Mutation Screening Of A β-Glucosidase-producing Strain And Purification Of β-Glucosidase

As a widespread enzyme in the world, P-glucosidase could hydrolyze the terminal non-reducing β-D-glucosidic bonds, and liberate β-D-glucoses and corresponding aglycones. There are quite bright prospects for the application of β-glucosidases from different sources in bioenergy, food industry, healthy products, agriculture and other areas. Especially, as a key component in cellulose, P-glucosidase plays a vital role in the process of cellulose degradation. Owing to the low level of P-glucosidase in the cellulose system produced by cellulose-producing strains, it is of crucial importance to screen the P-glucosidase high-producing strains. A Penicillium oxalicum C2’preserved in our lab was selected as the original strain, and several results are shown as follows:Mutant901B2was screened by compound mutation including both diethyl sulfate (DES) and ultraviolet-lithium chloride (UV-LiCl), with the Congo red screening plates. β-Glucosidase activity produced by901B2(0.69IU/mL) was2.16times higher than that by wild-type strain P. oxalicum C2’after submerged fermentation for5days in basal mediums.After being cultured for8days,901B2produced a large amount of P-glucosidase, whose activity was up to1.14IU/mL. The culture medium composition such as carbon sources, nitrogen sources, mineral salts, surfactants and the initial pH of medium were optimized by means of single factor experiments. As the prime factors influencing P-glucosidase production, the amount of wheat bran and (NH4)2SO4in culture medium predicted by Central Composite Design (CCD) was41.40g/L and3.11g/L. And the medium composition were wheat bran41.40g/L,(NH4)2SO43.11g/L, KH2PO42.0g/L, MgSO4·7H2O0.40g/L, FeSO4·7H2O0.40g/L, Tween-801.0%. Ultimately β-glucosidase produced by P. oxalicum901B2was50.0%increase than that in the basal medium (1.14IU/mL) and close to the maximum value (1.74IU/mL) of β-glucosidase activity predicted by Response Surface Methodology (RSM).β-Glucosidase in fermentation broth was purified by the method of ethanol fractional precipitation and HiTrap SP XL strong anion-exchange chromatography. It’s activity was9.89-fold higher than that of the crude enzyme, and the recovery of P-glucosidase was11.89%. It was demonstrated that the β-glucosidase collected finally was electrophoretically pure by the method SDS-PAGE. Protein point isolated by2-DE was highly expressed and in accordance with the molecular mass resulted by SDS-PAGE. The optimum reaction temperature for β-glucosidase produced by mutant P. oxalicum901B2was determined to be60℃, and there still was the excellent thermostability after being incubated within the limit of40～60℃for120min. The optimum pH for β-glucosidase was5.0, while it was stability under the acidic conditions in which pH was from3.0to6.0for24hrs. The β-glucosidase acticity was easily promoted by metal ions Mn2+and Fe2+and inhibited by Mg2+、Ca2+、K+and Cu2+, and it was also enhenced by the chelant EDTA.