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冯雁, Shiyu Wang, a Yan Feng, a, * Zuoming Zhang, a Baisong Zheng, a Na Li, a Shugui Cao, a Ikuo Matsui, b and Yoshitsugu Kosugib
Archives of Biochemistry and Biophysics 411(2003)56-62,-0001,():
-1年11月30日
Glutamate dehydrogenase from Pyrococcus horikoshii (Pho-GDH) was cloned and overexpressed in Escherichia coli. The cloned enzyme with His-tag was purified to homogeneity by affinity chromatography and shown to be a hexamer enzyme of 290±8 kDa (subunit mass 48 kDa). Its optimal pH and temperature were 7.6 and 90℃, respectively. The purified enzyme has outstanding thermostability (the half-life for thermal inactivation at 100℃ was 4h). The enzyme shows strict specificity for 2-oxoglutarate and L-glutamate and requires NAD(P)H and NADP as cofactors but it does not reveal activity on NAD as cofactor. Km values of the recombinant enzyme are comparable for both substrates: 0.2mM for L-glutamate and 0.53mM for 2-oxoglutarate. The enzyme was activated by heating at 80℃ for 1h, which was accompanied by the formation of its active conformation. Circular dichroism and fluorescence spectra show that the active conformation is heat-inducible and time-dependent.
Pyrococcus horikoshii, Hyperthermophilic archaea, Glutamate dehydrogenase, Thermostability, Heat activation
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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
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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【期刊论文】嗜下菌Aeropyum pernix K1酰基佬氨基酸释放酶/酯酶(APE1547)一种新晶型体学研究
冯雁, 王刚刚, 高仁钧, 杨海涛, 曹淑桂, 冯雁*, 饶子和*
科学通报,2002,47(22):1711~1713,-0001,():
-1年11月30日
应用悬滴汽相扩散法,用PEG4000作沉淀剂,获得了嗜热古菌Aeropyrum pernix 酰基氨基酸释放酶APE1547的一种新晶型,这种新晶型属于正交晶系,晶胞参数α=6.399nm, b=10.439nm. C=16.953nm、晶体空间群属于P212121。单位晶胞中每个晶体不对称单位含2个分子,Matthews常数为2.2×10-3nm3·-1,溶剂含量为43%,晶体衍射的最高分辨率达0.27nm,与以前获得的三斜晶系的晶体相比,这咱新晶型更适合于结构分析,可以加快结构解析的过程。
酰基的氨基酸释放酶 Aeropyrum pernix K1 正交晶系 不对称单位
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冯雁, 康伟, 王智
《生命的化学》,2005,25(2):94~97,-0001,():
-1年11月30日
植酸酶是一种能广泛应用于食品、饲料和医药工业的商业酶。目前较多的研究工作是通过随机突变、定点突变和晶体结构对照来阐明植酸酶的反应机制以及重要氨基酸的贡献。并以此为基础对酶分子进行改造。其中真菌和细菌分泌的植酸酶的最适pH较低.能够应用于生物体内,因此具有很大的商业前景。
植酸酶, 随机突变, 定点突变, 晶体结构
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冯雁, Binbin Liu, a, b Xuemei Li, a Renjun Gao, c Weihong Zhou, a Guiqiu Xie, c Mark Bartlam, a Hai Pang, a Yan Fengc and Zihe Raoa, b*
Acta Cryst.(2004). D60, 577-579,-0001,():
-1年11月30日
Inorganic pyrophosphatase (PPase; EC 3.6.1.1) from the hyperthermophile Pyrococcus horikoshii was crystallized by the hanging-drop vapour-diffusion method at pH 5.0 using polyethyleneglycol 4000 as the precipitant. The crystal belongs to space group P21212, with unitcell parameters a=71.7, b=86.5, c=92.5 A, α=β=γ=90°. There are two molecules in the asymmetric unit. The crystals were stable during exposure to X-rays and a full set of X-ray diffraction data was collected to 2.7 A Ê resolution in-house.
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