BIOTEC Central Research Unit, National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, 113 Phaholyothin Rd., Klong 1, Klongluang, Pathumthani, 12120, Thailand
Type
Article
Source Title
Bioscience, Biotechnology and Biochemistry
ISSN
09168451
Year
2008
Volume
72
Issue
6
Page
1448-1456
Open Access
All Open Access, Bronze
DOI
10.1271/bbb.70754
Format
PDF
Abstract
A gene encoding a thermostable pullulan-hydrolyzing enzyme was isolated from environmental genomic DNA extracted from soil sediments of Bor Khleung hot spring in Thailand. Sequence comparison with related enzymes suggested that the isolated enzyme, designated Env Npu193A, was most likely a neopullulanase-like enzyme. Env Npu193A was expressed in Pichia pastoris as a monomeric recombinant protein. The purified Env Npu193A exhibited pH stability ranging from 3 to 9. More than 60% of enzyme activity was retained after incubation at 60°C for 1 h. Env Npu193A was found to hydrolyze various substrates, including pullulan, starch, and γ-cyclodextrin. The optimal working condition for Env Npu193A was at pH 7 at 75°C with Km and Vmax toward pullulan of 1.22 ± 0.3% and 23.24 ± 1.7 U/mg respectively. Env Npu193A exhibited distinct biochemical characteristics as compared with the previously isolated enzyme from the same source. Thus, a culture-independent approach with sequence-basing was found to be an effective way to discover novel enzymes displaying unique substrate specificity and high thermostability from natural bioresources.
