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2011年02月18日

【期刊论文】基因组改组提高干酪乳杆菌耐酸性生产L-乳酸*

冯雁, 王玉华, , 李岩, 裴晓林, 冯雁**

中国生物工程杂志,2006,26(2):53~58,-0001,():

-1年11月30日

摘要

首先采用紫外线与亚硝基胍两种传统微生物诱变方法对干酪乳杆菌进行诱变,经低pH平板、碳酸钙平板和摇瓶试验获得了5株耐酸性提高的突变菌株。以获得的突变菌株为出发菌株,应用灭活双亲原生质体融合后致死损伤得到互补获得活性融合子的方法,对其进行基因组改组,经过低pH平板、碳酸钙平板和摇瓶筛选,获得4株可以在pH3.8平板上旺盛生长且产酸量较高的改组菌株。将改组菌株与原始菌株分别于pH 3.8和3.4的YE液体培养基中培养,改组菌株能够在原始菌株无法生存的pH条件(pH 3.4)下生长。在pH 3.8的条件下,对改组菌株与原始菌株的发酵特征进行比较,37℃发酵48小时后,改组菌株产酸量为原始菌株的2.4倍,表明基因组改组技术能有效提高多基因调控表型的进化。

基因组改组 干酪乳杆菌 耐酸性 L2乳酸

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2011年02月18日

【期刊论文】古核嗜热菌组蛋白基因多点突变的新方法

冯雁, 翁梁, 纪昕, 王智, 刘丹, 马吉胜, 李娜, 曹淑桂, 冯雁*

药物生物技术,2002,9(5):264~266,-0001,():

-1年11月30日

摘要

嗜热菌组蛋白基因序列中精氨酸密码子在大肠杆菌中的表达率极低,限制了组蛋白的大量获得。运用分子生物学技术,在传统PCR定点突变技术的基础上进行改进,参照古核嗜热菌组蛋白基因序列和大肠杆菌偏爱密码子表,设计了一对含有5个精氨酸突变密码子的单链DNA,全长为115nt,在3'端有20nt互补序列。将单链DNA在94℃变性,45℃复性,使其互补结合,72℃进行延伸反应,得到含有突变位点的组蛋白基因;再以此基因为模板设计相应的一对引物进行PER扩增反应,得到大量组蛋白突变基因。该研究为一些序列已知,片段较小基因的多点突变提供了一种简单、易行的方法。

多点突变, 聚合链式反应, 组蛋白

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2011年02月18日

【期刊论文】Structure Based Hyperthermostability of Archaeal Histone HPhA from Pyrococcus horikoshii

冯雁, Ti Li, Fei Sun, Xin Ji, Yan Feng* and Zihe Rao*

J. Mol. Biol.(2003)325, 1031-1037,-0001,():

-1年11月30日

摘要

The histone protein HPhA from the hyperthermophilic archaeon Pyrococcus horikoshii, shows hyperthermostability, as required for optimal growth of the organism at 95℃. The structure of recombinant P. horikoshii HPhA has been determined to 2.3A resolution by molecular replacement, and refined to Rwork and Rfree values of 20.7% and 27.3%, respectively. The HPhA monomer structure is characterized by the histone fold and assembles into a homodimer like other archaeal histones. There are four anions found in the dimer structure, giving rise to clues as to where DNA might bind. A detailed comparison of four known structures of archaeal histones, which evolve and exist under different temperatures, shows that the thermophilic archaeal histone HPhA has a larger hydrophobic contact area, an increased number of hydrogen bonds and a reduced solvent-accessible area. We also observe a unique network of tyrosine residues located at the crossover point of the two HPhA monomers, which locks them together and stabilizes the dimer. These factors together account for the increased thermal stability.

histone, HPhA, Pyrococcus horikoshii, archaea, authors hyperthermostability

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2011年02月18日

【期刊论文】Enantioselective lipase-catalyzed kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine

冯雁, Liangyu Zheng a, Suoqin Zhangb, Yan Fenga, Shugui Caoa, ∗, Jisheng Maa, Lifang Zhaoa, Gui Gaoa

Journal of Molecular Catalysis B: Enzymatic 31(2004)117-122,-0001,():

-1年11月30日

摘要

The enzyme (BSL2), a highly active lipase expressed from newly constructed strain of Bacillus subtilis BSL2, is used in the kinetic resolution of N-(2-ethyl-6-methylphenyl)alanine from the corresponding racemic methyl ester. Reaction conditions are optimized to enhance the enantioselectivity. The effects of various racemic alkyl esters, substrate concentration, operating temperature, pH of the aqueous medium and organic solvents on activity and enantioselectivity of BSL2 for kinetic resolution are also studied. A high enantiomeric ratio (E=60.7) is reached in diisopropyl ether/water (10%, v/v) and the enantioselectivity is about 22-fold higher than that in pure buffered aqueous solution. The results show that the reaction medium greatly influences BSL2 reaction and its enantioselectivity in the hydrolysis of racemic methyl ester.

Bacillus subtilis, BSL2, Kinetic resolution, Hydrolysis, (, S), -(, -), -N-(, 2-ethyl-6-ethylphenyl), alanine

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2011年02月18日

【期刊论文】Optimization of recombinant hyperthermophilic esterase production from agricultural waste using response surface methodology

冯雁, Xiaodong Ren, Dawei Yu, Siping Han, Yan Feng *

Bioresource Technology 97(2006)2345-2349,-0001,():

-1年11月30日

摘要

The aim of this work was to evaluate the capability of corn steep liquor being a low cost substrate on the recombinant protein production by cultivating recombinant Escherichia coli and increasing the production of hyperthermophilic esterase (HE). The effect of corn steep liquor, mineral salt and trace metals on hyperthermophilic esterase production was investigated by means of a five-level three-factor central composite rotatable design. Optimized values of the factors were determined and a maximum hyperthermophilic esterase production of 251.39 U/ml was obtained. This value equaled the yield by yeast extract and peptone medium on the whole.

Hyperthermophilic esterase, Corn steep liquor, Response surface methodology

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    上海交通大学,上海

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