Enzymatic characterization of an extracellular glucoamylase from Tricholoma matsutake and its cloning and secretory expression in Pichia pastoris. Issue 12 (2nd December 2018)

• Record Type: Journal Article
• Authors: Onuma, Hiroki; Uchiyama, Hiroto; Hara, Kento; Fukuta, Yasuhisa; Shirasaka, Norifumi
• Is Part Of: Bioscience, biotechnology, and biochemistry. Volume 82:Issue 12(2018)
• Page Start: 2180
• Page End: 2190
• Publication Date: 2018-12-02
• Subjects: Ectomycorrhizal fungi -- Glucoamylase -- heterologous expression -- Pichia pastoris -- Tricholoma matsutake; Biotechnology -- Periodicals; Biochemistry -- Periodicals; 660.6
• DOI: 10.1080/09168451.2018.1507723
• Languages: English
• ISSNs: 0916-8451

Abstract

ABSTRACT: A glucoamylase from the ectomycorrhizal fungus Tricholoma matsutake ( Tm GLA) was purified 33.2-fold to homogeneity as a single monomeric glycoprotein with a molecular mass of 63.9 kDa. Maximum activity was observed at 60°C and pH 5.0. The enzyme is active down to 50°C and in the pH range of 4.0–6.0, and its activity is strongly inhibited by Ag + . It degrades α-1, 4- and α-1, 6-glycosidic linkages in various polysaccharides. Its gene ( TmGlu1 ) was cloned using information from the enzyme's internal amino acid sequences and the whole genome sequence of T. matsutake NBRC 30605. The deduced amino acid sequence showed clear homology with those of GH family 15 proteins. Pichia pastoris transformed with TmGlu1 secreted the active enzyme in a glycosylated form, and its characteristics were the same as the native enzyme. Graphical abstract: : A glucoamylase from T. matsutake was purified and characterized. The gene was secretory expressed and induced by casamino acid in P. pastoris .