A novel saccharifying α‐amylase of Antarctic psychrotolerant fungi Geomyces pannorum: Gene cloning, functional expression, and characterization

Abstract

A DNA segment encoding a novel a-amylase was cloned from the Antarctic psychrotolerant fungi Geomyces pannorum. The gene has an open reading frame of 1761 bp, deduced 586 amino acids, revealing a relatively low homology to a-amylases from diverse fungal genera. The a-amylase gene was overexpressed in Pichia pastoris, and the recombinant a-amylase was purified and characterized. Enzymatic activity was optimal at pH 6.0 and 70°C, and the specific activity was 9.78 Â 10 3 U/mg when using soluble starch as substrate. Moreover, the enzyme retained more than 90% of its maximum activity over a wide pH range (6.0-9.0). Exceptionally, the main hydrolysis products of soluble starch catalyzed by the enzyme were glucose and maltose, of which glucose reached a concentration of 68.8% (w/w). This is the first report on identifying thermophilic fungal amylase from psychrotolerant fungi. Also, the preliminary study on the high-glucose-producing a-amylase with detailed enzymatic properties shows a potential application prospect in syrup industry.

Cold-adapted enzymes produced by fungi from terrestrial and marine Antarctic environments

“…Antarctic fungal strains representative of the genera Cryptococcus, Dioszegia, Glaciozyma, Holtermaniella, Leuconeurospora, Mrakia, Moesziomyces, Penicillium, Pseudogymnoascus, Rhodotorula, Phenoliferia, and Thelebolus were reported to be amylase producers [12,13,32,[62][63][64][65]99,118]. Amylase activity has been assayed by growing the selected strains on media supplemented with starch as a carbon source.…”

“…Therefore, the expression must be performed in other eukaryotic organisms; P. pastoris appears to be a good host for fungal cold-adapted enzyme expression. According to Gao et al [64], the a-amylase from Pseudogymnoascus pannorum R2-1 (formerly Geomyces pannorum) cloned in P. pastoris presented low homology to other a-amylases, with thermophilic characteristics (optimal activity at 70.0 C). In another study, Ramli et al [68] reported that the chitinase from G. antarctica PI12 expressed in P. pastoris was most active at 15.0 C, being stable in the range of 5.0-25.0 C. According to Feller and Gerday [58], a great contribution in this field could be associated with the use of a psychrophilic microorganism as a host for the expression of genes at low temperatures, preventing the formation of inclusion bodies and protecting heat-sensitive gene products.…”

“…The techniques used were diverse and yielded purification factors from 1.70-to 1568.00-fold and yield ranging from 0.65 to 86.20%. Enzymes secreted into the medium by Antarctic fungi are first concentrated by techniques, such as ultrafiltration [68,74,80,81,98,130], ammonium sulfate precipitation [63,64], ammonium sulfate and dialysis [79,85], or acetone precipitation [107]. After concentrating, the protein, purification can be carried out by a single or multiple steps of chromatography.…”

“…In studies carried out by Lario et al [81], Petrescu et al [98], and Ray et al [80], ion exchange chromatography was performed. On the other hand, in the studies reported by Abrashev et al [84], De Mot and Verachtert [63], Florczak et al [72], Gao et al [64], Turkiewicz et al [107], and Yu et al [79] size exclusion with ion exchange and/or affinity chromatography were combined.…”

Identification and magnetic immobilization of a pyrophilous aspartic protease from Antarctic psychrophilic fungus

“…To meet the increasing demands of biocatalytic tasks, extensive efforts have been made to isolate enzymes produced by extremophiles, owing to their special properties and significant potential economic values (González-Segura, Witte, McClard, & Hurley, 2007 ;Piette, Struvay, & Feller, 2011 ;Ueda et al, 2014 ). However, limited attention has been paid to the identification and characterization of these APs (Gao, Mao, Zhang, He, & Wei, 2016 ;Kasana & Gulati, 2011 ;Krishnan, Alias, Wong, Pang, & Convey, 2011 ;Mao et al, 2015 ).…”