Isolation Of A β-Glucosidase Gene From Metagenome Of Rabbit Cecum

With the population explosion,the global is facing a more and more seriouse food crisis.At the same time,the fossil energy sources,such as petroleum and coal,become increasingly exhausted, which makes it imminent for the human being to discover alternative and renewable sources.With the fetures of biomass abundance,regeneration,and degradation,natural cellulose is expected to be a promising source for solving these problemes.At the present time,the mehthods used for degrading cellulose in the industry are bothered with the problems such as high cost,energy consuming,and serious pollution to enviroment.However,these problems can be alleviated with the biodegradable approach by adding cellulase.No matter at home or abroad,the biodegradable apprpachs of using cellulase posseses the defects of low activity and little specieses.Thereforce the research and exploition of novel cellulases with higher activity,wider arange of substrates,and higher endurance against temprature and pH,is very important for industry and society.In the process of cellulose degrading,β-glucosidase can hydrolyze celiobiose or longer chains from the non-reducing by glucose units,and thusβ-glucosidase can prevent the cellobiose from accumulation.In the other word,β-glucosidase is the rate-limiting enzyme in the process of cellulose degradation.More than 99%of the microorganisms in nature are uncultureable,which are named "uncultrued microbes".Within these uncultured microbes,uncountable useful gnens remains to be exploitated. With the development of mocelular biology technology in recent years,an approach of research and development to uncultrued microbes has been bulit,which is metagenome research.Usually,the metagenome research starts with the extraction of total DNAs,which is called metagenomic DNA,from all the organisms within the sample.The obtained metagenomic DNA is subsequently cloned into certain of vectors to construct metagenemic libraries that are sreened for genes of interest by DNA-DNA hybridization,polymerase chain reaction(PCR)or activity screening.The digestive tracts of some kinds of animals are reported as a good resource for high efficient cellulase,since a lot of microbes' inhabitting there can produce lots of cellulase to digest the cellulosic feed.Such digestive tracts include the rumen of cow and sheep,the cecum of host and rabbit,and the intestinal tract of longicorn and termite.By an activity-based metagenomic approach,we cloned a novel cellulase gene from the microbes in rabbit cecum,named nglu007,and tried to analyze its enzymology properties.1.Extraction of Metagenomie DNA from rabbit cecumTo find a better and more efficient way for extracting microbial metagenomic DNA,we used three different methods after the cells collected from cecum contents.Then,we determined the OD_260/280.of these DNA samples,and digested them with various restriction endonucleases.The results displayed that the metagenomic DNAs obtained with PVPP-electroelution were of highest purity,relatively higher amount,and a better restriction endonucleases digestion;those obtained with phenol and choloroform were of the lowest purity,quitely low amount,and non-restriction endonucleases digestion;and obtained with genomic extraction kit,the metagenomic DNAs were of related high purity,the lowest content,and a bad reatriction enzyme digestion.Thus,we continue our research with DNA extracted by PVPP-electroelution.2.Construction of metagenomie DNA libraryAfter being digested with Sau3A I,the DNA fragments from 2.0 to 4.5 kb were recovered, subsequently cloned onto vector PUC118,which had been digested with BamH I and had been phosphorylated previously.Recombinant vectors were then transformated into competent cells of Escherichia coli to construct a metagenomic library.In our study,we successfully constructed a cecum metagenomic library containg about 9,000 transfromants containing inserted DNA fragments.3.Gene cloning and partially characterization of aβ-glucosidase gene After screeningβ-glucosidase-producing clones from the library by theβ-glucosidase screening plate,we separated aβ-glucosidase gene,named as nglu007.The ORF of nglu007 is 282 bp, encoding a 93 amino acidis protein with a predicted molecular of 10,545.90 Da.The GC percentage of the deduced protein was 60.3%,and its isoelectric point(pI value)was 5.30.The encoding protein possesed a membrane spaning domain between the 76th residue and the 81st residue,but no signal peptide sequence was found.After using the BLAST software,we found that no knownβ-glucosidase genes deposited in the GenBank showed high identity with nglu007.Therefore,we predicted that nglu007 might be a novelβ-glucosidase gene.Analysis of the cellulase activity expressed from E.coli showed that its optimum temperature and pH were 50℃and pH9.0,respectively.In neutral or basic enviroments,its activity kept ralatively high activity;while,at high temprature its activity kept high but unstable.Different metal ions showed different effects on its activities.Mg²⁺,Zn²⁺,and Mn²⁺enhanced its activity,and Ca²⁺, Cu²⁺had no obvious effect on its activity.While Na⁺,K⁺,Fe³⁺,EDTA,and urea caused obviouse inhibition.This enzyme had no specific subtract,and it showed activity towards a wide range of subtracts.It had a strong role on lactose and maltose,on chitin lower,and with invariable activity on filter paper,esculin and cellulose powder.By constructing a metagenomic library generated from microbes in rabbit cecum,we obtained a novelβ-glucosidase gene nglu007.The high-buck-tolerance characteriaztion of encoding product indicates that it has a potential application in the production of buck-tolerance enzyme.