Isolation and characterization of a novel thermostable neopullulanase-like enzyme from a hot spring in Thailand
| Autor: | Sutipa Tanapongpipat, Verawat Champreda, Lily Eurwilaichitr, Rutchadaporn Sriprang Kobayashi, Kittapong Tang |
|---|---|
| Rok vydání: | 2008 |
| Předmět: |
Glycoside Hydrolases
Starch Molecular Sequence Data Applied Microbiology and Biotechnology Biochemistry Hot Springs Analytical Chemistry law.invention Pichia pastoris chemistry.chemical_compound law Enzyme Stability Amino Acid Sequence Molecular Biology Conserved Sequence chemistry.chemical_classification Genome biology Organic Chemistry Temperature Pullulan General Medicine Hydrogen-Ion Concentration biology.organism_classification Thailand Enzyme assay genomic DNA Enzyme chemistry DNA glycosylase Recombinant DNA biology.protein Sequence Alignment Biotechnology |
| Zdroj: | Bioscience, biotechnology, and biochemistry. 72(6) |
| ISSN: | 1347-6947 |
| Popis: | 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 degrees C for 1 h. Env Npu193A was found to hydrolyze various substrates, including pullulan, starch, and gamma-cyclodextrin. The optimal working condition for Env Npu193A was at pH 7 at 75 degrees C with K(m) and V(max) 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. |
| Databáze: | OpenAIRE |
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