| Chitin as one of the most abundant natural polymeric compounds, is a linear chain polymer which is connected by N-Acetyl-β-D-glucosamine throughβ-1,4-glucosidic bond. Chitinase is widely spreading in animals, plants and microbes, and has a function of hydrolyzing chitin into chitin oligosaccharide and N-Acety glucosamine. Recently, as the wide application of degradation products from chitinase in medicine, food, agriculture, environmental protection, paper making, and chemistry, chitinase receives much attention as an important tool for chitin oligosaccharide.Many organisms capable of producing chitinase were screened, isolated, and purified both at home and abroad. However, Chitinases from fungal fermentation have many limitations in commercial process. Following the development of Protocols in Molecular Biology, induction, production and use of chitinase from microorganism is not met to the requirements of human. The process of encoding for the enzyme and expressed in the source at a high effective and low cost way, is undertaking. So, it's ideal that commercial process of chitinase, improved the activity and enhanced the production can be achieved using a method of recombinant microorganisms.Thermoascus aurantiacus var. aurantiacus is a wide distributed thermophilic fungus and a higher growth upper-limitation temperature, which can grow well up to 40-50 degrees. Degenerate primers based on the conserved sequences of the fungi chitinase and a chitinase gene named Chit-TAA was isolated for the first time from T. aurantiacus var. aurantiacus through RT-PCR and Tail-PCR. The full-length of DNA 1824bp contained an ORF of 1191bp encoding 396 amino acids. Accession number of the nucleotide sequence is GU338053 in the GenBank database. Sequence analysis of the deduced amino acid sequence revealed high homology with the catalytic domains of the Glycoside hydrolase family 18, contained 2 conserved motifs which were related with catalytic activity of chitinase. Among these conserve sequences, the invariant carboxylic amino acid residue Asp and Glu have been proved essential in the acid-base catalytisis of glycoside hydrolase of family 18.Chit-TAA was expressed in Pichia pastoris GS115. Gene recombination was used to construct the expression plasmid and then the plasmid was transformed into P. pastoris GS115. After screening for His+Mut+ transformants on MD and MM plates, PCR analysis of Pichia integrants and G418 screening determined the multicopy integrants to induce by methanol. These integrants were used to analyze expression levels every 24 hours of interest protein and determine the engineering strains with high expression level, such as GS-Chit-176.After six days induction using methanol the transformant strain had the highest efficient production and the expressing level was 0.433mg/mL. The activity of expressed protein was 7.25U/mL. The molecular mass of a single band of the enzyme was estimated to be 43.9 kDa. The optimum temperature of the enzyme was 60℃and its maximal activity was at pH 8.0. The enzyme was thermostable and could still keep 45% activity at 70℃after 30 min. The product was mainly chitobiose from the hydrolyzation of colloidal chitin by chitinases. To some extent, the chitinase revealed antifungal activity against the growthing of Alternaria alternata and Fusarium moniliforme and the sporegermination of Bipolar is maydis.The gene Chit-TAA was expressed in P. pastoris and the expression products had the activity of Chitinase. It implied the expression chtinase could be applied widely for the chitin industry and biological fields. Therefore, we hope to reconstruct the gene or optimize the expression condition to obtain the yeast engineering strains suitable for industrial application.Chitinase comprises signal peptide coding sequence, catalysis domain (CD), serine/threorine domain, Chitinase binding domain (BD), and c-terminal extension region. The existence of BD increases the concentration of enzymes on the surface of substrate, which helps CD hydrolyze substrate. CHBD of plants and bacterium is pretty similar to'CBD of cellulases; and cellulos as well as chitin are linear chain polymer connected throughβ-1,4-glucosidic bond, which show very similar structure between each other. This study introduced CBD of Cellulase gene chhl and cbh2 from Chaetomium thermophilum to C-and N-Tachit from T. aurantiacus var. aurantiacus, separately, via fusion PCR. The gene was expressed in P. pastoris. Two high expression strains were gotten from screening. The fused protein Tachit-CBD1 and CBD2-Tachit increased activity for colloidal chitin by 1.6-and 0.6-fold, separately, and by 0.32-and 0.9-fold, separately, for powdery chitin and they also had the thermostability. The optimum temperatures were both 60℃, and their maximal activity were both at pH 8.0.They kept relative stability at 50℃and the pH in the range of 5.0 to 8.0.Two mutants Tachit-L129F and Tachit-V134I were constructed by site-direct mutagenesis to amino acid in the CD of Tachit. The optimum temperature of Tachit-L 129 F was increased by 10℃, while the thermostable was almost the same. The optimum pH was decreased to 5.0, and kept stable under acid condition. The results of Tachit-V134I showed little effect of mutation on the optimum temperature of the enzyme. The mutant showed pretty basophilia, the maximal activity was at pH 11, and still kept 85% activity at pH 12.
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