Molecular mutagenesis at Tyr-101 of the amylomaltase transcribed from a gene isolated from soil DNA

Prikladnaia biokhimiia i mikrobiologiia
S WatanasatitarpaJ Kaulpiboon

Abstract

The wild-type (WT) amylomaltase gene was directly isolated from soil DNA and cloned into a pET19b vector to express in E. coli BL21(DE3). The ORF of this gene consisted of 1,572 bp, encoding an enzyme of 523 amino acids. Though showing 99% sequence identity to amylomaltse from Thermus thermophilus ATCC 33923, this enzyme is unique in its alkaline optimum pH. In order to alter amylomaltase with less coupling or hydrolytic activity to enhance cycloamylose (CA) formation through cyclization reaction, site-directed mutagenesis of the second glucan binding site involving in CA production was performed at Tyr-101. The result revealed that the mutated Y101S enzyme showed a small increase in cyclization activity while significantly decreased disproportionation, coupling and hydrolytic activities. Mutation also resulted in the change in substrate specificity for disproportionation reaction. The WT enzyme preferred maltotriose, while the activity of mutated enzyme was the highest with maltopentaose substrate. Product analysis by HPAEC-PAD demonstrated that the main CAs of the WT amylomaltase were CA29-CA37. Y101S mutation did not change the product pattern, however, the amount of CAs formed by the mutated enzyme tended to increase especi...Continue Reading

References

Mar 1, 1972·Clinica Chimica Acta; International Journal of Clinical Chemistry·I MiwaG Okuda
Apr 14, 1999·Proceedings of the National Academy of Sciences of the United States of America·K GesslerW Saenger
Sep 10, 2005·Applied and Environmental Microbiology·Thijs KaperLubbert Dijkhuizen
Mar 21, 2007·Journal of Bioscience and Bioengineering·Kazutoshi FujiiHiroki Kaneko
Aug 7, 2012·Applied and Environmental Microbiology·Wiraya SrisimaratPiamsook Pongsawasdi

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Citations

Aug 11, 2016·Biochemical and Biophysical Research Communications·Pitchanan NimpiboonPiamsook Pongsawasdi
Jan 18, 2017·The Korean Journal of Parasitology·Samasuk ThammachanthaPongwat Polpong

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