Targeted screening, recombinant expression and enzymatic properties of β-glucosidase Bgl747
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    Abstract:

    [Background] β-glucosidase (EC3.2.1.21) is one of the important components of the three cellulase enzymes. At present, most of the industrial cellulase are derived from fungi such as Trichoderma, and less derived from bacteria, and there are still problems in application such as narrow application range of reaction conditions (such as temperature, pH), low enzyme activity, and high acquisition cost. These greatly limit the application of β-glucosidase. Screening β-glucosidase from soil bacteria has a great possibility to screen out enzymes with better enzymatic properties, thus solving existing industrial problems. [Objective] Use functional screening method to screen β-glucosidase from soil, obtain a new type of β-glucosidase through gene recombination, expression optimization and protein purification, explore its enzymatic properties, and its industrial application lay the foundation. [Methods] The β-glucosidase was screened from the soil by the functional screening method. Because its full length is 747 bp, it was named Bgl747, and the recombinant expression plasmid pET-28a-Bgl747 was constructed with Escherichia coli BL21(DE3). Induced by IPTG to achieve soluble expression and optimize expression conditions, the purified enzyme was purified by His-tag protein purification kit, and its enzymatic properties were explored. [Results] β-glucosidase Bgl747 as part of the BglB superfamily, its molecular weight is 27.23 kD, and an optimal pH of 4.0, an optimal reaction temperature of 45℃; The optimal induction conditions are:when OD600 1.0, add the final concentration IPTG 0.6 mmol/L, the highest expression level of β-glucosidase Bgl747 protein was 1.82 mg/mL after being induced at 37℃ and 220 r/min for 10 h. The specific enzyme activity when the substrate is p-nitrophenyl-β-D- galactopyranoside (pNPG) is 225.07 U/mg, the Michaelis constant Km value and the maximum reaction rate are respectively 0.268 mmol/L, 547.23 μmol/(L·min); 1 mmol/L K+, 1 mmol/L and 10 mmol/L Fe2+, 30% methanol, 30% ethanol, 1 mmol/L and 10 mmol/L guanidine hydrochloride all have a promoting effect on enzyme activity, 30% TritonX-100 and 10 mmol/L SDS inhibits enzyme activity more obviously; The enzyme is feedback inhibition by the product glucose, the greater the glucose concentration, the more obvious the inhibitory effect, but when the glucose concentration is 1 mol/L, the enzyme activity remains above 50%. [Conclusion] Bgl747 has a wide and stable reaction temperature range and excellent enzymatic properties, and lays the foundation for its industrial applications such as cellulose degradation.

    Reference
    [1] Cairns JRK, Esen A. β-glucosidases[J]. Cellular and Molecular Life Sciences, 2010, 67(20):3389-3405
    [2] Ibrahim E, Jones KD, Taylor KE, Hosseney EN, Mills PL, Escudero JM. Recombinant E. coli cellulases, β-glucosidase, and polygalacturonase convert a Citrus processing waste into biofuel precursors[J]. ACS Sustainable Chemistry & Engineering, 2018, 6(6):7304-7312
    [3] Agrawal R, Verma AK, Satlewal A. Application of nanoparticle-immobilized thermostable β-glucosidase for improving the sugarcane juice properties[J]. Innovative Food Science & Emerging Technologies, 2016, 33:472-482
    [4] Kumar P, Ryan B, Henehan GTM. β-glucosidase from Streptomyces griseus:nanoparticle immobilisation and application to alkyl glucoside synthesis[J]. Protein Expression and Purification, 2017, 132:164-170
    [5] Xiang DJ, Man LL, Zhang CF, Zhang CL, Zhang D, Yin KD, Cao TX, Wang LN. Screening of a corn straw cellulose-degrading strains and optimization of the cellulase producing condition[J]. Aem Roducts Rocessing, 2014(4):37-39,43(in Chinese)向殿军, 满丽莉, 张春凤, 张彩玲, 张娣, 殷奎德, 曹天旭, 王丽娜. 玉米秸秆纤维素降解菌的分离纯化与产酶工艺优化[J]. 农产品加工·学刊:中, 2014(4):37-39,43
    [6] Zhang JF. Breeding of straw degradation engineering bacteria and development of quick-rot fungi in Northeast China[D]. Jilin:Doctoral Dissertation of Jilin Agricultural University, 2012(in Chinese)张建峰. 东北地区秸秆降解工程菌的选育及速腐菌剂的研制[D]. 吉林:吉林农业大学博士学位论文, 2012
    [7] Xia YL. Screening and preliminary characterization of a novel β-glucosidase gene[D]. Guangdong:Master's Thesis of Guangdong Pharmaceutical University, 2015(in Chinese)夏玉林. β-葡萄糖苷酶基因的筛选及性质分析[D]. 广东:广东药科大学硕士学位论文, 2015
    [8] Zheng FF, Wang JP, Lin Y, Wang ZL, Wei YT, Huang RB, Du LQ. Characterization of β-glucosidase from Streptomyces sp. and its molecular modification of glucose tolerance[J]. Acta Microbiologica Sinica, 2018, 58(10):1839-1852(in Chinese)郑芳芳, 王金佩, 林宇, 王子龙, 韦宇拓, 黄日波, 杜丽琴. 链霉菌GXT6β-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):1839-1852
    [9] Tang LL, Wang XM, Wu XL, Huang Q, Li H. Screening, cloning and characterization of a novel β-glucosidase from soil metagenomic library[J]. Food Science, 2018, 39(4):118-124(in Chinese)唐乐丽, 王晓萌, 吴秀玲, 黄庆, 李荷. 宏基因组文库新型β-葡萄糖苷酶的筛选、克隆及酶学性质分析[J]. 食品科学, 2018, 39(4):118-124
    [10] Liu Y, Zhang C, Peng H, Dong YN, Zhao WP. Recombinant expression and characterization of β-glucosidase from Thermoanaerobacterium thermosaccharolyticum[J]. Chinese Journal of Applied and Environmental Biology, 2020:1-9(in Chinese)刘洋, 张汆, 彭惠, 董艺凝, 赵维萍. 热解糖高温厌氧杆菌β-[J]. 应用与环境生物学报, 2020:1-9
    [11] Liu Y, Peng H, Zhang C, Dong YN, Sun X, Luo X, Cai H, Zhao WP. Recombinant expression and characterization of β-glucosidase from Bacillus thermoamylovorans[J]. Microbiology China, 2020, 47(7):2050-2059(in Chinese)刘洋, 彭惠, 张汆, 董艺凝, 孙星, 罗侠, 蔡华, 赵维萍. 嗜热淀粉芽孢杆菌来源β-葡萄糖苷酶的重组表达与酶学性质[J]. 微生物学通报, 2020, 47(7):2050-2059
    [12] Mai ZM, Yang J, Tian XP, Li J, Zhang S. Gene cloning and characterization of a novel salt-tolerant and glucose-enhanced β-glucosidase from a marine Streptomycete[J]. Applied Biochemistry and Biotechnology, 2013, 169(5):1512-1522
    [13] Li G, Jiang Y, Fan XJ, Liu YH. Molecular cloning and characterization of a novel β-glucosidase with high hydrolyzing ability for soybean isoflavone glycosides and glucose-tolerance from soil metagenomic library[J]. Bioresource Technology, 2012, 123:15-22
    [14] Hong MR, Kim YS, Park CS, Lee JK, Kim YS, Oh DK. Characterization of a recombinant beta-glucosidase from the thermophilic bacterium Caldicellulosiruptor saccharolyticus[J]. Journal of Bioscience and Bioengineering, 2009, 108(1):36-40
    [15] Xu H, Xiong AS, Zhao W, Tian YS, Peng RH, Chen JM, Yao QH. Characterization of a glucose-, xylose-, sucrose-, and D-galactose-stimulated β-glucosidase from the alkalophilic bacterium Bacillus halodurans C-125[J]. Current Microbiology, 2011, 62(3):833-839
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DAI Shuang, LI He. Targeted screening, recombinant expression and enzymatic properties of β-glucosidase Bgl747[J]. Microbiology China, 2021, 48(8): 2524-2533

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History
  • Received:November 23,2020
  • Adopted:March 11,2021
  • Online: July 30,2021
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