Screening Of Psychrotolerant Bacterium Producing Cold-active β-gakactisudase And Optimization For Producing Conditions Of β-galatosidase

Lactase, namelyβ-galactosidase which hydrolyzes the lactose to glucose and galactose can be used for the production of low-lactose dairy products to treat the "lactose intolerance". Following the fast development of science and technology, the role which lactase plays in the fields of environment, medicine, analyzing and so on is more and more important, in addition to its wide use in food industry. But the lactases that we usually use in dairy industry at present always have high optimal reaction temperatures, which raises cost and wastes energy. Therefore, it's necessary for us to obtain aβ-galactosidase with cold-activity, in order to reduce the cost and save energy.A psychrotolerant strain L3 capable of producing cold-activeβ-galactosidase was screened from soils near oil field in northeast of China, using X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) as indicator. According to the morphological and the molecular characterization, the strain L3 was preliminarily identified as Serratia proteamaculans and named as Serratia proteamaculans sp. L3. The results based on studies of its growth and morphological characteristics indicated that it growed well in the conditions of pH7.0-9.0, temperature ranging from 4℃to 35℃, and NaCl concentration of 0.5%-3%, and the optimal temperature, pH and concentration of NaCl were 25℃, pH7.5 and 1%, respectively.β-galactosidase from Serratia proteamaculans sp. L3 was exocellular enzyme. The studies concerning the activity as well as tolerability of enzyme showed that the optimal temperature and pH of the enzyme activity are 22℃and pH7.0, respectively which were consistent with optimal growth conditions of the strain L3. In addition , we also optimized the conditions for the strain L3 to produceβ-galactosidase using Response Surface Methodology. Combined with single factor experiment, we got the optimal condition in theory which is most favorable forβ-galactosidase production of the strain L3. The best cultivating conditions we obtained included 1.0g/L of glucose, 1.5g/L of yeast powder, 0.1g/L of(NH4)2SO4, 0.15g/L of K2HPO4, 0.50g/L of MgSO4, 0.76g/L of NaCl, pH 7.5 and temperature at 25℃. It was found that theβ-galactosidase producing ability of the strain L3 cultivated in the best condition is 1.1 time higher than before. This paper lays a solid foundation for application in the industry production.This paper obtained a plasmid p△LacZ as a cloning vector by removing LacZ from pUC19, preparing well for building the genomic DNA libraty of the strain L3.