Agrawal Ruchi, Satlewal Alok, Verma Ashok Kumar
Department of Biochemistry, College of Basic Sciences and Humanities, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, U.S. Nagar, 263145, Uttarakhand, India.
Department of Microbiology, College of Basic Sciences and Humanities, Govind Ballabh Pant University of Agriculture and Technology, Pantnagar, U.S. Nagar, 263145, Uttarakhand, India.
3 Biotech. 2013 Oct;3(5):381-388. doi: 10.1007/s13205-012-0095-z. Epub 2012 Oct 6.
The extracellular β-glucosidase from microorganisms is generally produced in low levels. Therefore, in this study, a β-glucosidase hyperproducing mutant was developed by multiple exposures of ethyl methyl sulfonate (EMS) and ultraviolet (UV) radiation (both individually and jointly) to Bacillus subtilis strain (PS). The developed mutants were screened, selected and characterized. The mutant, PS-UM1 developed after UV exposure alone, indicated a small increase in β-glucosidase production (718 U/l) in comparison to the wild-type strain, PS (675 U/l). The mutant, PS-CM5 developed after EMS exposure alone, displayed a slightly better production (762 U/l) than both the above strains. However, after exposure of the wild-type strain to both UV and EMS mutagens jointly, a better mutant (PS-CM5-UM3) was developed with 1.2-fold increase in production (806 U/l). Further, optimization of culture conditions by classical "one-variable-at-a-time" approach was done to determine the optimum, pH, temperature and nitrogen sources. The selected mutant (PS-CM5-UM3) produced up to 1,797 U/l enzyme and was found to be stable for ten generations. The β-glucosidase from the selected mutant (PS-CM5-UM3) was concentrated and purified using ammonium sulfate, dialysis and size-exclusion chromatography. The enzyme displayed maximal activity at 60 °C and it was found to be fairly stable at temperatures up to 70 °C for 30 min. Its molecular weight was determined to be around 60 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
微生物产生的细胞外β-葡萄糖苷酶通常产量较低。因此，在本研究中，通过对枯草芽孢杆菌菌株（PS）单独或联合多次暴露于甲基磺酸乙酯（EMS）和紫外线（UV）辐射，培育出了一株β-葡萄糖苷酶高产突变体。对培育出的突变体进行了筛选、选择和表征。单独经紫外线照射后培育出的突变体PS-UM1，与野生型菌株PS（675 U/l）相比，β-葡萄糖苷酶产量略有增加（718 U/l）。单独经EMS处理后培育出的突变体PS-CM5，其产量（762 U/l）略高于上述两种菌株。然而，野生型菌株经UV和EMS诱变剂联合处理后，培育出了一个更好的突变体（PS-CM5-UM3），产量增加了1.2倍（806 U/l）。此外，通过经典的“一次改变一个变量”方法对培养条件进行了优化，以确定最佳的pH、温度和氮源。所选突变体（PS-CM5-UM3）产生的酶高达1797 U/l，并且发现其在十代内保持稳定。使用硫酸铵、透析和尺寸排阻色谱法对所选突变体（PS-CM5-UM3）的β-葡萄糖苷酶进行了浓缩和纯化。该酶在60℃时表现出最大活性，并且发现在高达70℃的温度下30分钟内相当稳定。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）测定其分子量约为60 kDa。
