文献类型：期刊文献

中文题名：重组黄杆菌肝素酶Ⅲ的纯化、表征及培养条件对酶生产的影响(英文)

英文题名：Purification and properties of recombinant GST-haparinase III and optimizationm of cultivation conditions

作者：高兴[1];赵健[1];范立强[1];李素霞[1];王富军[2];姬胜利[3];袁勤生[1]

机构：[1]华东理工大学生物反应器工程国家重点实验室,上海200237;[2]浙江日升昌药业有限公司,浙江322100;[3]山东大学药学院,济南250012

年份：2009

卷号：25

期号：11

起止页码：1718

中文期刊名：生物工程学报

外文期刊名：Chinese Journal of Biotechnology

收录：CSTPCD;;Scopus;北大核心:【北大核心2008】；CSCD:【CSCD2011_2012】；PubMed;

基金：Supported by:Open Project Program of the State Key Laboratory of Bioreactor Engineering(No.F200-B-0232)~~

语种：中文

中文关键词：肝素酶Ⅲ;谷胱甘肽-S-转移酶;肝素黄杆菌;可溶性表达;表征

外文关键词：Heparinase III; glutathione-S-transferase（GST）; Flavobacterium heparinum; soluble expression; characterization

摘要：肝素酶III是一种特异性地裂解乙酰肝素的酶,在大肠杆菌中表达时容易形成包涵体。为实现肝素酶III的可溶性表达,利用谷胱甘肽-S-转移酶(GST)与肝素酶III融合性能,通过构建相应的表达质粒pGEX-heparinaseIII,在大肠杆菌中实现了肝素酶Ⅲ的可溶性表达。粗酶通过一步亲和纯化其纯度可达95%以上。通过对LB培养基摇瓶培养Escherichia coli BL21的诱导时机、诱导剂用量、诱导时间等培养条件的优化,确定了该可溶性肝素酶III融合蛋白的最适生产条件。通过对纯酶的最适反应温度、pH、Ca2+浓度等一系列性质研究,确定了该酶的最适反应条件。
Heparinase Ⅲ is an enzyme that specifically cleaves certain sequences of heparan sulfate.Previous reports showed that this enzyme expressed in Escherichia coli was highly prone to aggregation in inclusion bodies and lacks detectable biological activity.In this paper,we fused a glutathione-S-transferase（GST） tag to the N-terminus of heparinase III gene and expressed the fusion protein in Escherichia coli to develop an expression system of soluble heparinase III.As a result,approximately 80% of the fusion protein was soluble.The protein was then purified to near homogeneity via one-step affinity chromatography.A 199.4-fold purification was achieved and the purified enzyme had a specific activity of 101.7 IU/mg protein.This represented 32.3% recovery of the total activity of recombinant GST-heparinase III.The maximum enzyme production was achieved when bacteria were induced with 0.5 mmol/L isopropyl-β-D-thiogalactoside at 15°C for 12 h.The enzyme showed maximum activity at 30°C and pH 7.5.And the enzyme activity was stimulated by 1 mmol/L Ca2＋ and 150 mmol/L NaCl.