Abstract
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 3.0–7.0 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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
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 8:59–71
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 128:452–461
Dhawan S, Kaur J (2007) Microbial mannanases: an overview of production and applications. Crit Rev Biotechnol 27:197–216
Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356
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 25:271–276
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 86:482–487
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Li JF, Ma LP, Wu MC, Xia WS (2006a) Purification and some properties of an acidic β-mannanase from Aspergillus usamii. Food Ferment Ind 32:5–9 (in Chinese)
Li JF, Zhang JJ, Wu MC, Xia WS (2006b) Solid-state fermentation process of acidic β-mannanase and its crude properties. Food Sci 27:143–147 (in Chinese)
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 111:537–540
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 60:765–773
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 82:453–461
Ma S, Wang H, Wang Y, Bu H, Bai J (2011) Bio-hydrogen production from cornstalk wastes by orthogonal design method. Renew Energy 36:709–713
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 43:112–120
Sambrook J, Russell DW (2001) Molecular Cloning: A Laboratory Manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, pp 1595–1604
Shental-Bechor D, Levy Y (2008) Effect of glycosylation on protein folding: a close look at thermodynamic stabilization. Proc Natl Acad Sci USA 105:8256–8261
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 190:55–62
Srimathi S, Jayaraman G (2005) Effect of glycosylation on the catalytic and conformational stability of homologous α-amylases. Protein J 24:79–88
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 27:2767–2774
van Zyl WH, Rose SH, Trollope K, Görgens JF (2010) Fungal β-mannanases: mannan hydrolysis, heterologous production and biotechnological applications. Process Biochem 45:1203–1213
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 38:191–199
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 346:2149–2155
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 27:831–839
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 283:31551–31558
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 16:1196–1200
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 58:3184–3190
Acknowledgments
This work was financially supported by the National Nature Science Foundation of China (No. 31271811), Doctoral Research Funds of Jiangnan University (No. JUDCF11011) and Postgraduate Innovation Training Project of Jiangsu (No. CXZZ11_0480). We are grateful to Prof. Xianzhang Wu (School of Biotechnology, Jiangnan University) for providing technical assistance.
Author information
Authors and Affiliations
Corresponding author
Additional information
C.D. Tang and J. Guo, the two first authors, contributed equally to this work.
Rights and permissions
About this article
Cite this article
Tang, CD., Guo, J., Li, JF. et al. Enhancing expression level of an acidophilic β-mannanase in Pichia pastoris by double vector system. Ann Microbiol 64, 561–569 (2014). https://doi.org/10.1007/s13213-013-0689-7
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13213-013-0689-7