Cloning, Identification And Expression Of Cellulase Genes From Uncultured Microorganisms In Rabbit Cecum And Characterization Of The Expressed Products

Cellulose is the most abundant biomass in nature, composed of repeatingcellobiose units linked byβ-1,4- glucosidic bonds. Cellulose can be degraded toglucose through the synergistical hydrolysis of different families of cellulases,and glucose can be easily fermented into useful chemicals, such as ethanolwhich can be used as environmentally friendly bio-fuel. Due to the high cost andthe relatively low hydrolytic activity of the currently used cellulases, productionof fuel ethanol from lignocellulose has been run only in pilot plant and has notbeen commercialized yet. Therefore, a great deal of effort has being put into theexploitation of novel cellulases.Cellulase can be produced by a broad range of organisms includingmicrobes, plants and animals. The digestive tracts of herbivores harborsymbiotic microorganisms which help the hosts to digest the cellulosic feeds.Cecum is the most important digestive organ for some non-ruminants, such ashorse and rabbit. Symbiotic microorganisms in rabbit cecum produce fibrolyticenzymes to break down the plant cell walls. However, no study on cloningcellulase gene from the symbionts in rabbit cecum has been reported. The goalof this study was to clone novel cellulase genes from the microbes in rabbitcecum by an activity-based metagenomic approach, characterize the diversity of the cellulases in cecal system and try to search for cellulases with potentialapplication in biocatalysis.In this study, metagenomic DNA was isolated from the contents of therabbit cecum by direct lysis method without separating the microbial cells fromthe sample, purified by size exclusion chromatography using a columncontaining Sephadex G200 and PVPP. Analysis of the 16S rRNA genes revealedthat the metagenomic DNA extracted from the contents of the rabbit cecummainly contained the genomic DNA of Firmicutes, Proteobacteria andBacteroidetes. A metagenomic library that contained 3.25×10⁴ clones wasconstructed in Escherichia coli by cloning the purified DNA into a cosmidvector. The library had an average size of inserted fragment of 35.1 kb and atotal capacity of 1.14×10⁹ bp, and presented a good randomness of the clonedDNA.Functional screening was employed to search for positive clones expressingcellulase activities. Eleven independent clones expressing cellulase activities(four expressing endo-β-1,4-glucanase and seven expressingβ-glucosidase)were isolated.Subcloning and sequencing analysis identified 11 cellulase genes. Thepredicted products of these genes shared less than 50% identities and 70%similarities to cellulases in the databases, which indicated that all 11 cellulasegenes cloned from the rabbit cecum were novel. The high frequency of positiveclones isolated from the metagenomic library and the relatively low similaritiesof the genes' products to the known cellulases suggested that rabbit cecum is arich reservoir of novel cellulases.Sequence analyses indicated that all 4 endo-β-1,4-glucanases belonged toglycosyl hydrolase family 5 (GHF 5) and were predicted to have a GHF 5catalytic domain (cellulase domain) in their protein structures, and all 7β-glucosidases fell into glycosyl hydrolase family 3 (GHF 3) and were predictedto possess a catalytic GHF 3 domain and a GHF 3 C terminal domain. Phylogenetic analysis revealed that these cloned cellulases cluster into twoindependent groups seperated from the other known cellulases, but they arequite similar within the groups. The source bacteria of these genes remainedunknown.Crude enzymes of the 11 active clones were preliminarily characterized.Ten of the eleven cloned cellulases exhibited highest activities at pH 5.5～7.0and 40～55℃, a condition similar to that in the rabbit cecum. Only one13-glucosidase preferred to work in an alkaline condition (optimal pH: 7.5～8.5).All the four endo-β-1,4-glucanases could hydrolyze a wide range ofβ-1,4,β-1,4/β-1,3 orβ-1,3/β-1,6 linked polysaccharides. These results demonstratedthat the cloned enzymes had a good adaptation to the cecal system. Allendo-β-1,4-glucanases in this work could hydrolyze laminarin which is a glucanprimarily linked byβ-1,3 glucosidic bonds, whereas most of the GHF5endo-β-1,4-glucanases do not show this ability.One endo-β-1, 4-glucanase gene, umcel5G, and oneβ-glucosidase gene,umbgl3B, were overexpressed in E. coli and the purified recombinant enzymeswere characterized in more detail, pH and temperature optima of the twopurified recombinant enzymes were similar to those measured by using thecrude enzymes of the active clones, which confirmed the reliability of the dataobtained from the crude enzymes.Gene umcel5G was chosen to be expressed since its crude enzyme showedthe widest substrate range among the four cloned endoglucanases. Therecombinant Umcel5G was stable at pH 5.0～9.0 and temperatures below 55℃.The optimal pH and temperature of Umcel5G were 6.0～6.5 and 55℃,respectively. The purified recombinant Umcel5G cut randomly on the chain ofcello-oligosaccharides, revealing its endo-mode acting pattern. The activity ofthe recombinant Umcel5G was enhanced by CoCl₂, CaCl₂ and CrCl₂, andinhibited by CuCl₂, ZnCl₂, chelator EDTA and surfacant SDS. The Km and Vmax toward carboxymethyl cellulose (CMC) were 16.07 mg/ml and 417.5 U/mg protein, respectively.The recombinantβ-glucosidase, Umbgl3B, was stable at pH 4.5～9.0 andtemperatures below 35℃and showed the highest activity at pH 6.0～7.0 and 40℃. Umbgl3B was proved to be a typicalβ-glucosidase that hydrolyzedcellobiose to glucose and showed no activity toward cello-oligosaccharidecontaining more than 3 glucose molecules. The activity of the recombinantUmbgl3B was strongly enhanced by CoCl₂ and CrCl₂, and inhibited by CuCl₂,EDTA and SDS.Genes encoding cellobiohydrolase were not cloned in this work, and thepossible reasons were intensively discussed in the study.The enzymes cloned in this work represented at least some of the cellulasesoperating efficiently in the rabbit cecum. This is so far the first study on thecloning and the characterization of cellulase genes from the microbes in rabbitcecum, which helps to understand the properties, the diversity and the potentialbiotechnological applications of the cellulases in the cecal system of rabbit.