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。     

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。     

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。     

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。     

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。     

Coprinopsis cinerea 漆酶基因的克隆及其在毕赤酵母中的表达

本文通过RT-PCR从Coprinopsis cinerea okayama7#130中克隆得到laccase1,应用SignalP3.0Server软件分析其氨基酸序列后设计不含信号肽序列的新引物扩增得到不含信号肽的漆酶基因1（lac1）,并构建重组酵母表达载体pPIC9K-lac1,电击转化毕赤酵母GS1115并用甲醇诱导表达,之后研究重组酶的酶学性质。克隆得到的laccase1全长为1593bp,编码530个氨基酸,其中信号肽包含18个氨基酸。SDS-PAGE显示重组蛋白大小约为65KDa。酶学性质研究表明,该酶最适反应温度为45℃,最适pH为4.3。重组漆酶在45℃保存3h后活性基本保持不变,在pH4.0～10.0的范围内稳定性较好。成功分泌表达的漆酶活力达到1.108U/mL。     

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.     

Cloning and extracellular expression of inulin fructotransferase from Arthrobacter aurescens SK 8.001 in E. coli

BACKGROUND: Difructose anhydride (DFA) III is a natural and low‐calorie sweetener. It stimulates the absorption of calcium and other minerals. Inulin fructotransferase (IFTase; EC 4.2.2.18), catalysing inulin hydrolysis to DFA III, is considered to be the most promising enzyme for the production of DFA III. RESULTS: IFTase gene from Arthrobacter aurescens SK 8.001 was cloned and sequenced. Transformant with native IFTase signal peptide was a useful system for extracellular over‐expression of IFTase, and its extracellular IFTase activity reached 81.0 U mL^?1. This value was 4.1‐fold of that obtained with A. aurescens SK 8.001 for IFTase production. The recombinant IFTase was purified to electrophoretical homogeneity and characterized. The enzyme showed maximum activity at pH 6.0 and 55 °C, and retained 81.3% of its initial activity after incubation at 60 °C for 4 h. CONCLUSION: IFTase gene from A. aurescens SK 8.001 was cloned, sequenced and over‐expressed in E. coli. IFTase was reported for the first time to be over‐expressed extracellularly. The recombinant IFTase was purified and characterized, and shown to be a good candidate for potential application in DFA III production. Copyright © 2011 Society of Chemical Industry     

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.     

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。