铜绿假单胞菌中丙氨酸消旋酶的酶学特性及菌株检测
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摘要
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 alr_(Pa) 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 K_m and V_(max)of 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 V_(max)value of PaAlr was about 7-fold of that of Alr_(PAO1) 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.
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 alr_(Pa) 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 K_m and V_(max)of 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 V_(max)value of PaAlr was about 7-fold of that of Alr_(PAO1) 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.
引文
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[17]XUE Z W,HU Y,XU S J,et al.Characterization and Preliminary Mutation Analysis of a Thermostable Alanine Racemase from Thermoanaerobacter Tengcongensis MB4[J].Extremophiles,2013,17(4):611-621.doi:10.1007/s00792-013-0545-5
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[21]陆巧燕,廖彬.铜绿假单胞菌耐药性分析和治疗对策[J].临床合理用药杂志,2012,5(25):54-55.doi:10.15887/j.cnki.13-1389/r.2012.25.068LU Qiaoyan,LIAO Bin.Analysis of Drug Resistance of Pseudomonas aeruginosa and Study on Treatment Strategy[J].Chinese Journal of Clinical Rational Drug Use,2012,5(25):54-55.
[22]EMAMI S,NIKOKAR I,GHASEMI Y,et al.Antibiotic Resistance Pattern and Distribution of pslA Gene Among Biofilm Producing Pseudomonas aeruginosa Lsolated from Waste Water of a Burn Center[J].Jundishapur J Microbiol,2015,8(11):e23669.doi:10.5812/jjm.23669
[2]KIM S A,MOON H,LEE K,et al.Bactericidal Effects of Triclosan in Soap Both in Vitro and in Vivo[J].J Antimicrob Chemoth,2015,70(12):3345-3352.doi:10.1093/jac/dkv275
[3]刘晓岚,宋志军.铜绿假单胞菌的Quorum-sensing系统[J].中国医学文摘(肿瘤学),2006,20(2):180-181.LIU Xiaolan,SONG Zhijun.The Quorum-sensing System of Pseudomonas aeruginosa Strain[J].Journal of Chinese Medical Abstracts(Oncology),2006,20(2):180-181.
[4]朱玉莹,易勇,杨犀,等.多重耐药铜绿假单胞菌中Ⅰ型整合子新结构的发现及其与耐药的相关性[J].微生物学报,2013,53(9):927-932.doi:10.13343/j.cnki.wsxb.2013.09.003ZHU Yuying,YI Yong,YANG Xi,et al.Discovery of New Structure of ClassⅠIntegron in MDR Pseudomonas aeruginosa and Its Association with Drug Resistance[J].Acta Microbiologica Sinica,2013,53(9):927-932.
[5]CIUSTEA M,MOOTIEN S,ROSATO A E,et al.Thiadiazolidinones:A New Class of Alanine Racemase Inhibitors with Antimicrobial Activity Against Methicillin-resistant Staphylococcus Aureus[J].Biochem Pharmacol,2012,83(3):368-377.doi:10.1016/j.bcp.2011.11.021
[6]STRYCH U,DAVLIEVA M,LONGTIN J P,et al.Purification and Preliminary Crystallization of Alanine Racemase from Streptococcus Pneumoniae[J].BMC Microbiol,2007(7):40.doi:10.1186/1471-2180-7-40
[7]KOMBA E V,KIMBI E C,NGOWI H A,et al.Prevalence of Porcine Cysticercosis and Associated Risk Factors in Smallholder Pig Production Systems in Mbeya Region,Southern Highlands of Tanzania[J].Vet Parasitol,2013,198(3/4):284-291.doi:10.1016/j.vetpar.2013.09.020
[8]LEE Y,MOOTIEN S,SHOEN C,et al.Inhibition of Mycobacterial Alanine Racemase Activity and Growth by Thiadiazolidinones[J].Biochem Pharmacol,2013,86(2):222-230.doi:10.1016/j.bcp.2013.05.004
[9]邱伟,周学东,李明云.丙氨酸消旋酶的研究进展[J].国际口腔医学杂志,2016,43(2):228-232.doi:10.7518/gjkq.2016.02.025QIU Wei,ZHOU Xuedong,LI Mingyun.Research Progress on Alanine Racemase[J].International Journal of Stomatology,2016,43(2):228-232.
[10]MARTIN B,JOFR A,GARRIGA M,et al.Quantification of Listeria Monocytogenes in Fermented Sausages by MPN-PCR Method[J].Lett Appl Microbiol,2004,39(3):290-295.doi:10.1111/j.1472-765X.2004.01580.x
[11]MALATHI J,MURUGAN N,UMASHANKAR V,et al.Draft Genome Sequence of Multidrug-resistant Pseudomonas aeruginosa Strain VRFPA02,Lsolated from a Septicemic Patient in Lndia[J].Genome Announc,2013,1(4):e00425-13.doi:10.1128/genomeA.00425-13
[12]JU J S,XU S J,FURUKAWA Y,et al.Correlation Between Catalytic Activity and Monomer-dimer Equilibrium of Bacterial Alanine Racemases[J].J Biochem,2011,149(1):83-89.doi:10.1093/jb/mvq120
[13]冯利伟,郭娇洁,李辉欣,等.原玻璃蝇节杆菌D-氨基酸氧化酶及突变体的酶学特性[J].微生物学报,2014,54(8):897-904.doi:10.13343/j.cnki.wsxb.2014.08.007FENG Liwei,GUO Jiaojie,LI Huixin,et al.Characterization of D-amino Acid Oxidase and Its Mutantsfrom Arthrobacter Protophormiae[J].Acta Microbiologica Sinica,2014,54(8):897-904.
[14]FOTOUHI L,GANJAVI M,NEMATOLLAHI D.Electrochemical Study of Lodide in the Presence of Phenol and o-Cresol:Application to the Catalytic Determination of Phenol and o-Cresol[J].Sensors(Basel),2004,4(11):170-180.doi:10.3390/s041100170
[15]SODA K.Microdetermination of D-amino Acids and D-amino Acid Oxidase Activity with 3-methyl-2-benzothiazolone Hydrazone Hydrochloride[J].Anal Biochem,1968,25(1):228-235.
[16]HASHIMOTO A,NISHIKAWA T,OKA T,et al.Determination of Free Amino Acid Enantiomers in Rat Brain and Serum by High-performance Liquid Chromatography After Derivatization with N-tert-butyloxycarbonyl-L-cysteine and O-phthaldialdehyde[J].J Chromatogr,1992,582(1/2):41-48.
[17]XUE Z W,HU Y,XU S J,et al.Characterization and Preliminary Mutation Analysis of a Thermostable Alanine Racemase from Thermoanaerobacter Tengcongensis MB4[J].Extremophiles,2013,17(4):611-621.doi:10.1007/s00792-013-0545-5
[18]THOMPSON J D,GIBSON T J,PLEWNIAK F,et al.The CLUSTAL_X Windows Interface:Flexible Strategies for Multiple Sequence Alignment Aided by Quality Analysis Tools[J].Nucleic Acids Res,1997,25(24):4876-4882.
[19]ALTSCHUL S F,GISH W,MILLER W,et al.Basic Local Alignment Search Tool[J].J Mol Biol,1990,215(3):403-410.doi:10.1016/S0022-2836(05)80360-2
[20]TAMURA K,STECHER G,PETERSON D,et al.MEGA6:Molecular Evolutionary Genetics Analysis Version 6.0[J].Molecular Biology and Evolution,2013,30(12):2725-2729.doi:10.1093/molbev/mst197
[21]陆巧燕,廖彬.铜绿假单胞菌耐药性分析和治疗对策[J].临床合理用药杂志,2012,5(25):54-55.doi:10.15887/j.cnki.13-1389/r.2012.25.068LU Qiaoyan,LIAO Bin.Analysis of Drug Resistance of Pseudomonas aeruginosa and Study on Treatment Strategy[J].Chinese Journal of Clinical Rational Drug Use,2012,5(25):54-55.
[22]EMAMI S,NIKOKAR I,GHASEMI Y,et al.Antibiotic Resistance Pattern and Distribution of pslA Gene Among Biofilm Producing Pseudomonas aeruginosa Lsolated from Waste Water of a Burn Center[J].Jundishapur J Microbiol,2015,8(11):e23669.doi:10.5812/jjm.23669