β-葡萄糖苷酶的异源表达及与纤维素酶协同酶解竹纤维
收稿日期: 2013-11-06
修回日期: 2013-11-30
网络出版日期: 2013-12-27
基金资助
国家重点基础研究发展计划(2010CB732201);国家自然科学基金(31170067);中央高校基本科研业务费专项资金(201112G026)
Heterologous Expression of the β-Glucosidase and Its Synergistic Hydrolysis of Bamboo with Cellulase
Received date: 2013-11-06
Revised date: 2013-11-30
Online published: 2013-12-27
在毕赤酵母GS115中表达东方肉座菌EU7-22的β-葡萄糖苷酶基因(bgl?),获得基因工程菌株BP17。优化BP17发酵产酶条件后,重组β-葡萄糖苷酶活力达121 IU/mL。酶学性质研究表明,该酶最适反应温度为70℃,在60℃以下有较好的热稳定性;最适催化pH为5.0,在pH 3.0 ~ 8.0之间有较好的稳定性。将异源表达的β-葡萄糖苷酶添加到东方肉座菌的纤维素酶液中协同降解经过预处理的竹纤维,当纤维素酶添加量为FPA 20 IU/g底物,β-葡萄糖苷酶添加量为BG 6 IU/g底物时,纤维二糖浓度显著下降,酶解得率达到83.03%,表明重组β-葡萄糖苷酶的加入更有利于纤维素的酶解糖化。
成奕瑾 , 张婷 , 黎海龙 , 龙传南 , 刘健 , 龙敏南 . β-葡萄糖苷酶的异源表达及与纤维素酶协同酶解竹纤维[J]. 新能源进展, 2013 , 1(3) : 230 -235 . DOI: 10.3969/j.issn.2095-560X.2013.03.005
The β-glucosidase gene (bglI) from Hypocrea orientalis EU7-22 was cloned and effectively expressed in Pichia pastoris GS115. The ß-glucosidase activity expressed by recombinant strain BP17 reached 121 IU/mL. The expressed ß -glucosidase exhibited the optimum catalytic activity at 70°C and pH 5.0. The enzyme exhibited good stability at pH 3.0 ~ 8.0 and remained 65% of its original activity after 1 h at 60°C. The pretreated bamboo cellulose was synergistic hydrolyzed by the cellulases from Hypocrea orientalis EU7-22 and the recombinant β-glucosidase from strain BP17. Supplementing recombinant β-glucosidase greatly reduced the inhibitory effect caused by cellobiose, and the hydrolysis yield was improved to 83.03% with enhanced β-glucosidase activity of 6 IU/g substrate. The results indicated the recombinant β-glucosidase significantly boost the efficiency of saccharification.
[1] Lynd L R, Weimer P J, Van Zyl W H, et al. Microbial cellulose utilization: fundamentals and biotechnology[J]. Microbiology and Molecular Biology Reviews, 2002, 66(3): 506-577.
[2] Bhat M K, Bhat S. Cellulose degrading enzymes and their potential industrial applications[J]. Biotechnology Advances, 1997, (15): 583-620.
[3] Gruno M, Väljamäe P, Pettersson G, et al. Inhibition of the Trichoderma reesei cellulases by cellobiose is strongly dependent on the nature of the substrate[J]. Biotechnology and Bioengineering, 2004, 86: 503-511.
[4] Holtzapple M, Cognata M, Shu Y. Inhibition of Trichoderma reesei cellulase by sugars and solvents[J]. Biotechnology and Bioengineering, 1990, (36): 275-287.
[5] Andri? P, Meyer A S, A Jensen P, et al. Reactor design for minimizing product inhibition during enzymatic lignocelluloses hydrolysis: I. Significance and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes[J]. Biotechnology Advances, 2010, (28): 308-324.
[6] 龙传南, 成奕瑾, 邬小兵, 等. 东方肉座菌EU7-22纤维素酶基因的克隆及序列分析[J]. 生物技术通报, 2012, (11): 110-117.
[7] Wyk van JPH, Mohulatsi M. Biodegradation of waste paper by cellulase from Trichoderma viride[J]. Bioresource Technology, 2003, 86(1): 21-23.
[8] 姚秀清, 张全, 杨翔华, 等. 碱性双氧水法预处理木质纤维素[J]. 化学与生物工程, 2009, 26(3): 34-37.
[9] Chen P, Fu X Y, Tzi B N, et al. Expression of a secretory β-glucosidase from Trichoderma reesei in Pichia pastoris and its characterization[J]. Biotechnology Letters, 2011, 33: 2475-2479.
[10] Tiwari P, Misra B N, Sangwan N S. β-glucosidase from the fungus Trichoderma: An efficient cellulase machinery in biotechnological applications[J]. BioMed Research International, 2013, 2013: 1-10.
[11] Romanos M A, Scorer C A, Clare J J. Foreign gene expressing in yeast: A review[J]. Yeast, 1992, 8: 423-488.
[12] Yang B, Willies D M, Wyman C E. Changes in the enzymatic hydrolysis rate of avicel cellulose with conversion[J]. Biotechnology and Bioengineering, 2006, 94(6): 1122-1128.
[13] Kawai R, Yoshida M, Tani T, et al. Production and characterization of recombinant Phanerochaete chrysosporium β-glucosidase in the methylotrophic yeast Pichia pastoris[J]. Bioscience, Biotechnology and Biochemistry, 2003, 67(1): 1-7.
[14] Hong J, Tamaki H, Kumagai H, et al. Cloning and functional expression of thermostable β-glucosidase gene from Thermoascus aurantiacus[J]. Applied Microbiology and Biotechnology, 2007, 73: 1331-1339.
[15] Chen M, Xia L M, Xue P J. Enzymatic hydrolysis of corncob and ethanol production from cellulosic hydrolysate[J]. International Biodeterioration & Biodegradation, 2007, 59: 85-89.
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