In this study, barley β-amylase was heterologously expressed in Bacillus subtilis. The expression vector pP43NMK-amyB was constructed and transformed into B. subtilis WB800 and a recombinant B. subtilis WB-amyB was obtained. A maximum yield of 386 U/mL β-amylase was obtained in shaking flask fermentation. The purified enzyme had the specific activity of 613 U/mg. Its optimal reaction temperature and pH were 55°C and 5.0, respectively. The hydrolysis ability of recombinant β-amylase towards starch was similar to that of barley β-amylase. When it was combined with pullulanase, the maximum conversion rate of starch to maltose reached 81.8%, therefore, the recombinant barley β-amylase can completely replace barley β-amylase in industrial application.
WANG Xueliang
,
NIU Chengtuo
,
BAO Min
,
LI Qi
,
WANG Jinjing
. Heterologous expression of barley β-amylase in Bacillus subtilis[J]. Food and Fermentation Industries, 2019
, 45(14)
: 34
-40
.
DOI: 10.13995/j.cnki.11-1802/ts.019801
[1] 于轩. 不同来源淀粉的分子结构对其酶解性能影响的研究[D]. 无锡:江南大学, 2013.
[2] NIU C, ZHENG F, LI Y, et al. Process optimization of the extraction condition of β-amylase from brewer's malt and its application in the maltose syrup production [J]. Biotechnology & Applied Biochemistry, 2018,65(4):639-647.
[3] AIYER P V. Amylases and their applications [J]. African Journal of Biotechnology, 2013, 4(13): 1 525-1 529.
[4] 张剑, 林庭龙,秦瑛,等. β-淀粉酶研究进展[J]. 中国酿造, 2009, 28(4): 5-8.
[5] LAHMAR I, RADEVA G, MARINKOVA D, et al. Valorization of a plant β-amylase: Immobilization and dataset on the kinetic process [J]. Data in Brief, 2018, 16(C): 386-391.
[6] KANEKO T, KIHARA M, ITO K. Genetic analysis of β-amylase thermostability to develop a DNA marker for malt fermentability improvement in barley, Hordeum vulgare [J]. Plant Breeding, 2000, 119(3): 197-201.
[7] 韦玉琴. β-淀粉酶小罐发酵及酶学性质分析[D]. 南宁:广西大学, 2011.
[8] 张萧萧, 牛丹丹,沈微,等. β-淀粉酶的表达与酶学性质[J]. 食品科学, 2016, 37(3): 164-169.
[9] 许黎明, 成春燕,韦星明,等. 大豆β-淀粉酶基因在毕赤酵母中的高密度发酵表达[J]. 食品与发酵工业, 2017, 43(9):28-33.
[10] 朱培, 李崎,朱林江,等. 大麦β-淀粉酶基因在大肠杆菌中的异源表达[J]. 食品与发酵工业, 2016, 42(6): 31-35.
[11] YOSHIGI N, OKADA Y, MAEBA H, et al. Construction of a plasmid used for the expression of a sevenfold-mutant barley β-amylase with increased thermostability in Escherichia coli and properties of the sevenfold-mutant beta-amylase [J]. Journal of Biochemistry, 1995, 118(3): 562-567.
[12] 韩登兰, 王腾飞,汪俊卿,等. 一株芽孢表面稳定展示海藻糖合酶工程菌的构建[J]. 中国酿造, 2017, 36(3): 138-143.
[13] 赵婷, 姚粟,葛媛媛,等. 美国食品工业用菌种法规标准简介[J]. 食品与发酵工业, 2014, 40(4): 108-113.
[14] ZHANG X Z, CUI Z L, HONG Q, et al. High-level expression and secretion of methyl parathion hydrolase in Bacillus subtilis WB800 [J]. Applied & Environmental Microbiology, 2005, 71(7): 4 101-4 103.
[15] IRWIN C R, FARMER A, WILLER D O, et al. In-fusion® cloning with vaccinia virus DNA polymerase [J]. Methods in Molecular Biology, 2012, 890: 23-25.
[16] NIU C, ZHU L, XIN X, et al. Rational design of disulfide bonds increases thermostability of a mesophilic 1,3-1,4-β-glucanase from Bacillus terquilensis [J]. Plos One, 2016, 11(4): e0154036.
[17] LI X, YU H Y. Extracellular production of beta-amylase by a halophilic isolate, Halobacillus sp. LY9 [J]. Journal of Industrial Microbiology & Biotechnology, 2011, 38(11): 1 837-1 843.
[18] YOSHIGI N, OKADA Y, SAHARA H, et al. Expression in Escherichia coli of cDNA encoding barley beta-amylase and properties of recombinant beta-amylase [J]. Bioscience, Biotechnology, and Biochemistry, 1994, 58(6): 1 080-1 086.
[19] 张广操. 耐热大豆β-淀粉酶的筛选及其cDNA在大肠杆菌中的表达[D]. 北京:中国农业科学院, 2005.
[20] KOIDE T, OHNISHI Y, HORINOUCHI S. Characterization of recombinant beta-amylases from Oryza sativa [J]. Bioscience biotechnology and biochemistry, 2011, 75(4): 793-796.
[21] 亓旭辉.Bacillus flexus β-淀粉酶在短小芽孢杆菌中的重组表达、分子改造及应用研究[D]. 无锡:江南大学, 2017.
[22] 李猛, 陈利飞,杨建楼,等. 产β-淀粉酶菌株的筛选及β-淀粉酶基因在大肠杆菌中的克隆与表达[J]. 生物技术通报, 2014(12): 161-167.
[23] LI S, SING S, WANG Z. Improved expression of Rhizopus oryzae α-amylase in the methylotrophic yeast Pichia pastoris [J]. Protein Expression & Purification, 2011, 79(1): 142-148.
[24] 沈卫锋, 牛宝龙,翁宏飚,等. 枯草芽孢杆菌作为外源基因表达系统的研究进展[J]. 浙江农业学报, 2005, 17(4): 234-238.
[25] MA Y F, EVANS D E, LOGUE S J, et al. Mutations of barley β-amylase that improve substrate-binding affinity and thermostability [J]. Molecular Genetics and Genomics, 2001, 266(3): 345-352.
[26] MA Y F, EGLINTON J K, EVANS D E, et al. Removal of the four C-terminal glycine-rich repeats enhances the thermostability and substrate binding affinity of barley β-amylase [J]. Biochemistry, 2000, 39(44): 13 350-13 355.
