| β-galactosidase(EC2.2.1.23)i.e.lactase is commonly found in animal tissues,plants and microorganisms.It has the ability to decompose lactose into galactose and glucose,and tocatalyze the production of galactooligosaccharides(GOS).At present,in food industry and markets,these two enzyme properties of lactase have different practical significance.Lactose intolerance is a global pervasive physiological problem,and it is possible for people to become lactose intolerant patients from lactose tolerance groups for a variety of reasons,so no/low lactose dairy products have drawn more and more attention.In addition,with the rapid improvement of living conditions,high incidence of the diseases increases.Therefore,people pay more attention to dietary management and food safety.Studies have shown that GOS can balance intestinal flora,regulate intestinal pH,prevent constipation,improve the physiological function of the human intestinal tract,assist in calcium absorption,accelerate fat metabolism,protect liver function and so on,which makes GOS,the natural food additives,have a better market value.And these directions are all that lactase may involve in the market and industrial fields.There are many studies on the high temperature resistance of lactase and the direction of lactase hydrolysis,but the research on the direction of GOS synthesis has just started.In these studies,there has been little study on the bi-directional properties of the entire lactase by changing one amino acid at the activation center.Only limited groups in the world are working on the related research.Therefore,to study the impact of one amino acid change on lactase enzyme activity is of importance.In this study,the 415-site asparagine(Asn or N)ofβ-galactosidase[Pyrococcus furiosus DSM 3638]were mutated to serine(Ser or S)by point mutation.The mutant was constructed into the Pichia pastoris expression plasmid vector and was integrated into Pichia pastoris cells by electroporation.After drug selection,the selected cell strain was used for fermentation to prepare mutant enzymes of β-galactosidase.The results showed that the mutant β-galactosidase product had the highest enzymatic activity at 95 ℃ and pH5.5,which was resistant to high temperature.The thermostability of the lactase was good at 100 ℃ for 1h,The activity of the mutant enzyme is very stable in the low temperature storage environment.The mutant has better ability to hydrolyze the milk and can be used in the processing of low lactose milk and its related products.After the mutation,another feature ofβ－galactosidase,namely,the ability to produce gal actooli go saccharides was also improved,but not obvious,was not good enough for industrialization.Therefore,the lactase produced by the mutant strain can be used in the processing industry of dairy products,but can not be used to produce more galactose oligomers.At the same time,we found that the activity of the lactase was increased with the increase of the cell density through the production of the test grade,which was very beneficial for the industrial production of lactase and late purification.It is speculated that the lactase gene derived from Pyrococcus furiosus DSM 3638 is more resistant to heat.Because the site of amino acid changes affect the active center of the conformation,so that the stability of the enzyme has been greatly improved.The active site where the mutation site is located may impact more the process of hydrolyzing lactose,but it can impact less on the synthesis of GOS.It can be further speculated that perhaps the ability of the thermostable lactase to synthesize GOS would be affected by other sites. |
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