Cellulase (endo-1,4-β-D-glucanase)
(Talaromyces emersonii)

Several procedures are available for the measurement of endo-1,4-β-glucanase (EG). Primary methods employ defined oligosaccharides or highly purified polysaccharides and measure the rate of hydrolysis of glycosidic bonds using a reducing-sugar method. However, these primary methods are not suitable for the measurement of EG in crude fermentation broths due to the presence of reducing sugars and other enzymes active on these substrates. In such cases, dyed soluble or insoluble substrates are preferred as they are specific, sensitive, easy to use, and are not affected by other components, such as reducing sugars, in the enzyme preparation.

Over the past 8 years, we have been actively involved in the development of simple and reliable assay procedures, for the measurement of enzymes of interest to the cereals and related industries. In some instances, different procedures have been developed for the measurement of the same enzyme activity (e.g. α-amylase) in a range of different materials (e.g. malt, cereal grains and fungal preparations). The reasons for different procedures may depend on several factors, such as the need for sensitivity, ease of use, robustness of the substrate mixture, or the possibility for automation. In this presentation, we will present information on our most up-to-date procedures for the measurement of α-amylase, endo-protease, β-glucanase and β-xylanase, with special reference to the use of particular assay formats in particular applications.

Enzymatic degradation of carbohydrates is of major significance in the industrial processing of cereals and fruits. In the production of beer, barley is germinated under well-defined conditions (malting) to induce maximum enzyme synthesis with minimum respiration of reserve carbohydrates. The grains are dried and then extracted with water under controlled conditions. The amylolytic enzymes synthesized during malting, as well as those present in the original barley, convert the starch reserves to fermentable sugars. Other enzymes act on the cell wall polysaccharides, mixed-linkage β-glucan and arabinoxylan, reducing the viscosity and thus aiding filtration, and reducing the possibility of subsequent precipitation of polymeric material (Bamforth, 1982). In baking, β-amylase and α-amylase give controlled degradation of starch to fermentable sugars so as to sustain yeast growth and gas production. Excess quantities of α-amylase in the flour result in excessive degradation of starch during baking which in turn gives a sticky crumb texture and subsequent problems with bread slicing. Juice yield from fruit pulp is significantly improved if cell-wall-degrading enzymes are used to destroy the three-dimensional structure and water-binding capacity of the pectic polysaccharide components of the cell walls. Problems of routine and reliable assay of carbohydrate-degrading enzymes in the presence of high levels of sugar compounds are experienced with such industrial processes.

Enzymic degradation of carbohydrates is of major significance in the industrial processing of cereals and fruits. In the production of beer, barley is germinated under well defined conditions (malting) to induce maximum enzyme synthesis with minimum respiration of reserve carbohydrates. The grains are dried and then extracted with water under controlled conditions. The amylolytic enzymes synthesized during malting, as well as those present in the original barley, convert the starch reserves to fermentable sugars. Other enzymes act on the cell wall polysaccharides, mixed-linkage β-glucan and arabinoxylan, reducing the viscosity and thus aiding filtration, and reducing the possibility of subsequent precipitation of polymeric material. In baking, β-amylase and α-amylase give controlled degradation of starch to fermentable sugars so as to sustain yeast growth and gas production. Excess quantities of α-amylase in the flour result in excessive degradation of starch during baking which in turn gives a sticky crumb texture and subsequent problems with bread slicing. Juice yield from fruit pulp is significantly improved if cell-wall degrading enzymes are used to destroy the three-dimensional structure and water binding capacity of the pectic polysaccharide components of the cell walls. Problems of routine and reliable assay of carbohydrate degrading enzymes in the presence of high levels of sugar compounds are experienced with such industrial process.

Nanocellulose isolation from lignocellulose is a tedious and expensive process with high energy and harsh chemical requirements, primarily due to the recalcitrance of the substrate, which otherwise would have been cost-effective due to its abundance. Replacing the chemical steps with biocatalytic processes offers opportunities to solve this bottleneck to a certain extent due to the enzymes substrate specificity and mild reaction chemistry. In this work, we demonstrate the isolation of sulphate-free nanocellulose from organosolv pretreated birch biomass using different glycosyl-hydrolases, along with accessory oxidative enzymes including a lytic polysaccharide monooxygenase (LPMO). The suggested process produced colloidal nanocellulose suspensions (ζ-potential -19.4 mV) with particles of 7-20 nm diameter, high carboxylate content and improved thermostability (T_o = 301°C, T_max = 337°C). Nanocelluloses were subjected to post-modification using LPMOs of different regioselectivity. The sample from chemical route was the least favorable for LPMO to enhance the carboxylate content, while that from the C1-specific LPMO treatment showed the highest increase in carboxylate content.

Background: Production of value-added materials from lignocellulosic biomass residues is an emerging sector that has attracted much attention as it offers numerous benefits from an environmental and economical point of view. Non-digestible oligosaccharides represent a group of carbohydrates that are resistant to gastrointestinal digestion, and therefore, they are considered as potential prebiotic candidates. Such oligosaccharides can derive from the biomass cellulose fraction through a controlled enzymatic hydrolysis that eliminates the yield of monomers. Results: In the present study, hydrolysis of organosolv-pretreated forest residues (birch and spruce) was tested in the presence of four cellulases (EG5, CBH7, CBH6, EG7) and one accessory enzyme (LPMO). The optimal enzyme combinations were comprised of 20% EG5, 43% CBH7, 22% TtLPMO, 10% PaCbh6a and 5% EG7 in the case of birch and 35% EG5, 45% CBH7, 10% TtLPMO, 10% PaCbh6a and 5% EG7 in the case of spruce, leading to 22.3% and 19.1 wt% cellulose conversion into cellobiose, respectively. Enzymatic hydrolysis was applied on scale-up reactions, and the produced oligosaccharides (consisted of > 90% cellobiose) were recovered and separated from glucose through nanofiltration at optimized temperature (50 C) and pressure (10 bar) conditions, yielding a final product with cellobiose-to-glucose ratio of 21.1 (birch) and 20.2 (spruce). Cellobiose-rich hydrolysates were tested as fermentative substrates for different lactic acid bacteria. It was shown that they can efficiently stimulate the growth of two Lactobacilli strains. Conclusions: Controlled enzymatic hydrolysis with processive cellulases, combined with product recovery and purification, as well as enzyme recycling can potentially support the sustainable production of food-grade oligosaccharides from forest biomass.

Background: GH7 cellobiohydrolases (CBH1) are vital for the breakdown of cellulose. We had previously observed the enzyme as the most dominant protein in the active cellulose-hydrolyzing secretome of the hypercellulolytic ascomycete—Penicillium funiculosum (NCIM1228). To understand its contributions to cellulosic biomass saccharification in comparison with GH7 cellobiohydrolase from the industrial workhorse—Trichoderma reesei, we natively purified and functionally characterized the only GH7 cellobiohydrolase identified and present in the genome of the fungus. Results: There were marginal differences observed in the stability of both enzymes, with P. funiculosum (PfCBH1) showing an optimal thermal midpoint (T_m) of 68°C at pH 4.4 as against an optimal T_m of 65°C at pH 4.7 for T. reesei (TrCBH1). Nevertheless, PfCBH1 had an approximate threefold lower binding affinity (K_m), an 18-fold higher turnover rate (k_cat), a sixfold higher catalytic efficiency as well as a 26-fold higher enzyme-inhibitor complex equilibrium dissociation constant (K_i) than TrCBH1 on p-nitrophenyl-β-D-lactopyranoside (pNPL). Although both enzymes hydrolyzed cellooligomers (G2–G6) and microcrystalline cellulose, releasing cellobiose and glucose as the major products, the propensity was more with PfCBH1. We equally observed this trend during the hydrolysis of pretreated wheat straws in tandem with other core cellulases under the same conditions. Molecular dynamic simulations conducted on a homology model built using the TrCBH1 structure (PDB ID: 8CEL) as a template enabled us to directly examine the effects of substrate and products on the protein dynamics. While the catalytic triads—EXDXXE motifs—were conserved between the two enzymes, subtle variations in regions enclosing the catalytic path were observed, and relations to functionality highlighted. Conclusion: To the best of our knowledge, this is the first report about a comprehensive and comparative description of CBH1 from hypercellulolytic ascomycete—P. funiculosum NCIM1228, against the backdrop of the same enzyme from the industrial workhorse—T. reesei. Our study reveals PfCBH1 as a viable alternative for CBH1 from T. reesei in industrial cellulase cocktails.

Background: Many microorganisms possess enzymes that can efficiently degrade lignocellulosic materials, but do not have the capability to produce a large amount of ethanol. Thus, attempts have been made to transform such enzymes into fermentative microbes to serve as hosts for ethanol production. However, an efficient host for a consolidated bioprocess (CBP) remains to be found. For this purpose, a synthetic biology technique that can transform multiple genes into a genome is instrumental. Moreover, a strategy to select cellulases that interact synergistically is needed. Results: To engineer a yeast for CBP bio-ethanol production, a synthetic biology technique, called “promoter-based gene assembly and simultaneous overexpression” (PGASO), that can simultaneously transform and express multiple genes in a kefir yeast, Kluyveromyces marxianus KY3, was recently developed. To formulate an efficient cellulase cocktail, a filter-paper-activity assay for selecting heterologous cellulolytic enzymes was established in this study and used to select five cellulase genes, including two cellobiohydrolases, two endo-β-1, 4-glucanases and one beta-glucosidase genes from different fungi. In addition, a fungal cellodextrin transporter gene was chosen to transport cellodextrin into the cytoplasm. These six genes plus a selection marker gene were one-step assembled into the KY3 genome using PGASO. Our experimental data showed that the recombinant strain KR7 could express the five heterologous cellulase genes and that KR7 could convert crystalline cellulose into ethanol. Conclusion: Seven heterologous genes, including five cellulases, a cellodextrin transporter and a selection marker, were simultaneously transformed into the KY3 genome to derive a new strain, KR7, which could directly convert cellulose to ethanol. The present study demonstrates the potential of our strategy of combining a cocktail formulation protocol and a synthetic biology technique to develop a designer yeast host.

A model-based framework is described that permits the optimal composition of cellulase enzyme mixtures to be found for lignocellulose hydrolysis. The rates of hydrolysis are shown to be dependent on the nature of the substrate. For bacterial microcrystalline cellulose (BMCC) hydrolyzed by a ternary cellulase mixture of EG2, CBHI, and CBHII, the optimal predicted mixture was 1:0:1 EG2:CBHI:CBHII at 24 h and 1:1:0 at 72 h, at loadings of 10 mg enzyme per g substrate. The model was validated with measurements of soluble cello-oligosaccharide production from BMCC during both single enzyme and mixed enzyme hydrolysis. Three-dimensional diagrams illustrating cellulose conversion were developed for mixtures of EG2, CBHI, CBHII acting on BMCC and predicted for other substrates with a range of substrate properties. Model predictions agreed well with experimental values of conversion after 24 h for a variety of enzyme mixtures. The predicted mixture performances for substrates with varying properties demonstrated the effects of initial degree of polymerization (DP) and surface area on the performance of cellulase mixtures. For substrates with a higher initial DP, endoglucanase enzymes accounted for a larger fraction of the optimal mixture. Substrates with low surface areas showed significantly reduced hydrolysis rates regardless of mixture composition. These insights, along with the quantitative predictions, demonstrate the utility of this model-based framework for optimizing cellulase mixtures.