Study On Enzymatic Characteristics And Metabolic Enhancement Mechanism Of Cellulase Biosynthesis By Bacillus Subtilis Z2

Bacillus subtilis is a potential production strain of cellulase and hemicellulase because of its complete system for secreting glycoside hydrolase.In this study,Bacillus subtilis Z2 isolated and purified from nature was used as the parent strain to study the effect of graphene oxide（GO）stress on cellulase biosynthesis,and the differentially expressed genes of lignocellulolytic enzymes,extracellular enzymes and enzyme synthesis-related signaling pathways were analyzed by transcriptomics,respectively.On this basis,a modular strategy of synthesis biology was used to construct NAD⁺biosynthesis and NADH regeneration modules in B.subtilis Z2,which significantly improved the cellulase production,and the regulatory mechanism of cellulase synthesis was revealed.The main research work of this paper is listed as follows.Firstly,the effect of different carbon and nitrogen sources on the cellulase activities of B.subtilis Z2 were studied.The result indicates that the activities of FPase（total cellulase）,CMCase（endoglucanase）andβ-glucosidase were 0.57±0.04,5.54±0.11 and1.80±0.08 U/m L,respectively,with the pretreated straw as carbon source and the yeast extract as nitrogen source.On this basis,the enzyme production conditions were optimized using the response surface method.The result demonstrates that the maximal cellulase activity was 5.64±0.15 U/m L under the conditions of 6%（w/v）carbon source and 0.6%（w/v）nitrogen source,pH 7.0 and 40℃.In addition,using B.subtilis Z2 crude enzyme solution and commercial xylanase to hydrolyze alkali-pretreated straw,it was found that the maximal reducing sugar concentration was 84.27±5.13 mg/m L at 48 h.In order to verify the continuous hydrolysis capacity of endoglucanase produced by B.subtilis Z2,the prokaryotic expression system was used to express the endoglucanase EG5Z（including the catalytic domain and carbohydrate binding module CBM3）and EG5Z-1（lacking the CBM3 module）.The enzymatic properties and enzymatic hydrolysis ability of lignocellulose were also analyzed.The results show that both EG5Z and EG5Z-1 possessed the capacity of continuous hydrolysis.Moreover,EG5Z had better thermal stability,stronger binding ability to insoluble substrates,and high reducing sugar concentration was obtained using synergistic enzymolysis of lignocellulose with xylanase.This work focused on the effect of GO stress on expression regulation of the lignocellulolytic enzyme genes in B.subtilis Z2.The result indicates that the activities of FPase（0.71±0.01 U/m L）,CMCase（6.74±0.14 U/m L）andβ-glucosidase（1.89±0.12U/m L）under 10μg/m L GO stress were increased by 0.36-,0.64-and 0.24-fold,respectively,while those of the control group were 0.52±0.02 U/m L,4.12±0.19 U/m L and 1.52±0.08 U/m L.To reveal the mechanism by which the expression of lignocellulolytic enzyme-related genes was regulated in B.subtilis subjected to GO stress,the transcriptome analysis of B.subtilis Z2 strain was performed.The results show that four genes（cal J,cha A,tca B and spc F）,which were related to intracellular Ca²⁺homeostasis and calcium signaling transduction,were upregulated in the 10μg/m L GO stress group.The use of Ca²⁺inhibitors（LaCl₃ and EDTA）and deletion of gene spc F further demonstrated that the overexpression of lignocellulolytic enzyme genes was regulated via calcium signaling in B.subtilis subjected to GO stress.Transcriptome data showed that the transcription levels of genes related to NAD（H/⁺）synthesis and electron transport chain were also upregulated in the experimental group.Therefore,a modular engineering strategy via four modules was adopted to redirect metabolic flux towards NAD⁺biosynthesis（module1-3）and NADH regeneration（module 4）in B.subtilis Z2,and the effect of intracellular NAD（H/⁺）level on the cellulase synthesis was also analyzed.It was found that upon assembling the four genes（ycel,nad V,nad M and mdh）in B.subtilis Z2,the intracellular NADH level was increased 13.09-fold,and the cellulase activity（10.95±1.78 U/m L）in the engineered strain was 1.04-fold higher than the control（5.37±0.37 U/m L）.The use of LaCl₃ and deletion of gene spc F further demonstrated that the expression of cellulase genes was regulated by intracellular NAD（H/⁺）level via calcium signaling pathway.