地衣芽孢杆菌A.4041耐高温α-淀粉酶的纯化及性质

Purification and Properties of Thermostable α-Amylase from Bacillus Licheniformis A. 4041

地衣芽孢杆菌A.4041（Bacillus，licheniformisA．4041）耐高温α-淀粉酶，经硫铁沉淀和垂直板制备凝胶电泳纯化，得到三种电泳均一的组分，α－Ⅰ，α－Ⅱ和α－Ⅲ．其相对质量分数分别为14.7％，32.2％和52.9％；等电点为5．2，5．4和5．5；相对分子质量为48000，55000和60000．酶作用于可溶性淀粉的米氏常数分别为4.5mg／mL，3．4mg／mL和2．6mg／mL．最适作用温度皆为90℃；最适pH为6.0～6.5；稳定pH范围皆为4～11．5在温度高于70℃，保温30min条件下，三组分的活力开始下降，但α－Ⅱ和α－Ⅲ在100℃，保温30min时仍分别保持15％和20％的残余活力，表明该酶的热稳定性良好．Ca2+，Mg2+，K+对酶明显激活；Cu2+，Fe3+，Zn2+，Al3+，EDTA、甲醛、戊二醛对酶强烈抑制．

The three electrophoretically homogeneous fractions of α-amylase, α-Ⅰ, α-Ⅱ,α-Ⅲ were purified from bacillus licheniformis A. 4041 by means of ammonium sulfate precipitation and preparative slab gel electrophoresis. Their relative contents were 14. 7%, 32.2% and 52.9% respectively.The pI values were 5. 2, 5. 4 and 5. 5. The molecular weights were 48 000, 55 000 and 60 000 respectively.The Km values for soluble starch were 4. 5 mg/mL, 3. 4 mg/mL, 2. 6 mg/mL. The optimum temperature was all 90℃, the optimum pH was 6.0~6.5. The stable pH range was all 4~11. 5. The residual activities began to drop over 70℃ for 30 min, but those of α-Ⅱ and α-Ⅲ at 100℃ 30 min still remained 15% and 20% respectively. The activity was strongly enhanced by Ca2+, Mg2+, K+,while strongly inhibited by Cu2+, Fe3+, Zn2+, Al3+, EDTA, formaldehyde and glutaraldehyde.