Enzymatic Saccharification Research Articles

Purification and biochemical characterization of novel α-amylase and cellulase from Bacillus sp. PM06

Bacillus sp. PM06, previously isolated from sugarcane waste pressmud, could produce dual enzymes α-amylase and cellulase. The isolate’s crude enzymes were purified homogeneously using ammonium sulfate precipitation followed by High Quaternary amine anion exchange chromatography. Purified enzymes revealed the molecular weights of α-amylase and cellulase as 55 and 52 kDa, with a purification fold of 15.4 and 11.5, respectively. The specific activity of purified α-amylase and cellulase were 740.7 and 555.6 U/mg, respectively. It demonstrated a wide range of activity from pH 5.0 to 8.5, with an optimum pH of 5.5 and 6.4 for α-amylase and cellulase. The optimum temperature was 50 °C for α-amylase and 60 °C for cellulase. The kinetic parameters of purified α-amylase were 741.5 ± 3.75 µmol/min/mg, 1.154 ± 0.1 mM, and 589 ± 3.5/(s mM), using starch as a substrate. Whereas cellulase showed 556.3 ± 1.3 µmol/min/mg, 1.78 ± 0.1 mM, and 270.9 ± 3.8/(s mM) of V max, K m, K cat/K m, respectively, using carboxymethyl cellulose (CMC) as substrate. Among the various substrates tested, α-amylase had a higher specificity for amylose and CMC for cellulase. Different inhibitors and activators were also examined. Ca2+ Mg2+, Co2+, and Mn2+ boosted α-amylase and cellulase activities. Cu2+ and Ni2+ both inhibited the enzyme activities. Enzymatic saccharification of wheat bran yielded 253.61 ± 1.7 and 147.5 ± 1.0 mg/g of reducing sugar within 12 and 24 h of incubation when treated with purified α-amylase and cellulase. A more significant amount of 397.7 ± 1.9 mg/g reducing sugars was released from wheat bran due to the synergetic effect of two enzymes. According to scanning electron micrograph analysis, wheat bran was effectively broken down by both enzymes.

Shochu Koji Microstructure and Starch Structure during Preparation.

In this study, we investigated the changes in composition, microstructure, and starch molecular structure of shochu koji during preparation. We observed that the gelatinized and outer part of starch was decomposed in priority during the early and middle preparation stages. The gap between the starch granules increased with the delayed time. Finally, the koji microstructure became spongy. Shochu koji mold produced two α-amylases in different expression manners. Acid-labile α-amylase was produced in the early and middle preparation stages. Acid-stable α-amylase and saccharification power were produced in the middle and late stages. Throughout the koji preparation, reducing sugars content reached approximately 13-20 % of the total sugar content, with glucose representing over 70 % of the reducing sugars. α-Glucan fragments with C chains of degree of polymerization (DP) 4-73 were observed in the early and middle stages (<23 h), indicating the degradation of amylopectin at long B chains. In the latter stage, the amount of C chains of DP 6-30 decreased, while the longer C chains (DP 30<) did not change. These results showed that acid-labile α-amylase, acid-stable α-amylase, and saccharification enzymes including glucoamylase and α-glucosidase work preferentially on the amorphous regions of starch granules, and cooperative action of these enzymes during koji preparation contributes to the formation of the observed microstructure. Our study is the first report on the decomposition schemes of starch and the microstructure forming process in shochu koji.

Stable cellulolytic activity of Clostridium thermocellum against cellulosic biomass pretreated with ionic liquid 1-ethyl 3-methylimidazolium acetate

Using cellulosic biomass has considerable potential for the production of valuable chemicals while contributing to carbon neutrality. This study investigates the effect of biomass pretreatment with ionic liquid 1-ethyl 3-methylimidazolium acetate ([EMIM][Ac]) on the efficiency of enzymatic saccharification using the cellulolytic system of the thermophilic bacterium Clostridium thermocellum. Although [EMIM][Ac] pretreatment improves the efficiency of enzymatic saccharification, it also inhibits the cellulolytic enzyme. Following incubation with 30 % [EMIM][Ac], while the cellulolytic activity of the commercial cellulase cocktail (Celluclast) decreased to 15 % and that of C. thermocellum exoproteome remained stable, suggesting a relative tolerance of C. thermocellum cellulases to [EMIM][Ac]. The reducing sugars released from the pretreated cellulosic biomass corn hull by Celluclast were reduced by 24 % with supplementation of 5 % [EMIM][Ac] but remained unchanged for C. thermocellum exoproteome. These results highlight the potential of [EMIM][Ac] pretreatment of cellulosic biomass as a promising approach for enzymatic saccharification using C. thermocellum.

Enzymatic hydrolysis of organosolv-pretreated corncob and succinic acid production by Actinobacillus succinogenes

In this study, the conversion of organosolv-treated corncob into monosaccharides through enzymatic saccharification was investigated, with the resulting monosaccharides being utilized as a carbon source to produce succinic acid. The synergy between the cellulase and xylanase provided 76% cellulose and 64% xylan digestibility at 50 °C and pH 5.2. In separate hydrolysis and fermentation (SHF), Actinobacillus succinogenes produced 12.7 g/L of succinic acid from the hydrolysate with 0.12 g/g yield based on the pretreated corncob. Simultaneous saccharification and fermentation (SSF) demonstrated better performance with 16 g/L succinic acid titer and 0.24 g/g yield, though SHF provided a higher production rate. The condition in the SSF (37 °C and pH near neutral) was suboptimal for the enzymes, thus the succinic acid production was limited by the saccharification step. These findings emphasize the potential of organosolv-treated corncob to serve as an enzymatic hydrolysis substrate without neutralization and detoxification, supplying glucose and xylose for succinic acid production by A. succinogenes.

Effects of Anaerobic Germination and Enzymatic Saccharification on Chemical Compositions of Functional Drink from Riceberry Rice

Anaerobic germination and enzymatic saccharification significantly affected the chemical compositions of riceberry rice-based functional drink. Anaerobic germination significantly affected the moisture and carbohydrate contents in germinated riceberry rice (GRR), whereas the contents of crude protein, crude fat, ash and crude fiber were not changed significantly. During anaerobic germination, production of ATP was limited; therefore, the enzyme activity in the seed could be delayed in order to conserve the nutrients. With increasing germination time, gamma-aminobutyric acid (GABA) increased, reaching a maximum percentage of 58.6% in 96-hour GRR but total phenolics and total anthocyanins significantly decreased with a loss percentage of 26.5% and 44.4%, respectively. Enzymatic saccharification using a-amylase and a-glucoamylase significantly increased sugar (total sugar, reducing sugar and glucose) contents in GRR extract, depending on incubation duration of both enzymes. The 0.5 hour-incubation with a-amylase in combination with the 12 hours-incubation with a-glucoamylase was the condition under which the extract contained the highest amount of reducing sugar (22.5 g/L), glucose (11.1 g/L) and total phenolics (22.5 g gallic acid equivalent/L). As the result, anaerobic germination in combination with enzymatic saccharification might be applied as a green process for the production of a functional drink from riceberry rice.

Process development for fungal cellulase production and enzymatic saccharification of Parthenium hysterophorus for bioethanol production

Parthenium hysterophorus (PH), often known as parthenium weed, is one of the seventh most devastating weeds in the world.The primary aim of the study was to produce and optimize fungal FPase enzymes under solid-state fermentation (SSF) conditions for application in enzymatic hydrolysis to produce bioethanol. The impact of various physiological factors was verified through a systematic approach, initially using the one-factor-at-a-time (OFAT) method. Further optimization was carried out using statistical tools by response surface methodology (RSM), selecting three variables, i.e., lactose concentration, peptone concentration, and pH level. The outcomes of this research demonstrated that the RSM-based model led to the optimization of enzyme activity, resulting in 4.08U/ml of FPase, with a lactose concentration of 1.62 %, a peptone concentration of 1.5 %, and a pH level of 6 being the ideal conditions. When employing the FPase enzyme for biomass hydrolysis, the maximum yield of reducing sugar, at 0.37 ± 0.01 g/g, was achieved within a 96-hour period, using enzyme and substrate levels of 30 FPU/100 ml and 3 % (w/v), respectively. Fermentation of enzymatic hydrolysate was carried out by Saccharomyces cerevisiae at 30 °C, pH 6.0, and 120 rpm, which resulted in 0.45g/g ethanol yield after 48 h. Overall, our findings reveal thatPH biomass can be introduced into sugar-based biorefineries to produce biofuels and other valuable chemicals.

Potential Development and Economic Analysis of Bioethanol Fabrication Design from Oil Palm Biomass with Ionic Liquid Choline Acetate Using SuperPro Designer (SPD) Software

The increasing use of energy causes the availability of energy, especially in Indonesia, to be depleted. This condition encourages humans to develop renewable energy sources, one of which is bioethanol, with the availability of abundant raw materials in nature. However, the development of bioethanol is still ineffective because it requires a long time and has high costs. Therefore, a design for bioethanol fabrication from oil palm plantation products with ionic liquid pretreatment using SuperPro Designer (SPD) software was developed. The bioethanol production process was simulated through three stages: pretreatment, enzymatic saccharification, and fermentation. Pretreatment was carried out using the ionic liquid Choline Acetate, after which the results of saccharification and fermentation were analyzed. The results of saccharification showed that around 138 g/L of palm oil was converted into sugar for fermentation. The overall result of the simulation system is that the fermentation process for 12 hours produced 92 g/L ethanol or 96% of the theoretical yield. Meanwhile, ethanol production through fabrication for energy can reduce ethanol prices by 30% from their initial price. This ethanol fabrication design is very supportive of optimizing biomass into bioethanol. Therefore, this design can be used as a recommendation for economical bioethanol production to obtain optimal results in bioethanol production.

Hydrogen chloride treatment of rice straw for upcycling into nanofibrous products for sugar pool

The durability of the harvested wood products is currently contributing to the creation of a temporary carbon pool for the mitigation of global warming. To establish a new method to supply durable fabrication products from grass biomass, a hydrogen chloride treatment process was developed. Applying this treatment to rice straw particles reduced the xylan content and dramatically improved the disintegration efficiency to fibers. The treated and disintegrated particles exhibited a 6.3 times larger specific surface area than the untreated particles, and both particle disassembly into cells and cell surface fibrillation into nanofibrils were observed in microscopy images. The treatment of the particles also improved the susceptibility to enzymatic saccharification, enabling the fiber to be recycled into fermentable sugars after the use as structural materials. Thus, this process offers a method for bioresource management by converting grass into structural materials that pool carbon and sugars.

Physicochemical Characterization of Potassium Hydroxide Pretreated Chestnut Shell and Its Bioconversion to Lactic Acid by Lacticaseibacillus rhamnosus

Lactic acid (LA) is an important platform chemical with a wide range of applications, including bioplastic materials, and demand for it is growing rapidly. However, the high cost of feedstock for LA production is a major barrier to industrial production. This study designed a process to produce LA from chestnut shell (CS), a low-cost biomass. The entire process includes KOH pretreatment, enzymatic saccharification, and fermentation. This study investigated the chemical compositions and physicochemical properties of raw CS and KOH pretreated CS (KpCS) to evaluate the impact of the pretreatment process that enhances the conversion of cellulose into glucose. The results showed that KOH affected the lignin removal and surface morphological changes of CS, and FT-IR and TGA patterns correlated to increased cellulose fractions were found. In the fermentation process, Lacticaseibacillus rhamnosus was selected as a prominent LA producer, and the fermentation using KpCS hydrolysate was carried out. As a result, cell growth (27%), glucose consumption (23%), and LA production (21%) were all achieved higher than the control group. The LA production yield from our suggested process was estimated to be 187 g/kg CS, and we concluded that CS has a high potential as a feedstock for LA production.

Co-production of pigment and high value-added bacterial nanocellulose from Suaeda salsa biomass with improved efficiency of enzymatic saccharification and fermentation

This study evaluated the co-production of pigment and bacterial nanocellulose (BNC) from S. salsa biomass. The extraction of the beet red pigment reduced the salts and flavonoids contents by 82.7%–100%, promoting the efficiencies of enzymatic saccharification of the biomass and the fermentation of BNC from the hydrolysate. SEM analysis revealed that the extraction process disrupted the lignocellulosic fiber structure, and the chemical analysis revealed the lessened cellulase inhibitors, consequently facilitating enzymatic saccharification for 10.4 times. BNC producing strains were found to be hyper-sensitive to NaCl stress, produced up to 400.4% more BNC from the hydrolysate after the extraction. The fermentation results of BNC indicated that the LDU-A strain yielded 2.116 g/L and 0.539 g/L in ES-M and NES-M, respectively. In comparison to the control, the yield in ES-M increased by approximately 20.0%, while the enhancement in NES-M was more significant, reaching 292.6%. After conducting a comprehensive characterization of BNC derived from S. salsa through Scanning Electron Microscopy (SEM), Fourier Transform Infrared Spectroscopy (FTIR), X-ray Diffraction (XRD), and Thermogravimetric Analysis (TGA), the average fiber diameter distribution of these four BNC materials ranges from 22.23 to 33.03 nanometers, with a crystallinity range of 77%–90%. Additionally, they exhibit a consistent trend during the thermal degradation process, further emphasizing their stability in high-temperature environments and similar thermal properties. Our study found an efficient co-production approach of pigment and BNC from S. salsa biomass. Pigment extraction made biomass more physically and chemically digestible to cellulase, and significantly improved BNC productivity and quality.

Maximised bioethanol extraction from bamboo biomass through alkali pretreatment and enzymatic saccharification by application of ANN-NSGA-II-based optimisation method

Maximised bioethanol extraction from bamboo biomass through alkali pretreatment and enzymatic saccharification by application of ANN-NSGA-II-based optimisation method

Optimization, Scale-Up, and Economic Analysis of the Ethanol Production Process Using Sargassum horneri

Recently, the extensive spread of some algae along coastlines has surged into unmanageable thick decomposition layers. This study aimed to demonstrate the use of Sargassum horneri as a biomass resource for ethanol production through the continuous hydrolysis, enzymatic saccharification, and fermentation process. Sugars from S. horneri were obtained using a combination of thermal acid hydrolysis and enzymatic saccharification. The optimal conditions for thermal acid hydrolysis involved a 10% (w/v) S. horneri slurry treated with 100 mM H2SO4 at 121 °C for 60 min; enzymatic saccharification using 16 U/mL Cellic CTec2 further boosted the monosaccharide concentration to 23.53 g/L. Fermentation experiments were conducted with mannitol-adapted Saccharomyces cerevisiae BY4741 using S. horneri hydrolysate. Enhanced ethanol production was observed in the hydrolysate, particularly with mannitol-adapted S. cerevisiae BY4741, which yielded 10.06 g/L ethanol. Non-adapted S. cerevisiae produced 8.12 g/L ethanol, as it primarily utilized glucose and not mannitol. Regarding ethanol fermentation using 5 L- and 500 L-scale fermenters, the ethanol concentrations reached 10.56 g/L and 7.88 g/L with yields of 0.51 and 0.45, respectively, at 48 h. This study confirmed the economic viability of ethanol production using waste seaweed with optimized pretreatment conditions and the adaptive evolution of S. cerevisiae to mannitol.

The Conformational Change of the L3 Loop Affects the Structural Changes in the Substrate Binding Pocket Entrance of β-Glucosidase

β-glucosidase (Bgl) hydrolyzes cellobiose to glucose, thereby releasing non-reducing terminal glucosyl residues. Bgl is an essential enzyme belonging to the biomass-degrading enzyme family, which plays a vital role in enzymatic saccharification during biofuel production. The four loops above the Bgl substrate-binding pocket undergo a conformational change upon substrate recognition. However, the structural dynamism of this loop and how it is conserved among Bgl family members remain unknown. Herein, to better understand the four loops above the substrate-binding pocket of Bgl, four Bgl crystal structures in Thermoanaerobacterium saccharolyticum (TsaBgl) were determined at 1.5–2.1 Å. The L1, L2, and L4 loops of TsaBgl showed a rigid conformation stabilized by their neighboring residues via hydrogen bonds and hydrophobic interactions. The TsaBgl L3 loop showed relatively high flexibility and two different N-terminal region conformations. The conformational change in the TsaBgl L3 loop induced a change in charge and shaped at the substrate-binding pocket entrance. The amino acid sequences and structures of the TsaBgl L1–4 loops were compared with other 45 Bgl proteins, and a diversity of the L2 and L3 loops was observed. Differences in amino acids and lengths of Bgls L2–L3 loop induced differences in the conformation and structure of the Bgls substrate-binding pocket entrance. These findings expand our knowledge on the molecular function of the loops in the Bgl enzyme family.

Tween 80 reversed adverse effects of combined autohydrolysis and p-toluenesulfonic acid pretreatment on enzymatic hydrolysis of poplar

In this study, a combined pretreatment involving autohydrolysis and p-toluenesulfonic acid (p-TsOH) was performed on poplar to coproduce xylooligosaccharides (XOSs) and monosaccharides. The autohydrolysis (180 °C, 30 min) yielded 53.2 % XOS and enhanced the delignification efficiency in the subsequent p-TsOH treatment. Furthermore, considerably high glucan contents (64.1 %∼83.1 %) were achieved in the combined pretreated substrates. However, their enzymatic digestibilities were found to be extremely poor (9.6 %∼14.2 %), which were even lower than the single p-TsOH pretreated substrates (10.2 %∼35.8 %). The underlying reasons were revealed by systematically investigating the effects of the single and combined pretreatment strategies on substrate properties. Moreover, the Tween 80 addition successfully reversed the adverse effects of combined pretreatment on the enzymatic hydrolysis, achieving a high glucose yield of 99.3 % at an enzyme loading of 10 filter paper units/g (FPU/g) glucan. These results deepen the understanding of the synergy of combined pretreatment on biomass fractionation and enzymatic saccharification.

Chemical pretreatment in lignocellulosic biomass, anaerobic digestion, and biomethanation

The current impacts of climate change necessitate the promotion and use of renewable energy sources to avert the growing environmental and health concerns emanating from fossil fuels. Lignocellulosic biomass (LCB) is a promising, renewable, and sustainable energy source based on its abundance and feedstock properties. Anaerobic digestion (AD) involves a biochemical process that can convert LCB to biogas through hydrolysis and biomethanation processes through the action of microorganisms such as methanogens and sulfate-reducing bacteria. The hydrolysis of LCB releases various reducing sugars, which are essential in the production of biofuels such as bioethanol and biogas, organic acids, phenols, and aldehydes. The resultant biogas can complement energy needs while achieving economic, environmental, and health benefits. Enhancement of the AD process for LCB to bioenergy can be realized through appropriate pretreatment capable of disrupting the complex lignocellulosic structure and freeing cellulose and hemicellulose from the binding lignin for enzymatic saccharification and fermentation. Determining the optimal pretreatment technique for AD is critical for the success of the LCB energy production process. This study evaluated the application of chemical pretreatment to the improvement of LCB digestion for bioenergy production. The study reviews the LCB characteristics, AD processes, and the role of various chemical pretreatment techniques such as acid, alkali, organosolv, ozonolysis, and ionic fluids. The findings of this study create an understanding of the action methods and benefits of different LCB chemical pretreatment techniques while highlighting the outstanding drawbacks that require divergent strategies.

Analysis of Aureobasidium pullulans LB83 secretome reveals distinct carbohydrate active enzymes for biomass saccharification

Aureobasidium pullulans LB83 is a versatile biocatalyst that produces a plethora of bioactive products thriving on a variety of feedstocks under the varying culture conditions. In our last study using this microorganism, we found cellulase activity (FPase, 2.27 U/ml; CMCase, 7.42 U/ml) and other plant cell wall degrading enzyme activities grown on sugarcane bagasse and soybean meal as carbon source and nitrogen, respectively. In the present study, we provide insights on the secretome analysis of this enzymatic cocktail. The secretome analysis of A. pullulans LB83 by Liquid Chromatography coupled to Mass Spectroscopy (LC-MS/MS) revealed 38 classes of Carbohydrate Active enZymes (CAZymes) of a total of 464 identified proteins. These CAZymes consisted of 21 glycoside hydrolases (55.26%), 12 glycoside hydrolases harboring carbohydrate-binding module (31.58%), 4 carbohydrate esterases (10.53%) and one glycosyl transferase (2.63%). To the best of our knowledge, this is the first report on the secretome analysis of A. pullulans LB83.

A comparative study between the conventional and advanced biomass pretreatment methods applied on the Napier Grass used as a biomass

This paper presents a comparative analysis of the traditional and advanced pretreatment methods on the Napier Grass used as a biomass. It is a perennial C-4 type grass belongs to a sugarcane family, also called as Pennisetum Purpureum. It is found in abundance all across the world and traditionally used as a cattle feed stock in India.The author made an attempt for better effectiveness of the Napier Grass processing towards better productivity by using various pretreatment methods viz: Spectrophotometry, High Performance Liquid Chromatography, Enzyme Hydrolysis and Sonication.Napier Grass has lignocellulosic fibers with structural carbohydrates that requires pretreatment and enzymatically saccharified before they fermented into monomeric sugars and biofuel. The experimentation is carried out to examine the release of structural carbohydrate from Napier Grass using dilute acid pretreatment. In order to accelerate the process, the protein-rich fungus biomass Rhizopus Oligosporus has been added to the crude Napier Grass juice.It has been observed that, if green processed Napier Grass is treated with 5% concentrated sulfuric acid for a time span of 45 min at 120 °C, that results to yield the highest concentration of glucose from hemicellulose and cellulose with the highest quantum of xylose.The experimental result shows an increase in monomeric sugar that released from Napier Grass fibers after enzymatic saccharification process and the xylose and glucose concentrations achieved to 85.5% by dry weight of Napier grass.

Integrated multi-objective optimization of sodium bicarbonate pretreatment for the outer anatomical portion of corncob using central composite design, artificial neural networks, and metaheuristic algorithms

Effective pretreatment of raw biomass is crucial in establishing a successful biorefinery process, and response surface methodology (RSM), a statistical optimization method, is routinely employed for optimizing pretreatment methodologies. However, relying solely on statistical methods fails to capture the nonlinear relationships between pretreatment methodology and lignocellulose recalcitrance. Machine learning (ML) approaches excel at understanding these relationships. Therefore, combining statistical and ML optimization can establish better pretreatment parameters. This study presents a multi-stage approach to identify and optimize a chemical pretreatment for the outer anatomical portion of corncobs (CO). Various chemicals were screened using fixed factor screening and regular two-level factorial design (2FI), and among them NaHCO3 was chosen for further optimization, using central composite design (CCD). Moderate delignification (33.96%), minimum sugar loss (5.26 mg/g of CO), and a high enzymatic saccharification yield of up to 82% were achieved with the design. Three distinct metaheuristic algorithms, namely Teaching-Learning-Based Optimization (TLBO), Particle Swarm Optimization (PSO), and Genetic Algorithm (GA) were employed to optimize three specific hyperparameters of the artificial neural network (ANN), to create hybrid metaheuristic-optimized ANN models, which were subsequently used to validate the results obtained from the CCD. Among the different models, The TLBO-optimized ANN model provided the most accurate predictions than the CCD.

Natural lignin modulators improve bagasse saccharification of sugarcane and energy cane in field trials

AbstractThe burgeoning cellulosic ethanol industry necessitates advancements in enzymatic saccharification, effective pretreatments for lignin removal, and the cultivation of crops more amenable to saccharification. Studies have demonstrated that natural inhibitors of lignin biosynthesis can enhance the saccharification of lignocellulose, even in tissues generated several months post‐treatment. In this study, we applied daidzin (a competitive inhibitor of coniferaldehyde dehydrogenase), piperonylic acid (a quasi‐irreversible inhibitor of cinnamate 4‐hydroxylase), and methylenedioxy cinnamic acid (a competitive inhibitor of 4‐coenzyme A ligase) to 60‐day‐old crops of two conventional Brazilian sugarcane cultivars and two energy cane clones, bred specifically for enhanced biomass production. The resultant biomasses were evaluated for lignin content and enzymatic saccharification efficiency without additional lignin‐removal pretreatments. The treatments amplified the production of fermentable sugars in both the sugarcane cultivars and energy cane clones. The most successful results softened the most recalcitrant lignocellulose to the level of the least recalcitrant of the biomasses tested. Interestingly, the softest material became even more susceptible to saccharification.

Designing of recombinant hydrolytic enzymes cocktail for effective saccharification of delignified rice straw

Enzymatic saccharification is the most crucial and challenging step in lignocellulosic biorefineries for bioethanol production. The enzymatic saccharification requires a cocktail of hydrolytic enzymes, cellulases and hemicellulases for effective degradation of biomass. However, the complete bioconversion of biomass is governed by the proportion and synergistic action of hydrolytic enzymes present in the cocktail. Thus, this study aims to formulate an enzyme cocktail consisting of recombinant cellulases (bifunctional cellulolytic chimeric enzyme, CtGH1-L1-CtGH5-F194A and cellobiohydrolase, CtCBH5A) and xylanases (endo-1,4-β-xylanase, CtGH11A, and β-xylosidase, BoGH43), all in crude form (unpurified enzyme present in the total cellular proteins) for producing high yield of total reducing sugars using choline chloride:acetic acid pretreated rice-straw (CApRS). D-optimal mixture design approach was used for statistically designing the optimal proportion of crude recombinant enzyme cocktail for hydrolysis of delignified rice straw. The developed enzyme cocktail comprising chimera, CtCBH5A, CtGH11 and BoGH43 in ratio 35.1:41.8:10.0:13.1 resulted in cellulolytic enzyme activity, 5.6 FPU/mL and xylanase activity, 102 U/mL. Enzymatic saccharification of 3% (w/v) delignified CApRS using developed enzyme cocktail at 187 FPU/gCApRS, pH 5.8 and 35 °C, yielded total reducing sugar of 498 mg/gbiomass, glucose yield (410 mg/g) and xylose yield (71.3 mg/g) resulting in 72% saccharification efficiency. Whereas, significantly low total reducing sugar yield of 80 mg/g and 218 mg/g were released from raw RS and partially pretreated RS, respectively, by the developed cocktail under the same hydrolytic conditions, thereby inferring the effectiveness of the enzyme cocktail against CApRS biomass. The developed enzyme cocktail was compared with a commercial enzyme cocktail and prospects its applicability in lignocellulosic biorefineries.