| Wood-feeding termites can efficiently degrade lignocellulose with the aid of their symbionts and abundant cellulases and hemicellulases.In the intestinal tracts of higher or lower termites,there are a variety of symbiotic microorganisms,which have the ability to produce cellulases or hemicellulases and may play important roles in the degradation of lignocelluloses.With the application of high-throughput sequencing techniques,researchers have found numerous cellulase and hemicellulase genes from wood-feeding termites.However,until so far,very few stuides of the enzyme activities and characteristics have been made.In this study,the diversity of cellulase and hemicellulase genes from gut microorganisms of a lower termite,Reticulitermes chinensis Snyder and a higher termite,Nasutitermes hainanensis were investigated.Among them three cellulase and hemicellulase genes have been heterologous expressed and characterized.All of the results form the basis for further clarifying the degradation mechanisms of lignocelluloses in wood-feeding termites and make significance for the illustration of the symbiotic mechanisms between termites and their gut microorganisms.The main results are as following:1.The whole genome of a Verrucomicrobia strain isolated from Reticulitermes chinensis was sequenced and analyzed.The genome of Opitutaceae sp.TSB-47 is 7.29Mb in size,having 5520 coding genes,contains 62.88mol%G+C.Strain TSB-47 genome contains a number of cellulase genes,including 3 endoglucanase genes,14 glucosidase genes;It also contains a variety of hemicellulase genes,4 endo-?-1,4-xylanase genes,3 ?-xylosidase genes and 41 galactosidase genes.Among them 7 genes were heterologous expressed and only a xylanase Xyn1959 obtained soluble protein.Phylogenetic analysis showed that the Xynl959 gene was most closely related to Endo-?-1,4-xylanase gene detected from Pedosphaera parvula(62%).Xyn1959 exhibited maximal activity at pH 5.2 and retained more than 60%of the maximal activity between pH range of 4.4 to 7.6.The optimum temperature of the purified Xyn1959 was 35? and retained more than 50%of the maximal activity between 15? to 45?.The Xyn1959 revealed 37.91 U/mg in specific activity.The Km and Vmax values were 2.40 mg/ml and 65.03U/mg,respectively.The final hydrolysis products of Xyn1959 on beechwood xylan were mainly xylobiose(X2),xylotriose(X3),and xylotetrose(X4).These results indicated that the Xyn1959 was an endo-type xylanase.2.With a set of designed primer that based on GHF7 genes obtained from Reticulitermes chinensis,GHF7 cellulase gene libraries were constructed with the intestinal DNA and cDNA as template,respectivelly.7 GHF7 DNA sequences and 6 cDNA sequences were obtained.All these genes were 1356bp in length and without intron.Phylogenetic analysis showed that GHF7 cellulase genes were most closely related to symbiotic flagellates in R.chinensis.In comparison to these model structures by ClustalW2 multiple alignments,RcGHF7-RT33 is categorized as an exoglucanase.However,recombinant version of RcGHF7-RT33 exhibited(3-glucosidase activity instead of exoglucanase(CBH).The optimal reaction conditions of RcGHF7-RT33 were around pH 5.2 and 45?.RcGHF7-RT33 retained more than 70%of the maximal activity between pH range of 3.2 to 8.0,showing that it is an acidic enzyme.The RcGHF7-RT33 revealed 0.205 U/mg in specific activity.The Km and Vmax values were 0.4375 mg/ml and 1.49U/mg respectively.3.A fosmid metagenomic library of whole-gut from Nasutitermes hainanensis was constructed.According to the manual of fosmid library production kit,about 2000 clones were acquired.The insert size of clone ranged from 20 to 60 kb,with average insert size of 38.7kb.Based on different substrates,7 clones with the activity of endo-1,4-?-D-glucanase had been acquired and the representative clones were sequenced.Based on PCR amplification of designed degenerate primers,EG gene libraries were established with DNA extracts of the whole-guts,hindgut P3 region and P4 region of Nasutitermites hainanensis.From which five EG genes that belonged to GHF5 were obtained,which were named NSEG5a,NSEG5b,NSEG5c,NSEG5d,NSEG5e,respectively.The genes were 1152bp in length,encoding 383 amino acids.According to the RFLP analysis,the five genes were mainly from symbiotic bacteria of hindgut P3 region and P4 region,suggesting that hindgut microbes play an important role on lignocellulose degradation.The optimum temperature and pH of the purified NSEG5a was 35? and 6.0,respectively.NSEG5a was very stable over a wide pH range at pH 4.4 to 8.4 for 3 days at 4?.The NSEG5a revealed 3.75U/mg in specific activity.The Km and Vmax values on carboxymethyl cellulose(CMC)substrate were 1.72 mg/ml and 4.68 U/mg,respectively.The hydrolysis pattern analysis by TLC indicated that the main degradation products of cellotetraose(G4)and cellopentaose(G5)by NSEG5a were cellobiose(G2)and cellotriose(G3).In contrast,NSEG5a exerted no action on cellobiose and cellotriose.These results indicate that the NSEG5a was an endoglucanase. |
PDF Full Text Request