Bacillus cereus TKU027 所生產幾丁質酶,幾丁聚醣酶及蛋白酶之純化,定性與應用

Abstract

The chitinase, chitosanase and protease-producing strain TKU027 was isolated from soil in Changhua of Taiwan with shrimp head powder as the sole carbon/nitrogen source and identified as Bacillus cereus. The optimized culture conditions for chitinase and protease production were found to be shaken at 37℃ for 2 days in 50 mL and 100 mL of medium containing 1% shrimp head powder (SHP) , 0.1% K2HPO4 and 0.05% MgSO4．7H2O (pH 6). A chitinase CHI and a chitosanase CHS were purified from the culture supernatant by ammonium sulfate precipitation, DEAE-Sepharose and Sephacryl S-100. The molecular masses of CHI and CHS determined by SDS-PAGE were approximately 65 and 63 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of CHI and CHS were pH 6, 50℃, pH 5–8, <40℃ and pH 6, 60℃, pH 3–10, <50℃, respectively. The chitinase activity was inhibited by Mn2+ and PMSF. The chitosanase activity was inhibited by Cu2+, Mn2+ and EDTA. The culture supernatant obtained from B. cereus TKU027 in the optimized culture conditions enhanced the growth of L. paracasei TKU012 most obviously up to 175%, followed by L. paracasei 12193 up to 144% and L. kefir 14011 up to 132%. The crude enzyme from B. cereus TKU027 culture supernatant hydrolyzed water soluble chitosan to produce N-acetyl chitooligosaccharides. The N-acetyl chitooligosaccharides also exhibited activity of enhancing growth for L. paracasei 12193 up to 324% and L. kefir 14011 up to 174%. In addition, B. cereus TKU027 and SHP were added in soil respectively to investigate the biodegradation of SHP and change of bacteria flora by PCR-DGGE analysis. The highest reducing sugar（1442 mg/g soil）, total sugar（3511 mg/g soil）and total viable cell counts（6 × 107 CFU/g soil）were found at the 5th week incubation with B. cereus TKU027 and SHP in Tamsui mangrove river soil.