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Biotechnology Laboratory

Hachioji

Applied Chemistry and Chemical Engineering


Regents professor

  • Fumitaka OYAMA: Professor
  • Yasusato SUGAHARA: Associate Professor
  • Masayoshi SAKAGUCHI: Lecturer
  • Human diseases
  • Chitin and chitinase
  • Hydrolytic enzymes

Topics of research

1. Biomedical importance of mammalian chitinases
2. Novel method for preparing chitin oligomers
3. Structure-function relationship for hydrolytic enzymes

Research content

1. Biomedical importance of mammalian chitinases: Chitinases hydrolyze the β-1-4 glycosidic bonds of chitin, a major structural component of fungi, crustaceans and insects. Humans and mice express two active chitinases, chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase). Because the level of expression of these chitinases is increased in many inflammatory conditions, including Gaucher disease and mouse models of asthma, both chitinases may play important roles in the pathophysiologies of these and other diseases. In the beginning, we established a quantitative real-time PCR system that uses standard DNA produced by ligating the cDNA fragments of the target genes. We found that AMCase mRNA is synthesized at extraordinarily high levels in the mouse stomach. In contrast, the AMCase expression level in human stomach was significantly lower than the expression level observed in mouse stomach. Then, we produced mouse AMCase as a Protein A-AMCase-V5-His in in E. coli. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0. The recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N’-diacetylchitobiose fragments. These results suggest that AMCase can function as a digestive enzyme that breaks down polymeric chitin in the mouse stomach.

2. Novel method for preparing chitin oligomer: Recent researches have shown that chitin oligomers (N-acetyl chitooligosaccharides) are general elicitor inducing defense responses in plant and mammals. We are developing a novel method for efficient preparation of chitin oligomers using an organic solvent after hydrochloric acid hydrolysate of chitin.

3. Structure- function relationship for hydrolytic enzymes
a. Glucoamylse: This enzyme hydrolyzes starch, a biopolymer of α-1,4-linked glucose, and yields glucose. Thus glucoamylase is widely used in starch-processing industries. We are expressing glucoamylases and GH 15 family-related enzymes in E. coli, and comparing the functions such as substrate specificity and thermostability.
b. Aqualysin I: This is a thermophilic protease used as a constituent of washing agents. Comparing the structural factors for the psychrophilic protease, a homologue of aqualysin I, we are searching for the protein-stabilizing amino acid residues in active site and/or overall structure.