以多孔陶瓷球为载体固定化β-葡萄糖苷酶,对固定化条件进行优化,确定陶瓷球固载β-葡萄糖苷酶的条件为:5 g陶瓷球载体与3 mL酶液,在4 ℃摇床中振荡交联反应2 h,低温干燥后,用含有0.3 g油性胶黏剂的溶液对固定化酶进行包封,酶液中β-葡萄糖苷酶浓度为36.5 μmol/(mL·min),缓冲液pH为4.8,戊二醛体积分数0.5%。陶瓷球固定化β-葡萄糖苷酶的最适反应pH为4.8,游离酶pH为4.2,固定化和游离酶的最适反应温度均为60 ℃; 固定化酶的酸碱、热和储存稳定性优于游离酶; 固定化和游离酶的反应动力学常数Km分别为7.9 mmol/L和0.73 mmol/L; 利用固定化酶重复分批酶解纤维二糖-葡萄糖混合糖液,前5批的酶解得率在80%以上。
Abstract
With the objective of achieving practicable β-glucosidase immobilization, porous ceramic balls were taken as the carriers, and the conditions on β-glucosidase immobilization were studied. The optimum operation of β-glucosidase immobilization was as follows:5 g porous ceramic balls were put into 0.3 mL β-glucosidase solution(pH 4.8), in which the concentrations of β-glucosidase and glutaraldehyde were 36.5 μmol/(mL·min)and 0.5%, respectively. The mixture was shaken at 4 ℃ for 2 h and then dried under low temperature. The immobilized β-glucosidase was obtained after enveloped by a solution which contained 0.3 g oil-based adhesive. The optimum reaction pH of immobilized β-glucosidase was 4.8, whereas the reaction pH of free β-glucosidase was 4.2. The optimum reaction temperatures of immobilized and free β-glucosidase were 60 ℃. Immobilized β-glucosidase offered the better properties than free β-glucosidase in pH, thermal and storage tests. The Km of immobilized and free β-glucosidase were 7.9 mmol/L and 0.73 mmol/L, respectively. A sugar mixture containing cellobiose and glucose was enzymatically hydrolyzed by the immobilized β-glucosidase. The yields of cellobiose conversion could be higher than 80% in 5 repeated batch operations.
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参考文献
[1] Tangnu S K, Blanch H W, Charles R. Enhanced production of cellulase, hemicellulase and β-glucosidase by Trichoderma reesei (Rut C-30)[J]. Biotechnol Bioeng,1981,23:1837-1849.
[2] Knutsen J S, Davis R H. Cellulase retention and sugar removal by membrane ultrafiltration during lignocellulosic biomass hydrolysis[J]. Appl Biochem Biotechnol, 2004, 116:585-599.
[3] Gan Q, Allen S J, Taylor G. Design and operation of an integrated membrane reactor for enzymatic cellulose hydrolysis[J]. Biochem Eng J, 2002, 12:223-229.
[4] Srivastava S K, Gopalkrishnan K S, Ramachandran K B. Kinetic characterization of a crude β-D-glucosidase from Aspergillus wentii Pt 2804[J]. Enzyme and Microbial Technology, 1984, 6(11):508-512.
[5] Wen Z Y, Liao W, Chen S. Production of cellulose/β-glucosidase by the mixed fungi culture and on dairy manure[J]. Process Biochemistry, 2005, 40(9):3087-3094.
[6] Vishnu M, Gyan P, Asmita P, et al. Biocatalytic approach for the utilization of hemicellulose for ethanol production from agricultural residue using thermostable xylanase and thermotolerant yeast[J]. Bioresource Technology, 2010(101):5366-5373.
[7] Shin H J, Yang J W. Galactooligosaccharide synthesis from lactose by Penicillium-Funiculosum cellulase[J]. Biotechnology Letters, 1996, 18(2):143-144.
[8] 彭志英. 食品生物技术[M]. 北京:中国轻工业出版社, 1999.
[9] Ghose T K, Measurement of cellulase activities[J]. Pure Appl Chem, 1987, 59:257-268.
[10] 朱均均. β-葡萄糖苷酶的固定化及纤维素辅助水解技术[D]. 南京:南京林业大学, 2006. Zhu J J. Immobilization of β-glucosidase and its improvement in cellulose enzymatic hydrolysis[D]. Nanjing: Nanjing Forestry University, 2006.
[11] 张锐,陈振宁,方桂珍,等. 羧甲基纤维素-壳聚糖聚电解质复合物对乳糖酶的固定化研究[J]. 林产化学与工业,2009,29:138-148. Zhang R, Chen Z N, Fang G Z, et al. Immobilization of β-galactosidase on carboxymethyl celulose-chitosan polyelectrolyte complex[J]. Chemistry and Industry of Forest Products, 2009, 29:138-148.
[12] 谢宁昌. 生物化学实验多媒体教程[M]. 上海:华东理工大学出版社,2006.
[13] 杨静. 木质纤维原料分段酶水解技术的研究[D]. 南京:南京林业大学,2010. Yang J. Multi-stage enzymatic saccharification of lignocellulosic materials[D]. Nanjing: Nanjing Forestry University, 2010.
[14] 梁华正, 杨水平, 丁志刚, 等. 弱碱性大孔树脂固定化硫磺菌β-葡萄糖苷酶的实验研究[J]. 食品科技, 2008(2):8-11. Liang H Z, Yang S P, Ding Z G, et al. Experimental s tudy on β-glucosidase in Laetiporus sulphureus immobilized with weakly basic macroporous resin[J]. Food Science and Technology, 2008(2):8-11.
[15] 孙君社. 酶与酶工程及其应用[M]. 北京:化学工业出版社, 2006.
[16] 沈雪亮, 夏黎明. 固定化纤维二糖酶在纤维原料水解中的应用[J]. 浙江大学学报:工学版, 2005, 39(2):287-291. Shen X L, Xia L M. Application of immobilized cellobiase in cellulosic material hydrolysis[J]. Journal of Zhejiang University:Engineering Science, 2005, 39(2):287-291.
基金
收稿日期:2012-09-25 修回日期:2013-01-04
基金项目:国家林业公益性行业科研专项项目(201004001); 江苏高校优势学科建设工程资助项目(PAPD); 江苏高校科技创新团队资助项目
第一作者:韦策,工程师,博士生。*通信作者:余世袁,教授。E-mail: syu@njfu.edu.cn。
引文格式:韦策,陆青山,勇强,等. 多孔陶瓷球固定化β-葡萄糖苷酶的研究[J]. 南京林业大学学报:自然科学版,2013,37(3):110-116.
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