Stabilities And Catalytic Properties Of Cellulase In GH45 And GH12 Families

Cellulase with good stability and high catalytic efficiency has broad industrial application prospects.In this study,GH45 cellulase TaCel45 and GH12 cellulase NfEG12A were cloned from thermophilic fungus Thielavia arenaria XZ7 and Neosartorya fischeri P1.According to their characteristics,we revealed the mechanisms of their stability,high catalytic efficiency and substrate promiscuous.Furtherly,we studied the mechanisms of the catalytic promiscuous of enzymes in?/?family.Details are as follows:The specific activity of TaCel45 towards CMC-Na was 498 U/mg.TaCel45 had maximum activity at 60?65?C and a melting temperature?T_m?of 68.4?C.However,TaCel45 exhibited extraordinary thermostability at 90?C and 100?C,retaining more than 70%and 45%of its activity after a 1 h incubation,respectively.Seven mutants?C11S,C12S,C16S,C31S,C171S,C193S,and C203S?were then constructed to investigate the effects of each disulfide bond on the structure,activity and stability of TaCel45.As a result,six disulfide bonds?C11-C136,C16-C87,C31-C57,C88-C203,C90-C193 and C160-C171?were found to be indispensable for the folding,secretion,and activity of TaCel45,while C12-C48 was critical for thermal adaptation and refolding.The mutant C12S showed decreased optimal temperature and T_m values of 50?C and 60.2?C,respectively.Moreover,the second structure of C12S has changed.Overall,this study demonstrated that disulfide bonds play a vital role in the folding and refolding capability and thermostability of this GH45 cellulase.Acidophilic and thermophilic NfEG12A had a highest activity towards CMC-Na?2210 U/mg?known so far.Based on the multiple sequence alignment and homology modeling,two specific sequences?FN and STTQA?were identified in the loop 3 region of GH12 endoglucanases from fungi.To determine their functions,these sequences were introduced into NfEG12A,or the counterpart sequence STTQA was removed from EG.These modifications had no effects on the pH and temperature optima and substrate specificity,but changed the catalytic efficiency(k_cat/K_m)of these enzymes?NfEG12A[100%]<NfEG12A-FN[140%]<NfEG12A-STTQA[190%],and EG[100%]>EG?STTQA[41%]?.Molecular docking and dynamic simulation analyses revealed that the longer loop 3 in GH12 may strengthen the hydrogen-bonding interactions between the substrate and protein,and thereby increase the turnover rate(k_cat).This study provides a new insight to understand the vital roles of loop 3 for GH12 endoglucanases in catalysis.The family 12 of glycoside hydrolase?GH12?mainly have endoglucanase and xyloglucanase activities,however what really determines the substrate recognition and specificity remains elusive.To stress this question,corelated residues?hotspot wizard?and sequence consensus?Signisite?have been used to identify 18 residues correlated to phenotypes of GH12 enzymes.Then 18 mutants were constructed in the gene of an endoglucanase NfEG12A.Among them,three residues,Y7,Y111 and N18,were found to create xyloglucanase in NfEG12A.Furtherly,the functions of these three residues were testified in another endoglucanase AtEglD from A.terreus and a xyloglucanase NfXyG12A from N.fischeri P1.As a result,the catalytic efficiencies of N18Y on xyloglucan increased to 1.4² fold,while that on glucan decreased to 0.16⁰.36 fold compared to their wild types,respectively.Besides,the catalytic efficiencies of triple mutants NfEG12A-Y7W/N18Y/Y111W and AtEglD-Y7W/N18Y/Y111W on xyloglucan increased to 13.7 and 3.2 folds,while that on?-glucan decreased to 0.58 and 0.62 folds compared to their wild types,respectively.These results demonstrated that residues 7,18 and 111 are very important positions for substrates recognition and binding.Furtherly,in order to clearly reveal the different substrates binding modes of GH12 enzymes,we capture the crystal structures of NfEG12A and NfEG12A-N18Y.Molecular docking with oligopolymers of two substrates analysis demonstrated that hydrogen interaction networks of binding sites N22,Q67,N194 and Q196 were critical in binding glucan,and residues N22,Y18,N194 and Q196 were vital for xyloglucan,besides hydrophobic interactions were emphasized for recognition xyloglucan.?/?family is characterized by a variety of catalytic reaction mechanisms.It mainly contains the esterase?EST?and hydroxynitrile lyase?HNL?.In order to study the mechanism of catalytic promiscuity,we used EST1 and HNL1 as templates,both of which contain two activities.Sequence consensus and structure flexibility analysis were used to identify the differential regions of two enzymes.As a result,a HNL1 convert to EST1 by replacing 16 residues within 10.5?among the catalytic pocket.The catalytic efficiencies of mutant HNL1₁^st+2^nd 16 residues on pNPAc increased to 322-fold,while that on mandelonitrile decreased to 0.29-fold compared to their wild types,respectively.Moreover,residues'positions in the catalytic pocket of the mutant have changed,making it easier to bind esters as the position of pNPAc.This study proved that not only the catalytic residues play an important role in determining the enzyme activity,but also the residues outside the catalytic pocket will change the enzyme activity and create more efficient one.