Enzymatic Properties And In-vitro Directed Evolution Of Trehalase From Enterobacter Cloacae

Trehalose is a non-reducing disaccharide,which is widely distributed in nature.Trehalase is a disaccharidase that can specifically hydrolyze trehalose.Trehalase as food additives can also treat two sugar deficiency and some diagnosis of human kidney disease can be achieved through the detection of urinary trehalase activity.Therefore,trehalase has very high application value in medical?food and other fields.In addition,it is possible to develop novel enzyme inhibitors for insect control by studying their structure and function.We cloned a gene of trehalase called treE?1650bp,GC58%?from Enterobacter Cloacae strain AR₀002,the gene treE codes 549 amino acids.We detecte its isoelectric point?p I?is 4.93 and predict its molecular mass may be 63.5kDa by SDS-PAGE.Compared with the gene sequence in the NCBI database,it was found that treE was 88% consistent with the sequence derived from Enterobacter Cloacae ATCC 13047.The optimal temperature of treE is 50? and has best response at pH 6.Enzyme were treated in various pH buffer for 48 h at 4oC,in the range of pH5.5-pH9.0,treE retained more than 80% of its original activity.It is conclusioned that under neutral conditions,the enzyme has good stability.It is showed that treE could be elevated by Mn2+,Fe3+?1mM?5mM?,Co2+?1mM?,and inhibited by Cu2+,Fe3+?10mM?,Mg2+?10mM?,Co2+?10mM?.The Km,Vmax,kcat,kcat/ Km of treE were 3.318 mmol/L,0.445 mmol/L?min,1265.174s-1 and 381.303 L?mmol-1?s-1 respectively.In order to improve the catalytic efficiency of treE,we constructed a random mutant library and screened the mutants by the technique of T7 phage-lysis combined with 96 deep-hole plates.We obtained one mutant named 1-D8 from the mutant library containing 16000 mutants.There are two amino acid sites changed?position 22 and position 491?.1-D8 exhibited the optimal temperature and pH at 45?,pH 6.0,respectively;Compared with treE,the optimal temperature of 1-D8 was decreased 5?;after the enzyme were treated in various pH buffer for 48 h at 4oC,treE retained more than 80% of its original activity in the range of pH5.5-pH9.0.The Km,kcat,kcat/ Km of 1-D8 were 3.082 mmol/L,1819.651s-1 and 590.412 L?mmol-1?s-1 respectively.It could be concluded that the catalytic efficiency of 1-D8 is 54.8% higher than treE.We want to know the effect of the amino acid sites` changes in catalytic efficiency.So we performed a single point mutation at two sites?position 22 and position 491?.Meanwhile,through structural simulation and molecular docking,we preliminarily determined that R168 may be related to the catalytic efficiency of treE.So we decided to change the 168 th amino acids?R?G?.Based on the experimental results,we find the catalytic efficiency of Y22 C did not change much compared with that of treE.Moreover,the activity of W491 R is 48.6% higher than treE.For the mutant R168 G,we found that although its Km decreased,the kcat decreased more,this eventually leds to a decrease in the ratio of Km and kcat,this means that the activity of the mutant R168 G is only 58.5% that of wild type?treE?.In any case,we got a new trehalase and its mutant,which is better than the wild type.It provides materials and clues for further modification and improvement of enzyme activity.Moreover,the amino acid residues related to the catalytic efficiency were identified,which provided a reference for further exploring the relationship between structure and function.