|摘要: ||本研究利用烏賊軟骨作為Bacillus sp. TKU004生產幾丁聚醣酶及生物活性物質之唯一碳/氮源。
Bacillus sp. TKU004生產幾丁聚醣酶之較適培養條件為：含有3%烏賊軟骨粉、0.1% K2HPO4及0.05% MgSO4．7H2O之100 mL液態培養基(pH 7)於250 mL三角錐形瓶，經滅菌45分鐘，於30℃搖瓶培養二天。發酵所得離心上清液，經硫酸銨沈澱、DEAE-Sepharose、Macro-Prep DEAE、Sephacryl S-100等層析步驟，純化出一種幾丁聚醣酶，其活性回收率為20%，比活性為2.8 U/mg；以SDS-PAGE及膠體過濾層析測定分子量分別為29 kDa及25 kDa；TKU004幾丁聚醣酶胜肽質譜鑑定結果，與B. subtilis subsp. subtilis str. 168之幾丁聚醣酶(GenBank編號為 gi16079742)相似度為23%。幾丁聚醣酶之最適反應溫度、最適反應pH、熱安定性、pH安定性分別為37℃、pH 7、<40℃、pH 4~7；其活性會受5 mM之Cu2+及Fe2+所抑制，而在2% Tween 20、2% Tween 40、2% Triton X-100及2 mM SDS存在下，分別還維持原活性94%、96%、93%、95%。
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.