Bacillus sp. TKU004發酵烏賊軟骨所生產幾丁聚醣酶及生物活性物質之研究

Abstract

This study focused on the utilization of squid pen as the sole carbon/nitrogen source by Bacillus sp. TKU004 to produce chitosanase and bioactive materials. The optimized culture condition for chitosanase production was composed of 3% squid pen powder (SPP), 0.1% K2HPO4, 0.05% MgSO4．7H2O (pH 7), with autoclave treatment for 45 min, afterward, TKU004 was incubated in 100 mL of above liquid medium in an Erlenmeyer flask (250 mL) and kept shaking at 30℃ for two days. The chitosanase was purified from the culture supernatant by using ammonium sulfate precipitation, chromatography procedures of DEAE-Sepharose, Macro-Prep DEAE, and Sephacryl S-100. The overall activity yield of the purified chitosanase was 20%, with specific chitosanase activity of 2.8 U/mg. The molecular mass of TKU004 chitosanase determined by SDS-PAGE and gel filtration was approximately 29 kDa and 25 kDa, respectively. The results of peptide mass mapping indicated that TKU004 chitosanase matched to chitosanase from B. subtilis subsp. subtilis str. 168 (GenBank accession number gi16079742) with 23% sequence coverage. The optimum temperature, optimum pH, thermal stability, and pH stability of TKU004 chitosanase were 37℃, pH 7, ＜40℃, and pH 4~7, respectively. The chitosanase was inhibited by 5 mM Cu2+ and Fe2+, but retained 94%, 96%, 93%, 95% of its original activity in the presence of 2% Tween 20, 2% Tween 40, 2% Triton X-100, and 2 mM SDS, respectively. Additionally, TKU004 was cultivated for 1~6 days by using squid pen powder and shrimp head powder as the different carbon/nitrogen sources. The results indicated that culture supernatant (3% SPP) had higher DPPH free radical scavenging effect at the third day and better total phenolic contents, reducing activity, and Fe2+ chelating ability at the fifth day.