Characterization Of Glucose/Non-glucose Tolerant β-glucosidase From The Metatranscriptome In Compost

β-glucosidase catalyzes the conversion of cellobiose to glucose,andβ-glucosidase is a rate-limiting enzyme in the process of cellulose degradation.As an important part of the cellulase system,β-glucosidase plays an important role in the process of biomass conversion.However,mostβ-glucosidases are inhibited by the feedback of glucose,so findingβ-glucosidase with high glucose tolerance and deciphering the mechanism of glucose inhibition/tolerance has been the focus of research.Zhang et al identified glucose/non-glucose tolerantβ-glucosidase according to the presence or absence of conserved amino acid residues Trp168 and Leu173,and found that functional microbial communities producingβ-glucosidase differentially regulate the expression of glucose/non-glucose tolerantβ-glucosidase genes under carbon catabolite repression（CCR）conditions,which is a ubiquitous regulatory mechanism.However,the biological activity of a singleβ-glucosidase gene and its coding enzyme and its ecological effects under different CCR in a complex composting environment is not clear.Whether Trp168 and Leu173 can be used as criteria for the identification of sugar tolerance/non-sugar toleranceβ-glucosidase remains to be further confirmed.In this study,four representativeβ-glucosidase genes were selected from the composting metatranscriptome for biological activity characterization,and the ecological effects of a singleβ-glucosidase under different CCR conditions in a complex composting environment were analyzed,and the related mechanisms of glucose tolerance were explored through molecular docking and molecular dynamics simulation.Based on the analysis of the basic enzymatic characteristics of fourβ-glucosidases,it was found that the affinity of BGLA,BGLB and BGLC to PNPG was much greater than that to cellobiose.Mn²⁺and Ca²⁺could promote the enzyme activities of BGLA,BGLB and BGLC,while Zn²⁺and Fe³⁺significantly inhibited the activities of BGLA,BGLB and BGLC.As an effective glucosidase inhibitor,gluconolactone significantly inhibited the activities of BGLA,BGLB and BGLC,while BGLA had little effect.Under the influence of 10%ethanol,BGLA and BGLB inhibited 30%and40%of the activity,while BGLC was not affected.The enzyme activity of BGLA and BGLC in the presence of glucose was the same as that of glucose-stimulatedβ-glucosidase.The maximum glucose stimulates concentration of 250mM and 100mM was 1.8 and 1.2 times higher than that without glucose,while the hydrolytic activity of BGLB was significantly inhibited by glucose.After enzyme activity detection,BGLD did not catalyze the hydrolysis of cellobiose or PNPG,which may be due to the improper folding of the protein structure when expressed in E.coli.The results of molecular docking showed that the binding energy of BGLA,BGLB,BGLC and PNPG was less than that of cellobiose,and the lower the binding energy,the higher the affinity with the substrate,which was consistent with the results of our enzyme activity experiment.At the same time,the binding site of glucose and BGLA is located at the entrance of the active channel,while the binding site of glucose with BGLB and BGLC is located at the active center,so glucose will not compete with the substrate PNPG or cellobiose to inhibit the activity of BGLA.In molecular dynamics simulation,a large amount of glucose was accumulated in the catalytic region of BGLB and BGLC,but not observed by BGLA,which further confirmed the glucose tolerance of BGLA.These computational results of computational biology can well explain our experimental results.Combined with metatranscriptome analysis,BGLA had Trp168 and Leu173,and BGLA was a glucose tolerantβ-glucosidase.The hydrolysis activity of BGLA was stimulated in the presence of glucose and could tolerate glucose at a concentration of 1000 mM.At the same time,under the condition of CCR,the expression of BGLA was up-regulated by the functional microbial community to make up for the inhibition of non-glucose tolerantβ-glucosidase hydrolytic activity,to maintain the balance of carbon metabolism in the whole process.Although BGLB has Trp168 and Leu173,the hydrolytic activity of BGLB is highly inhibited by glucose,and the expression of BGLB is down-regulated by the functional microbial community producingβ-glucosidase under the condition of CCR,so BGLB is essentially a non-glucose-tolerantβ-glucosidase.The Trp168 of BGLC was replaced by Phe,and the hydrolytic activity and transcriptional regulation mechanism of BGLC were consistent with non-glucose tolerantβ-glucosidase.In summary,the biological activity characteristics of individualβ-glucosidase can explain the differential expression and regulation mechanism of glucose-tolerant/non-glucose-tolerantβ-glucosidases under different CCR conditions in a complex composting environment.Considering that BGLB has Trp168 and Leu173 related to glucose tolerance,the transcriptional regulation of BGLB under CCR is consistent with that of non-glucose tolerantβ-glucosidase.Therefore,although many individualβ-glucosidases have proved the key role of Trp168 and Leu173 in glucose tolerance,the presence or absence of conservative amino acid residues Trp168 and Leu173 could not fully be used to identify glucose-tolerantβ-glucosidase and non-glucose-tolerantβ-glucosidases in a complex composting environment.Through the analysis of computational biology,it is found that the biological activity ofβ-glucosidase is determined by its structural basis,which also provides evidence for the identification of sugar-tolerant/non-glucose-tolerantβ-glucosidase by computational biology.At the same time,BGLA is still uninhibited under the high glucose concentration of 1000mM,and can also carry out transglycosylation,which has the potential for industrial application.