脱枝酶产生菌的分离鉴定及酶学性质研究

目的：从中国高校工业微生物资源与信息中心（CICIM-CU）细菌库中分离具有产脱枝酶酶活的细菌并鉴定,进行酶学性质的研究。方法：通过碘显色平板法筛选产酶菌株,利用16S rDNA确定其属种。对每一株产脱枝酶的细菌进行初步的酶学性质研究。结果：从4005株细菌中筛选出45株产脱枝酶的细菌,建立了产脱枝酶细菌的细菌库。酶学性质表明CICIM B272、CICIMB1-30两株菌产生的脱枝酶,酶反应的最适温度分别为70℃6、0℃,最适pH分别为6.0、5.5,来源于上述两种不同属种的脱枝酶在30-70℃反应条件下,酶在pH 4.5-8.5范围内活性稳定,Li＋、Na＋、K＋、Mg2＋、Mn2＋对两者酶活均有显著的激活作用,而Cu2＋、Fe3＋及EDTA对两者均有显著的抑制作用,Mn2＋、Ca2＋分别对两者的热稳定性具有很好的提升作用。以支链淀粉为底物的动力学常数Km分别为352.883mg/mL、4.5814mg/mL,Vmax分别为30.03mg/min.mL、0.4575mg/min.mL。结论：不同属种的脱枝酶酶学性质差别显著。

Study on Screening Identification and Properities of Debranching Enzyme Producing Bacteria

Objective: The bacteria of producing debranching enzyme were screened from CICIM-CU, identified, and studied the characteristics of debranching enzyme. Method:Screened debranching enzyme producing strain, identified taxonomic status, determine debranching enzyme properties by iodine solution coloration plate and 16S rDNA sequence alignment method. Result:45 strains which can produce debranching enzyme were screened from 4005 strains and the bacteria bank capable to produce debranching enzyme was set up finally.The results showed that the debranching enzyme from CICIM B272 and CICIM B1-30 performed the maximal activity at 70℃, pH 6.0 and 60℃ pH 5.5, respectively. The enzyme had thermostability between 30℃ and 70℃ and pH stability from 4.5 to 8.5. The enzymes were strongly activated by Li~+, Na~+, K~+, Mg~(2+), Mn~(2+), whereas inhibited by Cu~(2+), Fe~(3+) and EDTA. Mn~(2+), Ca~(2+) significantly increased their thermostability, respectively. The kinetic constants of the enzyme was obtained, their K_m for amylopectin were 352.883mg/mL and 4.5814mg/mL, V_(max) were 30.03 mg/min·mL and 0.4575 mg/min·mL, respectively. Conclusion: The difference in the enzymatic property of debranching enzyme was significant between different genera.