Effect Of Residue 222 On The Synthesis Of Galacto-oligosaccharide By Tn0602

As a functional prebiotic,galactooligosaccharide(GOS)can be used as food supplements to enhance a variety of health-related physiological activities.Nowadays,the most common method to produce GOS is the enzymatic synthesis by?-galactosidase and?-glucosidase.However,GOS catalyzed by?-galactosidase has complex glycosidic bonds and polymerization degree,which make it difficult to separate and purify.Based on previous research,we found that?-glucosidase from Thermotoga naphthophila RUK10 can specifically catalyze the synthesis of GOS3.In order to explore its mechanism,we selected some potential sites in the active pocket for mutagenesis based on related literatures.Among them,the hydrolysis and transglycosidation activity of the N222Y/F were significantly decreased,indicating that this site may be a key residue for catalysis.Meanwhile,the three-dimensional structure of Tn0602 was obtained by homology modeling,and docking was carried out with the substrate.After that,the interaction between the enzyme and the substrate was analyzed.It was confirmed that N222 was located at the aglycone+1 Subsite and was connected to the active site E166 and E351.Therefore,the N222 was selected for saturation mutation,and the saturation mutants were successfully constructed and expressed.Ni²⁺-NTA affinity chromatography was used to purify the mutants,and then the hydrolysis and transglycosidation activity were charaterized.Under the optimum p H conditions for lactose hydrolysis by wild-type,the activity of all mutants decreased to varying degrees.Among them,the corresponding mutations(such as Ser,Gln,etc.)with similar side chains to Asn have similar viability to the wild;.Besides,when it was mutated into the smallest Gly,larger Leu and Ile,alkaline Arg and Lys,as well as aromatic Phe and Tyr,the hydrolysis activity towards lactose decreased significantly.However,although the N222S mutant has a slight decrease in activity at p H 5.0,the activity can be increased by 20%at p H 3.0.Compared with the wild-type enzyme,the mutant has a slight deviation in the optimal temperature and p H in the reaction of hydrolyzing lactose,and their kinetic constants indicate lower specificities towards lactose.Taken together,the mutations at position N222 will lead to varying degrees of reduction in the hydrolysis activity towards lactose,which confirmed that Asn222 is one of key residues involved in hydrolysis process,and it is also the optimal result through natural selection.In the catalytic synthesis of GOS3,the benificial mutants include N222W,N222D,and N222Q,whose yields are increased by 23%,36%,and 6%compared with the wild.In addition,N222W can increase the yield of catalytic synthesis of GOS4,which is 44%higher than that of the wild type.The above results indicate that some mutations at position 222 can enhance the transglycosylation of the enzyme.Although the rest mutants do not show the same effect o,they can also enhance the yield of GOS3 due to their dramatic weakening in lactose hydrolysis.Although the maximum yield of GOS3 has not increased,it can be maintained at a relatively high level.For example,the level of GOS3 synthesized by the N222G increase continuously,reaching 85%of the maximum amount of wild-type product at 24 h,it reaches about110%at 84 h.The maximum yield of GOS3 catalyzed by N222E is only 5%lower than that of the wild-type.The yield can be maintained at a high level from 8 to 12 h.With the processing of the reaction,although the yield is slightly reduced,it maintains at about 80%of the maximum value.The above results indicate that compared with wild-type enzymes,some mutants are more suitable for industrial production of GOS.The saturation mutation at position 222 not only creates beneficial mutants which show higher yield of GOS,but also proves the hypothesis that reducing the hydrolysis activitywhile increasing the function of transferase/hydratase through rational design is an effective way to improve its pratical value.In following studies,mutants with excellent properties can be superimposed to explore the effect of site synergy on the catalytic synthesis of GOS,and further improveits application potential.