南极微球菌产低温淀粉酶条件优化及酶学性质初步研究

采用单因素试验对南极微球菌(Micrococcus antarcticus)产低温淀粉酶的条件进行了初步优化,其发酵培养基组成为(g/L):Na2HPO42.0,KH2PO41.0,MgSO4.7H2O 0.1,NaCl 5.0,(NH4)2SO42.5,麦芽糖5.0,微量元素溶液5.0 mL,pH 8.0,500 mL三角瓶装液100 mL,12℃,160 r/min振荡培养64 h,在此条件下,发酵液酶活由优化前的0.24 U/mL提高到2.6 U/mL,是优化前的10.8倍.发酵液经Millipore超滤膜包浓缩、Hitrap Q阴离子交换层析、Superdex 200凝胶过滤层析纯化获得纯淀粉酶.该淀粉酶的最适作用温度为30℃,在10~15℃下仍具有较高活性,但对热敏感,属典型的低温酶;最适作用pH为6.0,在pH 6.0~10.0范围内较稳定(>70%);Ca2+、Mn2+、Co2+、Mg2+对该酶有较为明显的激活作用,而Zn2+、Ba2+、Ag+、Cu2+、Al3+、Fe2+、Fe3+、Hg2+、螯合剂EDTA、citrate则对该酶有不同程度的抑制作用,并且在Tween系列及TrintonX-1...

Optimization of Fermentation Conditions for Cold-adapted Amylase Production by Micrococcus antarcticus and Its Enzymatic Properties

By single factor experiments, the fermentation conditions for cold-adapted amylase production from Micrococcus antarcticus were determined as follows (medium g/L):Na_2HPO_4 2.0,KH_2PO_4 1.0, MgSO_4·7H_2O 0.l.NaCl 5.0, (NH_4 )_2S0_4 2.5,maltose 5.0,trace element solution 5.0 mL, pH 8.0,100 mL/Erlenmeyer flask (500 mL) ; cultivation was in a rotating shaker at 12 C. and 160 r/min for 64 h. Under those conditions, the highest total enzyme activity (2.6 U/mL) was obtained and increased by 10.8 fold compared with the original value of 0.24 U/mL before optimization. This amylase was purified by concentration with ultrafiltration membrane module, Hitrap Q anion exchange chromatography and Superdex 200 gel filtration chromatography.The optimal temperature and pH for the purified amylase were 30℃ and 6.0, respectively .It still showed high activity at low temperature 10-15℃ .It was sensitive to high temperature but was stable at pH 6.0-10.0 with at least 70% activity remained.These results indicated that it was a typical cold-adapted enzyme.The enzyme activity was stimulated by Ca~(2+) ,Mn~(2+) ,Co~(2+) and Mg~(2+) ; but inhibited by Zn~(2+), Ba~(2+) , Ag~(+) , Cu~(2+) , Al~(3+) ,Fe~(2+) , Fe~(3+) ,Hg~(2+) , EDTA and citrate. This cold-adapted amylase showed resistance to inactivation of 0.1% nonionic surfactants such as Tween 80,TrintonX-100,etc.Its K_m was 0.90 mg/mL.