Study On The Thermal Modification Of Exo-inulinase

Inulin is rich in various asteraceae plants such as Jerusalem artichoke and chicory,and is a rich source of renewable resources.Inulin can be hydrolyzed by inulinase to produce fructose or high-fructose syrup,inulin oligosaccharides,and fuel alcohol,which are widely used in food,healthcare,and bio-energy.InuAGN25DVS used in this paper is a low-temperature exo-inulinase.About 75% of the total area of the earth belongs to a low-temperature environment and is suitable for low-temperature enzyme action.Treatment at a low temperature can prevent nutrient loss and food quality degradation.Enzymes are required to have good thermal stability for production,while low temperature enzymes usually have poor thermal stability.It is necessary to increase the thermal stability while ensuring the activity.As the temperature decreases,the catalytic activity of enzymes tend to decrease.It is also necessary to improve the activity of enzymes at low temperature.In order to obtain InuAGN25 DVS mutants with improved thermostability at high temperatures or improved activity at low temperatures,three mutants of InuAGN25 DVS were designed based on rational design.The three mutants were MutE137?5 with 137 EEDRK 141 deletion,Mut8 S with 8-point mutation including N61 E,K156R,P236 E,T243K,D268 E,T277D,Q390 K,and R409 D,and MutQ23?3 with deletion of 3 amino acids at the N-terminal(Q23,T24,and G25).The wild-type enzyme and the mutant enzymes were heterologously expressed in E.coli,purified by affinity chromatography.After excising the His-tag of the purified enzymes,the enzymatic properties were measured.The results of the study were as follows:1.The optimum pH of the mutant MutE137?5 was 6.0,which was the same as that of the wild-type enzyme InuAGN25 DVS.The optimum temperature was 35°C,which was 10°C lower than that of the wild-type enzyme,and the relative enzyme activity was increased by about 20% than the wild-type enzyme at 30°C.As such,the activity of the mutant at low temperatures increased.After incubation at 50°C for one hour,the wild-type enzyme had about 65% relative enzyme activity,whereas the mutant MutE137?5 had about 40% of the enzyme activity.2.The optimum pH of the mutant Mut8 S was 6.5,which was 0.5 more than the wild-type enzyme.The optimum temperature was 40°C,which was 5°C lower than that of the wild-type enzyme.When incubated at 50°C for one hour,the wild-type enzyme had about 65% relative enzyme activity,whereas the mutant Mut8 S had about 100% of the enzyme activity.3.The optimum pH and temperature of the mutant MutQ23?3 were 6.0 and 45°C,respectively,which were the same as that of the wild-type enzyme InuAGN25 DVS.When incubated at 50°C for one hour,the wild-type enzyme had about 65% relative enzyme activity,whereas the mutant Mut8 S had about 100% of the enzyme activity.In this study,the method of rational design was used to obtain the exo-inulinase MutE137?5 with improved low-temperature activity,and the exo-inulinase Mut8 S and MutQ23?3 with improved thermal stability.This study finally improved InuAGN25 DVS for its potential applications.Furthermore,the study gives useful information for the thermal modification of glycoside hydrolase family 32 exo-inulinases as well as relative mechanisms.