Cellobiohydrolase I
(Trichoderma longibrachiatum)

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: One of the sustainable development goals focuses on the biomass-based production as a replacement for fossil-based commodities. A novel feedstock with vast potentials is tunicate biomass, which can be pretreated and fermented in a similar way to lignocellulose. Ciona intestinalis is a marine filter feeder that is cultivated to produce fish feed. While the inner tissue body is used for feed production, the surrounding tunic remains as a cellulose-rich by-product, which can be further separated into outer and inner tunic. Ethanol production from organosolv-pretreated whole-tunic biomass was recently validated. The aim of the present study was to evaluate the potential of organosolv pretreated outer-tunic biomass for the production of biofuels and cellobiose that is a disaccharide with prebiotic potential. Results: As a result, 41.4 g/L of ethanol by Saccharomyces cerevisiae, corresponding to a 90.2% theoretical yield, was achieved under the optimal conditions when the tunicate biomass was pretreated at 195°C for 60 min at a liquid-to-solid ratio of 50. In addition, cellobiose production by enzymatic hydrolysis of the pretreated tunicate biomass was demonstrated with a maximum conversion yield of 49.7 wt. %. Conclusions: The utilisation of tunicate biomass offers an eco-friendly and sustainable alternative for value-added biofuels and chemicals. The cultivation of tunicate biomass in shallow coastal sea improves the quality of the water and ensures sustainable production of fish feed. Moreover, there is no competition for arable land, which leaves the latter available for food and feed production.

The adsorption behaviors of two glucanases, TvEG and TrCel7A, on three lignins were investigated. Three lignins were isolated from raw aspen and its pretreated solid residue. The isolated lignins were labeled as Asp-MWL, DA-MWL (pretreated by dilute acid), and GL-MWL (pretreated by green liquor), respectively. The surface properties of lignins and spin-coated lignin films were characterized by zeta potential, atomic force microscope (AFM) and contact angle. The enzyme adsorption behavior was monitored by quartz crystal microbalance (QCM) and fluorescence spectrometer. TlCel7A had similar adsorption capacities on the three lignin films but were higher than those of TvEG. The TrCel7A adsorptions on the three lignin films were affected by synergistic effect of electrostatic and hydrophobic interaction while the TvEG adsorptions on the three lignin films were mainly dominated by hydrophobic action. The adsorption capacities of TlCel7A and TvEG on the three lignin films were decreased by adding SL. Plausible explanation was that the SL and glucanase formed a complex with more negative charges, which suppressed the adsorption of glucannase on lignin through electrostatic repulsion. It also explained the improved enzymatic hydrolysis efficiency of lignocellulose upon adding SL.

Objective: The development of an enzymatic assay for the specific quantification of the C1-oxidation product, i.e. gluconic acid of cellulose active lytic polysaccharide monooxygenases (LPMOs). Results: In combination with a β-glucosidase, the spectrophotometrical assay can reliably quantify the specific C1-oxidation product of LPMOs acting on cellulose. It is applicable for a pure cellulose model substrate as well as lignocellulosic biomass. The enzymatic assay compares well with the quantification performed by HPAEC-PAD. In addition, we show that simple boiling is not sufficient to inactivate LPMOs and we suggest to apply a metal chelator in addition to boiling or to drastically increase pH for proper inactivation. Conclusions: We conclude that the versatility of this simple enzymatic assay makes it useful in a wide range of experiments in basic and applied LPMO research and without the need for expensive instrumentation, e.g. HPAEC-PAD.

Lytic polysaccharide monooxygenases (LPMOs) have attracted vast attention because of their unique mechanism of oxidative degradation of carbohydrate polymers and the potential application in biorefineries. This study characterized a novel LPMO from Myceliophthora thermophila, denoted MtLPMO9L. The structure model of the enzyme indicated that it belongs to the C1-oxidizing LPMO, which has neither an extra helix in the L3 loop nor extra loop region in the L2 loop. This was confirmed subsequently by the enzymatic assays since MtLPMO9L only acts on cellulose and generates C1-oxidized cello-oligosaccharides. Moreover, synergetic experiments showed that MtLPMO9L significantly improves the efficiency of cellobiohydrolase (CBH) II. In contrast, the inhibitory rather than synergetic effect was observed when combining used MtLPMO9L and CBHI. Changing the incubation time and concentration ratio of MtLPMO9L and CBHI could attenuate the inhibitory effects. This discovery suggests a different synergy detail between MtLPMO9L and two CBHs, which implies that the composition of cellulase cocktails may need reconsideration.

β-glucosidases play a critical role among the enzymes in enzymatic cocktails designed for plant biomass deconstruction. By catalysing the breakdown of β-1, 4-glycosidic linkages, β-glucosidases produce free fermentable glucose and alleviate the inhibition of other cellulases by cellobiose during saccharification. Despite this benefit, most characterised fungal β-glucosidases show weak activity at high glucose concentrations, limiting enzymatic hydrolysis of plant biomass in industrial settings. In this study, structural analyses combined with site-directed mutagenesis efficiently improved the functional properties of a GH1 β-glucosidase highly expressed by Trichoderma harzianum (ThBgl) under biomass degradation conditions. The tailored enzyme displayed high glucose tolerance levels, confirming that glucose tolerance can be achieved by the substitution of two amino acids that act as gatekeepers, changing active-site accessibility and preventing product inhibition. Furthermore, the enhanced efficiency of the engineered enzyme in terms of the amount of glucose released and ethanol yield was confirmed by saccharification and simultaneous saccharification and fermentation experiments using a wide range of plant biomass feedstocks. Our results not only experimentally confirm the structural basis of glucose tolerance in GH1 β-glucosidases but also demonstrate a strategy to improve technologies for bioethanol production based on enzymatic hydrolysis.

Background: The high cost of enzymes is one of the key technical barriers that must be overcome to realize the economical production of biofuels and biomaterials from biomass. Supplementation of enzyme cocktails with lytic polysaccharide monooxygenase (LPMO) can increase the efficiency of these cellulase mixtures for biomass conversion. The previous studies have revealed that LPMOs cleave polysaccharide chains by oxidization of the C1 and/or C4 carbons of the monomeric units. However, how LPMOs enhance enzymatic degradation of lignocellulose is still poorly understood. Results: In this study, we combined enzymatic assays and real-time imaging using atomic force microscopy (AFM) to study the molecular interactions of an LPMO [TrAA9A, formerly known as TrCel61A) from Trichoderma reesei] and a cellobiohydrolase I (TlCel7A from T. longibrachiatum) with bacterial microcrystalline cellulose (BMCC) as a substrate. Cellulose conversion by TlCel7A alone was enhanced from 46 to 54% by the addition of TrAA9A. Conversion by a mixture of TlCel7A, endoglucanase, and β-glucosidase was increased from 79 to 87% using pretreated BMCC with TrAA9A for 72 h. AFM imaging demonstrated that individual TrAA9A molecules exhibited intermittent random movement along, across, and penetrating into the ribbon-like microfibril structure of BMCC, which was concomitant with the release of a small amount of oxidized sugars and the splitting of large cellulose ribbons into fibrils with smaller diameters. The dividing effect of the cellulose microfibril occurred more rapidly when TrAA9A and TlCel7A were added together compared to TrAA9A alone; TlCel7A alone caused no separation. Conclusions: TrAA9A increases the accessible surface area of BMCC by separating large cellulose ribbons, and thereby enhances cellulose hydrolysis yield. By providing the first direct observation of LPMO action on a cellulosic substrate, this study sheds new light on the mechanisms by which LPMO enhances biomass conversion.

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.

The compositions and physical properties of pretreated lignocellulose vary depending on pretreatment methods; therefore, enzyme cocktails specific to pretreatments are desired for efficient saccharification of lignocellulose. Here, enzyme cocktails consisting of three pure lignocellulolytic enzymes endoglucanase (EG), cellobiohydrolase (CBH) and endoxylanase (XN) with a fixed amount of β-glucosidase were tailored for acid- and alkali-pretreated sugarcane bagasse (ACID and ALKALI, respectively). Based on a mixture design, the optimal mass ratios of EG, CBH, and XN were determined to be 61.25:38.73:0.02 and 53.99:34.60:11.41 for ACID and ALKALI, respectively. The optimized enzyme cocktail yielded a higher or comparable amount of reducing sugars from the hydrolysis of ACID and ALKALI when compared to that obtained using commercial cellulase mixtures. Using the commercial and easily available pure enzymes, this simple method for the in-house preparation of an enzyme cocktail specific to pretreated lignocellulose consisting of only four enzymes with a high level of hydrolysis will be helpful for achieving enzymatic saccharification in the lignocellulose-based biorefinery.

Broussonetia papyrifera, known as paper mulberry, is a potential feed stock for bioethanol production because of its cellulose-rich composition. Lignin in the dilute acid pretreated Broussonetia papyriferawas was fractionated to three different fractions, and their physiochemical properties were determined by FT-IR, GPC and NMR analyses. Different structural characteristics were observed from each lignin fraction. Cellobiohydrolases I (CBH) adsorption to each lignin was understood by the lignin properties. The results showed that aliphatic hydroxyl groups in lignin showed positive correlations with the maximum binding ability of CBH onto lignin samples. Also, the contents of phenolic compounds such as p-hydroxyphenyl benzoate (PB), syringyl (S) and guaiacyl (G) units in the lignin influenced their CBH binding.

In this study, two selected hardwoods were subjected to sodium chlorite delignification and steam explosion, and the impact of pre-treatments on synergistic enzymatic saccharification evaluated. A cellulolytic core-set, CelMix, and a xylanolytic core-set, XynMix, optimised for glucose and xylose release, respectively, were used to formulate HoloMix cocktail for optimal saccharification of various pre-treated hardwoods. For delignified biomass, the optimized HoloMix consisted of 75%: 25%, while for untreated and steam exploded biomass the HoloMix consisted of 93.75%: 6.25% protein dosage, CelMix: XynMix, respectively. Saccharification by HoloMix (27.5 mg protein/g biomass) for 24 h achieved 70-100% sugar yields. Pre-treatment of the hardwoods, especially those with a higher proportion of lignin, with a laccase improved saccharification by HoloMix. This study provided insights into enzymatic hydrolysis of various pre-treated hardwood substrates and showed the same lignocellulolytic cocktail comparable to/if not better than commercial enzyme preparations can be used to efficiently hydrolyse different hardwood species.

The beneficial mechanisms of the lytic polysaccharide monooxygenase CelS2 (ScLPMO10C) from Streptomyces coelicolor on interactions between cellulose and the processive cellulase cellobiohydrolase I (Cel7A) were investigated by quartz crystal microbalance with dissipation. Initial binding of CelS2 to cellulose coated SiO₂ sensors at 40°C was rapid, but minimal, and was followed by modest positive changes in oscillation frequencies of the quartz crystal sensors that were attributed to mass loss from the cellulose surface. The presence of oxidized cellulose on the CelS2 treated sensors was verified by X-ray photoelectron spectroscopy. Subsequent binding of purified Cel7A from Trichoderma longibrachiatum at 23°C was significantly less in overall extent to CelS2 treated sensors than to untreated controls despite identical initial binding rates and dissipation-change: frequency-change ratios (an indication of the rigidity of the newly forming layer). Moreover, initial Cel7A binding to the treated sensors was followed by positive overall frequency changes indicating potential increased hydrolytic removal of cellulose mass by cellobiohydrolase action at 23°C. Furthermore, secondary binding of Cel7A to the control sensors was coupled with considerable changes in dissipation (indicating greater surface viscoelasticity) not observed during the initial binding phase; the extent of this secondary binding was observed to be negligible during Cel7A interaction with the CelS2 treated sensors. This drastic difference in more viscoelastic secondary binding suggests a reduction in loose non-productive cellobiohydrolase binding following CelS2 treatment.

Lipid production by oleaginous microorganisms is a promising route to produce raw material for the production of biodiesel. However, most of these organisms must be grown on sugars and agro-industrial wastes because they cannot directly utilize lignocellulosic substrates. We report the first comprehensive investigation of Mucor circinelloides, one of a few oleaginous fungi for which genome sequences are available, for its potential to assimilate cellulose and produce lipids. Our genomic analysis revealed the existence of genes encoding 13 endoglucanases (7 of them secretory), 3 β-D-glucosidases (2 of them secretory) and 243 other glycoside hydrolase (GH) proteins, but not genes for exoglucanases such as cellobiohydrolases (CBH) that are required for breakdown of cellulose to cellobiose. Analysis of the major PAGE gel bands of secretome proteins confirmed expression of two secretory endoglucanases and one β-D-glucosidase, along with a set of accessory cell wall-degrading enzymes and 11 proteins of unknown function. We found that M. circinelloides can grow on CMC (carboxymethyl cellulose) and cellobiose, confirming the enzymatic activities of endoglucanases and β-D-glucosidases, respectively. The data suggested that M. circinelloides could be made usable as a consolidated bioprocessing (CBP) strain by introducing a CBH (e.g. CBHI) into the microorganism. This proposal was validated by our demonstration that M. circinelloides growing on Avicel supplemented with CBHI produced about 33% of the lipid that was generated in glucose medium. Furthermore, fatty acid methyl ester (FAME) analysis showed that when growing on pre-saccharified Avicel substrates, it produced a higher proportion of C14 fatty acids, which has an interesting implication in that shorter fatty acid chains have characteristics that are ideal for use in jet fuel. This substrate-specific shift in FAME profile warrants further investigation.

Heterogeneous enzymatic reactions are used in many industrial processes including pulp and paper, food, and biofuel production. Industrially-relevant optimization of the enzymes used in these processes requires assaying them with insoluble substrates. However, platforms for high throughput heterogeneous assays do not exist thereby severely increasing the cost and time of enzyme optimization, or leading to the use of assays with soluble substrates for convenient, but non-ideal, optimization. We present an innovative approach to perform heterogeneous reactions in a high throughput fashion using droplet microfluidics. Droplets provide a facile platform for heterogeneous reactions as internal recirculation allows rapid mixing of insoluble substrates with soluble enzymes. Moreover, it is easy to generate hundreds or thousands of picoliter droplets in a small footprint chip allowing many parallel reactions. We validate our approach by screening combinations of cellulases with real-world insoluble substrates, and demonstrate that the chip-based screening is in excellent agreement with the conventional screening methods, while offering advantages of throughput, speed and lower reagent consumption. We believe that our approach, while demonstrated for a biofuel application, provides a generic platform for high throughput monitoring of heterogeneous reactions.

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.

The kinetic behavior of aqueous cellulase on insoluble cellulose is best quantified through surface-based assays on a well-defined cellulose substrate of known area. We use a quartz crystal microbalance (QCM) to measure the activity of binary mixtures of Trichoderma longibrachiatum cellobiohydrolase I (Cel7A) and endoglucanase I (Cel7B) on spin-coated cellulose films. By extending a previous surface kinetic model for cellulase activity, we obtain rate constants for competitive adsorption of Cel7A and Cel7B, their irreversible binding, their complexation with the cellulose surface, and their cooperative cellulolytic activity. The activity of the two cellulases is linked through the formation of cellulose chain ends by Cel7B that provide complexation sites from which Cel7A effects cellulose chain scission. Although the rate-limiting step in Cel7A activity is complexation, Cel7B activity is limited by adsorption to the cellulose surface. A 2:1 bulk mass ratio of aqueous Cel7A:Cel7B, corresponding to a 4:1 surface mass ratio, effects the greatest rate of cellulose degradation across a range of cellulase concentrations at 25°C. We find that surface chain-end concentration is a major predictor of Cel7A activity. Disruption of the hydrogen-bonding structure of cellulose by Cel7B enhances the activity of Cel7A on the cellulose surface.

Droplet-based microfluidics is a powerful technique allowing ultra-high-throughput screening of large libraries of enzymes or microorganisms for the selection of the most efficient variants. Most applications in droplet microfluidic screening systems use fluorogenic substrates to measure enzymatic activities with fluorescence readout. It is important, however, that there is little or no fluorophore exchange between droplets, a condition not met with most commonly employed substrates. Here we report the synthesis of fluorogenic substrates for glycosidases based on a sulfonated 7-hydroxycoumarin scaffold. We found that the presence of the sulfonate group effectively prevents leakage of the coumarin from droplets, no exchange of the sulfonated coumarins being detected over 24 h at 30°C. The fluorescence properties of these substrates were characterized over a wide pH range, and their specificity was studied on a panel of relevant glycosidases (cellulases and xylanases) in microtiter plates. Finally, the β-D-cellobioside-6,8-difluoro-7-hydroxycoumarin-4-methanesulfonate substrate was used to assay cellobiohydrolase activity on model bacterial strains (Escherichia coli and Bacillus subtilis) in a droplet-based microfluidic format. These new substrates can be used to assay glycosidase activities in a wide pH range (4–11) and with incubation times of up to 24 h in droplet-based microfluidic systems.

For the first time, the competitive adsorption of inhibited cellobiohydrolase I (Cel7A, an exoglucanase) and endoglucanase I (Cel7B) from T. longibrachiatum is studied on cellulose. Using quartz crystal microgravimetry (QCM), sorption histories are measured for individual types of cellulases and their mixtures adsorbing to and desorbing from a model cellulose surface. We find that Cel7A has a higher adsorptive affinity for cellulose than does Cel7B. The adsorption of both cellulases becomes irreversible on time scales of 30–60 min, which are much shorter than those typically used for industrial cellulose hydrolysis. A multicomponent Langmuir kinetic model including first-order irreversible binding is proposed. Although adsorption and desorption rate constants differ between the two enzymes, the rate at which each surface enzyme irreversibly binds is identical. Because of the higher affinity of Cel7A for the cellulose surface, when Cel7A and Cel7B compete for surface sites, a significantly higher bulk concentration of Cel7B is required to achieve comparable surface enzyme concentrations. Because cellulose deconstruction benefits significantly from the cooperative activity of endoglucanases and cellobiohydrolases on the cellulose surface, accounting for competitive adsorption is crucial to developing effective cellulase mixtures.

Cellulose is the most abundant biopolymer and a major reservoir of fixed carbon on earth. Comprehension of the elusive mechanism of its enzymatic degradation represents a fundamental problem at the interface of biology, biotechnology, and materials science. The interdependence of cellulose disintegration and hydrolysis and the synergistic interplay among cellulases is yet poorly understood. Here we report evidence from in situ atomic force microscopy (AFM) that delineates degradation of a polymorphic cellulose substrate as a dynamic cycle of alternating exposure and removal of crystalline fibers. Direct observation shows that chain-end-cleaving cellobiohydrolases (CBH I, CBH II) and an internally chain-cleaving endoglucanase (EG), the major components of cellulase systems, take on distinct roles: EG and CBH II make the cellulose surface accessible for CBH I by removing amorphous-unordered substrate areas, thus exposing otherwise embedded crystalline-ordered nanofibrils of the cellulose. Subsequently, these fibrils are degraded efficiently by CBH I, thereby uncovering new amorphous areas. Without prior action of EG and CBH II, CBH I was poorly active on the cellulosic substrate. This leads to the conclusion that synergism among cellulases is morphology-dependent and governed by the cooperativity between enzymes degrading amorphous regions and those targeting primarily crystalline regions. The surface-disrupting activity of cellulases therefore strongly depends on mesoscopic structural features of the substrate: size and packing of crystalline fibers are key determinants of the overall efficiency of cellulose degradation.

Significant amounts of cell wall degrading (CWD) enzymes are required to degrade lignocellulosic biomass into its component sugars. One strategy for reducing exogenous enzyme production requirements is to produce the CWD enzymes in planta. For this work, various CWD enzymes were expressed in maize (Zea mays). Following growth and dry down of the plants, harvested maize stover was tested to determine the impact of the expressed enzymes on the production of glucose and xylose using different exogenous enzyme loadings. In this study, a consolidated pretreatment and hydrolysis process consisting of a moderate chemical pretreatment at temperatures below 75°C followed by enzymatic hydrolysis using an in-house enzyme cocktail was used to evaluate engineered transgenic feedstocks. The carbohydrate compositional analysis showed no significant difference in the amounts of glucan and xylan between the transgenic maize plants expressing CWD enzyme(s) and the control plants. Hydrolysis results demonstrated that transgenic plants expressing CWD enzymes achieved up to 141% higher glucose yield and 172% higher xylose yield over the control plants from enzymatic hydrolysis under the experimental conditions. The hydrolytic performance of a specific xylanase (XynA) expressing transgenic event (XynA.2015.05) was heritable in the next generation, and the improved properties can be achieved even with a 25% reduction in exogenous enzyme loading. Simultaneous saccharification and fermentation of biomass hydrolysates from two different transgenic maize lines with yeast (Saccharomyces cerevisiae D5A) converted 65% of the biomass glucan into ethanol, versus only a 42% ethanol yield with hydrolysates from control plants, corresponding to a 55% improvement in ethanol production.