SHV-71型β-内酰胺酶的酶动力学研究  

Enzymological study of SHV-71 β-lactamase

作　　者：黄支密[1] 李文芳[2] 彭青 钱元恕 单浩[1] 储秋菊[1] 

机构地区：[1]解放军第98医院检验科 [2]汕头大学医学院药理教研室

出　　处：《浙江检验医学》2009年第2期11-15,共5页Zhejiang Journal of Laboratory Medicine

摘　　要：目的对临床分离的鲍曼不动杆菌所产SHV-71型β-内酰胺酶的编码基因进行序列分析,研究其对多种β-内酰胺类抗生素的酶动力学参数,并观察pH、温度、底物浓度及酶抑制剂对酶活性的影响。方法PCR扩增SHV型酶的编码基因。将SHV的PCR产物与pMD18-T载体连接,测定它们的核苷酸序列,并将SHV-71型酶基因克隆至表达载体pET-41b(+),转化至宿主菌大肠埃希菌BL21(DE3)中。以IPTG诱导其表达并纯化,SDS-聚丙烯酰胺凝胶电泳检测表达情况。用分光光度计测定SHV-71型酶对多种β-内酰胺类抗生素的动力学参数,以及pH、温度、底物浓度及酶抑制剂对酶活性的影响。结果SHV-71型酶对第一、二、三代头孢菌素类抗生素的相对最大水解速度及相对水解效率均较高,而对青霉素类和碳青霉烯类抗生素的相对最大水解速度及相对水解效率均较低。该酶在pH7.4、37℃的活性最高;底物浓度低于120μmol/L时,反应初速度随底物浓度增加而呈线性增加,底物浓度大于120μmol/L时,酶活性反而受抑;克拉维酸和三唑巴坦对SHV-71型酶有抑制作用。结论根据SHV-71型酶的酶学特点,我们推测SHV-71型酶为超广谱β-内酰胺酶。Objective To study the genetype of β-lactamases produced by clinically isolated Acinetobacter baumannii,the kinetic parameters of β-lactams hydrolysis of SHV and the activity of SHV-71 in different pHs and temperatures.Methods SHV β-lactamase encoding genes were detected by PCR and sequenced after being subcloned into pMD18-T vector. SHV gene was cloned into pET-41b(+) vector and the recombinant plasmid was transformed into E.coli BL21. Expression and purification of SHV were comfirmed by SDS-PAGE electrophoresis. SHV-71 kinetic parameters were measured using spectrophotometer. The activity of SHV-71 emzyme in different pHs and temperatures was observed by spectrotometer.Results One gene type was a new SHV β-lactamase gene nominated SHV-71 (GenBank number:DQ296194).After being inducted with IPTG,a 58 kDa recombinant fusion protein of GST and SHV was expressed in the pET-41b(+) system.. Cephalosporins can be efficiently hydrolysed by SHV-71. SHV-71 enzyme was most active in pH 7.4 and 37 ℃. The concentration of substrates is also an important factor to the activity of SHV-71. Clavulanic acid and tazobactam can inhibit the activity of SHV-71 enzyme.Conclusion We found a new subtype of SHV β-lactamase which may be an extend-spectrum β-lactamase according to its characteristics of enzyme.

关 键 词：鲍曼不动杆菌 Β-内酰胺酶 原核表达及纯化 动力学参数 

分 类 号：R516[医药卫生—内科学]