The Studies On The Endo-β-1,4-glucanases From The Mollusca, Ampullaria Crosseans

Cellulose is the most abundant renewable biopolymers on earth which is a β-1,4 linked linear polymer of glucose units. The traditional held view of cellulose digestion in higher animals and invertebrates has been that gut microbes confer the ability to degrade cellulose. In 1998, this traditional view was challenged by two reports of endogenous animal endo-β-1,4-glucanase genes from plant-parasitic nematodes and a termite. Since these discoveries, a number of other animal cellulase genes, such as, crayfish, abalone, beetle, and so on, have been reported and the researches of animal celluase have attracted much interest. We purified three endo-β-1,4-glucanases— EG65, EG45 and EG27, from the gastric juice of the herbivorous mollusca, A. crosseans by a series of column chromatography and studied the properties of these enzymes in relative detail in which EG65 and EG45 were glycoproteins.Further studies were focused on the endo-β-1,4-glucanase of EG65. Base on the N-terminal amino-acid sequence of intact enzyme, internal amino-acid sequences and two conserved regions of endo-P-l,4-glucanases in the abalone Haliotis discus hannai and the red claw crayfish Cherax quadricarinatus, 3 groups of endo-β-1,4-glucanase cDNAs, that is, eg65-a (eg65-a1), eg65-b and eg65-c (including three and four cDNAs with >98% identity in sequence of amino-acids, respectively), were amplified by the polymerase chain reaction from the A. crosseans stomach tissue cDNA library. The coding region gave an amino-acid sequence of 713,723 and 722 residues, respectively, including the initiation methionine. The analysis of amino acid sequences deduced from the genes indicated that they all consisted of a cellulose-binding domain (CBD) family Ⅱ and a catalytic domain belonging to glycosyl hydrolase family 9 from N-terminal to C-terminal. The N-terminal region of 16-18 residues in the deduced sequence was regarded as the signal peptide.The sequences encoding intact enzymes except for the putative signal peptide (corresponding to recombinant enzymes EG65-a-16, EG65-b-16, EG65-C-16) and enzymes except for the putative signal peptide and CBD (corresponding to recombinant enzymes EG65-a-137, EG65-b-148 and EG65-C-147) of eg65-a1,...