Industrial Enzyme Cocktails Research Articles

Screening of novel fungal Carbohydrate Esterase family 1 enzymes identifies three novel dual feruloyl/acetyl xylan esterases.

Feruloyl esterases (FAEs) and acetyl xylan esterases (AXEs) are important enzymes for plant biomass degradation and are both present in Carbohydrate Esterase family 1 (CE1) of the Carbohydrate-Active enZymes database. In this study, ten novel fungal CE1 enzymes from different subfamilies were heterologously produced and screened for their activity towards model and complex plant biomass substrates. CE1_1 enzymes possess AXE activity, while CE1_5 enzymes showed FAE activity. Two enzymes from CE1_2 and one from CE1_5 possess dual feruloyl/acetyl xylan esterase (FXE) activity, showing expansion of substrate specificity. The new FXEs from CE1 can efficiently release both feruloyl and acetyl residues from feruloylated xylan, making them particularly interesting novel components of industrial enzyme cocktails for plant biomass degradation.

Functional analysis of chimeric TrCel6A enzymes with different carbohydrate binding modules.

The glycoside hydrolase (GH) family 6 is an important group of enzymes that constitute an essential part of industrial enzyme cocktails used to convert lignocellulose into fermentable sugars. In nature, enzymes from this family often have a carbohydrate binding module (CBM) from the CBM family 1. These modules are known to promote adsorption to the cellulose surface and influence enzymatic activity. Here, we have investigated the functional diversity of CBMs found within the GH6 family. This was done by constructing five chimeric enzymes based on the model enzyme, TrCel6A, from the soft-rot fungus Trichoderma reesei. The natural CBM of this enzyme was exchanged with CBMs from other GH6 enzymes originating from different cellulose degrading fungi. The chimeric enzymes were expressed in the same host and investigated in adsorption and quasi-steady-state kinetic experiments. Our results quantified functional differences of these phylogenetically distant binding modules. Thus, the partitioning coefficient for substrate binding varied 4-fold, while the maximal turnover (kcat) showed a 2-fold difference. The wild-type enzyme showed the highest cellulose affinity on all tested substrates and the highest catalytic turnover. The CBM from Serendipita indica strongly promoted the enzyme's ability to form productive complexes with sites on the substrate surface but showed lower turnover of the complex. We conclude that the CBM plays an important role for the functional differences between GH6 wild-type enzymes.

Activity and Thermostability of GH5 Endoglucanase Chimeras from Mesophilic and Thermophilic Parents.

Cellulases from glycoside hydrolase family 5 (GH5) are key endoglucanase enzymes in the degradation of diverse polysaccharide substrates and are used in industrial enzyme cocktails to break down biomass. The GH5 family shares a canonical (βα)8-barrel structure, where each (βα) module is essential for the enzyme's stability and activity. Despite their shared topology, the thermostability of GH5 endoglucanase enzymes can vary significantly, and highly thermostable variants are often sought for industrial applications. Based on the previously characterized thermophilic GH5 endoglucanase Egl5A from Talaromyces emersonii (TeEgl5A), which has an optimal temperature of 90°C, we created 10 hybrid enzymes with elements of the mesophilic endoglucanase Cel5 from Stegonsporium opalus (SoCel5) to determine which elements are responsible for enhanced thermostability. Five of the expressed hybrid enzymes exhibit enzyme activity. Two of these hybrids exhibited pronounced increases in the temperature optimum (10 and 20°C), the temperature at which the protein lost 50% of its activity (T50) (15 and 19°C), and the melting temperature (Tm ) (16.5 and 22.9°C) and extended half-lives (t1/2) (∼240- and 650-fold at 55°C) relative to the values for the mesophilic parent enzyme and demonstrated improved catalytic efficiency on selected substrates. The successful hybridization strategies were validated experimentally in another GH5 endoglucanase, Cel5 from Aspergillus niger (AnCel5), which demonstrated a similar increase in thermostability. Based on molecular dynamics (MD) simulations of both the SoCel5 and TeEgl5A parent enzymes and their hybrids, we hypothesize that improved hydrophobic packing of the interface between α2 and α3 is the primary mechanism by which the hybrid enzymes increase their thermostability relative to that of the mesophilic parent SoCel5.IMPORTANCE Thermal stability is an essential property of enzymes in many industrial biotechnological applications, as high temperatures improve bioreactor throughput. Many protein engineering approaches, such as rational design and directed evolution, have been employed to improve the thermal properties of mesophilic enzymes. Structure-based recombination has also been used to fuse TIM barrel fragments, and even fragments from unrelated folds, to generate new structures. However, little research has been done on GH5 endoglucanases. In this study, two GH5 endoglucanases exhibiting TIM barrel structure, SoCel5 and TeEgl5A, with different thermal properties, were hybridized to study the roles of different (βα) motifs. This work illustrates the role that structure-guided recombination can play in helping to identify sequence function relationships within GH5 enzymes by supplementing natural diversity with synthetic diversity.

Penicillium subrubescens is a promising alternative for Aspergillus niger in enzymatic plant biomass saccharification

In industrial applications, efficient mixtures of polysaccharide-degrading enzymes are needed to convert plant biomass into fermentable sugars. Most of the commercially produced lignocellulolytic enzymes are from a limited number of filamentous fungi, such as Trichoderma and Aspergillus species. In contrast, the plant biomass-degrading capacity of Penicillia has been less explored. We performed growth profiling of several Penicillia on diverse plant biomass-related substrates demonstrating the capacity particularly of Penicillium subrubescens to degrade crude lignocellulose feedstock, as well as polysaccharides, and metabolise their monomeric components. We focussed on the lignocellulolytic potential of P. subrubescens FBCC1632, which produced a variable set of (hemi-)cellulolytic activities on plant biomass substrates with activity levels comparable to those of Aspergillus niger. The good ability of the extracellular enzyme mixtures produced by P. subrubescens to saccharify complex plant biomasses, wheat bran and sugar beet pulp, indicated a high potential for this strain as a producer of industrial enzyme cocktails.

Functional Characterization and Low-Resolution Structure of an Endoglucanase Cel45A from the Filamentous Fungus Neurospora crassa OR74A: Thermostable Enzyme with High Activity Toward Lichenan and β-Glucan.

Biomass is the most abundant and short-term renewable natural resource on Earth whose recalcitrance toward enzymatic degradation represents significant challenge for a number of biotechnological applications. The not so abundant but critically necessary class of GH45 endoglucanases constitutes an essential component of tailored industrial enzyme cocktails because they randomly and internally cleave cellulose molecules. Moreover, GH45 glucanases are core constituents of major-brand detergent formulations as well as enzymatic aid components in the cotton processing industry, clipping unwanted cellulosic fibers from cotton (cellulosic)-based tissues. Here we report on a recombinant high-yield Neurospora crassa OR74A NcCel45A production system, a single-band GH45 endoglucanase purification, and a complete enzyme functional characterization. NcCel45A is a bimodular endoglucanase showing maximum activity at pH 6.0 and 60 °C, while most active against lichenan and β-glucans and lesser active toward filter paper, carboxymethylcellulose, and phosphoric acid-swollen cellulose. Gluco-oligosaccharide degradation fingerprinting experiments suggest cellopentaose as the minimal length substrate and ThermalFluor studies indicate that NcCel45A displays excellent stability at elevated temperatures up to 70 °C and pHs ranging from 5 to 9. Remarkably, we show that NcCel45A is uniquely resistant to a wide-range of organic solvents and small-angle X-ray scattering show a monkey-wrench molecular shape structure in solution, which indicates, unlike to other known cellulases, a non-fully extended conformation, thus conferring solvent protection. These NcCel45A unique enzymatic properties maybe key for specific industrial applications such as cotton fiber processing and detergent formulations.

The Study of Cellulose Structure and Depolymerization Through Single-Molecule Methods

Renewable energy has gained importance due to rising energy demands and diminishing fossil resources. Lignocellulosic biomass, with a core consisting of crystalline cellulose, has the potential to become a renewable source of fermentable sugars for energy production. However, to utilize this resource, biomass has to be broken down through physical, chemical, or enzymatic treatments. The biochemical hydrolysis of cellulose by cellulases offers an economical alternative to hazardous chemicals. Thus, complete knowledge of the molecular interactions between cellulases and cellulose would help to optimize the efficiency of industrial enzyme cocktails. Single-molecule (SM) methods study molecular events by visualizing individual molecules instead of measuring averages, thereby providing a detailed view of nanoscale processes with high spatial and temporal resolution. SM fluorescence microscopy utilizes enzyme and cellulose labeling, along with localization and tracking algorithms, to yield particle or fluorophore positions with nanoscale precision. Similarly, high-speed atomic force microscopy utilizes a high aspect ratio probe that is brought into close proximity and scanned across the sample to visualize the surface topography and its evolution over time. Both SM techniques have been recently applied to the study of cellulase-cellulose interactions and used to probe enzyme-binding orientation, affinity and reversibility, non-catalytic and catalytic surface motion, and the effect of molecular crowding on enzyme mobility. This review aims to showcase SM techniques and how they have been applied to study cellulose structure and cellulose depolymerization by cellulases. While the study of cellulase-cellulose interactions and cellulose depolymerization through SM microscopy is still a young field, these methods have already contributed to our understanding of the nanoscale processes involved in biomass conversion. Further application of SM techniques could elucidate molecular mechanisms involved in enzyme synergism, as well as the molecular changes that take place as cellulose fibrils are converted into soluble sugars.

Transient Kinetics and Rate-Limiting Steps for the Processive Cellobiohydrolase Cel7A: Effects of Substrate Structure and Carbohydrate Binding Domain

Cellobiohydrolases are exoacting, processive enzymes that effectively hydrolyze crystalline cellulose. They have attracted considerable interest because of their role in both natural carbon cycling and industrial enzyme cocktails used for the deconstruction of cellulosic biomass, but many mechanistic and regulatory aspects of their heterogeneous catalysis remain poorly understood. Here, we address this by applying a deterministic model to real-time kinetic data with high temporal resolution. We used two variants of the cellobiohydrolase Cel7A from Hypocrea jecorina , and three types of cellulose as substrate. Analysis of the pre-steady-state regime allowed delineation rate constants for both fast and slow steps in the enzymatic cycle and assessment of how these constants influenced the rate of hydrolysis at quasi-steady state. Processive movement on the cellulose strand advanced with characteristic times of 0.15-0.7 s per step at 25 °C, and the rate was highest on amorphous substrate. The cellulose binding module was found to raise this rate on crystalline, but not on amorphous, substrate. The rapid processive movement signified high intrinsic reactivity, but this parameter had marginal influence on the steady-state rate. This was because dissociation and association were slower and, hence, rate limiting. Specifically, the dissociation from the strand was found to occur with characteristic times of 45-100 s. This meant that dissociation was the bottleneck, except at very low substrate loads (0.5-1 g/L), where association became slower.

Conversion of α-Chitin Substrates with Varying Particle Size and Crystallinity Reveals Substrate Preferences of the Chitinases and Lytic Polysaccharide Monooxygenase of Serratia marcescens

Industrial depolymerization of chitinous biomass generally requires numerous steps and the use of deleterious substances. Enzymatic methods provide an alternative, but fundamental knowledge that could direct potential development of industrial enzyme cocktails is scarce. We have studied the contribution of monocomponent chitinases (ChiA, -B, and -C) and the lytic polysaccharide monooxygenase (LPMO) from Serratia marcescens on depolymerization of α-chitin substrates with varying particle size and crystallinity that were generated using a converge mill. For all chitinases activity was positively correlated to a decline in particle size and crystallinity. Especially ChiC, the only nonprocessive endochitinase from the S. marcescens chitinolytic machinery, benefited from mechanical pretreatment. Combining the chitinases revealed clear synergies for all substrates tested. CBP21, the chitin-active LPMO from S. marcescens, increased solubilization of substrates with high degrees of crystallinity when combined with each of the three chitinases, but this synergy was reduced upon decline in crystallinity.