Directed evolution of Thermus maltogenic amylase toward enhanced thermal resistance

Young Wan Kim, Ji Hye Choi, Jung Wan Kim, Cheonseok Park, Jung Woo Kim, Hyunju Cha, Soo Bok Lee, Byoung Ha Oh, Tae Wha Moon, Kwan Hwa Park

Research output: Contribution to journalArticle

73 Citations (Scopus)

Abstract

The thermostability of maltogenic amylase from Thermus sp. strain IM6501 (ThMA) was improved greatly by random mutagenesis using DNA shuffling. Four rounds of DNA shuffling and subsequent recombination of the mutations produced the highly thermostable mutant enzyme ThMA-DM, which had a total of seven individual mutations. The seven amino acid substitutions in ThMA-DM were identified as R26Q, S169N, I333V, M375T, A398V, Q411L, and P453L. The optimal reaction temperature of the recombinant enzyme was 75°C, which was 15°C higher than that of wild-type ThMA, and the melting temperature, as determined by differential scanning calorimetry, was increased by 10.9°C. The half-life of ThMA-DM was 172 min at 80°C, a temperature at which wild-type ThMA was completely inactivated in less than I min. Six mutations that were generated during the evolutionary process did not significantly affect the specific activity of the enzyme, while the M375T mutation decreased activity to 23% of the wild-type level. The molecular interactions of the seven mutant residues that contributed to the increased thermostability of the mutant enzyme with other adjacent residues were examined by comparing the modeled tertiary structure of ThMA-DM with those of wild-type ThMA and related enzymes. The A398V and Q411L substitutions appeared to stabilize the enzyme by enhancing the interdomain hydrophobic interactions. The R26Q and P453L substitutions led potentially to the formation of genuine hydrogen bonds. M375T, which was located near the active site of ThMA, probably caused a conformational or dynamic change that enhanced thermostability but reduced the specific activity of the enzyme.

Original languageEnglish
Pages (from-to)4866-4874
Number of pages9
JournalApplied and Environmental Microbiology
Volume69
Issue number8
DOIs
Publication statusPublished - 2003 Aug 1

Fingerprint

glucan 1,4-alpha-maltohydrolase
Thermus
amylases
Hot Temperature
enzyme
heat
thermal stability
mutation
Enzymes
enzymes
DNA Shuffling
mutants
Mutation
substitution
enzyme activity
Temperature
hydrophobic bonding
melting point
amino acid substitution
differential scanning calorimetry

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

Cite this

Kim, Y. W., Choi, J. H., Kim, J. W., Park, C., Kim, J. W., Cha, H., ... Park, K. H. (2003). Directed evolution of Thermus maltogenic amylase toward enhanced thermal resistance. Applied and Environmental Microbiology, 69(8), 4866-4874. https://doi.org/10.1128/AEM.69.8.4866-4874.2003
Kim, Young Wan ; Choi, Ji Hye ; Kim, Jung Wan ; Park, Cheonseok ; Kim, Jung Woo ; Cha, Hyunju ; Lee, Soo Bok ; Oh, Byoung Ha ; Moon, Tae Wha ; Park, Kwan Hwa. / Directed evolution of Thermus maltogenic amylase toward enhanced thermal resistance. In: Applied and Environmental Microbiology. 2003 ; Vol. 69, No. 8. pp. 4866-4874.
@article{526d31c1c2324a3e9a2f00c7f47275af,
title = "Directed evolution of Thermus maltogenic amylase toward enhanced thermal resistance",
abstract = "The thermostability of maltogenic amylase from Thermus sp. strain IM6501 (ThMA) was improved greatly by random mutagenesis using DNA shuffling. Four rounds of DNA shuffling and subsequent recombination of the mutations produced the highly thermostable mutant enzyme ThMA-DM, which had a total of seven individual mutations. The seven amino acid substitutions in ThMA-DM were identified as R26Q, S169N, I333V, M375T, A398V, Q411L, and P453L. The optimal reaction temperature of the recombinant enzyme was 75°C, which was 15°C higher than that of wild-type ThMA, and the melting temperature, as determined by differential scanning calorimetry, was increased by 10.9°C. The half-life of ThMA-DM was 172 min at 80°C, a temperature at which wild-type ThMA was completely inactivated in less than I min. Six mutations that were generated during the evolutionary process did not significantly affect the specific activity of the enzyme, while the M375T mutation decreased activity to 23{\%} of the wild-type level. The molecular interactions of the seven mutant residues that contributed to the increased thermostability of the mutant enzyme with other adjacent residues were examined by comparing the modeled tertiary structure of ThMA-DM with those of wild-type ThMA and related enzymes. The A398V and Q411L substitutions appeared to stabilize the enzyme by enhancing the interdomain hydrophobic interactions. The R26Q and P453L substitutions led potentially to the formation of genuine hydrogen bonds. M375T, which was located near the active site of ThMA, probably caused a conformational or dynamic change that enhanced thermostability but reduced the specific activity of the enzyme.",
author = "Kim, {Young Wan} and Choi, {Ji Hye} and Kim, {Jung Wan} and Cheonseok Park and Kim, {Jung Woo} and Hyunju Cha and Lee, {Soo Bok} and Oh, {Byoung Ha} and Moon, {Tae Wha} and Park, {Kwan Hwa}",
year = "2003",
month = "8",
day = "1",
doi = "10.1128/AEM.69.8.4866-4874.2003",
language = "English",
volume = "69",
pages = "4866--4874",
journal = "Applied and Environmental Microbiology",
issn = "0099-2240",
publisher = "American Society for Microbiology",
number = "8",

}

Kim, YW, Choi, JH, Kim, JW, Park, C, Kim, JW, Cha, H, Lee, SB, Oh, BH, Moon, TW & Park, KH 2003, 'Directed evolution of Thermus maltogenic amylase toward enhanced thermal resistance', Applied and Environmental Microbiology, vol. 69, no. 8, pp. 4866-4874. https://doi.org/10.1128/AEM.69.8.4866-4874.2003

Directed evolution of Thermus maltogenic amylase toward enhanced thermal resistance. / Kim, Young Wan; Choi, Ji Hye; Kim, Jung Wan; Park, Cheonseok; Kim, Jung Woo; Cha, Hyunju; Lee, Soo Bok; Oh, Byoung Ha; Moon, Tae Wha; Park, Kwan Hwa.

In: Applied and Environmental Microbiology, Vol. 69, No. 8, 01.08.2003, p. 4866-4874.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Directed evolution of Thermus maltogenic amylase toward enhanced thermal resistance

AU - Kim, Young Wan

AU - Choi, Ji Hye

AU - Kim, Jung Wan

AU - Park, Cheonseok

AU - Kim, Jung Woo

AU - Cha, Hyunju

AU - Lee, Soo Bok

AU - Oh, Byoung Ha

AU - Moon, Tae Wha

AU - Park, Kwan Hwa

PY - 2003/8/1

Y1 - 2003/8/1

N2 - The thermostability of maltogenic amylase from Thermus sp. strain IM6501 (ThMA) was improved greatly by random mutagenesis using DNA shuffling. Four rounds of DNA shuffling and subsequent recombination of the mutations produced the highly thermostable mutant enzyme ThMA-DM, which had a total of seven individual mutations. The seven amino acid substitutions in ThMA-DM were identified as R26Q, S169N, I333V, M375T, A398V, Q411L, and P453L. The optimal reaction temperature of the recombinant enzyme was 75°C, which was 15°C higher than that of wild-type ThMA, and the melting temperature, as determined by differential scanning calorimetry, was increased by 10.9°C. The half-life of ThMA-DM was 172 min at 80°C, a temperature at which wild-type ThMA was completely inactivated in less than I min. Six mutations that were generated during the evolutionary process did not significantly affect the specific activity of the enzyme, while the M375T mutation decreased activity to 23% of the wild-type level. The molecular interactions of the seven mutant residues that contributed to the increased thermostability of the mutant enzyme with other adjacent residues were examined by comparing the modeled tertiary structure of ThMA-DM with those of wild-type ThMA and related enzymes. The A398V and Q411L substitutions appeared to stabilize the enzyme by enhancing the interdomain hydrophobic interactions. The R26Q and P453L substitutions led potentially to the formation of genuine hydrogen bonds. M375T, which was located near the active site of ThMA, probably caused a conformational or dynamic change that enhanced thermostability but reduced the specific activity of the enzyme.

AB - The thermostability of maltogenic amylase from Thermus sp. strain IM6501 (ThMA) was improved greatly by random mutagenesis using DNA shuffling. Four rounds of DNA shuffling and subsequent recombination of the mutations produced the highly thermostable mutant enzyme ThMA-DM, which had a total of seven individual mutations. The seven amino acid substitutions in ThMA-DM were identified as R26Q, S169N, I333V, M375T, A398V, Q411L, and P453L. The optimal reaction temperature of the recombinant enzyme was 75°C, which was 15°C higher than that of wild-type ThMA, and the melting temperature, as determined by differential scanning calorimetry, was increased by 10.9°C. The half-life of ThMA-DM was 172 min at 80°C, a temperature at which wild-type ThMA was completely inactivated in less than I min. Six mutations that were generated during the evolutionary process did not significantly affect the specific activity of the enzyme, while the M375T mutation decreased activity to 23% of the wild-type level. The molecular interactions of the seven mutant residues that contributed to the increased thermostability of the mutant enzyme with other adjacent residues were examined by comparing the modeled tertiary structure of ThMA-DM with those of wild-type ThMA and related enzymes. The A398V and Q411L substitutions appeared to stabilize the enzyme by enhancing the interdomain hydrophobic interactions. The R26Q and P453L substitutions led potentially to the formation of genuine hydrogen bonds. M375T, which was located near the active site of ThMA, probably caused a conformational or dynamic change that enhanced thermostability but reduced the specific activity of the enzyme.

UR - http://www.scopus.com/inward/record.url?scp=0042530128&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0042530128&partnerID=8YFLogxK

U2 - 10.1128/AEM.69.8.4866-4874.2003

DO - 10.1128/AEM.69.8.4866-4874.2003

M3 - Article

C2 - 12902281

AN - SCOPUS:0042530128

VL - 69

SP - 4866

EP - 4874

JO - Applied and Environmental Microbiology

JF - Applied and Environmental Microbiology

SN - 0099-2240

IS - 8

ER -