Expression and characterization of alkaline β-glucosidase in Pichia pastoris from Bacillus pumilus

Authors

KAN Jinsong, Department of Biological and Environmental Engineering, Hefei University, Hefei, Anhui 230601, China
ZHANG Min, Department of Biological and Environmental Engineering, Hefei University, Hefei, Anhui 230601, China
XU Tao, Department of Biological and Environmental Engineering, Hefei University, Hefei, Anhui 230601, China

Abstract

Engineered Pichia pastoris strain secreting and expressing β-glucosidase gene of Bacillus pumilus was constructed and enzymatic properties of recombinant enzyme were investigated. Based on the codon usage of Pichia pastoris, the codon of the β-glucosidase gene of Bacillus pumilus was optimized, the whole gene sequence was designed and synthesized, and the expression vector was constructed and transferred into Pichia pastoris. The results showed that the β-glucosidase gene of Bacillus pumilus had been successfully transferred into the yeast and secreted and expressed. The activity of the fermentation broth after 72 h induction could reach 25.39 U/mL. The optimum temperature and pH for the recombinant β-glucosidase were 45 ℃ and 9.0, and the K_m and V_max of recombinant enzyme were 1.26 mmol/L and 32.15 μmol/(min·mg), respectively. The relative activity of recombinant β-glucosidase to soybean glycoside hydrolysis was 248% of that of pNPG, and the transglycosylation activity catalyzed the enzymatic synthesis of gentiobiose yield of 34.25 g/L using 50% glucose as the substrate.

Publication Date

9-28-2019

First Page

39

Last Page

44

DOI

10.13652/j.issn.1003-5788.2019.09.008

Recommended Citation

Jinsong, KAN; Min, ZHANG; and Tao, XU (2019) "Expression and characterization of alkaline β-glucosidase in Pichia pastoris from Bacillus pumilus," Food and Machinery: Vol. 35: Iss. 9, Article 8.
DOI: 10.13652/j.issn.1003-5788.2019.09.008
Available at: https://www.ifoodmm.cn/journal/vol35/iss9/8

References

[1] 郑芳芳, 王金佩, 林宇, 等. 链霉菌GXT6 β-葡萄糖苷酶的酶学性质及葡萄糖耐受性分子改造[J]. 微生物学报, 2018, 58(10): 1 839-1 852.
[2] SINGH G, VERMA A K, KUMAR V. Catalytic properties, functional attributes and industrial applications of β-glucosidases[J]. 3 Biotech, 2016, 6(1): 3.
[3] AHMED A, NASIM FU H, BATOOL K, et al. Microbial β-Glucosidase: Sources, production and applications[J]. Journal of Applied & Environmental Microbiology, 2017, 5(1): 31-46.
[4] POCAN P, BAHCEGUL E, OZTOP M H, et al. Enzymatic hydrolysis of fruit peels and other lignocellulosic biomass as a source of sugar[J]. Waste Biomass Valorization, 2018, 9(6): 929-937.
[5] HU Sheng-lin, WANG Dong-mei, HONG Jiong. A simple method for beta-glucosidase immobilization and its application in soybean isoflavone glycosides hydrolysis[J]. Biotechnology and Bioprocess Engineering, 2018, 23(1): 39-48.
[6] VERVOORT Y, HERRERA-MALAVER B, MERTENS S, et al. Characterization of the recombinant Brettanomyces anomalus β-glucosidase and its potential for bioflavouring[J]. Journal of Applied Microbiology, 2016, 121(3): 721-733.
[7] BOUDABBOUS M, BEN HMAD I, SAIBI W, et al. Trans-glycosylation capacity of a highly glycosylated multi-specific β-glucosidase from Fusarium solani[J]. Bioprocess Biosystems Engineering, 2017, 40(4): 559-571.
[8] DA SILVA A S, MOLINA J F, TEIXEIRA R S S, et al. Synthesis of disaccharides using β-glucosidases from Aspergillus niger, A. awamori and Prunus dulcis[J]. Biotechnology Letters, 2017, 39(11): 1 717-1 723.
[9] ZHAO Xue-song, GAO Ling, WANG Juan, et al. A novel ginsenoside Rb1-hydrolyzing β-D-glucosidase from Cladosporium fulvum[J]. Process Biochem, 2009, 44(6): 612-618.
[10] QIN Yong-ling, ZHANG Yun-kai, HE Hai-yan, et al. Screening and identification of a fungal β-glucosidase and the enzymatic synthesis of gentiooligosaccharide[J]. Appl Biochem Biotechnol, 2011, 163(8): 1 012-1 019.
[11] GNDZ ERGN B, ALIK P J B, ENGINEERING B. Lignocellulose degrading extremozymes produced by Pichia pastoris: Current status and future prospects[J]. Bioprocess Biosyst Eng, 2016, 39(1): 1-36.
[12] TANG Zi-zhong, LIU Shan, JING Hai-jun, et al. Cloning and expression of A. oryzae β-glucosidase in Pichia pastoris[J]. Mol Biol Rep, 2014, 41(11): 7 567-7 573.
[13] LIU Dong-yang, ZHANG Rui-fu, YANG Xing-ming, et al. Characterization of a thermostable β-glucosidase from Aspergillus fumigatus Z5, and its functional expression in Pichia pastoris X33[J]. Microbial Cell Factories, 2012, 11(1): 25.
[14] BOONVITTHYA N, TANAPONG P, KANNGAN P, et al. Cloning and expression of the Aspergillus oryzae glucan 1,3-beta-glucosidase A (exgA) in Pichia pastoris[J]. Biotechnology Letters, 2012, 34(10): 1 937-1 943.
[15] 易晓男, 任清华, 程炜, 等. 高产β-葡萄糖苷酶工程菌株的构建及其在2,6-二甲氧基对苯醌发酵制备中的应用[J]. 食品科学, 2017, 38(8): 69-73.
[16] ZHANG Cong, WANG Xi-feng, ZHANG Wei-can, et al. Expression and characterization of a glucose-tolerant β-1,4-glucosidase with wide substrate specificity from Cytophaga hutchinsonii[J]. Appl Microbiol Biotechnol, 2017, 101(5): 1 919-1 926.
[17] AHMED A, ASLAM M, ASHRAF M, et al. Microbial β-glucosidases: Screening, characterization, cloning and applications[J]. Journal of Applied & Environmental Microbiology, 2017, 5(2): 57-73.
[18] 江民华, 林厚民, 尹金阳, 等. 差异柠檬酸杆菌GXW-1 β-葡萄糖苷酶的酶学性质及分子改造[J]. 微生物学报, 2017, 57(3): 363-374.
[19] 刘群, 管政兵, 蔡宇杰, 等. Bacillus altitudinis SYBC hb4 碱性β-葡萄糖苷酶基因的克隆表达及酶学性质的研究[J].食品与生物技术学报, 2017, 36(11): 1 203-1 209.