Directed evolution to enhance thermostability of galacto-N-biose/lacto-N- biose I phosphorylase

Yoshiyuki Koyama; Masafumi Hidaka; Mamoru Nishimoto; Motomitsu Kitaoka

doi:10.1093/protein/gzt049

Directed evolution to enhance thermostability of galacto-N-biose/lacto-N- biose I phosphorylase

Yoshiyuki Koyama, Masafumi Hidaka, Mamoru Nishimoto, Motomitsu Kitaoka

AGR - Agricultural Chemistry

Research output: Contribution to journal › Article › peer-review

14 Citations (Scopus)

Abstract

Galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) is the key enzyme in the enzymatic production of lacto-Nbiose I. For the purpose of industrial use, we improved the thermostability of GLNBP by evolutionary engineering in which five substitutions in the amino acid sequence were selected from a random mutagenesis GLNBP library constructed using error-prone polymerase chain reaction. Among them, C236Y and D576V mutants showed considerably improved thermostability. Structural analysis of C236Y revealed that the hydroxyl group of Tyr236 forms a hydrogen bond with the carboxyl group of E319. The C236Y and D576V mutations together contributed to the thermostability. The C236Y/D576V mutant exhibited 208C higher thermostability than the wild type.

Original language	English
Pages (from-to)	755-761
Number of pages	7
Journal	Protein Engineering, Design and Selection
Volume	26
Issue number	11
DOIs	https://doi.org/10.1093/protein/gzt049
Publication status	Published - 2013 Nov

Keywords

96-well microplate-based screening
Directed evolution
Galacto-N-biose
Lacto-N-biose I phosphorylase
Random mutation
Thermostability

ASJC Scopus subject areas

Medicine(all)

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10.1093/protein/gzt049

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title = "Directed evolution to enhance thermostability of galacto-N-biose/lacto-N- biose I phosphorylase",

abstract = "Galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) is the key enzyme in the enzymatic production of lacto-Nbiose I. For the purpose of industrial use, we improved the thermostability of GLNBP by evolutionary engineering in which five substitutions in the amino acid sequence were selected from a random mutagenesis GLNBP library constructed using error-prone polymerase chain reaction. Among them, C236Y and D576V mutants showed considerably improved thermostability. Structural analysis of C236Y revealed that the hydroxyl group of Tyr236 forms a hydrogen bond with the carboxyl group of E319. The C236Y and D576V mutations together contributed to the thermostability. The C236Y/D576V mutant exhibited 208C higher thermostability than the wild type.",

keywords = "96-well microplate-based screening, Directed evolution, Galacto-N-biose, Lacto-N-biose I phosphorylase, Random mutation, Thermostability",

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T1 - Directed evolution to enhance thermostability of galacto-N-biose/lacto-N- biose I phosphorylase

AU - Koyama, Yoshiyuki

AU - Hidaka, Masafumi

AU - Nishimoto, Mamoru

AU - Kitaoka, Motomitsu

PY - 2013/11

Y1 - 2013/11

N2 - Galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) is the key enzyme in the enzymatic production of lacto-Nbiose I. For the purpose of industrial use, we improved the thermostability of GLNBP by evolutionary engineering in which five substitutions in the amino acid sequence were selected from a random mutagenesis GLNBP library constructed using error-prone polymerase chain reaction. Among them, C236Y and D576V mutants showed considerably improved thermostability. Structural analysis of C236Y revealed that the hydroxyl group of Tyr236 forms a hydrogen bond with the carboxyl group of E319. The C236Y and D576V mutations together contributed to the thermostability. The C236Y/D576V mutant exhibited 208C higher thermostability than the wild type.

AB - Galacto-N-biose/lacto-N-biose I phosphorylase (GLNBP) is the key enzyme in the enzymatic production of lacto-Nbiose I. For the purpose of industrial use, we improved the thermostability of GLNBP by evolutionary engineering in which five substitutions in the amino acid sequence were selected from a random mutagenesis GLNBP library constructed using error-prone polymerase chain reaction. Among them, C236Y and D576V mutants showed considerably improved thermostability. Structural analysis of C236Y revealed that the hydroxyl group of Tyr236 forms a hydrogen bond with the carboxyl group of E319. The C236Y and D576V mutations together contributed to the thermostability. The C236Y/D576V mutant exhibited 208C higher thermostability than the wild type.

KW - 96-well microplate-based screening

KW - Directed evolution

KW - Galacto-N-biose

KW - Lacto-N-biose I phosphorylase

KW - Random mutation

KW - Thermostability

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DO - 10.1093/protein/gzt049

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ER -

Directed evolution to enhance thermostability of galacto-N-biose/lacto-N- biose I phosphorylase

Abstract

Keywords

ASJC Scopus subject areas

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