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β-甘露聚糖酶末端残基变体对其酶学特性的影响 被引量：3

Effects of Terminal Variants of β-Mannanase on the Enzymatic Properties
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摘要 本研究用宇佐美曲霉Aspergillus usamii的5家族β-甘露聚糖酶AuMan5A为母本,借助同源建模、分子对接及分子动力学模拟等理性设计方法,将AuMan5A的N-末端和C-末端分别截去3个无规则的氨基酸残基,构建出截短的β-甘露聚糖酶AuMan5AN3C3.将AuMan5A和AuMan5AN3C3的编码基因分别在毕赤酵母GS115中进行表达,对表达产物进行了初步纯化并分析比较了其酶学特性及各自的表达水平.结果表明,reAuMan5A和reAuMan5AN3C3的最适温度Topt均为70℃,reAuMan5AN3C3在60℃的半衰期t1/260为38 min,较reAuMan5A(t1/260=40 min)略有降低;在相同表达条件下,reAuMan5AN3C3上清液的β-甘露聚糖酶活性为73.4 U/mL,较reAuMan5A的52.8 U/mL提高了39.0%;纯化的reAuMan5AN3C3酶比活性为182.7 U/mg protein,较reAuMan5A的126.3 U/mg protein提高了44.7%.与reAuMan5A相比,reAuMan5AN3C3对角豆胶的Km值下降不明显,Vmax值有显著提高. The β-mannanase from Aspergillus usamii （AuMan5A） belongs to the glycoside hydrolase family 5. A truncated β-mannanase AuMan5AN3C3 without 3 N-terminal and 3 C-terminal amino acid residues was designed by in silico approach using the wild type as the reference. Both AuMan5A and AuMan5AN3C3 encoding genes were cloned and expressed in Pichia pastoris GS115. The protein products were purified for the analysis for enzymatic properties. The results showed that the temperature optima of AuMan5A and AuMan5AN3C3 were 70 ℃. The half-life of reAuMan5AN3C3 at 60 ℃ （ t1/2^60） was 38 min, which was shorter slightly than that of reAuMan5A （t1/2 ^60= 40 min）. The β-mannanase activity from the expression supernatant of reAuMan5AN3C3 was 73.4 U/mL, which was about 39.0% higher than the value （52.8 U/mL） of reAuMan5A. The specific activity of purified reAuMan5AN3C3 was 182.7 U/rag protein, higher than that of reAuMan5A （126.3 U/mg protein）. A significant increase of Vmax was detected for reAuMan5AN3C3 with no significant change in Km as compared to the wild type.
作者 唐存多 李剑芳 邬敏辰 魏喜换 赵梅
机构地区 江南大学生物工程学院 江南大学食品学院 江南大学无锡医学院
出处 《中国生物化学与分子生物学报》 CAS CSCD 北大核心 2013年第7期655-660,共6页 Chinese Journal of Biochemistry and Molecular Biology
基金 国家自然科学基金项目(No.31271811) 江南大学博士研究生培育基金(No.JUDCF11011) 江苏省研究生创新工程项目(No.CXZZ11_0480)~~
关键词 Β-甘露聚糖酶 理性设计 末端残基变体 表达 酶学性质 β-mannanase in silieo design terminal variants expression enzymatic properties
分类号 Q81 [生物学—生物工程]
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参考文献21

  • 1Moreira L R, Filho E X. An overview of mannan structure and mannan-degrading enzyme systems [ J ]. Appl Microbiol Biotechnol, 2008,79 (2) : 165-178.
  • 2Li J F, Zhao S G, Tang C D et al. Cloning and functional expression of an acidophilic β-mannanase gene (AnmanSA) from Aspergillus niger LW-1 in Pichia pastoris [ J ]. J Agric Food Chem, 2012, 60 ( 3 ) :765-773.
  • 3Chauhan P S, Puri N, Sharma P, et al. Mannanases: microbial sources, production, properties and potential biotechnological applications[ J]. Appl Microbiol Biotechnol,2012,93 ( 5 ) : 1817- 1830.
  • 4van Zyl W H, Rose S H, Trollope K, et al. Fungal β- mannanases: Mannan hydrolysis, heterologous production and biotechnological applications [ J ]. Process Biochem, 2010, 45 (8) : 1203-1213.
  • 5Tang C, Guo J, Wu Met al. Cloning and bioinformatics analysis of a novel acidophilic 13-mannanase gene, AumanSA, from Aspergillus usamii YL-01-78 [ J ]. World J Microbiol Biotechnol, 2011, 27(12) :2921-2929.
  • 6Luo H, Wang Y, Wang H, et al. A novel highly acidic β- mannanase from the acidophilic fungus Bispora sp. MEY-1 : gene cloning and o~erexpression in Pichia pastoris [ J ]. Appl Microbiol Biotechnol, 2009, 82 ( 3 ) :453-461.
  • 7Wu M, Tang C, Li J, et al. Bimutation breeding of Aspergillus niger strain for enhancing β-mannanase production by solid-state fermentation [J]. Carbohydr Res, 2011, 346(14): 2149-2155.
  • 8Heck J X, de Barros Soares L H, Ayub M A Z. Optimization of xylanase and mannanase production by Bacillus circulans strain BL53 on solid-state cultivation [ J]. Enzym Microb Tech, 2005, 37(4) :417-423.
  • 9Chen X, Cao Y, Ding Y, et al. Cloning, functional expression and characterization of Aspergillus sulphureus 13-mannanase in Pichia pastoris[ J]. J Biotechnol,2007,128(3 ) :452-461.
  • 10Roth R, Moodley V, van Zyl P. Heterologous expression and optimized production of an Aspergillus aculeatus endo-1,4-6- mannanase in Yarrowia lipolytica[J]. Mol Biotechnol, 2009, 43 (2) :112-120.

二级参考文献22

  • 1傅婷婷,倪孟祥.核苷类抗艾滋病药物研发近况[J].药学进展,2007,31(5):211-216. 被引量：11
  • 2Wang J Y, Ling H, Yang W, et al. Structure of a two-domain fragment of HIV-1 integrase: implications for domain organization in the intact protein[J]. EMBO J, 2001, 20(24) : 7333-7343.
  • 3Goldgur Y, Graigie R, Cohen G H, et al. Structure of the HIV-I integrase catalytic domain complexed with an inhibitor: A platform for antiviral drug design[J]. Proc Natl Acad Sci U S A, 1999, 96(23) : 13040-13043.
  • 4Chen J C, Krucinski J, Miercke L J, et al. Crystal structure of the HIV-1 integrase catalytic core and C-terminal domains: A model for viral DNA binding[J]. Proc Natl Acad Sci U S A, 2000, 97(15): 8233-8238.
  • 5Maignan S, Guilloteau J P, Zhou L Q, et al. Crystal structures of the catalytic domain of HIV-1 integrase free and complexed with its metal cofactor: high level of similarity of the active site with other viral integrases[J] .J Mol Biol, 1998, 252(2): 359-368.
  • 6Cai M, Zheng R, Caffrey M, et al. Solution structure of the N- terminal zinc binding domain of HIV-1 integrase[J]. Nat Struct Biol, 1997, 4(7): 567-577.
  • 7Bujacz G, Alexandratos J, Wlodawer A, et al. Binding of different divalent cations to the active site of avian sarcoma virus integrase and their effects on enzymatic activity[J]. J Biol Chem, 1997, 272(29) : 18161-18168.
  • 8Steriniger M, Adams C D, Marko J F, et al. Defining characteristics of Tn5 transposase non-specific DNA binding [ J]. Nucleic Acids Res, 2006, 34(9): 2820-2832.
  • 9Nowotny M, Gaidamakov S A, Crouch R J, et al. Crystal structures of RNase H bound to an RNA/DNA hybrid: substrate specificity and metal-dependent catalysis[J]. Cell, 2005, 121(7): 1005-1016.
  • 10Renisio J G, Cosquer S, Cherrak I, et al. Pre-organized structure of viral DNA at the binding-processing site of HIV-1 integrase [ J]. Nucleic Acids Res, 2005, 33(6):1970-1981.

共引文献5

同被引文献39

  • 1许牡丹,杨伟东,许宝红,柯蕾.微生物β-甘露聚糖酶的制备与应用研究进展[J].动物医学进展,2006,27(9):31-34. 被引量：24
  • 2Lv J,Chen Y, Pei H,et al. Cloning, expression, and char- acterization of/3-mannanase from Bacillus subtilis MAFIC- S11 in Pichia pastoris [ J ]. Appl Biochem Biotechnol,2013, 169(8) :2 326 -2 340.
  • 3Chauhan P, Puri N, Sharma P, et al. Mannanases : microbial sources, production, properties and potential biotechnologi- cal applications [ J]. Appl Microbiol Biotechnol, 2012,93 (5) :1 817 - 1 830.
  • 4WANG Meng-fan, YOU Sheng-ping, ZHANG Shuai-shuai, et al. Purification, characterization, and production of/3- mannanase from Bacillus subtilis TJ-102 and its application in gluco-mannooligosaccharides preparation [ J ] . Eur Food Res Technol, 2013,237 ( 3 ) : 399 - 408.
  • 5Turner P, Mamo G, Karlsson E. Potential and utilization of thermophiles and thermostable enzymes in biorefining [ J ].Microb Cell Fact,2007,6 (1) :9.
  • 6FENG Yao-yu, HE Zhi-min, SONG LF, et al. Kinetics of fl-mannanase fermentation by Bacillus licheniformis [ J ]. Biotechnol Lett,2003,25(14) :1 143 - 1 146.
  • 7LI Jianfang, WEI Xihuan, TANG Cunduo, et al. Directed modificationof the Aspergillus usamii β-mannanase to improve its substrateaffinity by in silico design and site-directed mutagenesis[J]. Journalof Industrial Microbiology and Biotechnology, 2014, 41(4): 693-700.DOI:10.1007/s10295-014-1406-7.
  • 8LI Jianfang, ZHAO Shunge, TANG Cunduo, et al. Cloning andfunctional expression of an acidophilic β-mannanase gene (Anman5A)from Aspergillus niger LW-1 in Pichia pastoris[J]. Journal ofAgricultural and Food Chemistry, 2012, 60(3): 765-773. DOI:10.1021/jf2041565.
  • 9CHAUHAN P S, PURI N, SHARMA P, et al. Mannanases: microbialsources, production, properties and potential biotechnologicalapplications[J]. Applied Microbiology and Biotechnology, 2012,93(5): 1817-1830. DOI:10.1007/s00253-012-3887-5.
  • 10TANG Cunduo, LI Jianfang, WEI Xihuan, et al. Fusing acarbohydrate-binding module into the Aspergillus usamii β-mannanaseto improve its thermostability and cellulose-binding capacity by insilico design[J]. PLoS ONE, 2013, 8(5): e64766. DOI:10.1371/journal.pone.0064766.

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中国生物化学与分子生物学报
2013年 第7期

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