high glucose and cell concentrations:an alternative to fed-batch cultures. Biotechnol Bioeng,2008,99(4): 893-901
[108]Cho S, Shin D, Ji GE, et al. High-level recombinant protein production by overexpression of Mlc in Escherichia coli. J Biotechnol,2005,119(2):197-203
[109]Bentley WE, Kompals DS. Optimal induction of protein synthesis in recombinant bacterial culture. Ann N Y Acad Sci,1990,589 (1):121-138
[110]Khalilzadeh R, Shojaosadati SA, Bahrami A, et al. Over expression of recombinant human interferon-gamma in high cell density fermentation of Escherichia coli. Biotechnology Letters,2003,25:1989-1992
[111]Wang MY, Kwong S, Bentley WE. Effects of oxygen glucose glutamine feeding on insect cell baculovirus protein expression:a study on epoxide hydrolase production. Biotechnol Prog,1993,9(4):355-361
[112]刘晓铭,林玉.G3基因工程菌发酵中pH值对包涵体形成及目的蛋白表达的影响.生物技术,1998,8(5)：16-18
[113]吴军,于公义,冯尔玲.乙酸积累对基因工程菌培养的影响及与pH的关系.微生物学报,1996,36(6)：433-437
[114]Sitnsy KC, Mann MB, Stearns GW, et al. Use of a modified Tryptophanase promoter to direct high-level expression of foreign proteins in E. coli. Ann N Y Acad Sci,1996,782: 297
[115]Allen BR, Luli GW. A gradient-feed process for E. coli fermentation. Biopharm,1987, 1:38
[116]Fiseschko J, Ritch T. Production of human alpha consensus interferon in recombinant Escherichia coli. Chem Eng Commun,1986,45:229-240
[117]孙爱友,魏东芝,宋建民,秦鲁敏.一株乳酸片球菌和乳酸片球菌素的生产方法.中国专利,公开号：CN 101050437A
[118]De Vuyst L, Vandamme EJ. Antimicrobial potential of lactic acid bacteria. In:de Vuyst L and Vandamme EJ (Eds), Bacteriocins of Lactic Acid Bacteria:Microbiology, Genetics and Applications. London:Blackie Academic and Professional.1994,91-142
[119]Ray B, Daeschel MA. Bacteriocins of starter culture bacteria. In:Dillon VM and Board RG (Eds), Natural Antimicrobial Systems and Food Preservation. CAB International, Wallingford.1994,133-165
[120]Berry ED, Hutkins RW, Mandlgo RW. The use of bacteriocin-producing Pediococcus acidilactici to control post processing Listeria monocytogenes contamination of frankfurters. J Food Prot,1991,54,681-686
[121]Joosten HMLJ, Nunel M. Prevention of histamine formation in cheese by bacteriocin-producing lactic acid bacteria. Appl Environ Microbial,1996,62: 1178-1181
[122]谢继辉,陈晓琳,张明.有关细菌素分离纯化方法的评价.安徽农学通报,2008,14(15)：60-61,14
[123]Coventry MJ, Gordon JB, Alexander M, et al. A food-grade process for isolation and partial purification of bacteriocins of lactic acid bacteria that uses diatomite calcium silicate. Appl Environ Microbial,1996,62:1764-1769
[124]De Vos WM. Future prospects for research and applications of nisin and other bacteriocins. In:Hoover DG and Steenson LR (Eds), Bacteriocins of Lactic Acid Bactena. San Diego:Academic Press.1993,250-265
[125]Yang R, Johnson MC, Ray B. Novel method to extract large amounts of bacteriocins from lactic acid bacteria. Appl Environ Microbial,1992,58(10):3355-3359
[126]Daba H, Lacroix C, Huang J, et al. Simple method of purification and sequencing of a bacteriocin produced by Pediococcus acidilacticiUL5. J. Appl. Bacteriol.,1994,77, 682-688
[127]Francisco BE, Wang JK, Dae YK. Rapid purification, partial characterization, and antimicrobial spectrum of the bacteriocin, Pediocin AcM, from Pediococcus acidilactici M. International Journal of Food Microbiology,1997,37:1-11
[128]Kim WJ, Hong SS, Cha SK, Koo YJ. Use of bacteriocinogenic pediococcus acidilactici in sausage fermentation. J Microbial Biotechnol,1993,3:199-203
[129]Ray B, Daeschel MA. Food Biopreservatives of Microbial Origin. Boca Raton:CRC Press,1992:57-80
[130]Lowry OH, Rosebrough NJ, Farr AL, et al. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry,1951,193:265-275
[131]Schagger H, von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Analytical Biochemistry,1987,166(2):368-379
[132]王旭,何冰芳,李霜等Tricine-SDS-PAGE电泳分析小分子多肽.南京工业大学学报,2003,25(2)：79-81
[133]邢芳芳,黄瑞林,张友明,等.改进Tricine-SDS-PAGE法分析重组牛乳铁蛋白肽.饲料工业,2007,28(9)：10-13
[134]Schneider R, Fernandez FJ, Aguilar MB. Partial characterization of a class Ⅱa pediocin produced by Pediococcus parvulus 133 strain isolated from meat. Food Control,2006,17: 909-915
[135]Bauer R, Chikindas ML, Dicks LMT. Purification, partial amino acid sequence and mode of action of pediocin PD-1, a bacteriocin produced by Pediococcus damnosus NCFB 1832. International Journal of Food Microbiologe,2005,101:17-27
[136]解树涛,宋晓妍,石梅,等.康宁木霉(Trichoderma koningii) SMF2分泌的pepaibols类抗菌肽Trichokonins抑菌活性研究.山东大学学报(理学版),2006,41：140-144
[137]Biswas SR, Ray P, Johnson MC, et al. Influence of growth conditions on the production of a bacterioein, pediocin AcH by Pediocoeeus acidilactici H. Applied and Enviromental Microbiology,1991,57:1265-1267
[138]Tichaczek PS, Meyer JN, Ness IF, et al. Characterization of the bacteriocins curvacin a from Lactobacllus curvatus LTH673. Systematic Applied Microbiology,1992,15: 460-468
[139]Campos CA, Rodriguse O, Mata CP. Preliminary characterization of bacteriocins from Lactococcus lactis, Enterococcus faecium and Enterococcus mundtii strain isolated from turbot (Psetta maxima). Food Research International,2006,39:356-364
[140]MIRHOSSEINI MAHBOUBEH, NAHVI 1RAJ, EMTIAZI GITI. Incidence and antibiotic susceptibility of bacteriocin-producing lactic acid bacteria from dairy products. International Journal of Dairy Technology,2008,61(4):391-396
[141]Davey GP, Richardson BC. Purification and Some Properties of Diplococcin from Streptococcus cremoris 346. Applied and Enviromental Microbiology,1981,41(1): 84-89
[142]Bhunia AK, Johnson MC, Ray B, et al. Mode of action of Pediocin AcH from Pediococcus acidilactici H on sensitive bacterial strains. J Appl Bacterial,1941,70, 25-33
[143]Kim WJ, Ha DM, Ray B. Plasmid linkage of bacteriocin production and sucrose fermentation phenotypes m Pediococcus acidilactici M. J. Microbial. Biotechnol. J., 1991,169-175
[144]Gi-Seong Moon, Yu-Ryang Pyun, Wang June Kim. Expression and purification of a fusion-typed pediocin PA-1 in Escherichia coli and recovery of biologically active pediocin PA-1. International Journal of Food Microbiology,2006,108:136-140
[145]Sambrook J, Russell DW. Molecular cloning:A laboratory manual, (3rd ed.) New York: Cold Spring Harbor Laboratory Press.2001
[146]赵永芳.生物化学技术原理及应用.北京：科学出版社.2002
[147]汪家政,范明.蛋白质技术手册.北京：科学出版社.2000
[148][澳]查理德J,辛普.蛋白质与蛋白质组学实验指南.化学工业出版社.2006
[149]McLoughlin AJ. Plasmid stability and ecological competence in recombinant cultures. Biotechnology Advances,1994,12(2):279-324
[150]Friehs K. Plasmid copy number and plasmid stability. Advances in Biochemical Engineering/Biotechnology,2004,86:47-82
[151]Studier FW. Use of bacteriophage T7 lysozyme to improve an inducible T7 expression system. Journal of Molecular Biology,1991,219:37-44
[152]Summers DK. The kinetics of plasmid loss. Trends in Biotechnology,1991,9(8): 273-278
[153]Pogliano J, Ho TQ, Zhong Z, et al. Multicopy plasmids are clustered and localized in Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America,2001,98(8):4486-4491
[154]Pan SH, Malcolm BA. Reduced background expression and improved plasmid stability with pET vectors in BL21 (DE3). Biotechniques.2000,29(6):1234-1238
[155]陈卫,葛佳佳.半乳糖苷酶基因在大肠杆菌中过量表达及IPTG诱导条件.无锡轻工业大学学报,2002,21(5)：492-495
[156]Fischer B, Sumner I. Isolation, renaturation, and formation of disulfide bonds of eukaryotic proteins expressed in E. coli as inclusion bodies. Biotech Bioeng,1993, 41(1):3-13
[157]金元昌,陈春岚,苏晓艳,等GST-GnRH/TRS融合蛋白(包涵体)的分离、纯化与复性研究.西北农业学报,2006,15(4)：173-176
[158]刘永庆,陈溥言,杜念兴,等.生长抑素(SS)与硫氧还原蛋白(Thioredoxin)相融合生成的2种基因工程化融合蛋白SS-N/X和：SS-N/S的表达特性.中国兽医学报,2003,23(2)：172-176
[159]Miroux B, Walker JE. Over-production of proteins in Escherichia coli:Mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. Journal of Molecular Biology,1996,260(3):289-298
[160]安平,董志伟,寿成超.可溶性VEGF受体Flt-1的基因克隆及其活性产物在原核中的表达.中国生物化学与分子生物学报,2000,16(1)：62-65
[161]Kleman GL, Strohl WR. Acetate metabolism by Escherichia coli in High-cell-density fermentation. Appl Environ Microbiol,1994,60 (11):3952-3958
[162]Corchero JL, Villaverde D. Plasmid maintenance in Escherichia coli recombinant culture is dramatically, steadily, and specifically influncened by features of the encoded proteins. Biotechnol Bioeng,1998,58(60):625-632
[163]Jacques N, Guillerez J, Dreyfus M. Culture conditions differentially affect the translation of individual Escherichia coli mRNA. J Mol Biol,1992,226:597-608
[164]Stratton J, Chiruvolu V, Meagher M. High cell-density fermentation. Methods Mol Biol, 1998,103(1):107-120
[165]Van de Walle M, Shiloach J. Proposed mechanism of acetate accumulation in two recombinant Escherichia coli strains during high density fermentation. Biotechnol Bioeng,1998,57(1):71-78
[166]Cheng CY, Huang YL, Yang ST. A novel feeding strategy for enhanced plasmid stability and protein production in recombinant yeast Fed-batch fermentation. Biotechnol Bioeng,1997,56(1):23-31
[167]Mizutani S, Mori H, Shimizu, et al. Effect of amino acid supplement on cell yield and gene product in Escherichia coli harboring plasmid. Biotechnol Bioeng,1986,28: 203-211
[168]潘春梅,孙西玉,樊耀亭.气相色谱法测定秸秆水解液中的糠醛和乙酸含量.酿酒科技,2008,5：108-110
[169]李毅,蒲勤,赵忠良,等.溶氧反馈-分批补料高密度培养人骨形成蛋白-2工程菌.生物工程学报,2002,18(6)：718-723
[170]钟根深,石炳兴,王海东,等.表达重组葡激酶-水蛭素融合蛋白的大肠杆菌工程菌的高密度培养.中国生物工程杂志,2006,26(9)：11-15
[171]Yin L, Jian C, Ying YM, et al. Effect of additives and fed-batch culture strategies on the production of glutathione by recombinant Escherichia coli. Proc Biochem,1998,33: 709-714
[172]Castan A, Enfors SO. Characteristics of a DO-controlled fed-batch culture of Escherichia coli. Bioproc Eng,2000,22:509-515
[173]Shen YL, Zhang Y, Sun AY. High-level production of soluble tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) in high-density cultivation of recombinant Escherichia coli using a combined feeding strategy. Biotechnol Lett,2004, 26:981-984
[174]Luli GW, Strohl WR. Comparison of growth, acetate production and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations. Appl Environ Microbiol,1990,56:1004-1011
[175]Suarez DC, Kilikian BV. Acetic acid accumulation in aerobic growth of recombinant Escherichia coli. Process Biochem,2000,35:1051-1055.
[176]Jana S, Deb JK. Strategies for efficient production of heterologous proteins in Escherichia coli. Appl Microbiol Biotechnol,2005,67:289-298.
[177]储炬,李友荣.现代工业发酵调控学.北京：化学工业出版社.2002
[178]Schultz T, Martinez L, de Marco A. The evaluation of the factors that cause aggregation during recombinant expression in E. coli is simplified by the employment of an aggregation-sensitive reporter. Microbial Cell Factories,2006,5:28
[179]Belin P, Dassa J, Drevet P, et al. Toxicity-based selection of Escherichia coli mutants for functional recombinant protein production:Application to an antibody fragment. Protein Engineering, Design and Selection,2004,17(5):491-500