Characterization of a Maltase from an Early-Diverged Non-Conventional Yeast Blastobotrys adeninivorans.

Triinu Visnapuu,Karin Ernits,Kristina Põšnograjeva,Aivar Meldre,Tiina Alamäe,Katrin Viigand

doi:10.3390/ijms21010297

Triinu Visnapuu, Karin Ernits + Show 4 more

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https://doi.org/10.3390/ijms21010297

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Abstract

Genome of an early-diverged yeast Blastobotrys (Arxula) adeninivorans (Ba) encodes 88 glycoside hydrolases (GHs) including two α-glucosidases of GH13 family. One of those, the rna_ARAD1D20130g-encoded protein (BaAG2; 581 aa) was overexpressed in Escherichia coli, purified and characterized. We showed that maltose, other maltose-like substrates (maltulose, turanose, maltotriose, melezitose, malto-oligosaccharides of DP 4‒7) and sucrose were hydrolyzed by BaAG2, whereas isomaltose and isomaltose-like substrates (palatinose, α-methylglucoside) were not, confirming that BaAG2 is a maltase. BaAG2 was competitively inhibited by a diabetes drug acarbose (Ki = 0.8 µM) and Tris (Ki = 70.5 µM). BaAG2 was competitively inhibited also by isomaltose-like sugars and a hydrolysis product—glucose. At high maltose concentrations, BaAG2 exhibited transglycosylating ability producing potentially prebiotic di- and trisaccharides. Atypically for yeast maltases, a low but clearly recordable exo-hydrolytic activity on amylose, amylopectin and glycogen was detected. Saccharomyces cerevisiae maltase MAL62, studied for comparison, had only minimal ability to hydrolyze these polymers, and its transglycosylating activity was about three times lower compared to BaAG2. Sequence identity of BaAG2 with other maltases was only moderate being the highest (51%) with the maltase MalT of Aspergillus oryzae.

Highlights

IntroductionArxula adeninivorans) belongs to a basal clade of Saccharomycotina subphylum and diverged in the evolution of fungi long before Saccharomyces [1,2,3,4,5]
According to annotations provided at the MycoCosm website [26], the genome of Blastobotrys (Arxula) adeninivorans [2] encodes 185 carbohydrate-active enzymes, including 88 glycoside hydrolases (GHs) assigned to different families
Both of these proteins were predicted to lack a signal peptide and to locate intracellularly. We confirmed this by using the SignalP program. Aside from these two GH13 proteins, three putative extracellular α-glucosidases of GH31 family were detected in B. adeninivorans genome

Summary

Introduction

Arxula adeninivorans) belongs to a basal clade of Saccharomycotina subphylum and diverged in the evolution of fungi long before Saccharomyces [1,2,3,4,5]. A recent study states that the divergence of basal Saccharomycotina from Saccharomyces cerevisiae took place between 200 and 400 million years ago [4]. B. adeninivorans has several biotechnologically relevant properties: accumulation of lipids [6], salt tolerance, temperature-induced filamentation that promotes protein secretion and the ability to use a wide range of carbon and nitrogen sources, including purines, tannin and butanol, that are unusual nutrients for yeasts [2,7]. A highly active endo-inulinase of B. adeninivorans was cloned and recently characterized [11]. The genome of B. adeninivorans was sequenced in 2014 [2]

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