Functional Study Of GH12 Family Glycosidic Hydrolase Cel12A

Cellulose as the most abundant renewable resource on earth,it is degradated slowly in natural environment because of its stable structure.Currently,the enzymatic conversion of cellulose resources by cellulase is a hotspot of research.In our laboratory,GH12 family enzyme gene cel12 A has been cloned from Bacillus licheniformis ATCC 14580 strain,besides preliminary heterologous expression and enzymatic properties have been studied.Based on this,on the one hand,the endocellulase function of Cel12 A was studied,the key amino acid sites which play an important role in the enzyme activity were identified by sequence alignment and mutant construction,the possible mechanism was studied further more.On the other hand,taking Cel12 A as the object,the possible non-hydrolytic expansion function of GH12 family enzymes as small molecular protein was investigated by electron microscopy and synergistic experiments.The main experimental results of this paper are as follows:(1)Fifteen conserved amino acids related to the function of GH12 family cellulase were identified by sequence alignment,moreover,alanine screening was performed on these sites,the results of mutant enzyme activity assay showed that these amino acids had different effects on Cel12 A activity.After mutation of nucleophilic group E155 and acid-base catalytic group E243,the recombinant enzyme activity was completely lost.D137 mutant also lost almost all enzyme activity,suggesting that D137 may play a role as the third member of the "catalytic triad".In addition,the enzyme activity of N51 A,W53A,Y89 A and N133 A was lost nearly 80%,the enzyme activity of M157 A and W159 A was lost nearly 70%,the enzyme activity of V86 A and W139 A was lost nearly 60%,the enzyme activity of P167 A and F245 A was lost nearly 30%,the enzyme activity of V199 A and I189 A was lost less than 20%.(2)Furthermore,the functions of N51,W53,Y89 and N133,which had greater effect on enzyme activity,were studied.Mutations in the direction of aspartate D and alanine A were carried out on N51,increasing the distance between amino acid sites and substrates,thereby destroying hydrogen bonds,the residual enzyme activity was less than 30% after mutation,suggesting that N51 may play a role by binding with substrates to form hydrogen bonds.Mutations in the direction of tyrosine Y and histidine H were carried out on W53,changing the groove structure of the substrate binding site,the residual enzyme activity was less than 30% after mutation,suggesting that W53 may play an important role in the recognition and binding process of GH12 family enzyme molecules and substrates.After mutation of Y89 in the direction of phenylalanine F with only one hydroxyl difference,the activity of protoenzyme was basically maintained,suggesting that Y89 was not involved in the formation of hydrogen bonds,and may play a role in maintaining the correct conformation of sugar rings in the catalytic process.Mutations in the directions of aspartic acid D and phenylalanine F were carried out on N133 to weaken or even destroy the hydrogen bond formed between the amino acid and the acid-base catalytic group E243,the results showed that the activity of mutant enzymes decreased gradiently,suggesting that the hydrogen bond formed between N133 and E243 was the structural basis for the recognition and catalysis of cellulose substrates by Cel12 A.In addition,the optimal pH of the N133 mutant shifted to the acidic direction by nearly 0.4 units,suggesting the site could affect the catalytic environment of the catalytic residues,thereby affecting the maintenance of enzyme activity and the optimum pH of catalytic bond breaking.(3)The insoluble cellulose substrate pretreated by Cel12 A was observed to be obviously fluffy by scanning electron microscope,it was also found that Cel12 A and commercial cellulase had synergistic effects with these substrates.The results showed that Cel12 A,as a small molecular protein,had the expansion function similar to ExPansin and Swollenin.(4)To make further exploration of the expansion mechanism of Cel12 A,Cel12A inactivated mutant with complete loss of cellulase activity was used for expansion experiment.The results showed that the inactivated mutant Cel12 A could also exert expansion function to some extent,but the expansion function decreased compared with wild-type Cel12 A,suggesting that the expansion function of Cel12 A may be related to the activity of endocellulase and the characteristics of small molecule proteins.In this paper,the molecular mechanism of Cel12 A and the expansion function as non-enzymatic factors were discussed,which provided theoretical and experimental information for the protein engineering modification of GH12 cellulase family and the construction of extracorporeal efficient complex enzyme system.