Purification And Molecular Structure Of The β-D-galactosidase From Penicillium Decumbens

Beta-D-galactosidase （EC3.2.1.23）, which is commonly known as lactase, not onlycan catalyse hydrolysis of lactose to generate galactose and glucose, but also have atransfer glycosylation activity to produce galacto-oligosaccharide （GOS）, it plays animportant role in the field of food, medicine and analytical chemistry. In recent years,with the development of functional glycobiology, β-galactosidase enzyme graduallybecome an important tool for the hydrolysis of lactose and oligosaccharides synthesis,and it has been unprecedented favor of the peopleThe research is based on the Penicillium decumbens L-7, which is screened by ourlaboratory. The medium compositions and culture conditions of the strain L-7wereoptimized detailed through single factor and orthogonal experimental. As follows: thelactose concentration was70g/L, yeast extract concentration10g/L, peptone10g/L,K2HPO4was2.5g/L, the optimal incubation temperature was30℃, the optimalculture conditions were initial pH6.0, medium volume was50mL/250mL, the bestinoculum size was106/mL and rotation was180r/min. Under this condition, the activityof beta-D-galactosidase in the strain which was cultured by32h was up to153.27U/g,and was5times before optimization. The result of galactooligosaccharide yield in thefermentation broth under such fermentation conditions was analysed to30.2g/L by highperformance liquid chromatography （HPLC）.The enzyme purification was done by means of ultrasonic cell crusher, ammoniumsulfate faction, dialysis, desalination, DEAE ion-exchange chromatography, SephadexG-100gel filtration. The purified enzyme identified by SDS-PAGE indicated that asingle protein band was obtained. Its molecular weight is about110KDa. It exhibitedoptimum activity at60℃and pH6.0,and have an highly instability under60℃and pH3.0-7.0.The enzyme activity was significantly activated by Mg²⁺, Mn²⁺, and wasstrongly inhibited by Cu²⁺,Fe²⁺, Zn+.The β-galactosidase produced by the Penicillium decumbens was intracellularenzyme, and also have a low activity produced by the original strain, can not be used forindustrial production. Therefore, the β-galactosidase was also cloned by constructinggenetically engineered bacteria, to meet the demand of the industrial production.