菊糖果糖转移酶基因在毕赤酵母中的分泌表达

将PCR获得的不包含信号肽序列的菊糖果糖转移酶基因（ift）与毕赤酵母分泌表达载体pPIC9K进行连接,构建重组载体pPIC9K-IFTase。重组载体经线性化后电转入毕赤酵母GS115感受态细胞,利用G418抗性梯度筛选及PCR鉴定,获得一株高拷贝菊糖果糖转移酶重组毕赤酵母菌株。该重组菌株经甲醇诱导,能够分泌具有活性的菊糖果糖转移酶,且60h时酶活为10.3U/mL。结果表明,菊糖果糖转移酶可以实现在毕赤酵母的分泌表达。      

烟曲霉壳聚糖酶在毕赤酵母中的分泌表达

为了筛选高效表达壳聚糖酶的毕赤酵母菌株。重组质粒pPIC9K-CSN经SalⅠ线性化后,电击转化毕赤酵母GS115,表型筛选Mut+转化子,PCR鉴定后,用G418梯度浓度筛选多克隆子,用甲醇诱导摇瓶分泌表达。经PCR分析,质粒转到宿主菌GS115,在G418为4mg/mL时,筛选到了多克隆子,经SDS-PAGE分析表明分泌表达蛋白的相对分子量约为25kDa,酶活为14.59 U/mL。     

甜蛋白Monellin在毕赤酵母中的分泌表达

本研究采用毕赤酵母偏爱密码子,人工合成了高甜度Monellin基因,并构建了重组分泌型酵母表达载体pPIC9M,通过电击转化获得了可高效分泌表达有甜味活性高甜度Monellin的重组毕赤酵母GS115/pPIC9M.通过PCR检测证实Monellin基因已经整合进酵母基因组,SDS-PAGE和Western blot免疫杂交知所表达的蛋白是目的蛋白,最后通过双盲测定证实表达的蛋白存在正常活性.     

纳豆激酶原基因在毕赤酵母中的分泌表达

为构建纳豆激酶原基因的重组酵母菌分泌型表达载体pPRONK2,并在毕赤酵母菌中进行表达,将来源于豆豉的纳豆芽孢杆菌的纳豆激酶原基因克隆至毕赤酵母菌分泌型表达载体pHBM905A上,得到重组质粒pPRONK2,再将其经SalI酶切后分别转化3株毕赤酵母菌KM71、GS1 15、SMD1 168,在MD平板上筛选得到重组酵母菌.经检测重组酵母菌发酵液中具纳豆激酶纤溶活性.表明在毕赤酵母菌中成功地实现了纳豆激酶的分泌表达.     

米黑根毛霉脂肪酶基因在毕赤酵母中的高效表达

脂肪酶是主要工业用酶制剂之一,在食品、洗涤剂和制药等领域广泛应用。将编码米黑根毛霉(Rhizo-mucor miehei)脂肪酶RML的基因克隆到pPIC9K载体中,构建了分泌型表达载体pPIC9K-RML,载体经线性化后转化Pichia pastorisGS115,G418梯度筛选获得了分泌表达RML的重组毕赤酵母工程菌,SDS-PAGE分析显示表达的脂肪酶分子量大小与预期一致。初步研究表明,重组脂肪酶最适温度为40℃,最适pH值为8.0,以橄榄油为底物时,发酵上清液酶活最大可达102 U/mL,表明构建的重组毕赤酵母工程菌具有较好的工业化生产潜力。     

人乳铁蛋白在毕赤酵母中的高效分泌表达

人乳铁蛋白（rhLf）具有抗微生物活性、抗炎症、抗肿瘤及参与免疫调节等多种生物学活性,在食品、化妆品以及饲料添加剂中具有广阔的应用前景。从乳液中提取乳铁蛋白的成本较高,制约乳铁蛋白的大规模生产及应用,通过构建高产细胞工厂获取rhLf有望解决这一问题。该研究首先对基因表达元件中的启动子信号肽进行优化,人乳铁蛋白表达菌株上清总蛋白从215.00 mg/L提升至849.52 mg/L,表达元件的优化将人乳铁蛋白表达菌株上清总蛋白提高近3倍。进一步,过表达蛋白生产分泌途径相关的促分泌蛋白因子,调节细胞胁迫反应提高rhLf的产量。过表达调控谷胱甘肽氧化还原系统的Yap1和增加氧化耐受的Msn2,平衡酵母细胞生产压力下的氧化还原状态,人乳铁蛋白表达菌株上清总蛋白从849.52 mg/L提升至1 055.68 mg/L。最后,对目的蛋白进行半定量,目的蛋白从34.82 mg/L提升至197.30 mg/L,提高近4.8倍。该研究通过多种组合策略大大提升了人乳铁蛋白表达菌株的蛋白生产能力,并为毕赤酵母高效分泌表达提供指导。     

明胶-海藻酸钠固定化菊糖果糖转移酶

采用明胶和海藻酸钠为包埋载体,进行菊糖果糖转移酶固定化的初步研究,探究明胶与海藻酸钠浓度、Ca Cl2浓度、包埋时间等因素对固定化效果的影响,并对固定化酶的酶学性质进行了研究。结果表明,明胶浓度为20 g/L、海藻酸钠浓度为20 g/L、Ca Cl2浓度为40 g/L、包埋时间5 h,酶的包埋率为95.6%,重复操作8次后相对酶活力保留在50%以上。与游离酶相比,固定化酶的最适反应p H为5.5⁶.0,最适反应温度为656.0,最适反应温度为65⁷0℃,具有良好的操作稳定性。     

内切葡聚糖酶与木聚糖酶在毕赤酵母中的共分泌表达

为实现内切葡聚糖酶和木聚糖酶在毕赤酵母中的共分泌表达,进而降低酶制剂的生产成本,构建含木聚糖酶基因的重组表达载体pPICZαA-Aoxyn11A,经SacI线性化后,电转化至含内切葡聚糖酶基因Aucel12A的重组毕赤酵母GSC7中,获得双重重组毕赤酵母GSCX8。经甲醇诱导表达后,GSCX8发酵上清液中内切葡聚糖酶和木聚糖酶的活性分别为47.77IU/mL和192.71IU/mL,为单独表达菌株GSC7和GSX5的85%和80%。酶学性质分析显示,内切葡聚糖酶的最适pH为4.0,在pH3.0~8.5稳定;最适温度为50℃,在60℃以下稳定。木聚糖酶的最适pH为5.5,在pH3.0~10.0稳定;最适温度为55℃,在50℃以下稳定。GSCX8遗传稳定性测试结果表明,内切葡聚糖酶和木聚糖酶在毕赤酵母中实现了稳定的共表达。     

酵母分泌表达重组人神经生长因子

目的研究人神经生长因子基因工程制备工艺。方法构建了表达质粒pPIC9K/rhNGF,转化毕赤酵母,筛选多拷贝转化子,进行摇瓶表达,正交试验选择表达条件。结果得到高表达高外泌的工程菌,并确定了纯化步骤,经疏水层析和阳离子交换层析纯化后,取得高活性高纯度的rhNGF。结论为该药的规模生产奠定了基础。     

黑曲霉糖化酶基因在毕赤酵母中的克隆和表达

从高产糖化酶的黑曲霉的cDNA文库中筛选出糖化酶基因,并研究在毕赤酵母中的表达情况。运用RT-PCR从黑曲霉cDNA文库中克隆糖化酶基因的cDNA片段与载体pPIC9K相连,构建重组载体,电转化毕赤酵母GS115,筛选阳性克隆并进行研究。阳性克隆在MM培养基中发酵72 h和1%的甲醇的诱导的情况下,重组毕赤酵母产生的糖化酶酶活最大为15.6 U/mL。测定结果显示,其糖化酶大小为1 908 bp,编码636个氨基酸残基组成的蛋白质。经柱分离纯化其发酵上清液后,用SDS-PAGE电泳方法,测得分子质量大约为80 ku。黑曲霉糖化酶基因在毕赤酵母GS115中成功得到了表达。     

DFA III production from inulin with inulin fructotransferase in ultrafiltration membrane bioreactor

An ultrafiltration membrane bioreactor was used for the production of DFA III from enzymatic conversion of inulin. Compared with the traditional batch reactor, the productivity and purity of DFA III could be markedly enhanced and product inhibition was removed and IFTase could be continuously used for six runs in the UF membrane bioreactor. When the substrate concentration was 100 g/L, the concentration of DFA III was about 78.4 g/L, while the productivity and purity of DFA III could attain about 2385 and 92%, respectively.     

Secretory production of recombinant human chymase as an active form in Pichia pastoris

We succeeded in expressing in a Pichia pastoris (P. pastoris) host a cDNA encoding a mature human chymase (h-chymase) which was secreted directly into the culture medium. Recombinant human heart chymase (rh-chymase) was purified from the culture medium via a single one-step heparin-agarose column chromatography tracing, using succinyl-Ala-Ala-Pro-Phe-para-nitroanilide (Suc-AAPF-pNA) hydrolysing activity. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the rh-chymase showed a diffused protein band with molecular weight of 32-37 kDa. After deglycosylation, however, rh-chymase changed to a sharp protein band with molecular weight 28 kDa, which is equal in size to deglycosylated h-chymase. The rh-chymase had an activity to convert one of the natural substrates, angiotensin I, to angiotensin II. Double reciprocal plot analysis revealed that the K(m) value ofrh-chymase against Suc-AAPF-pNA was approximately 5.1 mM, which is close to that of purified h-chymase.     

杂合木聚糖酶的设计及在毕赤酵母中的表达

人工设计合成木聚糖酶,为木聚糖酶分子改造研究提供新思路。采用合成生物学思路,以黑曲霉XZ-3S木聚糖酶Xyn ZF-2基因序列为基础,将N端48个氨基酸替换成Ev Xyn11 N端的34个氨基酸;引入芳香族氨基酸P9Y和H14F;在C端引入二硫键Cys38-Cys191;α-螺旋及cord区域分别引入疏水性氨基酸K164M、G166A、N160I、V111A以及G109A,设计杂合酶Xyn ZL。基因全合成后,构建毕赤酵母重组表达质粒p PIC9K-ZL,将其转化酵母菌GS115,对工程菌GS115-XynZL进行单因素发酵条件优化及重组酶酶学性质测定。最佳发酵培养基为土豆培养基,最佳诱导温度为28℃,最佳种龄为20 h,最佳诱导时间为168 h,最佳诱导起始pH值为6. 5,甲醇诱导最佳体积分数为15 mL/L。重组酶酶学性质显示:最佳反应温度为55℃,最适pH值为5. 0,杂合酶Xyn ZL在毕赤酵母中表达后具有特定性质和功能,其设计方法拓宽了木聚糖酶分子改造研究的思路。     

Efficient induction of inulin fructotransferase by inulin and by difructose anhydride III in Arthrobacter aurescens SK 8.001

Inulin fructotransferase (IFTase; EC 4.2.2.18) has received great attention mainly due to its application in producing difructose anhydride III (DFA III), which is a novel functional sweetener. The object of this study was to investigate the induction of IFTase in Arthrobacter aurescens SK 8.001 with various carbon sources, especially inulin and DFA III. IFTase production could be significantly promoted by the supplement of inulin (5–50 g/L) and DFA III (5–20 g/L). Inulin at high initial concentrations gave no indication of catabolite repression, whereas 30 and 40 g/L DFA III intensely inhibited cell growth and IFTase activity. No fructose was detected in broth throughout the cultivation with inulin, and inulin was converted into DFA III and minor fructooligosaccharides. And when DFA III was the carbon source, DFA III was the only sugar detected in the broth. In conclusion, both DFA III and inulin are effective for IFTase induction, and inulin with higher IFTase activity proved to be a more potent inducer.     

一种密码子优化的酸性普鲁兰酶基因在巴斯德毕赤酵母中的高效表达

根据毕赤酵母密码子偏好性对来源于Bacillus deramificans的普鲁兰酶基因进行优化并构建在细胞内表达普鲁兰酶的重组毕赤酵母。优化后普鲁兰酶编码基因Bd P4在巴斯德毕赤酵母GS115中的表达水平平均比优化前的普鲁兰酶基因Bd P提高4倍以上。筛选到1株表达水平较高的重组菌Pichia pastoris GS115/p PIC9K-Bd P4 WB54。在5 L发酵罐获得了初步优化的发酵条件:发酵液p H 4.5,在菌体量达到87 g/L时开始甲醇流加,采用溶氧(DO)恒定策略控制甲醇流加,DO控制在25%的水平,甲醇流加速率为9.9 m L/(L·h),搅拌转速控制在最大值800 r/min,通风量2 V/(v·m)。在优化条件下重组菌发酵酶活在2 000 U/m L以上。重组酶最适p H为4.0,最适作用温度50℃,在50和55℃下酶活半衰期分别为32和18 h。     

Heterologous expression and purification of Zea mays transglutaminase in Pichia pastoris

Transglutaminases (TGases) are a family of enzymes that catalyze the cross-linking of proteins and are widely used in the food industry to improve the texture of dairy, meat, and bread products. Zea mays transglutaminase (TGZ) is a new type of TGase with a wide potential. TGZ was expressed in the yeast Pichia pastoris under an alcohol oxidase promoter. Maximal expression of recombinant TGZ was achieved by inducing recombinant GS115 (pPIC9K-tgz) in BMMY medium using 1.5% methanol for 96 h. Secreted TGZ was initially separated using Superdex 200 resin and further purified on cation exchange resin. The activity of TGZ following purification was 0.32 U/mg of protein. The polymerization effect of TGZ on casein catalyzed by recombinant TGZ was slightly lower than the effect of microbial transglutaminase (MTG). TGZ is a new potential additive for the food industry.     

巴斯德毕赤酵母(Pichia pastoris)高效异源表达脂肪酶研究进展

脂肪酶作为一种常见的工业用酶,广泛应用于食品、化妆品等与生活密切相关的工业领域,但较高的生产成本和使用成本,在一定程度上限制了其进一步应用。通过巴斯德毕赤酵母(Pichia pastoris)异源表达系统重组表达脂肪酶,成为解决限制脂肪酶发展的方法之一。本文介绍了脂肪酶在P.pastoris表达系统中的异源表达及其优化策略,并对毕赤酵母表面展示异源脂肪酶的技术及应用进行了归纳。     

超高压加工对菊糖果糖转移酶活力和构象的影响

运用高效液相、荧光光谱和圆二色谱等方法研究了超高压加工对菊糖果糖转移酶活力和构象的影响,并分析了压力处理后的酶构象变化与酶活力大小之间的联系。结果表明：在80℃的条件下,与未处理组相比,经压力处理后的菊糖果糖转移酶活力随处理压力的升高及保压时间的延长先上升后下降,200MPa处理30rain后相对酶活力达最高,为124．08％。菊糖果糖转移酶的内源荧光主要来自Trp残基,压力处理后酶的相对荧光强度和相对酶活力之间呈正线性相关,表明酶活力变化与压力处理后的Trp微环境的变化有关。圆二色谱的结果显示该酶仅在219nm有负峰,表明β-折叠是菊糖果糖转移酶的主要二级结构;219nm负峰的峰值大小与相对酶活力呈负线性相关,表明β-折叠是该酶发挥催化活力的结构基础。压力引起酶构象变化的研究为超高压加工提高酶活力的机理研究提供了一定的理论依据．     

Heterologous expression of endo-1,4-beta-xylanaseC from Phanerochaete chrysosporium in Pichia pastoris

The cDNA of endo-1,4-β-xylanaseC, isolated from Phanerochaete chrysosporium, was expressed in Pichia pastoris, under the control of the alcohol oxidase I promoter. Using either the intrinsic leader peptide of xylanaseC or the α-factor signal peptide of Saccharomyces cerevisiae, xylanaseC is efficiently secreted into the medium, at a maximum concentration of 2500 U·l(-1).     

Cloning of levansucrase from Leuconostoc mesenteroides and its expression in Pichia pastoris

A gene (m1ft) encoding levansucrase from Leuconostoc mesenteroides has been previously cloned in Escherichia coli. Presently, m1ft was cloned and secretively expressed in Pichia pastoris. Methanol induction of recombinant M1FT resulted in the production of active levansucrase (PM1FT). PM1FT-5 was expressed as an active form, but the protein accumulated mainly inside the cells, representing about 5% of total cell proteins. M1FT was secreted into the culture supernatant with a maximum yield of 14,400 U/L using fed-batch fermentations. P. pastoris-derived M1FT displayed catalytic activities comparable to those of the E. coli-derived M1FT. PM1FT was glycosylated at its 2 potential N-glycosylation sites.