Generation of thermostable enzyme genes using spontaneous mutations in thermophile Geobacillus kaustophilus HTA426

H. Suzuki; J. Kobayashi; K. Wada; M. Furukawa; K. Doi

Generation of thermostable enzyme genes using spontaneous mutations in thermophile Geobacillus kaustophilus HTA426

In: Industrial, medical and environmental applications of microorganisms

Authors:

H. Suzuki

H. Suzuki ¹Functional Genomics of Extremophiles, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan; hirokazusuzuki@agr.kyushu-u.ac.jp

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J. Kobayashi

J. Kobayashi ¹Functional Genomics of Extremophiles, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan; hirokazusuzuki@agr.kyushu-u.ac.jp

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K. Wada

K. Wada ²Applied Molecular Microbiology and Biomass Chemistry, Bioscience and Biotechnology, Graduate School, Kyushu University, Fukuoka 812-8581, Japan

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M. Furukawa

M. Furukawa ¹Functional Genomics of Extremophiles, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan; hirokazusuzuki@agr.kyushu-u.ac.jp

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K. Doi

K. Doi ³Microbial Genetic Division, Institute of Genetic Resources, Faculty of Agriculture, Graduate School, Kyushu University, Fukuoka 812-8581, Japan

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Type:: Chapter

Pages:: 441–446

DOI:: https://doi.org/10.3920/9789086867950_078

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While enzymes catalysts have been widely used in industry fields, many enzymes are not practical in spite of their catalytic activities that meet industry demand. A major reason for this is enzyme instability. Because enzyme stability can be commonly improved by enhancing enzyme thermostability, techniques to confer thermostability on enzymes could expand opportunities for enzyme utilization as industrial catalysts. Geobacillus kaustophilus HTA426 is an aerobic, high growth, Gram-positive thermophile that grows between 42 °C and 74 °C. In a previous study, we found that strain HTA426 exhibits frequent spontaneous mutations under certain conditions. In this study, we constructed two G. kaustophilus strains, MK242 and MK480. Strain MK242 lacked pyrF gene involved in uracil biosynthesis and thus was auxotrophic for uracil. Strain MK480 was constructed from strain MK242 by deleting five genes for DNA repair. Mutation assay confirmed frequent and various mutations in strain MK480. Two mesophile genes, Bacillus subtilis pyrF (BSpyrF) and Streptomyces azureus tsrR (SAtsrR) encoding thiostrepton resistance protein, were introduced in strain MK480 and propagated under uracil-starving and thiostrepton-containing conditions, respectively. After successive cultivations, the gene sequences in grown cells were analyzed to obtain two mutant genes, BSpyrFe1 (with C497T mutation) and SAtsrRe1 (with C772T mutation). BSpyrFe1 conferred prototrophy for uracil on strain MK242 at 70 °C, whereas BSpyrF did not. Although SAtsrR was not functional over 50 °C, SAtsrRe1 conferred thiostrepton resistivity on strain MK242 even at 55 °C. These results suggest that spontaneous mutation in G. kaustophilus MK480 provides a new approach to generate thermostable enzymes.

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Industrial, medical and environmental applications of microorganisms

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E-Book ISBN:: 9789086867950

Publisher:: Wageningen Academic

Print Publication Date:: 01 Nov 2014

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- Life Sciences

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Generation of thermostable enzyme genes using spontaneous mutations in thermophile Geobacillus kaustophilus HTA426

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Industrial, medical and environmental applications of microorganisms

Current status and trends

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