Expression And Molecular Modification Of Glutaminase From Bacillus Subtilis

Glutaminase?EC 3.5.1.2?can catalyze the?-glutamyl hydrolysis of glutamine to produce glutamic acid with umami taste.It has good applied potential in the processing of traditional brewed foods such as soy sauce.In view of the high salt and high temperature characteristics of soy sauce production process,this study used glutaminase?YbgJ?derived from Bacillus subtilis 168 as the research object.Based on B.subtilis WB600 expression system,the strategies of saturation mutation and N-terminal fusion self-assembling amphipathic peptides?SAPs?were used to modify the heat tolerance and salt tolerance of glutaminase in order to improve the application effect.The main research results were as follows:?1?Expression and properties analysis of glutaminaseThe YbgJ gene was cloned into carrier pP43NMK,and the expression plasmid pP43NMK-YbgJ was constructed and transformed into B.subtilis WB600 for expression.The enzyme activity test showed that the intracellular glutaminase activity reached the highest value of 149 U?mL^-1 when the recombinant B.subtilis WB600 was fermented for 15 h.SDS-PAGE analysis showed that the recombinant enzyme had a molecular band of approximately 37 kDa,which was consistent with the theoretical molecular weight of YbgJ.The active YbgJ was purified by nickel column affinity chromatography and the enzymatic properties were analyzed.The results were as follows:specific enzyme activity of YbgJ was 563 U?mg^-1;the optimum reaction temperature and 55 ^oC half-life(t_1/2)were 50 ^oC and 10.79 min,respectively;optimum pH was 9.0,after 6 h at pH 7-9,40 ^oC,residual enzyme activity was over 80%.In the soy sauce fermentation simulation system at pH 5,the addition of YbgJ?per 5 mL of model system adding 2 U enzyme?increased the glutamate content by 54%compared to the control group?not adding YbgJ?.The above results indicated that recombinant YbgJ has potential application in the flavoring process of soy sauce.?2?Improved thermal stability of glutaminase through saturated mutationsIn order to improve thermal stability of YbgJ,we identified the key amino acid E3,E55 and D213 that affecting the thermal stability of YbgJ through virtual saturation mutation of 49 amino acids with adverse interactions using Discovery studio 2017.In contrast to the wild type enzyme,The t_1/2 of the mutants E3C,E55F and D213T were increased by 58%,69%and 41%at 55oC,respectively.The t_1/2 of the composite mutants E3C/E55F/D213T,E3C/E55F,E3C/D213T and E55F/D213 were increased by 173%,144%,122%and 97%,respectively.Among them,the specific activity of E3C/E55F was enhanced by 23%,reaching 693 U?mg^-1.The structure analysis indicated that the hydrogen bonds of E3C/E55F/D213T,E3C/E55F,E3C/D213T and E55F/D213T were increased by30,23,11,and 8,respectively;and their adverse interactions were decreased by 5,4,4 and 3,respectively.The results indicated that the substitution of key amino acids in the enzyme molecule have a great influence on the thermal stability of YbgJ,and the decreases in intramolecular adverse interaction and the increases in hydrogen bond may account for the enhancement of thermal stability.?3?Improved salt tolerance of glutaminase through N-terminal fusion SAPsIn order to improve the salt tolerance of YbgJ,based on the B.subtilis WB600 expression system,two kinds of amphiphilic peptides?AEAK,LELK?and PT-Linker were fused to N terminal of the mutant E3C/E55F/D213T,respectively.The fusion enzyme AEAK-E3C/E55F/D213T and LELK-E3C/E55F/D213T were obtained.It was worth noting that LELK fused to YbgJ was mainly expressed in the form of active inclusion bodies.Compared with E3C/E55F/D213T,the residual enzyme activities of AEAK-E3C/E55F/D213T and LELK-E3C/E55F/D213T treated in 18%NaCl solution for 120 min increased by 6.4 times and 7 times,respectively.55 ^oC t_1/2/2 of AEAK-E3C/E55F/D213T and LELK-E3C/E55F/D213T Increase by 58%and 69%,respectively.In the soy sauce fermentation simulation system at pH 5,adding AEAK-E3C/E55F/D213T and LELK-E3C/E55F/D213T?per 5 mL of model system adding 2 U enzyme?made the content of glutamic acid increased by 69%and 114%,compared with the control group?adding E3C/E55F/D213T?.The fusion enzymes with improved salt tolerance and thermal stability obtained in the study will contribute to the industrial application of glutaminase in soy sauce processing.