Elsevier

Biochimie

Volume 168, January 2020, Pages 231-240
Biochimie

Research paper
Cloning, purification and study of recombinant GH3 family β-glucosidase from Penicillium verruculosum

https://doi.org/10.1016/j.biochi.2019.11.009Get rights and content

Highlights

  • The bglI gene of the filamentous fungus P. verruculosum was sequenced for the first time.

  • rPvBGL was successfully expressed into P. canescens under the control of a xylA promoter.

  • The remarkable ability of the rPvBGL to hydrolyze laminarin and a mixed β −1,3-1,4-glucan was demonstrated.

  • This study has shown the possibility of using rPvBGL for the construction of balanced cellulase enzyme preparations based on the fungus P. verruculosum.

Abstract

A novel bgl1 gene, encoding GH3 family β-glucosidase from Penicillium verruculosum (PvBGL), was cloned and heterologously expressed in P. canescens RN3-11-7 (niaD-) strain under the control of the strong xylA gene promoter. The recombinant rPvBGL was purified and their properties were studied in comparison with those of rAnBGL from Aspergillus niger expressed previously in the same fungal host. The rPvBGL had an observed molecular mass of 90 kDa (SDS-PAGE data) and displayed the enzyme maximum activity at pH 4.6 and 65 °C. The enzyme half-life time at 60 °C was found to be 87 min. Unlike the rAnBGL, the rPvBGL was not adsorbed on microcrystalline cellulose, which gives the latter enzyme an advantage in cellulose conversion with a longer time of hydrolysis.

Introduction

The enzymatic saccharification of polysaccharides from lignocellulose biomass is a crucial stage in the production of second-generation biofuels (ethanol, butanol, etc.), organic acids (lactic acid, fumaric acid), polymeric materials, food and feed additives and other useful products [[1], [2], [3]]. Cellulose is converted to glucose under the synergistic action of at least three types of glycoside hydrolases (endoglucanases, exo-cellobiohydrolases and β-glucosidases) [4]. β-Glucosidase (BGL) is the key enzyme that completes the saccharification process, converting cellobiose to glucose [5,6].

BGLs (also known as β-D-glucoside glucohydrolases, EC 3.2.1.21), catalyze the hydrolysis of the β-glucosidic linkages in β-linked oligosaccharides as well as in alkyl and aryl β-glucosides with the release of glucose [7]. They are classified into GH1 and GH3 families of glycoside hydrolases based on sequence and folding similarities [8,9].

Due to the wide substrate specificity and their ability to both hydrolyze and synthesize glycosidic bonds, BGLs have found a very broad range of applications. Considering industrial applications, they are currently used in the production of biodegradable nonionic surfactants and other compounds [10]; synthesis of diverse oligosaccharides, glycoconjugates, alkyl- and amino-glycosides [11]; detoxification of cassava [12]; removal of cyanogenic glucosides from sorghum malt during the production of African beer [13]; enzymatic release of aroma compounds from glucosidic precursors present in fruit juices during winemaking [14]; enhancement of aroma in tea extracts [15], production of anti-cancer compounds [16]. BGLs also play an important role in the generation of potentially sustainable energy sources (e.g., glucose, ethanol, hydrogen, methane, etc.) from biomass conversion [17].

The enzyme is ubiquitously present in nature and found in bacteria [18], fungi [19], yeasts [20], plants [21], and animals [22]. Many fungal species may produce high levels of the BGL activity, including Penicillium verruculosum, whose mutant strains are able to secrete highly-active extracellular cellulolytic cocktails, rich in the homologously or heterologously expressed BGL, with up to 50 g/L of the total protein in the culture broth [23,24].

In this paper, a novel bgl1 gene of P. verruculosum (anamorph Talaromyces verruculosus) was cloned and heterologously expressed in Penicillium canescens RN3-11-7 host strain. The recombinant BGL (rPvBGL) was purified to a homogeneous state and its properties were studied in comparison with those of a recombinant BGL from Aspergillus niger (rAnBGL) that has been previously expressed in the same (P. canescens) host [25].

Section snippets

Microbial strains and enzyme preparatios

P. canescens RN3-11-7 strain [26] and P. verruculosum B1-537 strain [23,27], both deficient in the nitrate-reductase gene (ΔniaD), were used as a host strain for transformation and DNA preparation, respectively. Basal cellulose enzyme preparation was produced by unmodified P.verruculosum B1-537 strain and used as a reference.

Cloning of the bgl1 gene

The previously described protocol [28] was applied to clone the bgl1 gene of P. verruculosum. Briefly, amplification of the bgl1 gene was carried out by polymerase chain

Expression of recombinant β-glucosidases

The P. verruculosum BGL was cloned and heterologously expressed in P. canescens RN3-11-7 recipient strain. The corresponding recombinant enzyme is referred here as rPvBGL. Several clones of P. canescens (PC1, PC2 and PC11) with a highest activity of the culture filtrates against pNPG and cellobiose were selected after screening of the transformants in shake flasks. The recombinant BGL of A. niger PC19 (rAnBGL) was cloned and heterologously expressed in the same host strain previously [25]. The

Conclusions

A novel gene bgl1, encoding a β-glucosidase from P. verruculosum was cloned and successfully expressed in the recipient fungal strain of P. canescens under the control of the strong xylA gene promoter. The recombinant rPvBGL was isolated in a homogeneous form. The enzyme exhibited a high activity against pNPG, cellobiose and other cellooligosaccharides. Surprisingly, it also demonstrated a remarkable ability to hydrolyze laminarin and a mixed β −1,3-1,4-glucan from barley. rPvBGL converted both

Author contributions

P.V. designed plasmids, expressed and characterized of recombinant GH3 family β-glucosidase (rPvBGL) from Penicillium verruculosum. A.R. was responsible for the concept of the study and the planning of the experiments. I.Z. purified homogeneous proteins and edited a foreign language of manuscript. A.S. discussed the results and revised manuscript.

Funding

This work was partially supported by the RFBR (Grant number: 18-29-07070).

Declaration of competing interest

The authors declare no conflict of interest.

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