Preliminary Study On Cellulases And Hemicellulases From Symbiotic Bacteria Harbored In The Gut Of Batocera Horsfieldi Larvae

Cellulases and hemicellulases have great potential for application in various industries, including the feed industry. The larvae of longhorned beetles (Cerambycidae) can develop deep within sapwood, and are thought to be a potential source harboring diverse and novel symbiotic microorganisms producing cellulases and hemicellulases. So far enzymes from symbiotic microorganisms in the gut of longhorned beetles still remain extremely limited.Using the homologous cloning strategy with degenerate primers, 26 gene fragments encoding cellulases or hemicellulases were cloned from the 34 bacterial strains isolated from the gut of Batocera horsfieldi larvae. These cellulases or hemicellulases are glycosyl hydrolase family (GH) 3β-glucosidases, GH 48 cellobiohydrolases, GH 10 and GH 11 endo-xylanases, GH 52β-xylosidases, GH 67α-glucuronidases, GH 16β-1,3(4)-endoglucanases, GH 2β-galactosidases, and GH 36α-galactosidases. Environmental DNA of luminal contents (TNG) of B. horsfieldi larvae was extracted and used as the template to clone gene fragments encoding cellulases or hemicellulases. As a result, some gene fragments related with GH 5 and GH 45β-1,4-endoglucanases, GH 48 cellobiohydrolases, GH 10 endo-xylanases, GH 39 and GH 52β-xylosidases, and GH 67α-glucuronidases were isolated. Furthermore, gene fragments of GH 5 and GH 10 were chosen for diversity analysis. Their Shannon indexes were 2.68 (GH 5) and 2.09 (GH 10), respectively. Phylogenetic analysis indicated that the GH 10 and GH 5 protein fragments from TNG were novel and diverse, and present at limited breadth of bacterial phyla but much greater in depth in terms of genera and species. Significant difference (p < 0.001) between GH 10 or GH 5 protein fragments from TNG and that from the wormhole niche of Monochamus alternatus, and among GH 5 protein fragments from gastrointestinal habitats (TNG, termite gut or rumen) revealed environment-specific distributions of GH 10 and GH 5 proteins in TNG.In this study, the conventional thermal asymmetric interlaced-PCR (TAIL-PCR) technique was improved for efficacy and ready-to-use, and GC TAIL-PCR specific for GC-rich genes was developed. As a result, a chromosomal segment containing 25 genes related to xylan and xylose metabolism and showing xylanolytic activity, 6 cellulases or hemicellulases encoding genes, and 2 xylanases encoding genes (xynA119 and xynB119) with 67.8% and 67.3 GC content, respectively, were obtained. Among them, 11 enzymes were selected for further study, including the GH 16β-1,3(4)-endoglucanase GluA111 from Sphingobacterium sp. TN111; GH 3β-glucosidase BglA49 from Serratia sp. TN49; GH 10 endo-xylanases XynA19 and XynB19, GH 43 arabinofuranosidase Gh43A19, and GH 36α-galactosidase GalA19 from Sphingobacterium sp. TN19; GH 10 endo-xylanase XynA119 and GH 11 endo-xylanase XynB119 from Streptomyces sp. TN119; GH 2β-galactosidase BgaA105 and GH 36α-galactosidase GalA105 from Sphingobacterium sp. TN105; and GH 36α-galactosidase GalA17 from Flavobacterium sp. TN17. The deduced amino acid sequences of these 11 enzymes showed the highest identity of 38.8–80.1% with their counterparts in public databases. Phylogenetic analysis revealed that endo-xylanase XynA19 andα-galactosidase GalA17 were more closely related to their counterparts from symbiotic bacteria in the gastrointestinal habitats than to that from bacteria belonged to the same phylum. The GH 3β-glucosidase BglA49 had no such characteristic. GH 36α-galactosidases from symbiotic bacteria shared two putative catalytic motifs KWD and SDXXDXXXR instead of KXD and RXXXD motifs of knownα-galactosidases. Site-directed mutagenesis indicated four essential amino acid residues (D480 in KWD; S548, D549, and R556 in SDXXDXXXR) involved in theα-galactosidase activity, among which D480 and D549 were the putative catalytic residues.The eleven genes mentioned above were inserted into pET vector and expressed successfully in Escherichia coli BL21 (DE3). All recombinant enzymes showed activity except for gluA111. The recombinant enzymes XynA119, XynB119, XynA19, BglA49 and GalA17 were purified to electrophoretic homogeneity by Ni2+-NTA metal chelating affinity chromatography and subjected to biochemical characterizations. All of the five enzymes showed optimal activity at neutral pH (pH 5.5–7.5) and low temperature (35–60°C), and were more stable at neutral pH and temperatures lower than 50°C. These common features are putatively dependent on the living environment and gut physiological environment of B. horsfieldi larvae. GalA17 was resistant to various proteases (trypsin, collagenase, proteinase K andα-chymotrypsin). XynB119 remained stable over a broad pH range (pH 1.0–11.0), and had strong protease resistance (pepsin, trypsin, collagenase, proteinase K andα-chymotrypsin). XynA19 remained active at low temperatures (retained ~40% xylanase activity at 10°C), and was more stable than known low-temperature endo-xylanases at 40°C. BglA49 was also active at low temperature (remained >20% activity at 10°C). Compared to the thermophilic and mesophilic counterparts, XynA19 and BglA49 had fewer hydrogen bonds and salt bridges. These properties suggest that GalA17, XynB119, XynA19 and BglA49 have various potential applications in food and feed industries.It is the first report to explore the diversity, phylogenesis, structure and properties of cellulases and hemicellulases from symbiotic bacteria harbored in the gut of B. horsfieldi larvae. The information improves our knowledge of cellulases and hemicellulases in the gut of wood-feeding insect and promotes the development of novel industrial enzymes.