Study For Thermo And Salt Tolerance Of Exo-inulinase InuAMN8

Inulin is a non-food crop with very good resistance to adversity.Exo-inulinase can degrade inulin in one step to obtain fructose syrup with a sugar concentration of up to 95%.Fructose syrup can also be used as a substrate to produce alcohol,sorbitol,lactic acid and succinic acid.The exo-inulinase with low temperature and high salt adaptability can be used in the field of biotechnology that requires low temperature and high salt environment,and has the advantages of avoiding the generation of by-products,saving energy and reducing consumption,and reducing microbial pollution.At present,the studies of exo-inulinase mutations has focused on conservative sites,mutations of catalytic amino acids and substrate binding amino acids,but there are few studies on heat tolerance mutations,and salt tolerance mutation studies have not been reported yet.The research object of this article,InuAMN8,is the low-temperature and NaCl-resistant exo-inulinase obtained in the early stage of our laboratory.Excavating the loop area through modeling,we found short loop regions at positions 122-126,169-172,and 243-247,and long loops at positions 268-280,360-368,and 376-380 near the catalytic pocket.The mutants with the Loop region deleted were named Mut A122?5,Mut G169?4,Mut G243?5,Mut V268?13,Mut V376?5 and Mut G360?9,respectively.In addition,by comparing the Y115 to Q131 fragment sequence of InuAMN8 with the GH32 family sequence,it was found that the sequence of this region is relatively diverse,poorly conservative,located in the catalytic pocket,and corresponding to the influence of the exo-inulinase InuAGN25 that has been studied in the laboratory thermally adaptable loop area(137EEDRK141).Based on this,the121-128 loop region of InuAMN8 was selected for loop region substitution and point mutation,and 8 mutants were designed and named Mut AP122EK5,Mut DL121EK5,Mut DL121ET6,Mut DR121EH9,Mut S120 R,Mut S120 D,Mut P124 G,and Mut P126 R.Meanwhile,the 117-119 region in a ?-sheet strand that close to the 121-128 loop region was selected for mutation,which include deletion sequences and point mutations,then 13 mutants were designed and named Mut S117?3,Mut Y119 H,Mut Y119 V,Mut S117 Q,Mut S117 D,Mut S117 G,Mut A118 P,Mut A118 N,Mut A118 H,Mut Y119 D,Mut Y119 T,Mut Y119 N,Mut S117,respectively.After the wild enzyme InuAMN8 and all the above mutant enzymes were heterologously expressed,purified and dialyzed in E.coli,the properties of heat adaptability and salt adaptability were determined.Mutants Mut G243?5,Mut V376?5 and Mut A118 P are not expressed in E.coli,while mutants Mut S117?3,Mut S117 Q and Mut S117 D are expressed in E.coli but have no activity.The results of other mutant studies are as follows:(1)The optimal temperature of wild enzyme InuAMN8 and mutant enzymes Mut A122?5,Mut G360?9,and Mut A118 N was 35?;the optimal temperature of mutant enzymes Mut G169?4 and Mut P126 R was 40?;the optimal temperature of the other 16 mutant enzymes was reduced,with a range of 20? to 30?,and the activity at low temperature was higher than that of the wild enzyme.(2)After incubation at 50? for 1 h,the relative enzyme activities of wild enzymes InuAMN8 and mutant enzymes Mut A122?5,Mut P126 R,and Mut P124 G were greater than85%,and the relative enzyme activities of mutant enzymes Mut V268?13,Mut G360?9,Mut DR121EH9,Mut Y119 D and Mut Y119 N were lower than 20%.Mut V268?13 and Mut G360?9 was inactivated,and the relative enzyme activities of other mutant enzymes were in the range of 40% to 80%.(3)In 15%(w/v)NaCl solution,the enzyme activities of wild enzyme InuAMN8 and mutant enzymes Mut A118 N,Mut S120 R,and Mut S120 D were about 50%,and the enzyme activities of other mutant enzymes were less than 50%.(4)After incubating for 1 h in 30%(w/v)NaCl solution,the wild enzyme InuAMN8 and mutant enzymes Mut A122?5,Mut A118 N,Mut P126 R,Mut G169?4,Mut Y119 H,and Mut S120 R remained stable and their activities were almost unaffected,while the relative enzyme activity of the mutant enzymes Mut V268?13 and Mut G360?9 was less than 40%,and the relative enzyme activity of other mutant enzymes was in the range of 40%-90%.In summary,this study performed loop deletion,substitution and point mutation of the exo-inulinase InuAMN8.The optimal temperature of the 14 mutant enzymes was reduced,the thermal stability was poor,and the activity and stability in NaCl were reduced.This study revealed 4 loop regions that related to the thermosalinity of exo-inulinase InuAMN8,including the loop regions corresponding to amino acids 121-129,169-172,268-280,and360-368,revealed 6 key the amino acid positions,117 th,118th,119 th,120th,124 th and126th positions,respectively,and provided the basis for the study of the hot-salt adaptability mechanism and rational design of exo-inulinase.