Structure and function of a novel thermostable pullulanase.
- Author:
Jie ZHEN
;
Zheng HU
;
Shufang LI
;
Jianyong XU
;
Hui SONG
- Publication Type:Journal Article
- MeSH:
Anoxybacillus;
classification;
enzymology;
China;
Glycoside Hydrolases;
metabolism;
Hydrogen-Ion Concentration;
Phylogeny;
RNA, Ribosomal, 16S;
genetics;
Temperature
- From:
Chinese Journal of Biotechnology
2014;30(1):119-128
- CountryChina
- Language:Chinese
-
Abstract:
Research on novel pullulanase has major significance on the domestic industrialization of pullulanase and the breakdown of foreign monopoly. A thermophilic bacteria LM 18-11 producing thermostable pullulanase was isolated from Lunma hot springs of Yunnan province. It was identified as Anoxybacillus sp. by 16S rDNA phylogenetic analysis. Full-length pullulanase gene was cloned from Anoxybacillus sp. LM18-11. The optimum temperature of the pullulanase was between 55 and 60 degrees C with a half-life as long as 48 h at 60 degrees C; and its optimum pH was between 5.6 and 6.4. V(max) and K(m) of the pullulanase was measured as 750 U/mg and 1.47 mg/mL, which is the highest specific activity reported so far. The pullulanase crystals structure showed a typical alpha-amylase family structure. The N-terminal has a special substrate binding domain. Activity and substrate binding were decreased when the domain was deleted, the V(max) and K(m) were 324 U/mg and 1.95 mg/mL, respectively. The pullulanase was highly heterologous expressed in Bacillus subtilis by P43 promoter. The extracellular enzyme activity was 42 U/mL, which increased more than 40 times compared to the initial strain. This pullulanase has good application prospects.
