重组大肠杆菌产角质酶-CBM摇瓶发酵优化及分泌表达研究

在TB培养基的基础上,通过单因素分析和正交设计对重组大肠杆菌产角质酶-CBM发酵进行优化,得到最适培养基的组分为:甘油5 g/L,蛋白胨 16 g/L,MgSO₄·7H₂O 2.5 mmol/L,K₂HPO₄ 13.7 g/L,KH₂PO₄ 1.53 g/L,菌体生长至对数前中期时添加终浓度为1 g/L乳糖 和0.75 g/L 甘氨酸,30℃发酵48 h,角质酶-CBM产量可达63 U/ml,较TB培养(20 U/ml)提高了近3倍。考察了热激作用、渗透调节物质及温度两控制对角质酶-CBM分泌表达的影响,在添加Lactose和Glycine后,发现在添加终浓度为75 mmol/L的L-脯氨酸,37℃热激1 h或47℃热激0.5 h,变温至25℃发酵,角质酶-CBM产量可达90 U/ml,较TB恒温培养提高了近四倍。     

分散泛菌蔗糖异构酶在大肠杆菌中的表达及发酵优化

为了提高分散泛菌Pantoea dispersa UQ68J来源的蔗糖异构酶产量,研究了不同信号肽及发酵条件对蔗糖异构酶在大肠杆菌中重组表达的影响。将携带天然信号肽的蔗糖异构酶基因优化后,转入大肠杆菌Escherichia coli BL21(DE3)构建重组表达菌株——ORI菌株,摇瓶发酵总酶活和胞外酶活分别为85 U/m L、65 U/m L。从天然信号肽开始第22位氨基酸作为成熟蛋白的起始,连接Pel B或Omp A信号肽构建P22和O22菌株,其中P22菌株发酵总酶活提高至138 U/m L,是ORI菌株总酶活的1.6倍;而O22菌株发酵总酶活和ORI菌株无明显差别。采用3.0 g/L的乳糖诱导,P22菌株的蔗糖异构酶总酶活提高至168 U/m L。在3 L发酵罐中,研究甘氨酸浓度和诱导时间对蔗糖异构酶分泌的影响,当补加0.5%甘氨酸,DCW为18 g/L(OD_(600)=30)开始诱导,P22菌株的蔗糖异构酶胞外酶活最高达1 981 U/m L,同时蔗糖异构酶总酶活达到2 640 U/m L,是已报道大肠杆菌重组表达蔗糖异构酶的最高水平。     

均匀设计法优化烟管菌产漆酶培养基

应用均匀设计、二次多项式逐步回归分析对烟管菌(Bjerkandera adusta)WZFF.W-Y11产漆酶液态发酵培养基进行优化。结果表明,培养基组成为麸皮水解液1%、淀粉24.0 g/L、葡萄糖24.0 g/L、豆饼粉4.8 g/L、NH₄Cl 3.2g/L、KH₂PO₄ 3.2 g/L、MgSO₄·7H₂O 0.2 g/L、CuSO₄ ·5H₂O 0.006 g/L,起始pH6.5,在28℃、150r/min、250mL的摇瓶培养条件下可以稳定地获得9672U/L的漆酶活力。     

枯草芽胞杆菌甘露聚糖酶发酵条件优化

旨在以枯草芽胞杆菌Bacillus subtilis J为生产菌株,发酵生产β-甘露聚糖酶,通过优化产酶条件,以达到提高β-甘露聚糖酶产量的目的。利用DNS比色法检测β-甘露聚糖酶活力,采用单因素试验,研究碳氮源种类及碳氮源浓度、温度、pH、接种量和装液量对菌株Bacillus subtilis J发酵产β-甘露聚糖酶的影响,结合响应面试验设计确定菌株Bacillus subtilis J发酵产甘露聚糖酶的最优发酵培养条件。单因素试验和响应面试验得到最优的发酵条件为魔芋粉28 g/L,胰蛋白胨21 g/L,K₂HPO₄ 6 g/L,MgSO₄·7H₂O 1 g/L,温度31 ℃,pH值 8.5,接种量1%（体积分数）,装液量50 mL/250 mL,发酵周期24 h。利用优化后的培养基生产β-甘露聚糖酶,其酶活力达到84.38 U/mL,是初始发酵培养基产酶活力的3.36倍。通过对发酵条件的优化,大幅度提高了β-甘露聚糖酶的产量,为其工业生产提供数据参考。     

带His-tag的解脂耶氏酵母脂肪酶Lip2在毕赤酵母中的表达及纯化

将去自身信号肽并且N-端带6×His标签的YlLip2基因克隆至表达载体pPIC9K中,电转化GS115获得高效表达脂肪酶His₆-YlLip2的基因工程菌。筛选到的阳性克隆子摇瓶发酵脂肪酶活力最高为400U/ml。对重组毕赤酵母在10 L发酵罐中表达His₆-YlLip2的分批补料发酵工艺进行了初步优化,探讨了培养基、pH、温度对生物量和重组蛋白表达量的影响。结果表明:采用FM22培养基,诱导温度为25℃,pH 5.0,甲醇诱导114 h后His₆-YlLip2的最高酶活力达到3160U/ml。SDS-PAGE分析表明,蛋白的分子量大约为38kDa。重组的His₆-YlLip2经镍柱一步纯化后的纯度达到95.43%,比酶活达到4250U/mg。     

深海低温脂肪酶基因工程菌LIP001发酵条件的优化

对从深海沉积物宏基因组文库中获得的产低温脂肪酶基因工程菌LIP001进行了发酵条件优化。通过单因素试验对LIP001产脂肪酶的主要影响条件进行了探讨,确定了培养条件为30℃、pH7.0、接种量5%、装液量50ml。在单因素的基础上通过正交试验优化了影响重组菌LIP001产酶主要因素:橄榄油、酵母粉、磷酸盐、MgSO₄,确定了培养基为橄榄油1%、酵母粉0.5%、蛋白胨1%、硫酸铵0.5%、磷酸盐0.5%、MgSO₄为0.2%、氯霉素12.5μg/ml,优化后的脂肪酶活为1980U/ml,比优化前提高了54.7%,为大规模发酵奠定了基础。采用5升发酵罐方法试验,酶活达到2420U/ml。     

利用响应面分析法优化胶质类芽胞杆菌初级种子培养基

采用响应面分析法（RSM）对胶质类芽胞杆菌的初级种子培养基进行了优化,以提高其菌体密度和后续发酵效率。首先采用单因子试验筛选出提升菌体密度的适宜碳源和氮源,分别为麦芽糖和胰蛋白胨,在此基础上采用Plackett Burman(PB)试验设计法,对9种影响菌体生长繁殖的因素进行了评价,结果表明,麦芽糖和MgSO₄·7H₂O对菌体繁殖影响最为显著。用快速登高试验逼近关键因素的最大响应区域,通过中心组合试验和验证试验,获得胶质类芽胞杆菌优化培养基成分为麦芽糖 2.5 g/L,胰蛋白胨1.0 g/L,MgSO₄·7H₂O 0.73 g/L,K₂HPO₄·3H₂O 0.4 g/L,NaCl 0.06 g/L,FeCl₃ 0.6 mg/L,水杨酸10 mg/L,CaCO₃ 1.0 g/L。使用该优化培养基可将菌体密度较优化前提高了近10倍,含量达到4.12×10² cfu/mL。结果为提高胶质类芽胞杆菌的生产发酵水平和保证产品质量提供依据。     

重组大肠杆菌产γ-环糊精葡萄糖基转移酶的摇瓶发酵优化

为了实现来源于碱性芽孢杆菌Alkalophilic Bacillus clarkii 7364的γ-环糊精葡萄糖基转移酶的高效胞外表达,对OmpA信号肽介导的E.coli BL21(DE3)/pET20b(+)-γcgt基因工程菌进行发酵培养基及发酵条件的优化,并进行正交试验,获得最优培养基:甘油5g/L、蛋白胨6g/L、酵母膏24g/L、钙离子6mmol/L、镁离子2mmol/L、甘氨酸0.75%、PO₄^3- 0.1mol/L;在此基础上最适发酵条件:pH6.5、25℃培养、装液量30ml/250ml、转速220r/min、0.02%SDS、在发酵10h时利用5g/L乳糖进行诱导,使得酶活从初始的5189.2U/ml提高到20268.8U/ml。研究结果得到高效表达的培养条件,为实现该酶的工业化应用打下了基础。     

红树林植物促生菌SZ7-1 菌株的培养基优化

采用响应面分析法对红树林植物促生菌SZ7-1 菌株发酵培养基进行了优化。首先利用Plackett-Burman (PB)设计对影响SZ7-1 生长的11 个营养因素进行评价, 并筛选出显著影响因子:玉米糖浆、酵母膏和K₂HPO₄; 其次用最陡爬坡实验逼近以上三因素最优水平; 最后采用响应面法对该3 个显著因素的最佳水平范围进行研究, 得到的最佳浓度为: 玉米浆28 g/L、酵母膏14 g/L和K₂HPO₄ 2.2 g/L。在此条件下, 培养20 h 后SZ7-1 菌数达到2.6×10¹⁰ CFU/mL, 与优化前基础培养基、LB 和牛肉膏蛋白胨培养基中生长的菌数相比, 分别提高了12.4、2.4 和5.5 倍。     

变色栓菌产锰过氧化物酶的条件优化*

研究了多种培养基组分及培养条件对变色栓菌产锰过氧化物酶(MnP)的影响。当培养基中果糖浓度为20g/L,酒石酸铵浓度为10mmol/L,吐温80浓度为1.0g/L,MgSO₄·7H₂O为0.43g/L,最终pH为4.5,500mL三角瓶装液量为100mL,接种量为10片(8mm)菌苔,培养温度为30℃,转速为280r/min时,MnP的活力有了很大程度的提高,最高酶活力可达2,270U/L。     

重组大肠杆菌产Streptomyces sp. FA1来源木聚糖酶的摇瓶发酵优化

为了实现来源于Streptomyces sp. FA1的木聚糖酶的高效胞外分泌表达,对E.coli BL21(DE3)/pET20b(+)/coe/xynA基因工程菌的发酵产酶诱导条件进行优化,获得最优的诱导条件为25 ℃发酵6 h后添加终浓度为0.4 mmol/L的IPTG。在此基础上对发酵培养基进一步优化,得到最优培养基成分为:甘油11 g/L,酵母粉24 g/L,蛋白胨8 g/L,磷酸盐浓度89 mmol/L,镁离子4 mmol/L。最终酶活达到780.2 U/ml,为未优化前的2.2倍,是目前大肠杆菌摇瓶发酵产木聚糖酶的最高表达水平,为实现该酶的工业化生产奠定基础。     

流加发酵对重组Bacillus subtilis发酵生产角质酶的影响

为实现基因工程菌Bacillus subtilis WSHB06-07生产角质酶的高产,在3L发酵罐中考察了不同初糖浓度对菌体生长和产酶的影响,并在选择38 g/L初始蔗糖浓度的基础上,进行碳源的分批流加和恒速流加,结果表明发酵16 h开始流加碳源,采用总补糖量60g/L,蔗糖平均流速为4g/(L·h)的恒速补料方式,角质酶酶活在31h可达到最大545.87U/ml,比分批发酵酶活提高67.8%,并获得较高的角质酶生产强度,满足工业化生产要求。     

Medium optimization for keratinase production in hair substrate by a new <Emphasis Type="Italic">Bacillus subtilis</Emphasis> KD-N2 using response surface methodology

Culture medium for keratinase production from hair substrate by a new Bacillus subtilis strain, KD-N2, was optimized. Effects of culture conditions on keratinase production were tested, and optimal results were obtained with 10% inocula (v/v), 16 g/L hair substrate, an initial pH value of 6.5 and a culture volume of 20 mL. Several carbon sources (sucrose, cornflour) and nitrogen sources (yeast extract, tryptone and peptone) had positive effects on keratinase production, with sucrose giving optimal results. To improve keratinase yield, statistically based experimental designs were applied to optimize the culture medium. Fractional factorial design (FFD) experiments showed that MgSO₄ and K₂HPO₄ were the most significant factors affecting keratinase production. Further central composite design (CCD) experiments indicated that the optimal MgSO₄ and K₂HPO₄ concentrations were 0.91 and 2.38 g/L, respectively. Using an optimized fermentation medium (g/L: NaCl 1.0, CaCl₂ 0.05, KH₂PO₄ 0.7, sucrose 3, MgSO₄ 0.91, K₂HPO₄ 2.38), keratinase activity increased to 125 U/mL, an approximate 1.7-fold increase over the previous activity (75 U/mL). Human hair was degraded during the submerged cultivation.     

麦芽糖诱导软化芽孢杆菌α-环糊精葡萄糖基转移酶在枯草杆菌中的表达

通过PCR扩增软化芽孢杆菌α-环糊精葡萄糖基转移酶基因,将基因片段克隆到大肠杆菌-枯草杆菌穿梭载体pGJ103中,转化枯草杆菌WB600得基因工程菌进行外源表达。在1.5%的麦芽糖初始发酵培养基上摇瓶培养,48 h后重组枯草杆菌产酶活性为6.1U/ml。通过单因素分析和响应面分析对重组枯草杆菌产CGT酶摇瓶发酵条件进行优化。分析得到培养基关键组分麦芽糖,玉米淀粉和酵母粉三者最佳浓度分别为:15.5g/L,13g/L和20g/L。在此条件下,摇瓶培养36h后α-CGT酶活性为17.6U/ml,5L罐分批发酵30h后酶活达到20U/ml (水解活性为1.4×10⁴ IU/ml)。     

利用重组大肠杆菌生产α-环糊精葡萄糖基转移酶

将来源于软化类芽孢杆菌（Paenibacillus macerans）的α-环糊精葡萄糖基转移酶（α-CGT）基因插入含pelB信号肽的质粒pET-20b（＋）中,构建了表达载体pET-20b（＋）／cgt,并将其转化表达宿主E.coli BL21（DE3）。对重组菌E．coli BL21／pET-cgt进行摇瓶发酵条件的优化,确定了其胞外表达α-CGT酶的最适条件：葡萄糖8g/L,乳糖0．5g/L,蛋白胨12g/L,酵母膏24g/L,K2HPO472mmol／L,KH2PO417mmol／L,CaCl2 2．5mmol／L;初始pH为7．0,诱导温度为25℃。在该条件下培养90h后最终α-CGT酶的胞外比活达到22．1u／mL,与来源菌Pmacerans所产天然酶比活相比提高了42倍,实现了α-CGT酶的高效生产。将基因工程菌在上述条件下于3L发酵罐中发酵,90h后胞外酶比活达到22．6U／mL,证实了工业化放大的可能性。     

Coexpression of folding accessory proteins for production of active cyclodextrin glycosyltransferase of Bacillus macerans in recombinant Escherichia coli

Coexpression of folding accessory proteins, molecular chaperones, and human peptidyl-prolyl cis-trans isomerase (PPIase) increased production of active cyclodextrin glycosyltransferase (CGTase) of Bacillus macerans, which is otherwise mainly expressed as inclusion body in recombinant Escherichia coli. The best partner for soluble expression of CGTase was found to be human PPIase followed by coexpression of DnaK-DnaJ-GrpE together with GroEL-GroES. Such a significant enhancement by human PPIase coexpression seemed to be due to dual functions of chaperone and peptidyl-prolyl cis-trans isomerization. Coexpression of GroEL-GroES or minichaperone alone did not influence the specific CGTase activity. For production of active CGTase in large amounts, a high cell density culture was achieved using a pH-stat fed-batch strategy. The optimized fed-batch fermentation resulted in dry cell weight of 103.4 g/L and CGTase activity of 1200 U/mL. Combination of human PPIase expression at a gene level and cell culture optimization at a process scale exerted a synergistic effect on the product yield of soluble CGTase expression in recombinant E. coli.     

RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples

Levan fructotransferase (LFTase) from Arthrobacter ureafaciens K2032 was expressed with N-terminal fusion of a LacZ-derived secretion motif (TMITNSSSVP) using the lac promoter system in recombinant Escherichia coli JM109 [pUDF-A81]. In flask cultures, recombinant enzyme activity was detected in culture media, and sequence analysis of N-terminal residues showed that about 40% of the extracellular recombinant LFTase had an authentic N-terminus. In a fed-batch bioreactor containing recombinant E. coli at high cell concentrations (OD(600)>200), the extracellular LFTase accumulated to 46000 U ml(-1) (approximately 2.0 g l(-1)) which was almost 40% of total (intra- and extracellular) recombinant LFTase. The synthesized recombinant enzyme was secreted soon after gene expression was induced by IPTG. Prolonged high secretion caused cell lysis and growth inhibition during the production phase in fed-batch cultures. When lactose was added by continuous feed mode, the secretion of recombinant LFTase and hence the cell lysis were significantly delayed in spite of the increased synthesis level. Therefore the induced cell culture of recombinant E. coli could grow up to a much higher cell concentration with continuing recombinant enzyme synthesis. In the case of the controlled feed of lactose, the maximum activities (U ml(-1)) of total and extracellular LFTase were nearly 100% and 70% higher, respectively.