嗜热菌来源的生淀粉酶分离纯化及其酶学性质

Purification and Characterization of Thermostable Amylases from Two Bacterial Species

摘要:

从嗜热菌库中分离到两株能水解生淀粉的菌株173和174, 通过扩增和测定两株菌的16S rDNA序列并进行比对结果表明, 所分离两株菌属于Geobacillus属的细菌。液体摇瓶发酵菌株173、174, 其产生的生淀粉酶(简称RSDE173、RSDE174)活力分别达14.5 U/mL和12.9 U/mL。通过生淀粉吸附－熟淀粉洗脱系统和TOYOPEARL HW-55F系统进行分离纯化, 得到纯化的RSDE173和RSDE174, 纯化倍数分别为50和29, 活力回收率分别为34%和41%。有关RSDE173和RSDE174酶学性质研究显示, 对熟淀粉水解的最适作用温度均为70℃, 而对生淀粉水解则分别在50℃~60℃和40℃~60℃下表现出高水解活力; 对不同底物的最适作用pH值均为5.0~5.5; 它们对大多数试验离子的敏感性较低, 但个别离子如Co2+、Cu2+对RSDE173或Cu2+对RSDE174的酶活力有一定的抑制作用。纯化的这两种生淀粉酶对不同来源生淀粉的底物专一性并不相同。RSDE173底物专一性顺序为红薯淀粉＞小麦淀粉＞玉米淀粉＞木薯淀粉＞糯米淀粉; 而RSDE174的糯米淀粉＞小麦淀粉＞红薯淀粉＞玉米淀粉＞木薯淀粉。RSDE173对生红薯淀粉有很好的降解, 其水解糊化淀粉与生红薯淀粉的比值为1.48; 而RSDE174优先降解生糯米淀粉, 其相应比值为1.69。

Abstract:

Two thermophilic bacterial isolates, strain 173 and strain 174, with raw starch-digesting activities were selected from thermophilic bacteria growing in hot spring of Tengchong County, Yunnan Province, China. By amplification, sequencing and alignment analysis of 16S ribosomal DNAs, they were identified as members of genus Geobacillus. In shaker flask culture Geobacillus sp. 173 produced 14.5 U/mL amylase and for Geobacillus sp. 174 with 12.9 U/mL . Both amylases were purified by starch absorption-desorption and chromatograph. The amylases from strain 173 and strain 174 purified to about 50 and 29 folds were respectively achieved with an overall yield of 34% and 41%. The maximum gelati-nized-starch-digesting activity of the purified amylases were at 70°C and pH 5.0~5.5. The high raw-starch-digesting ac-tivity of these enzymes were observed at 50°C~60°C ( from strain 173) and 40°C ~60°C (from strain 174). Both enzymes were not sensitive to ions including mental ions (Na+, K+, Mg2+, Ca2+, Mn2+, Zn2+) and others (EDTA, Citrate), but were slightly inhibited by ions such as Co2+, Cu2+ for amylase from strain 173 and Cu2+ for amylase from strain 174. Both en-zyme had specificity of starch substrates.