摘要
目的:表达SHV-4型超广谱β-内酰胺酶,对纯化的SHV-4酶进行特性研究。方法:将SHV-4基因克隆到pET26b表达质粒,重组质粒转入E.coli BL21(DE3)构建工程菌,重组工程菌经摇瓶发酵后获得种子菌,种子菌转入发酵罐发酵,当菌体密度OD600为0.8时,加入诱导剂IPTG至终浓度为1.0mmol/L,于37℃培养6h。培养液离心收获菌体沉淀,其超声裂解上清液进行Ni2+亲和层析,获取的SHV-4酶进行分子量、纯度、等电点、最适条件、米氏常数、催化常数等特性进行分析。结果:SDS-PAGE电泳,等电聚焦电泳及系列的酶动力学实验表明SHV-4酶分子量约为30kD,纯度大于95%,等电点为7.9,最适pH值为6.0～8.0,最适温度为37℃,以头孢硝噻吩为底物测定的Km值为2.61×10-6mol/L,Kcat为258s,Kcat/Km为9.88×107mol-1·s-1·vL。结论:重组的SHV-4酶初步满足制成商品酶的条件。
Objective：To express SHY-4 extended-spectrum-β-lactamase （ESBL） in pET26b/BL21 （DE3） system and to research the characterization of the purified SHY-4 ESBL. Methods： The SHY-4 genes were cloned into pET26b expression plasmids,and the recombinant plasmids were transformed into E.Coli BL21（DE3） to bui1d the engineer bacteria. The engineer bacteria were fermented in LB broth in flask to obtain seed bacteria. Subsequently, the seed bacteria were fermented in fermenter, the expression of SHY-4 β-lactamase was induced at OD600 of 0.8 with 1.0 mmol/L IPTG for 6h.Cell culture was centrifuged, the pellet was disrupted by ultrasonic and was centrifuged,then the supernatant was purified by Ni^2＋ affinity resin to get SHY-4 β-lactamase.The characterization of purified SHY-4 β- lactamase was analyzed by molecular weight（MW）, isoelectric point （pI）, Kin, Kcat and so on. Results： SDS-PAGE, isoelectric focusing electrophoresis and a series of enzyme kinetics experiments indicated that SHV-4 β-lactamase MW was 30.0 kD, pI 7.9, purity more than 95 percentage, optimum pH was 6.0- 8.0,optimum temperature 37℃ ,Kin of nitrocefin 2.61 × 10^-6mol/L, Kcat of nitrocefin 258 s^-1 and Kcat/ Km of nitrocefin 9.88 × 10^7 mol^-1· s^-1·L. Conclusion： The recombinant SHY-4 β -lactamase satisfies the fundamental conditions for producting a commodity of enzyme.
出处
《温州医学院学报》
CAS
2007年第1期1-4,共4页
Journal of Wenzhou Medical College
基金
浙江省科技厅科研基金重点资助项目(2004C23018)。
作者简介
王震（1980-），男，浙江仙居人，助教，医学硕士。
通讯作者：吕建新，教授，硕士生导师，Email：1jx@wzmc．net。
