【作者】 宋建民；

【导师】 魏东芝；

【作者基本信息】 华东理工大学 ， 发酵工程， 2011， 博士

【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)。更多还原

【Abstract】 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.更多还原