热稳定性α-淀粉酶的特性研究。来自地芽孢杆菌 IIPTN。

Characterization of hyperthermostable alpha-amylase from Geobacillus sp. IIPTN.

机构信息

Biotechnology Area, Indian Institute of Petroleum, Dehradun, 248005, India.

出版信息

Appl Microbiol Biotechnol. 2010 May;86(6):1857-66. doi: 10.1007/s00253-009-2430-9. Epub 2010 Jan 22.

Abstract

A newly isolated Geobacillus sp. IIPTN (MTCC 5319) from the hot spring of Uttarakhand's Himalayan region produced a hyperthermostable alpha-amylase. The microorganism was characterized by biochemical tests and 16S rRNA gene sequencing. The optimal temperature and pH were 60 degrees C and 6.5, respectively, for growth and enzyme production. Although it was able to grow in temperature ranges from 50 to 80 degrees C and pH 5.5-8.5. Maximum enzyme production was in exponential phase with activity 135 U ml(-1) at 60 degrees C. Assayed with cassava as substrate, the enzyme displayed optimal activity 192 U ml(-1) at pH 5.0 and 80 degrees C. The enzyme was purified to homogeneity with purification fold 82 and specific activity 1,200 U mg(-1) protein. The molecular mass of the purified enzyme was 97 KDa. The values of K(m) and V(max) were 36 mg ml(-1) and 222 micromol mg(-1) protein min(-1), respectively. The amylase was stable over a broad range of temperature from 40 degrees C to 120 degrees C and pH ranges from 5 to 10. The enzyme was stimulated with Mn(2+), whereas it was inhibited by Hg(2+), Cu(2+), Zn(2+), Mg(2+), and EDTA, suggesting that it is a metalloenzyme. Besides hyperthermostability, the novelty of this enzyme is resistance against protease.

摘要

一种从印度北阿坎德邦喜马拉雅山温泉中分离出的新的地杆菌(IIPTN,MTCC 5319)产生了一种超耐热的α-淀粉酶。该微生物通过生化试验和 16S rRNA 基因测序进行了鉴定。生长和酶生产的最佳温度和 pH 值分别为 60°C 和 6.5。尽管它能够在 50-80°C 和 pH5.5-8.5 的温度范围内生长,但最大酶产量出现在指数生长期,在 60°C 时酶活为 135 U/ml。以木薯为底物进行酶活测定时,该酶在 pH5.0 和 80°C 时显示出最佳活性 192 U/ml。该酶经纯化至均一性,纯化倍数为 82,比活为 1200 U/mg 蛋白。纯化酶的分子量为 97 kDa。K(m)和 V(max)的值分别为 36mg/ml 和 222μmol/mg 蛋白 min。该酶在 40-120°C 的宽温度范围和 5-10 的宽 pH 范围内稳定。该酶受 Mn(2+)刺激,受 Hg(2+)、Cu(2+)、Zn(2+)、Mg(2+)和 EDTA 抑制,表明它是一种金属酶。除了超耐热性之外,这种酶的新颖之处在于对蛋白酶的抗性。

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