菌株TKU031係以烏賊軟骨粉為唯一碳/氮源，篩選自台灣淡水三芝土壤，經鑑定為Bacillus cereus之幾丁聚醣酶及蛋白酶生產菌。幾丁聚醣酶較適生產條件為，以含有1％烏賊軟骨粉、0.1 % K2HPO4、0.05 % MgSO4．7H2O之100mL液態培養基( pH 7)，於37℃搖瓶 ( 150 rpm ) 培養2天。所得發酵液經離心、硫酸銨沉澱以及 DEAE–Sepharose與 Macro-PrepR DEAE Cartridge陰離子交換層析等步驟，純化出的一種經SDS–PAGE測定分子量為43 kDa幾丁聚醣酶CHS。CHS的最適反應pH、最適反應溫度、pH安定性以及熱安定性分別為pH 5、50℃、pH 5–9以及<50℃。CHS的酵素活性活性會受1mM的 Zn2+、Cu2+、Fe2+及5mM 的Mn2+、EDTA所抑制。
於較適培養，添加0.1%硼砂進行培養發現可促進B. cereus TKU031菌體生長達122%，若添加0.1%硼砂及0.05%硼酸則可促進B. cereus TKU031幾丁聚醣酶之生產，相對活性分別是未添加之培養基培養第3天177％及第2天的 195％。 Strain TKU031, a chitosanase– and protease– producing bacteria, was isolated from the soil of Sanjhih, Taiwan with squid pen powder (SPP) as the sole carbon/nitrogen source and identified as Bacillus cereus. Optimized culture condition for chitosanase production was found when the organism was cultured at 37℃ for two days in 100mL medium (pH 7) containing 1%SPP (w/v), 0.1 % K2HPO4、0.05 % MgSO4．7H2O.
One chitosanase (CHS) was purified from the culture supernatant of B. cereus TKU031 by ammonium sulfate precipitation, DEAE–Sepharose chromatography and Macro-PrepR DEAE Cartridge chromatography. The molecular weight of CHS was determined by SDS-PAGE approximately to be 43kDa. The optimum pH, optimum temperature, pH stability, and thermal stability of CHS was pH 5, 50℃, pH 5–9, <50℃. CHS was inhibited by EDTA and completely inactivated by 1mM Zn2+、Cu2+、Fe2+, and 5mM Mn2+.
In addition, the growth of B. cereus TKU031 was found to be promoted up to 1.22-fold by adding 0.1% sodium tetraborate in the culture medium. The productivities of chitosanase were also increased by adding 0.1% sodium tetraborate and 0.05% boric acid, which enhanced 1.77- and 1.95- folds, respectively.
