Characterization of a Novel Recombinant ß-Lactamase from Bacillus subtilis R5

被引:1
|
作者
Ali, Amjed [1 ]
Tayyab, Muhammad [1 ]
Hashmi, Abu Saeed [2 ]
Nadeem, Asif [3 ]
Hanif, Shumaila [4 ]
Firyal, Sehrish [1 ]
Saeed, Shagufta [1 ]
Awan, Ali Raza [1 ]
Wasim, Muhammad [1 ]
机构
[1] Univ Vet & Anim Sci, Inst Biochem & Biotechnol, Abdul Qadir Jillani Rd, Lahore, Pakistan
[2] Riphah Int Univ, Raiwind Rd Campus, Lahore, Pakistan
[3] Virtual Univ Pakistan, Dept Biotechnol, Lahore, Pakistan
[4] Univ Agr Faisalabad, Dept Chem, Faisalabad, Pakistan
关键词
ss-lactamase; Antibiotic resistance; Bacillus subtilis R5; Antibiotic; susceptibility; Diagnostics; A BETA-LACTAMASE; BIOCHEMICAL-CHARACTERIZATION; ANTIBIOTIC-RESISTANCE; CLONING; PURIFICATION; EXPRESSION; MECHANISM; GENE;
D O I
10.17582/journal.pjz/20210718130706
中图分类号
Q95 [动物学];
学科分类号
071002 ;
摘要
Current study deals with the characterization of recombinant ss-lactamase from a locally isolated strain Bacillus subtilis R5. The study was an initial step for the fulfillment of commercial needs of ss-lactamase in Pakistan. The 1 kb ss-lactamase gene was amplified by PCR using the genomic DNA of B. subtilis R5 as template. The purified PCR product was cloned in pTZ57R/T and sub-cloned in pET21a. Expression of recombinant protein was examined in BL21 CodonPlus (DE3) cells. SDS-PAGE confirmed the size of recombinant protein as 34 kDa. Recombinant ss-lactamase was produced optimally when the BL21 CodonPlus (DE3) cells were induced with 0.6 mM IPTG with a post induction time of 5h at 37 degrees C. The characterization studies demonstrated the maximal enzyme activity at 37 degrees C in 50 mM sodium phosphate buffer pH 7. The presence of EDTA in the activity assay mixture reduced the ss-lactamase activity to 91% while Zn2+, Co2+, Mn2+ enhanced the enzymatic activity to 144, 121 and 108% when used at a final concentration of 1 mM. The ionic and non-ionic detergents showed slight inhibitory impact on the recombinant ss-lactamase activity. The enzyme exhibited the K (m) and V (max) values of 2.27 mM and 45.45 mu mol/min, respectively when benzylpenicillin was utilized as substrate. The degradative ability of recombinant ss-lactamase to hydrolyze a variety of ss-lactam ring containing antibiotics makes it a suitable candidate for its utilization as positive control in diagnostics and in antibiotic susceptibility testing experiments.
引用
收藏
页码:2301 / 2308
页数:8
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