Isolation of a Multi-functional Endogenous Cellulase Gene from Mollusc, Ampullaria crossean

Key Laboratory of Proteomics, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China.
Sheng wu hua xue yu sheng wu wu li xue bao Acta biochimica et biophysica Sinica 11/2003; 35(10):941-6.
Source: PubMed


The cellulase genes of some animals, most coding for endo-beta-1,4-glucanases, were found and cloned. There has been no reports about genes encoding exo-beta-1,4-glucanase or endo- -1,4-xylanase from animal. Here we cloned the cDNA of a cellulase designated as EGX from mollusc, Ampullaria crossean, and expressed it in Pichia pastoris for the first time. The cellulase EGX is a multi-functional beta cellulase with the activities of exo-beta-1,4-glucanase, endo-beta-1,4-glucanase and endo-beta-1,4-xylanase. The opening reading frame of EGX cDNA is 1185 bp and encodes 395 amino acids. The EGX gene can also be amplificated from the genomic DNA by PCR, which verified the endogenous origin of this gene. This EGX gene was the first multi-functional cellulase gene that was directly isolated from animals.

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    • "on the basis of hydrophobic cluster analysis for amino-acid sequences (Henrissat et al., 1989; Henrissat, 1991; Henrissat and Bairoch, 1993). Invertebrate cellulases are enrolled in five families, i.e., GHF5 (nematodes: Globodera rostochiensis and Heterodera glycines; Smant et al., 1998), GHF6 (sea squirt: Ciona savignyi; Matthysse et al., 2004), GHF9 (termite: Reticulitermes speratus, Watanabe et al., 1998; abalone: Haliotis discus hannai, Suzuki et al., 2003; sea urchin: Strongylocentrotus nudus, Nishida et al., 2007), GHF10 (freshwater snails: Ampullaria crossean, Wang et al., 2003; Pomacea canaliculata, Imjongjirak et al., 2008), and GHF45 (bivalve: Mytilus edulis, Xu et al., 2001; freshwater snail: A. crossean, Guo et al., 2008; freshwater bivalve: Corbicula japonica , Sakamoto and Toyohara, 2009). Among these cellulases, GHF9-type cellulases appear to be most widespread in nature and well characterized (Davison and Blaxter, 2005). "
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    ABSTRACT: The common sea hare Aplysia kurodai is known to be a good source for the enzymes degrading seaweed polysaccharides. Recently four cellulases, i.e., 95, 66, 45, and 21 kDa enzymes, were isolated from A. kurodai (Tsuji et al., 2013). The former three cellulases were regarded as glycosyl-hydrolase-family 9 (GHF9) enzymes, while the 21 kDa cellulase was suggested to be a GHF45 enzyme. The 21 kDa cellulase was significantly heat stable, and appeared to be advantageous in performing heterogeneous expression and protein-engineering study. In the present study, we determined some enzymatic properties of the 21 kDa cellulase and cloned its cDNA to provide the basis for the protein engineering study of this cellulase. The purified 21 kDa enzyme, termed AkEG21 in the present study, hydrolyzed carboxymethyl cellulose with an optimal pH and temperature at 4.5 and 40°C, respectively. AkEG21 was considerably heat-stable, i.e., it was not inactivated by the incubation at 55°C for 30 min. AkEG21 degraded phosphoric-acid-swollen cellulose producing cellotriose and cellobiose as major end products but hardly degraded oligosaccharides smaller than tetrasaccharide. This indicated that AkEG21 is an endolytic β-1,4-glucanase (EC A cDNA of 1013 bp encoding AkEG21 was amplified by PCR and the amino-acid sequence of 197 residues was deduced. The sequence comprised the initiation Met, the putative signal peptide of 16 residues for secretion and the catalytic domain of 180 residues, which lined from the N-terminus in this order. The sequence of the catalytic domain showed 47-62% amino-acid identities to those of GHF45 cellulases reported in other mollusks. Both the catalytic residues and the N-glycosylation residues known in other GHF45 cellulases were conserved in AkEG21. Phylogenetic analysis for the amino-acid sequences suggested the close relation between AkEG21 and fungal GHF45 cellulases.
    Full-text · Article · Aug 2014 · Frontiers in Chemistry
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    • "is required for complete de-polymerization of cellulose to glucose. These enzymes have been isolated from bacteria and fungi [1], plants [2], molds [3], microbes from animal intestines [4], and herbivorous invertebrates such as arthropods [5] [6] [7] [8] [9] [10], nematodes [10], mollusks [11] [12] [13] [14] [15] [16] and an echinoderm [17]. "
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    ABSTRACT: We previously identified the cellulase SnEG54 from Japanese purple sea urchin Strongylocentrotus nudus, the molecular mass of which is about 54kDa on SDS-PAGE. It is difficult to express and purify a recombinant cellulase protein using bacteria such as Escherichia coli or yeast. In this study, we generated mammalian expression vectors encoding SnEG54 to transiently express SnEG54 in mammalian cells. Both SnEG54 expressed in mammalian cells and SnEG54 released into the culture supernatant showed hydrolytic activity toward carboxymethyl cellulose. By using a retroviral expression system, we also established a mammalian cell line that constitutively produces SnEG54. Unexpectedly, SnEG54 released into the culture medium was not stable, and the peak time showing the highest concentration was approximately 1-2days after seeding into fresh culture media. These findings suggest that non-mammalian sea urchin cellulase can be generated in human cell lines but that recombinant SnEG54 is unstable in culture medium due to an unidentified mechanism.
    Full-text · Article · Apr 2010 · Biochemical and Biophysical Research Communications
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    • "Gut extracts of freshwater molluscs and crustaceans were found to strongly hydrolyze cellulose (Monk, 2006). Endogenous cellulase genes have been cloned from clam Corbicula japonica (Sakamoto et al., 2007), abalone (Suzuki et al., 2003), snail (Wang et al., 2003), mussel (Xu et al., 2001) and crayfish (Byrne et al., 1999) proving direct digestion of cellulose by those aquatic invertebrates. Digestive enzymes could be used as a complementary tool for determining which dietary components are effectively metabolized (Johnston and Freeman, 2005) but it remains insufficient to determine to what extent these components contributed to the organism's diet. "
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    ABSTRACT: a b s t r a c t We examined the feeding niche of four species of molluscs by analysis of their stable isotope signatures and cellulase activities to determine if they could utilize terrestrial organic matter. The molluscs and potential food sources were collected from the upper, middle and lower estuary of the Yura River from spring 2007 to winter 2008. All species showed positive cellulase activity which highlighted their potential to digest terrestrial organic matter. Consumption and assimilation of terrestrial organic matter by estuarine molluscs however varied spatially and temporally, reflecting species-specific differences in feeding niche and in response to variations in food availability in the estuary. Thus, terrestrial primary production in the catchment area supports secondary production of molluscs in the Yura River estuary.
    Full-text · Dataset · Feb 2010
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