CAJ | 학술논문

目的对Pr0167位点突变型CTX-M-14超广谱β-内酰胺酶(ESBL)的动力学特征进行分析与评价.方法以携带CTX-M-14基因的大肠埃希菌临床株为模板,克隆目的基因,重组工程菌,并表达CTX-M-14型ESBL.进而采用基于重叠延伸PCR法的定点突变技术,将CTX-M-14型ESBL的167位点Pro(P)分别突变为Gly(G)、Gln(Q)、Ser(S)和Thr(T),重组构建P167G、P167Q、P167S和P167T四株突变型的CTX-M-14工程菌.表达与纯化野生型、重组型和突变型CTX-M-14型ESBL,检测其水解β-内酰胺类抗菌素的酶动力学参数(Kcat、Km和Km).结果对野生型与重组型CTX-M-14型ESBL的酶动力学参数进行配对t检验,结果显示两者Kcat(t=1.796,P=0.123)、Km(t=0.559,P=0.596)、Kcat/Km(t=0.893,P=0.406)间的差异无统计学意义(P>0.1).与重组CTX-M-14型ESBL相比,P167S突变型酶对头孢他啶的Km值大幅降低,为突变前的1/16(8.39/134.85);Kcat和Km值分别为突变前的2.87倍(1.81/0.63)和43.6倍(0.218/0.005);且对青霉素、氨苄西林、头孢唑啉、头孢呋辛、头孢曲松和头孢噻肟的Kcat/Km值都呈现出显著减小的趋势(P<0.05).与重组CTX-M-14 ESBL相比,P167Q和P167G突变型酶对头孢他啶的Kcat值亦大幅减小(P<0.01),而P167T突变型对头孢他啶的酶动力学参数无明显变化.结论重组型与野生型CTX-M-14 ESBL之间各项酶动力学参数的差异无统计学意义(P>0.1).而P167S位点的突变,不仅增强了CTX-M-14型ESBL对头孢他啶的亲和力,也加快了酶-底物复合物的转换速率.通过对Pr0167位点4种突变型酶的动力学参数比较,初步说明突变型CTX-M ESBL对头孢他啶所具备的高水解活性机制,不能简单地归结于Pro167位点被小侧链氨基酸残基取代而导致酶活性中心空间扩大的解释.
목적 대Pr0167위점돌변형CTX-M-14초엄보β-내선알매(ESBL)적동역학특정진행분석여평개.방법 이휴대CTX-M-14기인적대장애희균림상주위모판,극륭목적 기인,중조공정균,병표체CTX-M-14형ESBL.진이채용기우중첩연신PCR법적정점돌변기술,장CTX-M-14형ESBL적167위점Pro(P)분별돌변위Gly(G)、Gln(Q)、Ser(S)화Thr(T),중조구건P167G、P167Q、P167S화P167T사주돌변형적CTX-M-14공정균.표체여순화야생형、중조형화돌변형CTX-M-14형ESBL,검측기수해β-내선알류항균소적매동역학삼수(Kcat、Km화Km).결과 대야생형여중조형CTX-M-14형ESBL적매동역학삼수진행배대t검험,결과현시량자Kcat(t=1.796,P=0.123)、Km(t=0.559,P=0.596)、Kcat/Km(t=0.893,P=0.406)간적차이무통계학의의(P>0.1).여중조CTX-M-14형ESBL상비,P167S돌변형매대두포타정적Km치대폭강저,위돌변전적1/16(8.39/134.85);Kcat화Km치분별위돌변전적2.87배(1.81/0.63)화43.6배(0.218/0.005);차대청매소、안변서림、두포서람、두포부신、두포곡송화두포새우적Kcat/Km치도정현출현저감소적추세(P<0.05).여중조CTX-M-14 ESBL상비,P167Q화P167G돌변형매대두포타정적Kcat치역대폭감소(P<0.01),이P167T돌변형대두포타정적매동역학삼수무명현변화.결론 중조형여야생형CTX-M-14 ESBL지간각항매동역학삼수적차이무통계학의의(P>0.1).이P167S위점적돌변,불부증강료CTX-M-14형ESBL대두포타정적친화력,야가쾌료매-저물복합물적전환속솔.통과대Pr0167위점4충돌변형매적동역학삼수비교,초보설명돌변형CTX-M ESBL대두포타정소구비적고수해활성궤제,불능간단지귀결우Pro167위점피소측련안기산잔기취대이도치매활성중심공간확대적해석.
Objective To analyze and evaluate the characteristics of enzyme kinetics of CTX-M-14 type extended-spectrum β-lactamase(ESBL) with Pro167 residue substitution. Methods By molecular cloning and PCR techniques, CTX-M-14 gene was directionally cloned into plasmid pET28a( + ) from a clinical E. coli isolate and formed an expression vector to transform competent E. coli BL21 (DE3 ). Prol67 residue substitutions of P167G, P167Q, P167S and P167T were introduced to CTX-M-14 by site-directed muta-genesis based on overlap extension PCR with the former recombinant plasmid as PCR template, respectively.The wild-type CTX-M-14, recombinant CTX-M-14 protein and its variants were expressed and purified, then their steady-state kinetic parameters (Kcat, Km and Kcat/Km ) against β-lactam antibiotics were determined.Results The kinetic parameters of wild-type and recombinant CTX-M-14 had no statistically significant differences (P>0.1). The 1/Km, Kcat and Kcat/Km values of P167S variant against ceftazidime were 16-fold, 2.87-fold and 43.6-fold higher than those of recombinant CTX-M-14, respectively, and the Kcat/Km value of P167S variant against penicillin, ampicillin, cefazolin, cefuroxime, ceftriaxone, cefotaxime decreased( < 0.05). Compared with the kinetic parameters of recombinant CTX-M-14, the kinetic parameters of P167T variant against ceftazidime had no significant change, but the Kcat values of P167Q and P167G variants decreased dramatically(P<0.01). Conclusion There was no difference between the enzyme activities of wild-type and recombinant CTX-M-14. P167S variant could not only promote the enzyme affinity of CTX-M-14 to ceftazidime but also improve the conversion rate of enzyme-substrate complex in the ceftazidime hydrolysis. The comparison of the kinetic parameters of CTX-M-14 and its variants with Pro167 residue substitution showed that the increased activity of CTX-M ESBL variants against ceftazidime could not be simply explained with the enlarged cavity in active site that may be caused by the replacement of Pro167 residue by smaller amino acids.