| The presence of lignin hinders the degradation of roughage and its utilization by ruminants,the buffalo rumen microorganisms can degrade and utilize lignin in prelaboratory studies,and the isolated lignin-degrading bacteria contained the gene encoding laccase(lignin-degrading enzyme).In order to investigate the potential of buffalo rumen microorganisms for the degradation of lignin and the enzymatic properties of clonally expressed laccase derived from buffalo rumen bacteria,we first collected rumen contents from three adult fistulated buffaloes and examined microorganisms,enzymes,and pathways associated with lignin degradation in buffalo rumens by macro-genome sequencing;By analyzing the whole genome sequences of two lignin-degrading bacteria isolated from buffalo rumen in the previous stage,Ochrobactrum pseudintermedium and Bacillus cereus,five laccase genes were mined,and recombinant expression bacteria were constructed using Escherichia coli BL21(DE3)strain as the host to investigate the enzymatic properties of recombinant laccase.The objective is to provide scientific parameters for developing rumen-derived enzymes for lignin degradation and researching buffalo roughage tolerance mechanisms.The main findings were as follows:1.Metagenomics analysis revealed that the buffalo rumen contains lignin-degrading bacteria,enzymes and metabolic pathways that can be greatly exploited.(1)The major microorganisms responsible for lignin degradation in the rumen of buffalo originate from Bacteroidetes,Prevotella,Butyrivibrio,Fibrobacter,Eubacterium,etc.(2)Glycoside hydrolase is the most abundant carbohydrate enzyme in the rumen of buffalo,and there were a variety of enzymes found that degrade lignin,mainly including laccase,peroxidase,ferulic acid esterase,etc.(3)Carbohydrate metabolism and amino acid metabolism are the most important metabolism in the rumen of buffalo,and several metabolic pathways related to lignin degradation have been identified,mainly including lignin catabolic metabolic process,phenylalanine metabolism,benzoate metabolism,aromatic compound metabolic process,etc.2.Sodium lignosulfonate can be degraded by recombinant laccase Lac3833 of Bacillus cereus origin from buffalo rumen.Five prokaryotic expression strains were constructed by ligating two laccase(lac01691,lac03031)genes from Ochrobactrum pseudintermedium and three laccase(lac3833,lac1866,lac118)genes from Bacillus cereus into the p ET28a(+)vector and finally transformed into BL21(DE3)strain,respectively.Studies on clone expression and enzyme properties revealed that:(1)Among five recombinant laccases induced by different isopropyl-β-D-thiogalactoside(IPTG)concentrations and temperature gradients,only Lac01691 and Lac3833 were well soluble and highly expressed.Lac01691 was optimally expressed at 16℃ and 0.4 mmol/L IPTG,and Lac3833 was optimally expressed at 16℃ and 0.2 mmol/L IPTG.Optimal imidazole elution concentrations for Lac01691 range from 60 mmol/L to 180 mmol/L,and for Lac3833 range from 40mmol/L to 160mmol/L.(2)Compared with Lac01691,Lac3833 had significantly higher enzyme activity,Lac3833 exerted maximum enzyme activity at 40℃ for all three substrates: 2,2’-Azinobis-(3-ethylbenzthiazoline-6-sulphonate)(ABTS),2,6-dimethoxyphenol(2,6-DMP)and guaiacol.A relative activity of Lac3833 was highest at p H 4.5 when ABTS and 2,6-DMP were used as substrates,and at p H 5.5 when guaiacol was used as substrate.The stability of Lac3833 is higher at 40℃ than 45℃,and in p H 5.0 buffers than p H 4.0 and p H 4.5 buffers,and sodium lignosulfonate exhibited the highest degradation rate at 8.39% after 24 h reaction.,In summary:(1)The metagenomics analysis revealed that the buffalo rumen lignin-degrading microorganisms mainly came from Bacteroides,Prevotella,and Butyrivibrio etc.,which contains genes coding for laccase,oxidase,and other enzymes that degrade lignin.(2)A laccase of Bacillus cereus of buffalo rumen origin,expressed by BL21(DE3)strain as a heterologous host,is well soluble and highly expressed,temperature and p H stability are good and sodium lignosulfonate can be degraded using it. |
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