Abstract
Psychrophiles are organisms that thrive in cold environments. One of the strategies for their cold adaptation is the ability to synthesize cold-adapted enzymes. These enzymes usually display higher catalytic efficiency and thermolability at lower temperatures compared to their mesophilic and thermophilic counterparts. In this work, a psychrophilic bacterial isolate codenamed π9 was selected for the cloning of the gene encoding triose phosphate isomerase (TIM), an enzyme in the glycolytic pathway. Based on 16S rRNA gene sequence analysis, this isolate was identified as a species of the genus Pseudomonas under the P. fluorescens group. The cloning of a 816 bp fragment of TIM gene which covers the 756 bp open reading frame was achieved by a combination of degenerate and splinkerette PCRs. The partial sequence of this gene was first PCR amplified by using degenerate primers and the flanking sequences were subsequently amplified by splinkerette PCR technique. Amino acid sequence of the cloned TIM was 97% identical to TIM from Pseudomonas fluorescens and shared 51% identity with the TIM from psychrophilic Vibrio sp. This work demonstrated the use of multiple PCR techniques to clone a gene without prior knowledge of its sequence. The cloning of the TIM gene by PCR was more rapid and cost effective compared to the traditional genomic library construction and screening method. Homology model of the TIM protein in this study was generated based on Escherichia coli TIM crystal structure. The model could serve as a hypothetical TIM structure from a psychrophilic microorganism for further investigation into areas that showed deviations from the known mesophilic TIM structures.
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Acknowledgments
We wish to thank Professor Nazalan Najimudin and Dr. Rashidah Abdul Rahim for sharing their Antarctic bacterial isolates with us. This work was supported by Fundamental Research Grant Scheme 203/PPSK/6170012.
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See Too, W.C., Few, L.L. Cloning of triose phosphate isomerase gene from an antarctic psychrophilic Pseudomonas sp. by degenerate and splinkerette PCR. World J Microbiol Biotechnol 26, 1251–1259 (2010). https://doi.org/10.1007/s11274-009-0295-9
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DOI: https://doi.org/10.1007/s11274-009-0295-9