铜绿假单胞菌中丙氨酸消旋酶的酶学特性及菌株检测

Enzymatic Characterization of Alanine Racemase from Pseudomonas aeruginosa and Strain Detection of P.aeruginosa

铜绿假单胞菌为条件致病菌,是目前各种医院内感染最广泛、最严重的致病菌之一.以分离自公共场所洗手液的菌株6-3为研究对象,其16S rRNA基因与Pseudomonas aeruginosa DSM 50071的16S rRNA基因的一致性为99.8%,初步确定其为铜绿假单胞菌;根据同源蛋白的核苷酸序列设计菌株6-3中丙氨酸消旋酶的扩增引物,通过PCR从6-3菌株中克隆获得了丙氨酸消旋酶基因(alrPa),基因序列与P. aeruginosa PAO1中丙氨酸消旋酶Alr_(PAO1)的氨基酸同源率为98.9%,存在4个不一致的氨基酸位点;经原核表达、Ni-NTA亲和层析法纯化获得了蛋白PaAlr,酶学特性分析表明,重组蛋白PaAlr的最适反应温度为40℃,最适反应pH=10.0;最适底物是L-Ala,具有严格的底物特异性,K_(m,L-Ala)=10.96mmol/L,V_(max,L-Ala)=1 562μmol/(L·min),K_(m,D-Ala)=8.45mmol/L,V_(max,D-Ala)=881.9μmol/(L·min),其最大反应速率约为Alr_(PAO1)的7倍;以铜绿假单胞菌中丙氨酸消旋酶为检测对象,以PCR和Western-blotting 2种方法对铜绿假单胞菌进行检测,实验发现,在目的基因序列已知的前提下,采用PCR法具有较好的检测灵敏性和专一性.

Pseudomonas aeruginosa,a kind of conditional pathogen,is one of the most widespread and serious pathogenic bacteria in hospitals.In this study,the strain 6-3 was isolated from hand soap in public places.Its 16S rRNA gene showed a high sequence similarity(99.8%)with the species of genus P.aeruginosa DSM 50071,indicating that strain 6-3might be P.aeruginosa.The alanine racemase gene alrPa from strain 6-3 was cloned and sequenced.The protein sequence deduced from the DNA sequence displayed 98.9% sequence identity with the alanine racemase from P.aeruginosa PAO1,including 4 different amino acid residues.The protein PaAlr was then expressed and purified by Ni-NTA affinity column chromatography.The recombinant PaAlr showed high specificity to L-alanine and exhibited optimal racemization pH and temperature at 10.0 and 40℃,respectively.The kinetic parameters Km and Vmaxof PaAlr at 40℃ and pH 10.0,determined by HPLC,were 10.96mmol/L and 1 562μmol/(L·min)for L-alanine,and 8.45mmol/L and 881.9μmol/(L·min)for D-alanine,respectively.The Vmaxvalue of PaAlr was about 7-fold of that of AlrPAO1 fromP.aeruginosa PAO1.With alanine racemase of P.aeruginosaas detection target,PCR and Western-blotting were performed to detect P.aeruginosa.The results showed that PCR method had better sensitivity and specificity when the target gene sequence was known.