Efficient production of recombinant tannase in Aspergillus oryzae using an improved glucoamylase gene promoter

Kyotaro Ichikawa, Yoshihito Shiono, Tomoko Shintani, Akira Watanabe, Hiroshi Kanzaki, Katsuya Gomi, Takuya Koseki

Research output: Contribution to journalArticle

Abstract

A tannase-encoding gene, AotanB, from Aspergillus oryzae RIB40 was overexpressed in A. oryzae AOK11 niaD-deficient mutant derived from an industrial strain under the control of an improved glucoamylase gene promoter PglaA142. The recombinant tannase, designated as rAoTanBO, was produced efficiently as an active extracellular enzyme. Purified rAoTanBO showed a smeared band with a molecular mass of approximately 80–100 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The rAoTanBO had a molecular mass of 65 kDa, after treatment with endo-β-N-acetylglucosaminidase H. Purified rAoTanBO exhibited maximum activity at 30–35°C and pH 6.0. The tannase activity of purified rAoTanBO towards natural and artificial substrates was 2–8 folds higher than that of the recombinant enzyme produced by Pichia pastoris, designated as rAoTanBP. N-terminus of the mature rAoTanBP had six more amino acids than the N-terminus of the mature rAoTanBO. Kinetic analyses showed that rAoTanBO had higher catalytic efficiency (kcat/Km) than rAoTanBP. rAoTanBO was stable up to 60°C and higher thermostability than rAoTanBP. N-linked oligosaccharides had no effect on the activity and stability of rAoTanBO and rAoTanBP.

Original languageEnglish
JournalJournal of Bioscience and Bioengineering
DOIs
Publication statusAccepted/In press - Jan 1 2019

Fingerprint

tannase
Aspergillus oryzae
Glucan 1,4-alpha-Glucosidase
Aspergillus
Molecular mass
Enzymes
Genes
Oligosaccharides
Gene encoding
Sodium dodecyl sulfate
Polyacrylates
Electrophoresis
Amino acids
Acetylglucosaminidase
Pichia
Gels
Sodium Dodecyl Sulfate
Kinetics
Polyacrylamide Gel Electrophoresis
Substrates

Keywords

  • AotanB gene
  • Aspergillus oryzae
  • Efficient production
  • Over-expression
  • Tannase

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

Cite this

Efficient production of recombinant tannase in Aspergillus oryzae using an improved glucoamylase gene promoter. / Ichikawa, Kyotaro; Shiono, Yoshihito; Shintani, Tomoko; Watanabe, Akira; Kanzaki, Hiroshi; Gomi, Katsuya; Koseki, Takuya.

In: Journal of Bioscience and Bioengineering, 01.01.2019.

Research output: Contribution to journalArticle

Ichikawa, Kyotaro ; Shiono, Yoshihito ; Shintani, Tomoko ; Watanabe, Akira ; Kanzaki, Hiroshi ; Gomi, Katsuya ; Koseki, Takuya. / Efficient production of recombinant tannase in Aspergillus oryzae using an improved glucoamylase gene promoter. In: Journal of Bioscience and Bioengineering. 2019.
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