冯雁
主要从事酶学和酶工程研究。涉及以下领域:微生物功能基因组学;酶结构-功能关系;酶分子进化;酶生物技术及其应用。
个性化签名
- 姓名:冯雁
- 目前身份:
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学术头衔:
博士生导师, 教育部“新世纪优秀人才支持计划”入选者
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学科领域:
生物化学
- 研究兴趣:主要从事酶学和酶工程研究。涉及以下领域:微生物功能基因组学;酶结构-功能关系;酶分子进化;酶生物技术及其应用。
冯雁
1986年毕业于吉林大学分子生物学系;1989年于吉林大学获生物化学与分子生物学硕士学位;1994年于白求恩医科大学获医学博士学位。1989年在吉林大学分子酶学工程教育部重点实验室任教,历任讲师、副教授、教授;2006年任重点实验室主任。先后在日本国立生命科学和技术研究所(NIBH)、日本国立产业技术融合研究所(NAIR)和美国国立健康总署癌症研究所(NCI)从事博士后和特聘研究员工作。2009年起任上海交通大学特聘教授,博士生导师。2010年起任上海交通大学生命科学技术学院生物科学与技术系主任。《Molecular Catalysis B,Enzymatic》、《中国生物化学与生物物理学报》等刊物编委;中国微生物学会酶工程专业委员会副主任;中国生物化学与分子生物学会酶学专业委员会副主任;中国生物化学与分子生物学会蛋白质化学专业委员会理事;中国生物物理学会理事。
主要致力于酶学和酶工程领域的研究。系统建立了在医药、食品及化工等领域有重要影响的酶(脂肪酶、蛋白酶及多糖水解酶等)基因资源库;解析了多种嗜热酶的晶体结构,并从酶学、生物信息学和生物物理学等角度探讨了酶活性、稳定性及底物选择性的复杂关系;率先在国内开展了酶分子改造的研究工作,利用定向进化、合理设计及分子模拟等技术构建了功能明显改变的进化酶。以上研究得到国家重点基础研究发展计划(973计划)、国家自然科学基金、中日双边合作项目及省部级科研项目的支持,并与美国、欧洲、日本、韩国等多个实验室保持密切的合作关系。
已在J. Biol. Chem., J. Mol. Biol., Biochem. Biophys. Acta等国内外学术刊物上发表论文80余篇,研究成果获省级科技进步一等奖和二等奖各一项;作为副主编编写《酶工程》专著一部。曾获教育部优秀青年教师资助计划(2003年)、中共吉林省委吉林省人民政府“吉林省优秀留学回国人员” (2005年)、教育部新世纪优秀人才(2006年)等称号。
主要从事酶学和酶工程研究。涉及以下领域:
(1) 微生物功能基因组学;
(2) 酶结构-功能关系;
(3) 酶分子进化;
(4) 酶生物技术及其应用。
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主页访问
1175
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关注数
0
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成果阅读
1006
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成果数
20
冯雁, Qiuyan Wang, Guangyu Yang, Yanli Liu, and Yan Feng
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 281, NO.27, pp. 18618-18625, July 7, 2006,-0001,():
-1年11月30日
It has been shown that highly conserved residues that form crucial structural elements of the catalytic apparatus may be used to account for the evolutionary history of enzymes. Using saturation mutagenesis, we investigated the role of a conserved residue (Arg526) at the active site of acylaminoacyl peptidase from hyperthermophilic Aeropyrum pernix K1 in substrate discrimination and catalytic mechanism. This enzyme has both peptidase and esterase activities. The esterase activity of the wild-type enzyme with p-nitrophenyl caprylate as substrate is 7 times higher than the peptidase activity with Ac-Leu-p- nitroanilide as substrate. However, with the same substrates, this difference was increased to 150-fold for mutant R526V. A more dramatic effect occurred with mutant R526E, which essentially completely abolished the peptidase activity but decreased the esterase activity only by a factor of 2, leading to a 785-fold difference in the enzyme activities. These results provide rare examples that illustrate how enzymes can be evolved to discriminate their substrates by a single mutation. The possible structural and energetic effects of the mutations on kcat and Km of the enzyme were discussed based on molecular dynamics simulation studies.
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【期刊论文】Thermolysis of recombinant Escherichia coli for recovering a thermostable enzyme
冯雁, Xiaodong Ren, Dawei Yu, Siping Han, Yan Feng ∗
Biochemical Engineering Journal 33(2007)94-98,-0001,():
-1年11月30日
The development of recombinant DNA has made it feasible to produce a wide range of valuable protein products in the bacterium Escherichia coli. Extraction of intracellular protein from E. coli is traditionally achieved by mechanical, chemical or enzymatic disruption technology. In this study, thermolysis, which differs from the traditional ones, is presented for disruption of E. coli cells to release recombinant thermostable enzyme. Heat treatment of E. coli at 80℃ is highly effective to destroy the integrity of the bacterial cell wall and release the recombinant thermostable enzyme. At the same time of disruption, the recombinant thermostable enzyme was partially purified. Moreover, thermolysis was carried out in fermentation broth in situ, which may make it a more applicable approach for industrial-scale processes.
Bioseparations, Cell risruption, Downstream processing, Protein recovery, Thermolysis, Escherichia coli
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【期刊论文】Overexpression and characterization of a lipase from Bacillus subtilis
冯雁, Jisheng Ma, Zuoming Zhang, Baijing Wang, Xiangju Kong, Yuguo Wang, Shugui Cao*, Yan Feng*
Protein Expression and PuriWcation 45(2006)22-29,-0001,():
-1年11月30日
A novel plasmid, pBSR2, was constructed by incorporating a strong lipase promoter and a terminator into the original pBD64. A mature lipase gene from Bacillus subtilis strain IFFI10210, an existing strain for lipase expression, was cloned into the plasmid pBSR2 and transformed into B. subtilis A.S.1.1655. Thus, an overexpression strain, BSL2, was obtained. The yield of lipase is about 8.6mg protein/g of wet weight of cell mass and 100-fold higher than that in B. subtilis strain IFFI10210. The recombinant lipase was puriWed in a three-step procedure involving ammonium sulfate fractionation, ion exchange, and gel Wltration chromatography. Characterizations of the puriWed enzyme revealed a molecular mass of 24 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, maximum activity at 43℃ and pH 8.5 for hydrolysis of p-nitrophenyl caprylate. The values of Km and Vm were found to be 0.37mM and 303 μmolmg-1·min-1, respectively. The substrate speciWcity study showed that p-nitrophenyl caprylate is a preference of the enzyme. The metal ions Ca2+, K+, and Mg2+ can activate the lipase, whereas Fe2+, Cu2+, and Co2+ inhibited it. The activity of the lipase can be increased about 48% by sodium taurocholate at the concentration of 7mM and inhibited at concentrations over 10mM.
Bacillus subtilis, Lipase, Overexpression, Characterization
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冯雁, Xiaodong Ren, Dawei Yu, Siping Han, Yan Feng *
Bioresource Technology 97(2006)2345-2349,-0001,():
-1年11月30日
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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冯雁, Guirong Zhang a, b, Renjun Gaoa, Liangyu Zheng a, Aijun Zhang a, Yuanhong Wang a, c, Qiuyan Wang a, Yan Feng a, Shugui Caoa, ∗
Journal of Molecular Catalysis B: Enzymatic 38(2006)148-153,-0001,():
-1年11月30日
To enhance the enantioselectivity of a hyperthermophilic esterase from archaeon Aeropyrum pernix K1 (APE1547), a directed evolution approach is employed to generate mutant library from the native enzyme. A mutation (TBC26) is identified after one round of epPCR. The enantioselectivity of TBC26 is increased up to 2.6-fold compared to that of wild type enzyme. TBC26 contains five amino acid substitutions (R11G, L36P, V223A, I551L, A564T). The five mutation sites are spatially distant to the catalytic center. According to the published crystal structure ofWTand considering the changes of secondary and tertiary structure, here we try to explain the change of enantioselectivity of the TBC26. The results suggest that the change of enantioselectivity of enzyme has a close relationship to the configuration of the enzyme.
Directed evolution, Enantioselectivity, Screening, 2-Octanol acetate, Configuration, Mutant
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【期刊论文】An archaeal histone-like protein as an efficient DNA carrier in gene transfer
冯雁, Liang Wenga, Dan Liub, Yuanyuan Lia, Shugui Caoa, Yan Fenga, *
Biochimica et Biophysica Acta 1702(2004)209-216,-0001,():
-1年11月30日
HPhA, a recombinant histone-like protein from Pyrococcus horikoshii OT3 strain, has compacting activity with DNA as previously reported. The extreme stability and DNA packaging activity of the HPhA make it a candidate as a DNA carrier. Here, the plasmid DNA-HPhA complexes were fully characterized by gel retardation assay and DNase resistance assay. It was further proved that HPhA has in vitro DNA transfection activity. HPhA-mediated transfection efficiency was dependent on the mass ratio of HPhA to DNA, the incubation time and the presence of calcium. A protocol for HPhA-mediated transfection in vitro was established to improve transfection efficiency. The optimal mass ratio of HPhA to DNAwas 6:1, and the incubation time required for the DNA-HPhA complex to be in contact with the cell was 4 h. In addition, the presence of 2 mM CaCl2 in the cell culture medium was required for efficient transfection. Serum did not show inhibition of HPhA-mediated transfection. Most importantly, the cytotoxicity of HPhA is lower than that of commonly used cationic liposome-based gene delivery systems, and HPhA-mediated transfection in NIH 3T3, HEK 293, HL-7702, HepG2 and Cos 7 cell lines in vitro has a higher efficiency and reproducibility. These results demonstrate that the HPhA is a new, potentially widely applicable and highly efficient gene carrier.
Gel retardation assay, Transfection, Histone-like protein, Lipofectamine, Serum resistance
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冯雁, Liang Weng, a Yan Feng, a, * Xin Ji, a Shugui Cao, a Yoshitsugu Kosugi, b and Ikuo Matsuib
Protein Expression and Purification 33(2004)145-152,-0001,():
-1年11月30日
A histone-like gene, PHS051 from hyperthermophilic archaeon Pyrococcus horikoshii OT3 strain, was cloned, sequenced, and expressed in Escherichia coli. The recombinant histone, HPhA, encodes a protein of 70 amino acids with a molecular weight of 7868 Da. Amino acid sequence analysis of HPhA showed high homology with other archaeal histones and eukaryal core histones. The HPhA was purified to homogeneity by heat precipitation and affinity chromatography. Gel electrophoresis mobility shift assays demonstrate that the purified HPhA has high affinity to DNA. The complex of the HPhA and DNA allows DNA to be protected from cleavage by the restriction enzyme TaqI at 65℃. Circular dichroism spectra reveal that the conformation of the recombinant histone HPhA becomes looser when temperatures increase from 25 to 90℃. The HPhA has inherited a remarkable thermostability especially in the presence of 1M KCl and retained DNA binding activity at extreme temperature, which is consistent with our previous report about its structure stability analyzed by X-ray crystallography.
Histone, Archaea
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【期刊论文】A novel phospholipase A2/esterase from hyperthermophilic archaeon Aeropyrum pernix K1
冯雁, Baijing Wang, Dongmei Lu, Renjun Gao, Zhen Yang, Shugui Cao, and Yan Feng*
Protein Expression and PuriWcation 35(2004)199-205,-0001,():
-1年11月30日
An open reading frame of the hyperthermophilic archaeon Aeropyrum pernix K1 APE2325, which composed of 474 bases, was cloned and expressed in Escherichia coli BL21 (DE3) Codon Plus-RIL. The recombinant protein was puriWed by Ni-chelation aYnity chromatography. It showed a single band with a molecular mass of 18kDa in SDS–PAGE. The puriWed enzyme exhibited both phospholipase A2 and esterase activities with the optimal catalytic temperature at 90℃. The enzyme activity was Ca2+-independent. Kinetic analysis revealed its Km, kcat, and Vm for the p-nitrophenyl propionate substrate were 103μM, 39s-1, and 249μmol/min/mg, respectively. The recombinant protein was thermostable and its half-life at 100℃ was about 1h.
Aeropyrum pernix K1, Phospholipase A2, Esterase, Thermostability
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【期刊论文】Crystal Structure of an Acylpeptide Hydrolase/Esterase from Aeropyrum pernix K1
冯雁, Mark Bartlam, , Ganggang Wang, Haitao Yang, Renjun Gao, Xiaodong Zhao, Guiqiu Xie, Shuigui Cao, Yan Feng, and Zihe Rao, *
Structure, Vol. 12, 1481-1488, August, 2004,-0001,():
-1年11月30日
Acylpeptide hydrolases (APH; also known as acylam-ino acid releasing enzyme) catalyze the removal of an N-acylated amino acid from blocked peptides. The crystal structure of an APH from the thermophilic arch-aeon Aeropyrum pernix K1 to 2.1 A resolution confirms it to be a member of the prolyl oligopeptidase family of serine proteases. The structure of apAPH is a sym-metric homodimer with each subunit comprised of two domains. The N-terminal domain is a regular seven-bladed β-propeller, while the C-terminal domain has a canonical α/β hydrolase fold and includes the active site and a conserved Ser445-Asp524-His556 catalytic triad. The complex structure of apAPH with an organo- phosphorus substrate, p-nitrophenyl phosphate, has also been determined. The complex structure unam- biguously maps out the substrate binding pocket and provides a basis for substrate recognition by apAPH. A conserved mechanism for protein degradation from archaea to mammals is suggested by the structural features of apAPH.
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冯雁, Binbin Liu, *‡ Mark Bartlam, *‡ Renjun Gao, † Weihong Zhou, * Hai Pang, * Yiwei Liu, * Yan Feng, † and Zihe Rao*‡
Biophysical Journal Volume 86 January 2004 420-427,-0001,():
-1年11月30日
A homolog to the eubacteria inorganic pyrophosphatase (PPase, EC 3.6.1.1) was found in the genome of the hyperthermophilic archaeon Pyrococcus horikoshii. This inorganic pyrophosphatase (Pho-PPase) grows optimally at 88℃. To understand the structural basis for the thermostability of Pho-PPase, we have determined the crystal structure to 2.66 A resolution. The crystallographic asymmetric unit contains three monomers related by approximate threefold symmetry, and a hexamer is built up by twofold crystallographic symmetry. The main-chain fold of Pho-PPase is almost identical to that of the known crystal structure of the model from Sulfolobus acidocaldarius. A detailed comparison of the crystal structure of Pho-PPase with related structures from S. acidocaldarius, Thermus thermophilus, and Escherichia colishows significant differences that may account for the difference in their thermostabilities. A reduction in thermolabile residues, additional aromatic residues, and more intimate association between subunits all contribute to the larger thermophilicity of Pho-PPase. In particular, deletions in two loops surrounding the active site help to stabilize its conformation, while ion-pair networks unique to Pho-PPase are located in the active site and near the C-terminus. The identification of structural features that make PPases more adaptable to extreme temperature should prove helpful for future biotechnology applications.
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