| Miscanthus sinensis is one of the most potential fiber producing energy plants at present.The rumen is one of the most efficient cellulose degradation systems in nature,which is rich in cellulose degradation microorganism and its encoded cellulase.It is one of the important ways to effectively solve the biodegradation of the energy crop and accelerate its development and utilization by intensive study of the Miscanthus degradation mechanism in the rumen microorganism and searching for the new gene resources for high efficiency degradation of cellulose.β-glucosidase play a key role in the glycosylated hydrolysis of cellulose.β-glucosidase Unglu135B12 is a highly active beta glucosidase from the metagenomic library of miscanthus domesticated cattle.However,it has a poor thermal stability.In this paper,the rumen microorganism of mixed feed(Rumen1)and miscanthus domesticated cattle(Rumen2)was used to study the microbial community structure,functional genes and the composition of cellulase in the rumen microorganism of the domesticated cattle by high throughput sequencing technology.Meanwhile,β-glucosidase Unglu135B12 as the research object,by the technique of error prone PCR,site-directed mutation and computer aided simulation,for the thermal stable molecular modification,and provide a reference for the study of molecular modification in vitro for microbial sources of beta-glucosidase.The results of high throughput sequencing of the cattle rumen metagenomic showed that Rumen1 obtained a total of 10.86G data,GC content of 50.64%,assembly of 244320 Contigs,and 659076 ORFs.Rumen2 obtained 11.65G data volume,GC content 50.29%,assembled 282938 Contigs,and predicted 764177 ORFs.Species annotation analysis showed that the rumen was rich in cellulose degrading microorganisms,and the species diversity of Rumen1 was higher than that of Rumen2.Proteobacteria,Bacteroides and Firmicutes were the dominant bacteria in the rumen microorganisms.The species abundance of Bacteroides and Firmicutes of Rumen1were higher than that from the Rumen2.The abundance of Acinetobacter and przewalskii in Rumen2 were higher than that in Rumen1.Functional gene annotation showed that rumen microbes were mainly involved in carbohydrate metabolism,amino acid metabolism and energy metabolism.Rumen1 and Rumen2 were different in the abundance of functional genes in glycolysis,the KREBS cycle,pentose phosphate pathway,methane metabolism and nitrogen cycle.The carbohydrate active enzyme annotation analysis showed that Rumen1contains 5070 glycoside hydrolases(GHs),3254 glycosyltransferase(GTs),179polysaccharide cleft synthase(PLs),2079 carbohydrate esterase(CEs),313 auxiliary oxidoreductase(AAs),2140 carbohydrate binding module(CBMs),and 255 dockerin,54 Cohesin and 231 S-layer homologous domains,a total of 13575 carbohydrate active enzymes.Rumen2 contains 6847 GHs,4042 GTs,269 PLs,2608 CEs,312 AAs,3033 CBMs,253 Dockerin,60,and 205.GH family analysis showed that GH5,GH9,GH44,GH45 and GH48 played a major role in cellulose degradation.The abundance of GH1,GH8,GH9,GH10,GH11,GH30,GH35,GH42,GH57,GH74,GH42,GH57,GH74,and GH124 of Rumen2 is more than 1.5 times higher than that in Rumen1.10new genes of the rumen metagenomic GH3 family were cloned,and 6 genes of them showed theβ-glucoside activity.The mutant library was constructed by the error prone PCR technology,and the mutant beta glucosidase ep-mut21(R72Y/N273A/V520P)and ep-mut48(W232F/T389G)were screened to improve the thermal stability.The optimum temperature for ep-mut21 is 46.8°C,the optimum pH is 5.8,the Km value is 0.142mmol/L,the catalytic efficiency is increased by 2.7 times,the 50°C half-life is increased by 2.5 times(14.6 min),and the enzyme activity is increased by 2.6 times(5.72×10~3U/mg).The optimum temperature of ep-mut48 is 43.2°C,the optimum pH5.2 and the Km value are 0.207 mmol/L,the catalytic efficiency is increased by 1.9times,the 50°C half-life reaches 13.5 min,and the enzyme activity is increased by2.1 times(4.62×10~3U/mg).The mutant D86Q,K191D and W271Y with better thermal stability than wild type were screened by site directed saturation mutagenesis.The optimum temperature of mutant D86Q is 46.8°C,the optimum pH5.2 is 2.7times,the catalytic efficiency is increased by 2.7 times,the 50°C half-life is 14.6 min,and the enzyme activity reaches 4.84×10~3U/mg.The optimum temperature of mutant K191D is 46.8°C,the optimum pH5.2,50°C half-life is 11.2 min,enzyme activity 1.95×10~3U/mg.The optimum temperature of W271Y was 46.8°C,the optimum pH5.8 and Km value were 0.143 mmol/L,the catalytic efficiency increased by 1.5 times,the half-life of 50°C was 8.9 min,and the enzyme activity reached 2.86×10~3U/mg.After the mutation of the nucleophilic group D270 and acid/base group E477 alanine,the enzyme activity almost lost and the mutant was glycine,the affinity of the mutant enzyme and substrate was enhanced,the catalytic efficiency was reduced,and the activity of the enzyme was reduced.The mutant structure analysis showed that the thermal stability of the mutant ep-mut21 may be related to the change of the potential of R27Y,the spatial structure of N273A and the rigidity of V520P.The increase of the thermal stability of the mutant ep-mut48 may be related to the caption-pi bond formed by W232F,the hydrophobicity of T389G,and the increase of the thermal stability of K191D and W271Y.It is related to the action of hydrogen bond.In conclusion,the rumen contains enriched in lignocellulose degradation microbes and enzymes,miscanthus domesticated changed microbial community composition and the abundance of cellulose enzyme,which provides guidance for the screening of enzyme genes with potential for industrial application.The modified Unglu135B12 recombinase has improved thermostability and maintained good enzyme activity,providing a basis for industrial production and application. |
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