| β-Galactosidase(EC 3.2.1.23),usually known as lactase,is a crucial enzyme involved in carbohydrate metabolism in animals,plants,and microorganisms.When the subsrate is lactose,the enzyme can hydrolyze β-(1,4)-glycosidic bonds and catalyze transgalactosylation to transfer free galactoside with other receptors for synthesizing galactooligosaccharides(GOS).Industrial production depends on the import from Japan or the USA in order to meet the requirement of lactase.Hence β-galactosidase which has excellent characteristics is a hot topic for research and development now.A new fungal strain of Penicillium oxalicum was isolated from a soil sample collected near a milk factory.The β-galactosidase from Penicillium oxalicum has the ability of hydrolysis activity and transgalactosylation,but the enzyme is an endoenzyme.So it is difficult to extract,isolate and purify this enzyme.In this study,the β-galactosidase gene from Penicillium oxalicum was cloned and the enzyme was expressed successfully in Pichia pastoris GS115.Fermentation process optimization,enzymatic isolation and purification and enzymatic properties were investigated.In addition,the application of the enzyme was researched.Those results laid the foundation for the industrial production and utilization.The main results of this study are as follows:Firstly,total RNA was extracted and reverse transcription polymerase chain reaction(RT-PCR)was carried out to obtain the gene of β-galactosidase(GalA).GalA gene was inserted into the pPICZαA plasmid and then introduced into Pichia pastoris GS115.Positive clones were selected by using YPDS plates containing different concentrations of Zeocin.The protein molecular weight estimated to be approximately 110 kDa by SDS-PAGE and the specific activity of 12.34 U·mg-1,so the strain(P.pastoris GS115/pPICZαA-GalA)was built successfully.Secondly,through single factor test,the expression conditions of the β-galactosidase was optimized.As a result,the recombinant β-galactosidase activity reached the level of 33.16 U·m L-1 under the optimized conditions of inoculum size 8%,initial pH value 6.0,induction temperature 29℃,methanol concentration 1.5%,and induction period 120 h.Its activity was 2.69 times as high as before unoptimized.After enzymatic isolation and purification,the specific enzyme activity of recombinant β-galactosidase toward o-nitrophenyl-β-D-galactopyranoside(ONPG)reached 100.31 IU·mg-1 which was 2.3 times as high as crude enzymes.Maximal activity was observed at pH 5.0 and 60℃.The purified β-galactosidase was stable over a broad pH range of 4.5~8.5 and a temperature range of under 55℃.The β-galactosidase activity was almost unaffected by EDTA and vast majority metal ions tested,but its activity was significantly inhibited by Cu2+ and SDS.The kinetic properties Km and Vmax for the artificial substrate ONPG were 1.32 mmol·L-1 and 17.54 mmol·L-1·min-1,respectively.The kinetic properties Km and Vmax for the artificial substrate lactose were 1.15 mmol·L-1 and 13.38 mmol·L-1·min-1,respectively.Finally,the recombinant β-galactosidase was compared with β-galactosidase from Escherichia coli and Aspergillus oryzae,the hydrolysis efficiency were 90.3%,75.4%,64.6% respectively for the artificial substrate lactose.The optimized synthesis GOS conditions of lactose as substrate by β-galactosidase were as follows: 140 g·L-1 lactose,8 U·m L-1 β-galactosidase dosage and 14 h enzyme catalyzed reaction time.HPLC analysis verified that the recombinant β-galactosidase yielded GOS of 33.89%.LC-MS analysis suggested that recombinant β-galactosidase yielded major products of disaccharides,trisaccharides and tetrasaccharides.HPLC analysis revealed that the major products of GOS were different from human milk oligosaccharides(HMO)in the core domain. |
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