Biorefineries are currently searching for alternative renewable energy resources due to the depletion of fossil fuels and the negative environmental impact of their combustion [15]. Lignocellulosic biomass produced by the pulp and paper industry is one of the most abundant renewable resources on earth [2, 37, 43]. Agricultural residues and sludge waste from the paper manufacturing process consist of 37–50% and 50–70% cellulose, respectively [20]. Therefore, biorefineries have considerable interest in converting these resources into fermentable sugars for biofuel production [46]. This is one of the most important biotechnology alternatives for sustainable biofuel production.
For efficient hydrolysis of cellulose to fermentable sugars the synergistic action of three cellulases are required [7]. Exoglucanases that act on the ends of the cellulose chain releasing β-cellobiose while endoglucanases attack the O-glycosidic bonds resulting in different glucan chain lengths [2], β-Glucosidases then act specifically on the β-cellobiose by hydrolysing the β-1-4 glycosidic bonds to produce glucose monomers [24]. This is a rate-limiting step as β-glucosidases are often inhibited by their product glucose resulting in feedback inhibition [31].
β-Glucosidases are classified based on three characteristics (i) substrate specificity, (ii) nucleotide sequence, and (iii) amino acid sequence. Based on substrate specificity β-glucosidases are classified into three groups aryl-β-D-glucosidases which have a strong affinity for aryl-β-D-glucosides such as 4-nitrophenyl-β-D-glucopyranoside; cellobiases that hydrolyse only disaccharides; and broad specificity glucosidases that exhibit activity on many substrate types and are the most common [44]. Classification by nucleotide sequence includes β-glucosidases and phospho-β-glucosidases from bacteria and mammals (BGA); and β-glucosidases from yeasts, moulds, and rumen bacteria (BGB) [44]. The third classification group includes β-glucosidases with structural similarity and conserved amino acid sequence motifs [42].
Currently, there are 133 glycoside hydrolase (GH) families in the Carbohydrate Active enZYme database which are further subdivided into clans based on the similarity of their catalytic domain structures and amino acids based on common ancestry [48]. Sixty-two β-glucosidases originating from archaebacteria, plants and animals belong to GH family one and 44 originating from bacteria fungi and yeast belong to GH family three, however, these enzymes may also be found in GH families five, nine, 13 and 116 [55]. β-Glucosidases can be intracellular, extracellular, or cell-bound enzymes. The GH three family β-glucosidases are extracellular, or cell bound whilst those from GH family one is predominantly intracellular [2]. GH one and three family β-glucosidases are known to display tolerance to glucose [48].
Numerous microbes including bacteria, fungi and actinomycetes are ubiquitous in nature and the endo and exogenous microbial enzymes from many of these organisms have been widely explored [4, 10, 15, 39]. Specifically, β-glucosidases from several fungal species including Aspergillus, Fusarium, and Trichoderma have been explored for glucose-tolerant β-glucosidases [15, 39, 54]. β-Glucosidases from Aspergillus and Trichoderma sp. have been commercialised and are currently used in industrial applications including cellulose hydrolysis, however, their commercial preparations are a huge cost factor for industries [33]. Therefore, there is a need to search for a novel thermophilic, glucose tolerant β-glucosidase producer able to withstand basic to slightly acidic environments.
Both the bacterial and fungal enzyme producers studied produce several β-glucosidases, however, these enzymes do not display tolerance to glucose due to feedback inhibition at high glucose concentrations [4]. Enzyme production is costly, therefore, to meet industrial demand, a low-cost growth medium is required for enzyme production. Two methods may be used to produce microbial β-glucosidases: solid-state fermentation and submerged fermentation. Submerged fermentation has an advantage which includes shorter fermentation periods for enzyme production [50]. β-Glucosidase production is influenced by medium composition and culture conditions. Physical and chemical parameters known to influence β-glucosidase culture conditions include incubation time, pH, incubation temperature, agitation speed, nitrogen, and carbon sources [21]. Temperature and pH are the most important factors governing microbial growth. Carbon and nitrogen source supplementation provides an enriched environment for microbial growth thus increasing enzyme production [13]. Screening and optimisation of growth conditions are crucial to ensure maximal enzyme production with the potential to reduce β-glucosidase production costs [23].
Optimization of growth conditions can be done via two approaches the classical One Variable at a Time (OVAT) and statistical Plackett Burman Design (PBD) and Box Behnken Design (BBD) [14]. The OVAT technique allows for the optimisation of one factor at a time, however, the disadvantage of this method is that it is laborious, time consuming and does not allow one to study the interaction of variables thus making it impossible to detect the true optimum when multiple different variables come together [32]. Therefore, statistical methods such as PBD and BBD are used to eliminate the limitations of the OVAT optimization process [28]. PBD is a screening technique used to screen media components in shake flasks reducing the total number of experiments, thus determining the most important factors [18]. Response surface methodology using a BBD is an effective method to evaluate the interactions between variables by assessing the effect of independent variables on enzyme production [49, 52].
Cellulose hydrolysis by current commercial cellulase cocktails has been achieved, however, these cocktails require supplementation of β-glucosidases as the cocktails do not contain sufficient β-glucosidases for the complete hydrolysis of cellulose to glucose [58]. β-Glucosidases present in these cocktails are also inhibited by their product glucose thus reducing yields [41]. Current commercial β-glucosidases from T. reesei are very expensive due to high production costs and cannot withstand acidic environments, it is, therefore, necessary to search for a native fungal producer of glucose-tolerant β-glucosidases for supplementation of existing cellulase cocktails. Therefore, to meet industrial demand, there is a need to optimize the production of enzymes by optimization of growth parameters to produce high levels of β-glucosidases. There are various reports on the optimization of growth parameters using PBD and BBD to increase β-glucosidase yields [57, 35, 32]. β-Glucosidase production from Aspergillus terreus strain EMOO 6 − 4 and Paecilomyces variotii was increased by optimization using the two statistical methods mentioned above [32, 19].
Although there were various reports on the production and characterization of β-glucosidases from multiple fungal species there were very few reports on glucose-tolerant β-glucosidases. Therefore, the present study optimized glucose-tolerant β-glucosidase production by PBD and BBD by a novel glucose-tolerant β-glucosidase producer Neofusicoccum parvum strain F7. We also report the purification and characterization of the crude extract produced under optimal conditions for the enzyme’s potential application in cellulose hydrolysis as a supplement to existing commercial cellulase cocktails.