金属β-内酰胺酶GOB-54的发现与表征  

Discovery and biochemical characterization of GOB-54 metallo-β-lactamase

作　　者：章升霞 王绍琛[1] 吕云斌 冯治洋[1] ZHANG Shengxia;WANG Shaochen;LYU Yunbin;FENG Zhiyang(College of Food Science and Technology,Nanjing Agricultural University,Nanjing 210095,China)

机构地区：[1]南京农业大学食品科技学院,江苏南京210095

出　　处：《食品与发酵工业》2024年第24期44-50,共7页Food and Fermentation Industries

基　　金：国家自然科学基金项目(32370089)。

摘　　要：该文旨在从土壤中挖掘新型β-内酰胺类抗生素耐药基因,分析耐药机制,做好临床预警,为开发新型β-内酰胺类药物提供信息。利用功能筛选策略,从土壤宏基因组文库中筛选获得阳性克隆,通过亚克隆构建和序列测定,得到耐药基因序列。利用序列比对、系统发育树分析、三维结构建模等生物信息学方法对耐药基因进行分析。使用微量肉汤稀释法评估β-内酰胺酶的耐药活性,利用生化实验分析酶水解抗生素的能力。鉴定出一个β-内酰胺抗生素耐药基因,与已知的β-内酰胺酶基因最高只有52.30%氨基酸序列一致性,命名为gob-54,编码β-内酰胺酶GOB-54。SWISS-MODEL三维建模发现GOB-54与临床来源的GOB-18具有高度相似的结构。动力学参数表明在所测试的β-内酰胺抗生素中,GOB-54对哌拉西林具有最高的亲和力和催化活性,动力学参数Km和Kcat/Km分别为121.82μmol/L和49.15 L/(mmol·s)。该研究利用功能宏基因组学方法挖掘出土壤来源的β-内酰胺抗生素耐药基因gob-54,编码广谱β-内酰胺酶GOB-54,为临床上监测和防控β-内酰胺类抗生素耐药基因的传播提供借鉴。This study aimed to investigate novelβ-lactam resistance genes in soil and elucidate the underlying mechanisms of resistance,thus providing valuable clinical alerts,and insights that contribute to the development of newβ-lactam drugs.The positive clone was screened from soil metagenomic library,and resistance gene sequence was obtained by subclone construction and sequencing.Sequence alignment,phylogenetic tree and three-dimensional structure modeling were used to analyze the drug resistance gene.Microbroth dilution method was used to evaluate drug resistance.The gene was expressed heterologously,and the hydrolysis ability of the purified protein was tested.Sequence analysis of the subclone revealed that pZF700 contained one novelβ-lactamase gene,gob-54,exhibiting 52.3%amino acid sequence identity with the knownβ-lactamase genes.This gene encodes the broad-spectrumβ-lactam enzyme GOB-54.The three-dimensional modeling of GOB-54 generated using SWISS-MODEL,revealed striking structural similarities with GOB-18 from clinical sources.The kinetic parameters indicated that GOB-54 had the highest affinity and catalytic activity for piperacillin among theβ-lactam antibiotics tested.The K m and K cat/K m values were found to be 121.82μmol/L and 49.15 L/(mmol·s),respectively.In this study,the novelβ-lactam resistance gene gob-54 was discovered from soil by functional metagenomics serves as a valuable reference for clinical monitoring and prevention of the dissemination ofβ-lactam antibiotic resistance genes.

关 键 词：Β-内酰胺酶 土壤微生物 功能宏基因组学 抗生素耐药基因 

分 类 号：R914[医药卫生—药物化学]