Enhancing expression level of an acidophilic β-mannanase in Pichia pastoris by double vector system

详细信息    查看全文

• 作者：Cun-Duo Tang (1)
  Jing Guo (2)
  Jian-Fang Li (3)
  Xi-Huan Wei (3)
  Die Hu (1)
  Shu-Juan Gao (2)
  Xin Yin (1)
  Min-Chen Wu (4)
  
• 关键词：Aspergillus usamii ; β ; Mannanase ; Pichia pastoris ; Double vector system ; Expression optimization
• 刊名：Annals of Microbiology
• 出版年：2014
• 出版时间：June 2014
• 年：2014
• 卷：64
• 期：2
• 页码：561-569
• 全文大小：
• 参考文献：1. Bien-Cuong D, Thi-Thu D, Berrin J-G, Haltrich D, Kim-Anh T, Sigoillot J-C, Yamabhai M (2009) Cloning, expression in / Pichia pastoris, and characterization of a thermostable GH5 mannan endo-1,4- / β- mannosidase from / Aspergillus niger BK01. Microb Cell Factories 8:59-1 CrossRef
  2. Chen XL, Cao YH, Ding YH, Lu WQ, Li DF (2007) Cloning, functional expression and characterization of / Aspergillus sulphureus β-mannanase in / Pichia pastoris. J Biotechnol 128:452-61 CrossRef
  3. Dhawan S, Kaur J (2007) Microbial mannanases: an overview of production and applications. Crit Rev Biotechnol 27:197-16 CrossRef
  4. Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350-56 CrossRef
  5. Duruksu G, Ozturk B, Biely P, Bakir U, Ogelm ZB (2009) Cloning, expression and characterization of endo- / β-1,4-mannanase from / Aspergillus fumigates in / Aspergillus sojae and / Pichia pastoris. Biotechnol Prog 25:271-76 CrossRef
  6. Katakura Y, Zhang W, Zhuang G, Omasa T, Kishimoto M (1998) Effect of methanol concentration on the production of human / β ₂-glycoproteinI domain V by a recombinant / Pichia pastoris: a simple system for the control of methanol concentration using a semiconductor gas sensor. Ferment Bioeng 86:482-87 CrossRef
  7. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-85 CrossRef
  8. Li JF, Ma LP, Wu MC, Xia WS (2006a) Purification and some properties of an acidic / β-mannanase from / Aspergillus usamii. Food Ferment Ind 32:5- (in Chinese)
  9. Li JF, Zhang JJ, Wu MC, Xia WS (2006b) Solid-state fermentation process of acidic / β-mannanase and its crude properties. Food Sci 27:143-47 (in Chinese)
  10. Li J, Tang C, Shi H, Wu M (2011) Cloning and optimized expression of a neutral endoglucanase gene ( / ncel5A) from / Volvariella volvacea WX32 in / Pichia pastoris. J Biosci Bioeng 111:537-40 CrossRef
  11. Li JF, Zhao SG, Tang CD, Wang JQ, Wu MC (2012) Cloning and functional expression of an acidophilic / β-mannanase gene ( / Anman5A) from / Aspergillus niger LW-1 in / Pichia pastoris. J Agric Food Chem 60:765-73 CrossRef
  12. Luo H, Wang Y, Wang H, Yang J, Yang Y, Huang H, Yang P, Bai Y, Shi P, Fan Y, Yao B (2009) A novel highly acidic / β-mannanase from the acidophilic fungus / Bispora sp. MEY-1: gene cloning and overexpression in / Pichia pastoris. Appl Microbiol Biotechnol 82:453-61 CrossRef
  13. Ma S, Wang H, Wang Y, Bu H, Bai J (2011) Bio-hydrogen production from cornstalk wastes by orthogonal design method. Renew Energy 36:709-13 CrossRef
  14. Roth R, Moodley V, Zyl P (2009) Heterologous expression and optimized production of an / Aspergillus aculeatus endo-1,4- / β-mannanase in / Yarrowia lipolytica. Mol Biotechnol 43:112-20 CrossRef
  15. Sambrook J, Russell DW (2001) Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 1595-604
  16. Shental-Bechor D, Levy Y (2008) Effect of glycosylation on protein folding: a close look at thermodynamic stabilization. Proc Natl Acad Sci USA 105:8256-261 CrossRef
  17. Sreekrishna K, Brankamp RG, Kropp KE, Blankenship DT (1997) Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast / Pichia pastoris. Gene 190:55-2 CrossRef
  18. Srimathi S, Jayaraman G (2005) Effect of glycosylation on the catalytic and conformational stability of homologous / α-amylases. Protein J 24:79-8 CrossRef
  19. Tan Z, Li J, Wu M, Tang C, Zhang H, Wang J (2011) High-level heterologous expression of an alkaline lipase gene from / Penicillium cyclopium PG37 in / Pichia pastoris. World J Microbiol Biotechnol 27:2767-774 CrossRef
  20. van Zyl WH, Rose SH, Trollope K, G?rgens JF (2010) Fungal / β-mannanases: mannan hydrolysis, heterologous production and biotechnological applications. Process Biochem 45:1203-213 CrossRef
  21. Wu MC, Qian ZK, Jiang PH, Min TS, Sun CR, Huang WD (2003) Cloning of an alkaline lipase gene from / Penicillium cyclopium and its expression in / Escherichia coli. Lipids 38:191-99 CrossRef
  22. Wu MC, Tang CD, Li JF, Zhang HM, Guo J (2011a) Bimutation breeding of / Aspergillus niger strain for enhancing / β-mannanase production by solid-state fermentation. Carbohydr Res 346:2149-155 CrossRef
  23. Wu MC, Wang JQ, Zhang HM, Tang CD, Gao J, Tan ZB (2011b) Cloning and sequence analysis of an acidophilic xylanase (XynI) gene from / Aspergillus usamii E001. World J Microbiol Biotechnol 27:831-39 CrossRef
  24. Zhang YL, Ju JS, Peng H, Gao F, Zhou C, Zeng Y, Xue YF, Li Y, Henrissat B, Gao GF, Ma YH (2008) Biochemical and structural characterization of the intracellular mannanase AaManA of / Alicyclobacillus acidocaldarius reveals a novel glycoside hydrolase family belonging to clan GH-A. J Biol Chem 283:31551-1558 CrossRef
  25. Zhang JH, Wu D, Chen J, Wu J (2011) Enhancing functional expression of / β-glucosidase in / Pichia pastoris by co-expressing protein disulfide isomerase. Biotechnol Bioproc Eng 16:1196-200 CrossRef
  26. Zhao JQ, Shi PJ, Luo HY, Yang PL, Zhao H, Bai YG, Huang HQ, Wang H, Yao B (2010) An acidophilic and acid-stable / β-mannanase from / Phialophora sp. P13 with high mannan hydrolysis activity under simulated gastric conditions. J Agric Food Chem 58:3184-190 CrossRef
  
• 作者单位：Cun-Duo Tang (1)
  Jing Guo (2)
  Jian-Fang Li (3)
  Xi-Huan Wei (3)
  Die Hu (1)
  Shu-Juan Gao (2)
  Xin Yin (1)
  Min-Chen Wu (4)
  
  1. School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, People’s Republic of China
  2. School of Pharmaceutical Science, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, People’s Republic of China
  3. School of Food Science and Technology, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, People’s Republic of China
  4. Wuxi Medical School, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu, 214122, People’s Republic of China
  
• ISSN：1869-2044

文摘

An acidophilic β-mannanase-encoding gene (Auman5A) from Aspergillus usamii YL-01-78 was amplified and inserted into pPIC9K and pPICZαA vectors. The resulting recombinant vector, pPIC9K-Auman5A, was transformed into Pichia pastoris GS115. One strain having the highest recombinant β-mannanase activity of 54.6?U/ml, labeled GSKM4-8, was chosen from the first-batch P. pastoris transformants. Then, the pPICZαA-Auman5A was transformed into GSKM4-8 again. From the second-batch transformants, one strain (GSKZαM4-2) with the highest β-mannanase activity of 78.1?U/ml was obtained, and used to optimize expression conditions. As GSKZαM4-2 was induced under the optimized conditions (initial pH value 6.5, induction period 120?h, methanol concentration 1.5?%, and induction temperature 32?°C), β-mannanase activity reached 162.8?U/ml. Protein and carbohydrate assays showed that the β-mannanase, a glycoprotein with an apparent molecular weight of 49.8?kDa and a carbohydrate content of 21.3?%, was extracellularly expressed. It displayed the maximum activity at pH?3.0 and 70?°C, and was stable at a pH range of 3.0-.0 and at 60?°C. Its activity was not significantly affected by metal ions tested and EDTA, but inhibited by Ag+ and Hg2+. Its most favorable substrate was locust bean gum, followed by konjac flour and guar gum. The K m and V max towards locust bean gum were 1.36?mg/ml and 415.8?U/mg, respectively. These results suggested that the β-mannanase can be expressed with higher level and possesses superior enzymatic properties, making it a good candidate in industrial processes.