The Studies On Endo-β-1,3-glucanase And The Coding Gene From Debaryomyces Hansenii WC43-3

β-1,3-glucanase can cleave theβ-1, 3-glycosidic bond inβ-glucans and it exists in a variety of fungi.β-1,3-glucanase plays an important role in fungi metabolism and morphogenesis with great potential applications in industry. According to the hydrolysis mode,β-1,3-glucanase can be divided into exo-β-1,3-glucanase and endo-β-1,3-glucanase . A variety extracellularβ-1,3-glucanases of fungi and yeasts have been reported, but information on extracellular endo-β-1,3-glucanase from marine yeasts still lacks.A marine yeast WC43-3 producing high level endo-β-1,3-glucanase was screened from our marine yeast collections. Routine identification and molecular identification results show that the strain is a Debaryomyces hansenii. It was found that the yeast strain WC43-3 produced over 0.45U/ml of endo-β-1,3-glucanase within 58h under optimum conditions. The optimum conditions for endo-β-1,3-glucanase production by strain WC43-3 were: the medium composed of glucose 5%(w/v), peptone 2 %(w/v), yeast extract 0.5%(w/v), calcium carbonate 0.4%(w/v), initial pH 6.0 and the growth conditions of a speed of 180rpm and temperature of 28 oC.The endo-β-1,3-glucanase was purified by chromatography. According to the data from SDS-PAGE,the enzyme is a single chain protein with a molecular weight of 65.0kDa. The optimal pH and temperature of the purifiedβ-1,3-glucanase were 5.0 and 40°C, respectively. The enzyme was activated by Ni²⁺, but inhibited by Mn²⁺, Ca²⁺, Mg²⁺, Co²⁺, Zn²⁺, Hg²⁺, Cu²⁺, Fe³⁺ and Fe²⁺. The enzyme was inhibited by phenylmethylsulfonyl fluoride, iodoacetic acid, ethylenediamine tetraacetic acid, ethylene glycol bis (2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid and 1,10-phenanthroline. The K_m and V_max values of the purified endo-β-1,3-glucanase for laminarin were 2.31 mg/ml and 1.65 mg/(min*ml), respectively.The endo-β-1,3-glucanase structural gene (DhENG1 gene, accession number: HQ699479) of strain WC43-3 was isolated by degenerate PCR and inverse PCR. An open reading frame of 1,830 bp encoding a 609 amino-acid protein with calculated molecular weight of 65.11kDa was characterized. The promoter of the intronless gene was located from -90 to -135 and had one TATA box, while its terminator contained the sequence AATAAA. The protein had the Family81 glycoside hydrolase signature EESTSED and the second and last Glu (E) residues may be potential catalytic sites. The DhENG1 gene was cloned into pINA1317 expression vector and expressed in Yarrowia lipolytica Po1h and the transformant named A-5 with high endo-β-1,3-glucanase activity was obtained. The gene DhENG1 encoding endo-β-1,3-glucanase has been verified.