| ?-galactosidase,as an exoglycosidase,can efficiently catalyze the removal of terminal galactose residue from the non-reducing end of glycans and release free galactose.In 2020,the expected amount of lactose-free milk will reach more than €1 billion.Due to the existing technology,the domestic ?-galactosidase production capacity cannot meet China's demand.Each year,a large amount of ?-galactosidase is imported,which is expensive.And the demand for low and medium temperature galactosidase has also increased.Therefore,the development of a novel ?-galactosidase is an urgent task.?-galactosidase derived from Lactobacillus brevis ATCC 367 was focused on.Lactobacillus brevis was commonly found in fermented cabbage or sauerkraut.It is a probiotic with high safety.And the ?-galactosidase was composed of two different subunits.In this project,different prokaryotic expression vectors were constructed,and finally co-expression vector with efficient capacity of expressing the heterodimeric galactosidase was selected.The biochemical parameters and application were studied and discussed.The research details were as follows:1.Two subunits of heterodimer from Lactobacillus brevis were synthesized.Expression vectors were constructed and expressed,including subunits,co-expression protein,fusion protein.The glutamic acid at 468th and 536th positions of the bigger subunit were site-mutated to alanine.All the expression vector constructs were transformed into E.coli BL21(DE3,LacZ-)chemical competent cells.2.Enyzme activities of subunits,mixtures of subunits,mutants,co-expressed proteins and fusion proteins were all evaluated.co-expressed protein from one vector,called LbGalAB,had better activity than co-expressed protein from two vector.The activity of fusion proteins was comparable to LbGalAB.Except for the lower activity of"Linker 3",the activity of the fusion proteins increases as linker gets longer.However,the others were inactive.3.Recombinant enzymes were identified by SDS-page after purification.Fusion proteins can not be purified.Co-expressed protein LbGalAB had two obvious bands on gel,and the molecular weights of them are approximately 71 KDa and 35 KDa,respectively.4.LbGalAB was determined for characterization and its optimum pH and temperature were 7.5 and 30 ?.And it could maintain its activity for a long time at 16?.It is not a metal ion-dependent enzyme.Magnesium ions can significantly increase its activity while surfactant SDS inhibits it.The specific activity of the enzyme is 366 mU/mg,the kinetic parameters for different substrates are as follows:Km value for pNP-?-galactose is 1.1 mM and Vmax is 405 ?M/min,for lactose is 5.6 mM and Vmax is 49.4 ?M/min,and for N-acetyl-D-lactosamine is 8.7 mM and Vmax is 13.6 ?M/min.5.LbGalAB reacted with human,bovine or goat milk samples.It was shown that the lactose in milk was quantitatively(98%)removed at 16? within 1 h.Commerical?-galactosidases originating from Aspergillus and Kluyveromyces species generally require incubation time of more than 24 h to achieve such high lactose removal rates.The good enzymatic stability of LbGalAB at 16? combined with its neutral pH optimum suggests that this enzyme may also be a promising candidate for the hydrolysis of lactose in dairy products. |
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