氨基化细菌纤维素载体的制备及其固定β-半乳糖苷酶

程浩， 陈勇， 应汉杰

(南京工业大学生物与制药工程学院， 江苏南京211800)

摘要：利用二氯亚砜和乙二胺对细菌纤维素薄膜进行氯化和氨基化制备氨基化细菌纤维素薄膜，并以该薄膜作为载体固定β-半乳糖苷酶，研究载体的结构性质和固定化酶的制备条件及相关酶学性质。通过元素分析、红外光谱和X线光电子能谱等分析方法来表征载体性质。结果显示，有大量氨基接入细菌纤维素表面。最佳的固定化酶的条件：戊二醛添加量4 g/L，固定化时间3 h，酶添加量3 mg/mL，pH7.0和交联时间90 min，此条件下，最高酶活回收率为78.4%，吸附酶量为63.1 mg/g。与游离酶相比，固定化酶的最适温度为40 ℃，比游离酶高10 ℃，最适pH提高0.5，有向碱性方向移动的趋势，重复使用7次后剩余77.8%的相对酶活力。

关键词：氨基化; 细菌纤维素; 固定化;β-半乳糖苷酶

doi:10.3969/j.issn.1672-3678.2015.06.007

收稿日期：2014-11-06

基金项目：国家杰出青年基金(21025625);国家重点基础研究发展计划(973计划)(2013CB733602);国家高技术研究发展计划(863计划)(2012AA021203);国家科技支撑计划(2012BAI44G01);国家自然科学基金面上基金(21376118);国家自然科学基金青年基金(21106070)；长江学者和创新发展计划(PCSIRT)；江苏省自然科学基金(SBK20150207)

作者简介：程浩(1988—)，男，河南南阳人，硕士研究生，研究方向：生物化工;应汉杰(联系人)，教授，E-mail:yinghanjie@njtech.edu.cn

中图分类号：Q814

文献标志码：A

文章编号：1672-3678(2015)06-0036-07

Abstract：Sulfoxide chloride and ethylenediamine were used to conduct the chlorination and amination of bacterial cellulose for the immobilization of β-galactosidase. Immobilization conditions, the enzymatic properties and microstructure of immobilized β-galactosidase were studied. Elemental analysis,infrared spectrum and X-ray photoelectron spectroscopy confirmed the presence of abundant amino groups on the surface of bacterial cellulose. Results showed that the maximum recovery of enzyme activity and the amount of immobilized β-galactosidase were 78.4% and 63.1 mg/g,respectively,under the optimal immobilization conditions: glutaraldehyde concentration 4 g/L, immobilization time 4 h and enzyme concentration 3 mg/mL,pH 7.0 and crosslinking time 90 min. Compared with the free β-galactosidase,the optimal reaction temperature was increased from 30 to 40 ℃,and the optimal pH increased by 0.5. In addition,the immobilized β-galactosidase remained 77.8% of its original activity after its 7`(th) repeated use.

Keywords：amination;bacterial cellulose;immobilization;β-galactosidase

Preparation of bacterial cellulose membrane with amino group as carrier and its immobilization of β-galactosidase

CHENG Hao,CHEN Yong,YING Hanjie

(College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University,Nanjing 211800,China)

细菌纤维素(BC)和植物纤维素有相似的基本结构，都是由D-葡萄糖以β-1,4糖苷键组成的无分支结构的高分子，具有很多优良性能，其持水性、结晶度、机械性能和纯度均非常高，独特的纳米纤维网络使其有良好的生物亲和性[1-2]，但其难溶解、难加工且官能团单一导致的其他功能不足等弱点，限制了其进一步应用。……