重组大肠杆菌高效分泌表达β-木糖苷酶发酵条件的优化

[目的]嗜热拟青霉β-木糖苷酶基因在大肠杆菌中高效分泌表述重组β-木糖哥酶摇瓶发酵条件优化,及5 L发酵罐放大培养.[方法]通过单因素试验对诱导剂种类及其添加量、诱导起始 OD600、培养温度、培养时间进行优化研究.[结果]摇瓶优化结果表明:2％乳糖为诱导剂、培养温度为33℃C、OD600控制在0.8-0.9时诱导为最佳产酶条件,在此条件下培养48 h后胞外酶活达到103.9 U/mL,胞外分泌的比例高达99％以上.进行5L发酵罐放大培养,发酵48 h胞外酶活达到最高值392.5 U/mL,蛋白含量为10.1 g/L.[结论]该重组大肠杆菌高效分泌β-木糖苷酶,具有较好的工业化生产前景.     

毕赤嗜甲醇酵母工程菌高密度培养表达黑曲霉菊粉内切酶的研究

目的:对毕赤嗜甲醇酵母工程菌inu-26高密度培养表达黑曲霉菊粉内切酶的条件进行优化,找出最佳的外源蛋白表达条件。方法:在摇瓶优化培养的基础上进行发酵罐高密度培养,优化最佳产酶条件。结果:以葡萄糖为碳源、微量元素添加量100～200mL/L、甲醇浓度1g/L、pH6.0～7.0、诱导时间96h时酶的表达量最高;摇瓶模拟高密度培养表明影响酵母生长的最主要因素葡萄糖和硫酸铵的最佳浓度分别为20～45和11.5g/L;利用培养基F1进行高密度培养优于其他培养基,工程菌生长符合指数生长曲线,细胞生长延迟期为1.36h,比生长速率μ为0.4846h-1。结论:以葡萄糖为碳源,采用葡萄糖-甲醇混合诱导和100%甲醇单一诱导相结合,在菌体鲜重约为280g/L时连续诱导96h,菌体生长良好,不会出现自溶,且酶的表达量最高,为摇瓶培养的3倍多,酶活最高可达540 U/mL。     

重组毕赤酵母生产β-葡萄糖苷酶发酵条件初步研究

为提高重组毕赤酵母(P.pastoris KM71/pPIC9K-bgl)生产β-葡萄糖苷酶的产量,在摇瓶条件下对重组P.pastoris产β-葡萄糖苷酶的发酵过程进行了优化,得到最佳的条件:生长阶段甘油浓度为30 g/L,接种量为10%,诱导阶段甲醇的初浓度为4%,过程补加甲醇0.5%,诱导温度30℃,pH7.5,诱导周期120 h,酶活可达到245 U/mL。在此基础上,在3 L发酵罐上进行初步放大,流加甘油提高细胞密度至OD_(600)为170,开始流加甲醇诱导,最终BGL酶活达到1 175 U/mL。比摇瓶提高了4.8倍,为β-葡萄糖苷酶工业化生产打下了坚实的基础。     

Optimization of fermentation conditions of xylanase produced by recombinant Pichia pastoris

采用单因素实验确定重组毕赤酵母产木聚糖酶生长相的最适条件,然后利用Plackett-Burman实验设计对诱导相培养基成分和培养条件的10个因素进行筛选,方差分析结果表明,影响木聚糖酶表达的主要因子为酵母膏、诱导pH和摇床转速;在此基础上,用Box-Behnken的响应面方法对3个因素进行进一步优化,当酵母膏为11.13 g/L,pH为6.38,摇床转速为228 r/min时酶活有最大值,为262.77 U/mL,较优化前提高了175.44％.优化后的摇瓶发酵条件应用于7L发酵罐并连续诱导培养120 h,发现诱导72 h后的木聚糖酶酶活最高,为2054.89U/mL.     

黄杆菌肝素酶Ⅱ重组菌培养条件的优化及高密度发酵

黄杆菌肝素酶Ⅱ(HepⅡ)是一类可特异性切割肝素、硫酸乙酰肝素类分子内连接键的酶。文中对黄杆菌肝素酶Ⅱ重组菌的诱导时机、诱导剂添加量、诱导温度、诱导时间等诱导产酶条件进行优化。经过优化最佳摇瓶发酵产酶条件为:37℃培养重组菌至对数生长前期,添加诱导剂IPTG至终浓度为0.3 g/L,20℃下诱导10 h,酶活达到最高,为570 U/L。在此基础上通过发酵罐高密度培养手段将菌体浓度OD600进一步提高到98,酶活大幅度提高到9 436 U/L,该研究结果为HepⅡ的工业化生产与应用奠定了良好的基础。     

培养方式对Xenorhabdus nematophila生长与抗菌活性的影响

为了明确不同培养方式对Xenorhabdus nematophilaYL001生长和抗菌活性的影响,提高YL001菌株的抗菌活性。采用分批发酵方式研究了摇瓶与发酵罐培养对置nematophila YL001生长和抗菌活性的影响。实验结果表明：在通气量2．5L／min、搅拌转速300r／min条件下,发酵罐的体积氧传递系数KLa明显高于摇瓶,通气及供氧状况好于摇瓶,细胞生长及代谢旺盛,细胞生长量较摇瓶发酵增加了23．9％;但由于pH变化幅度大,抗菌物质的活性单位仅达到摇瓶的发酵水平,为229U／mL。发酵罐pH控制初步研究表明,发酵过程中控制pH为7．5时,细胞生长量和抗菌活性达到23．71g/L和290U／mL,较不控制pH分别增加了21％和25％。通风对X.nematophila YL001生长和抗菌物质的产生有较大的影响,发酵罐培养时通气及供氧状况好于摇瓶,有利于YL001菌株的生长和抗菌物质的产生。     

N+离子注入诱变选育中性蛋白酶高产菌株及发酵条件的研究

对产中性蛋白酶的枯草芽孢杆菌(Bacillus subtilis)AS1.398进行离子注入诱变,从正突变率较高的注入剂量30～50 × 1014 ions/cm2范围内,筛选出一株高产菌株ZC-7.该菌株在优化了的摇瓶培养基中,培养42h,产酶可达16900U/mL.在7L发酵罐上控制pH6.0～7.0,溶氧10%～20%.以32℃3～40℃～30℃变温发酵42h,酶活力最高可迭19680U/mL,为初始菌株的2.1倍.     

重组酪氨酸酚裂解酶发酵工艺的研究

左旋多巴是治疗帕金森氏病的首选药物,生物酶法合成左旋多巴具有工艺简单、条件温和、立体选择性高和环境友好等优点。本论文以实验室前期构建的表达具核梭杆菌(Fusobacterium nucleatum) TPL (Fn-TPL)的重组大肠杆菌为基础,采用单因素实验通过对5 L发酵罐发酵工艺优化以及补料策略的研究,确定了分批发酵的工艺参数:pH 6. 5,诱导温度30℃,诱导剂乳糖20 g/L。在5 L发酵罐中,进一步研究了10 mL/h、20 mL/h、30 mL/h三个速率的恒速流加对菌体生物量和TPL酶活的影响。结果表明,补料速率为20 mL/h时,生物量最高为30. 43 g dcw/L,体积酶活最高为9 420 U/L,较摇瓶发酵培养活力提高了3. 3倍。     

一株产几丁质酶菌株的筛选及其产酶条件的研究

采用平板透明圈法从土壤中分离筛选到一株产几丁质酶放线菌株L12,用250mL摇瓶发酵初筛和复筛,酶活力为0.63U/mL。通过产酶条件实验,初步确定了该菌株较适产酶培养基和摇瓶发酵条件。条件优化后,30℃、250mL摇瓶发酵48h,几丁质酶活力达到1.06U/mL。     

弧菌外膜蛋白ompk工程菌规模化培养表达初探

目的:为Ompk亚单位疫苗生产提供参数。方法:利用振荡和发酵培养,测定不同培养时间的菌液浊度和蛋白诱导表达效果。结果:工程菌30℃摇瓶培养10h,种子罐培养8～10h,较为合适;摇瓶和5L发酵罐诱导表达培养,升温42℃诱导表达,7h效果较佳;经5批次50L、500L发酵罐诱导表达培养,均可得到重组蛋白表达的工程菌体,50L和500L发酵罐最高生物量分别为5.32g/L和6.38g/L,平均可达到3.75 g/L和4.74 g/L;适当延迟升温诱导前的培养时间,可提高工程菌的得率。结论:初步确定了重组蛋白工程菌规模化诱导表达培养方法,利用500L发酵罐培养可得到诱导表达的工程菌体。     

Functionalization of cellulose acetate fibers with engineered cutinases

In the present work, we describe for the first time the specific role of cutinase on surface modification of cellulose acetate fibers. Cutinase exhibits acetyl esterase activity on diacetate and triacetate of 0.010 U and 0.007 U, respectively. An increase on the hydroxyl groups at the fiber surface of 25% for diacetate and 317% for triacetate, after a 24 h treatment, is estimated by an indirect assay. Aiming at further improvement of cutinase affinity toward cellulose acetate, chimeric cutinases are genetically engineered by fusing the 3′‐end coding sequence with a bacterial or a fungal carbohydrate‐binding module and varying the linker DNA sequence. A comparative analysis of these genetic constructions is presented showing that, the superficial regeneration of cellulose hydrophilicity and reactivity on highly substituted cellulose acetates is achieved by chimeric cutinases. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

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。     

Application of factorial designs for optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae pneumoniae AS-22.

Production of cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae pneumoniae AS-22 was optimized in shake flasks using a statistical experimental design approach. Effect of various components in the basal medium, like carbon, nitrogen, phosphorus, and mineral sources as well as initial pH and temperature, were tested on enzyme production. The optimum concentrations of the selected media components were determined using statistical experimental designs. Two level fractional factorial designs in five variables, namely, dextrin, peptone, yeast extract, ammonium dihydrogen orthophosphate, and magnesium sulphate concentrations were constructed. The optimum medium composition thus found consisted of 49.3 g/L dextrin, 20.6 g/L peptone, 18.3 g/L yeast extract, 6.7 g/L ammonium dihydrogen orthophosphate, and 0.5 g/L magnesium sulphate. The maximum CGTase activity obtained was 21.4 U/mL in 28 h of incubation. The cell growth and CGTase production profiles were studied with the optimized medium in shake flasks and in 1-L fermenters. It was observed that the enzyme production was growth associated both in shake flask and in fermenter, although it was slower in shake flask. The maximum CGTase activity obtained in the fermenter was 32.5 U/mL in 16 h. The optimized medium resulted in about 9-fold increase in the enzyme activity as compared to that obtained in the basal medium in shake flask as well as in fermenter.     

pH控制下绿色木霉（Trichoderma viride）流加发酵生产纤维素酶

绿色木霉（Trichodermaviride）在pH控制发酵条件下,采用流加葡萄糖发酵策略,可显著提高综合滤纸酶活力（FPA）和内切酶（endo—β—1,4-glucanase,EG）、外切酶exo—β-1,4-glucanase,CBH）、纤维二糖酶（cellobiase,CB）酶活。在5L发酵罐中采用pH控制和流加葡萄糖工艺,可提高CB酶含量,改变酶组分之间的比例,使得FPA、EG、CB和CBH酶活分别达到50．0U／mL,210．0U／mL,4．0U／mL和2．5U／mL,比摇瓶发酵分别提高了6．7．4．2、19、2．5倍。     

重组毕赤酵母产木聚糖酶的发酵条件优化研究

采用单因素实验确定重组毕赤酵母产木聚糖酶生长相的最适条件,然后利用Plackett—Bur—man实验设计对诱导相培养基成分和培养条件的10个因素进行筛选,方差分析结果表明,影响木聚糖酶表达的主要因子为酵母膏、诱导pH和摇床转速;在此基础上,用Box—Behnken的响应面方法对3个因素进行进一步优化,当酵母膏为11．13彰L,pH为6．38,摇床转速为228r／min时酶活有最大值,为262．77u／mL,较优化前提高了175．44％。优化后的摇瓶发酵条件应用于7L发酵罐并连续诱导培养120h,发现诱导72h后的木聚糖酶酶活最高,为2054．89u／mL。     

pH两阶段控制策略发酵生产重组角质酶

为提高基因工程菌Bacillus subtilis WSHB06-07发酵生产角质酶的产量和生产强度,考察了pH（5.5~8.0）对菌体生长和产酶的影响。基于不同pH发酵过程中菌体比生长速率及比产物合成速率的变化,确定了pH两阶段控制策略,即0~4 h时控制pH 7.5,4 h后将pH调至6.5。通过采用这一优化策略,角质酶酶活有了较大的提高,达170 U/mL,生产强度为16.9 kU/(L·h),比恒定pH 7.5控制模式下分别提高了122.6%和123.2%。     

Enhanced cutinase production with Thermobifida fusca by two-stage pH control strategy

A mutant of Thermobifida fusca ATCC 27730 was used for cutinase production. Acetate was the most suitable carbon source for cell growth and cutinase production compared with others. The pH was one of the most important factors affecting cutinase yield and productivity. Batch cutinase fermentations by mutant Thermobifida fusca WSH04 at various pH values ranging from 7.0 to 7.9 were studied. Based on the effects of different pH values on the specific cell growth rate and specific cutinase formation rate, a two-stage pH control strategy was developed, in which the pH was set at 7.3 for the first 20 h, and switched to 7.6 afterwards. By applying this two-stage pH control strategy for cutinase fermentation, the maximal cutinase activity reached 19.8 U/mL.     

Engineered Thermobifida fusca cutinase with increased activity on polyester substrates

A bacterial cutinase from Thermobifida fusca, named Tfu_0883, was genetically modified by site-directed mutagenesis to enhance its activity on poly(ethylene terephthalate) (PET). The new mutations tailored the catalytic site for PET, increasing the affinity of cutinase to this hydrophobic substrate and the ability to hydrolyze it. The mutation I218A was designed to create space and the double mutation Q132A/T101A was designed both to create space and to increase hydrophobicity. The activity of the double mutant on the soluble substrate p-nitrophenyl butyrate increased two-fold compared to wild-type cutinase, while on PET both single and double mutants exhibited considerably higher hydrolysis efficiency. The replacement of specific amino acids at the active site was an effective approach for the improvement of the Tfu_0883 cutinase capacity to hydrolyze polyester surfaces. Thus, this study provides valuable insight on how the function and stability of enzymes can be improved by molecular engineering for their application in synthetic fiber biotransformation.     

A dual enzyme system composed of a polyester hydrolase and a carboxylesterase enhances the biocatalytic degradation of polyethylene terephthalate films

TfCut2 from Thermobifida fusca KW3 and the metagenome‐derived LC‐cutinase are bacterial polyester hydrolases capable of efficiently degrading polyethylene terephthalate (PET) films. Since the enzymatic PET hydrolysis is inhibited by the degradation intermediate mono‐(2‐hydroxyethyl) terephthalate (MHET), a dual enzyme system consisting of a polyester hydrolase and the immobilized carboxylesterase TfCa from Thermobifida fusca KW3 was employed for the hydrolysis of PET films at 60°C. HPLC analysis of the reaction products obtained after 24 h of hydrolysis showed an increased amount of soluble products with a lower proportion of MHET in the presence of the immobilized TfCa. The results indicated a continuous hydrolysis of the inhibitory MHET by the immobilized TfCa and demonstrated its advantage as a second biocatalyst in combination with a polyester hydrolase for an efficient degradation oft PET films. The dual enzyme system with LC‐cutinase produced a 2.4‐fold higher amount of degradation products compared to TfCut2 after a reaction time of 24 h confirming the superior activity of his polyester hydrolase against PET films.     

Purification and characterization of cutinase from Bacillus sp. KY0701 isolated from plastic wastes

A total of approximately 400 bacterial strains were isolated from 73 plastic wastes collected from 14 different regions. Nineteen isolates that form clear zones both on tributyrin and poly ε-caprolactone (PCL) agar, were identified based on 16S rRNA gene sequences. Among these, Bacillus sp. KY0701 that caused the highest weight loss of PCL films in minimal salt medium, was selected for cutinase production. The highest enzyme activity (15 U/mL) was obtained after 4 days of incubation at 50°C, pH 7.0 and 200?rpm in a liquid medium containing 1.5% (w/v) apple cutin and 0.1% (w/v) yeast extract. The purified enzyme was stable at high temperatures (50–70°C) and over a wide pH range (5.5–9.0). The relative activity of cutinase was at least 75% in the percent of various organic solvents. The apparent K_m and V_max values of the cutinase for p-nitrophenyl butyrate were 0.72?mM and 336.8?µmol p-nitrophenol/h/g, respectively. In addition, it showed high stability and compatibility with commercial detergents. These features of cutinase obtained from Bacillus sp. KY0701 make it a promising candidate for application in the detergent and chemical industries. In our best knowledge, this is the first report for cutinase production and characterization produced by a Bacillus strain.