Pure Cellulase Research Articles

Evaluation of free and immobilized cellulase on chitosan-modified magnetic nanoparticles for saccharification of sorghum residue.

Enzymatic hydrolysis plays a pivotal role in transforming lignocellulosic biomass. Addressing alternate techniques to optimize the utilization of cellulolytic enzymes is one strategy to improve its efficiency and lower process costs. Cellulases are highly specific and environmentally benign biocatalysts that break down intricate polysaccharides intosimple formsofsugars. In contrast to the most difficult and time-consuming enzyme immobilization processes, in this research, we studied simple, mild, and successful techniques for immobilization of pure cellulase on magnetic nanocomposites using glutaraldehyde as a linker and used in the application of sorghum residue biomass. Fe3O4 nanoparticles were coated with chitosan from the co-precipitation method, which served as an enzyme carrier. The nanoparticles were observed under XRD, Zeta Potential, FESEM, VSM, and FTIR. The size morphology results presented that the Cs@Fe3O4 have 42.2nm, while bare nanoparticles (Fe3O4) have 31.2nm in size. The pure cellulase reaches to 98.07% of loading efficiency and 71.67% of recovery activity at optimal conditions. Moreover, immobilized enzyme's pH stability, thermostability, and temperature tolerance were investigated at suitable conditions. The kinetic parameters of free and immobilized enzyme were estimated as Vmax; 29 ± 1.51 and 27.03 ± 2.02µmolmin-1mg-1, Km; 4.7 ± 0.49mM and 2.569 ± 0.522mM and Kcat; 0.13s-1, and 0.89s-1. Sorghum residue was subjected to 2% NaOH pre-treatment at 50℃. Pre-treated biomass contains cellulose of 64.8%, used as a raw material to evaluate the efficiency of reducing sugar during hydrolysis and saccharification of free and immobilized cellulase, which found maximum concentration of glucose 5.42g/L and 5.12g/L on 72h. Thus, our study verifies the use of immobilized pure cellulase to successfully hydrolyze raw material, which is a significant advancement in lignocellulosic biorefineries and the reusability of enzymes.

Synthetic microbial consortia bioprocessing integrated with pyrolysis for efficient conversion of cellulose to valuables

Synthetic microbial consortium (SynCONS) based bioprocessing integrated with pyrolysis is successfully demonstrated as an advance in our quest to convert cellulose to valuables. Comparative metabolic analysis of multiple microbes allowed robust design of SynCONS aimed at consolidated bioprocessing of cellulose to ethanol and lactate. The microbial partners of Trichoderma reesei - Parageobacillus thermoglucosidasius (fungal-bacterial) and Thermobifida fusca - P. thermoglucosidasius (bacterial-bacterial, thermophilic) showed promising cellulose degradation and total yields of 9 % and 23 % respectively. Further, higher ethanol yields were achieved in thermophilic SynCONS using engineered fermentative partner P. thermoglucosidasius TM242 (33 %) or pure cellulases for saccharification (51 %). Finally, pyrolyzing the residual cellulose and microbial biomass resulted in carbon material with desirable physicochemical properties as revealed by Scanning Electron Microscopy, X-ray Diffraction and Raman spectroscopy. Overall, the integration of SynCONS bioprocessing with pyrolysis confirmed a promising strategy for conversion of cellulose to chemicals, biofuels, and carbon suitable for several industrial applications.

Enzimatic Kinetics of Cellulose Hydrolysis using Cellulase from Goat Rumen Fluids

In line with the depletion of petroleum reserves from fossils, humans have forced people to look for alternative renewable energy sources. Nipah plant stems are organic material that contains a lot of lignocellulose. Cellulose in lignocellulose can be hydrolyzed to glucose to produce bioethanol. Problems arise because hydrolysis using pure cellulase requires relatively high costs, so it is necessary to find an alternative source of cheap cellulase. One of the cheap sources of cellulase is goat rumen fluid. Until now, there is not much information regarding the reaction kinetics and cellulase characteristics of goat rumen fluid. The purpose of this study was to isolate crude cellulase extract, determine the KM and Vmax values ​​and determine the optimum temperature and pH. The research was carried out at the Unsoed Agricultural Technology Laboratory. The samples used were seven rumen fluids. The study began with isolation followed by enzymatic kinetics studies. Fractionation was carried out by adding ammonium sulfate salt (50, 60,70, and 80 %), and centrifugation at 7,000 rpm at 4 oC for 15 minutes. Crude cellulase extract with the highest enzyme activity value was used for the study of enzymatic reaction kinetics and characterization. The crude cellulase resistance test to temperature was carried out at 45, 55 and 65 oC and the pH was carried out at pH 4, 5, 6, 7 and 8. The KM and Vmax values ​were determined by measuring the activity of crude cellulase extract at various concentrations of CMC (1, 1 ,5, 2, 2.5, and 3 %). The results showed that crude cellulase extract had an average specific activity of 1.7356 IU/mg. The highest enzyme activity was 0.9854 IU/ml. The optimum crude cellulase extract was at pH 6 with an activity of 1.1015 IU/ml and a temperature of 60 oC with an activity of 0.7829 IU/ml. At a CMC concentration of 2.5% crude cellulase extract had an activity of 0.3179 IU/ml with a Vmax value of 0.0045 IU/ml and a KM of 0.0252 %.

Enzimatic Kinetics of Cellulose Hydrolysis using Cellulase from Goat Rumen Fluids

In line with the depletion of petroleum reserves from fossils, humans have forced people to look for alternative renewable energy sources. Nipah plant stems are organic material that contains a lot of lignocellulose. Cellulose in lignocellulose can be hydrolyzed to glucose to produce bioethanol. Problems arise because hydrolysis using pure cellulase requires relatively high costs, so it is necessary to find an alternative source of cheap cellulase. One of the cheap sources of cellulase is goat rumen fluid. Until now, there is not much information regarding the reaction kinetics and cellulase characteristics of goat rumen fluid. The purpose of this study was to isolate crude cellulase extract, determine the KM and Vmax values ​​and determine the optimum temperature and pH. The research was carried out at the Unsoed Agricultural Technology Laboratory. The samples used were seven rumen fluids. The study began with isolation followed by enzymatic kinetics studies. Fractionation was carried out by adding ammonium sulfate salt (50, 60,70, and 80 %), and centrifugation at 7,000 rpm at 4 oC for 15 minutes. Crude cellulase extract with the highest enzyme activity value was used for the study of enzymatic reaction kinetics and characterization. The crude cellulase resistance test to temperature was carried out at 45, 55 and 65 oC and the pH was carried out at pH 4, 5, 6, 7 and 8. The KM and Vmax values ​were determined by measuring the activity of crude cellulase extract at various concentrations of CMC (1, 1 ,5, 2, 2.5, and 3 %). The results showed that crude cellulase extract had an average specific activity of 1.7356 IU/mg. The highest enzyme activity was 0.9854 IU/ml. The optimum crude cellulase extract was at pH 6 with an activity of 1.1015 IU/ml and a temperature of 60 oC with an activity of 0.7829 IU/ml. At a CMC concentration of 2.5% crude cellulase extract had an activity of 0.3179 IU/ml with a Vmax value of 0.0045 IU/ml and a KM of 0.0252 %.

Production, Purification and Characterization of Cellulase from Cossus cossus Larvae

This study aimed to purify and characterize the biochemical properties of cellulase from bacterial isolates of C. cossus larvae, to determine the effect of adding crude extract cellulase and pure enzymes to the hydrolysis of rice straw. The research steps included purification of enzymes by ion exchange chromatography and gel filtration chromotography, as well as SDS-PAGE electrophoresis analysis. Pure cellulases are characterized by their biochemical properties and applied in the hydrolysis of cellulose. The results showed that the characteristics of an optimum pH of 5.6, an optimum temperature of 70⁰C and a substrate concentration of 1.5% CMC, and a molecular weight of 36.49 kDa. 

Endo-1,4-D-glucanohydrolase assisted extraction of essential oil from the seed kernels of Nutmeg by using a two-step protocol

The cell walls of plants are made up of cellulose as the major composite and the hydrolysis of this polysaccharide has proven to be a major step in the extraction of many biomolecules. This research was on both pure and partially purified endo-1,4-D-glucanohydrolase, EC 3.2.1.4 (cellulase) assisted extraction of essential oil from the seed kernels of Nutmeg by using a two-step protocol that involved the pretreatment of the sample with cellulase followed by the distillation of the essential oil using Clevenger apparatus. The essential oil obtained after pretreatment with pure cellulase was 8.2 % while 5.8 % of essential oil was obtained when treatment prior to hydro-distillation of oil was done with partially purified cellulase. An oil yield of 3.8 % was observed when no enzyme pretreatment was carried out. The GC-MS analysis of the essential oil obtained from the two-step protocol showed the presence of 25 and 24 components from the sample pretreated with the partially purified cellulase and pure cellulase respectively. There were three major groups of components observed from the essential oils. These are the monoterpenes made up of sabinene, α-pinene, and β-pinene; the sesquiterpenes made up of safrole and alpha-copaene; and the phenylpropanoid/aromatic compounds, composed of myristicin and methyl eugenol. The presence of these principal compounds in the major groups has given nutmeg essential oil improved value due to the possibility of incorporating them as functional ingredients in several products with possible applications in the pharmaceutical, agricultural, fragrance, flavour, and cosmetic industries

A glucose oxidase peroxidase-coupled continuous assay protocol for the determination of cellulase activity in the laboratory: the Abuajah method

This present study reports a novel laboratory protocol, the glucose oxidase peroxidase (GOPOD)-coupled continuous assay (GcCA) which is based on the general principle that all cellulases generate β-d-glucose as one of their hydrolytic products. Resultantly, the β-d-glucose generated serves as a substrate to an auxiliary enzyme, GOPOD, coupled to the cellulase hydrolytic process, in a simultaneous secondary reaction which continuously quantifies the concentration of glucose generated as a measure of incremental cellulase activity in the laboratory. The protocol is initial hydrolytic rate-driven. Thus, making it very suitable for kinetic studies of pure cellulase samples in the laboratory. GcCA protocol is simple, rapid, specific, highly sensitive and accurate, non-laborious and saves cost in terms of time and materials. It is robust and dynamic and could be modified or adapted to suit either standard, 1000 μL cuvette or 96-well microtiter plate formats. Its optimized total assay reaction mixture of 500 μL constitutes a substrate, enzyme, buffer, distilled water and coupled GOPOD components. The GcCA protocol was validated for linearity, applicability, sensitivity, precision and compared with a standard and widely used assay method for optimized enzyme activity. It was further compared with the most widely used routine assay procedure for determination of reducing sugars, the DNS method. The GcCA protocol is a high throughput technique which enables the assay of over 120 samples in one day.

Improved production of cellulase by Trichoderma reesei (MTCC 164) from coconut mesocarp-based lignocellulosic wastes under response surface-optimized condition.

Experimental investigations were carried out to develop economic production process of cellulase using coconut mesocarp as an inexpensive lignocellulosic inducer while replacing commercial cellulose. Cellulase production was initially investigated from commercial cellulose in different submerged conditions using Trichoderma reesei (MTCC 164). Maximum enzyme production was achieved 6.3g/l with activity level 37 FPU/ml in the condition where cellulose to water content ratio was maintained at 5:35 (W/V). To achieve similar maximum production of cellulase from coconut mesocarp, response surface methodology was implemented to optimize most influencing parameters. Most influencing nutritional parameters such as coconut mesocarp, glucose and peptone were optimized in the concentration ranges of 35g/l, 35g/l and 25g/l, respectively. Selecting optimized parameter values, fermentations were conducted inside the fermenter with 2L operating volume to ensure high concentration and activity profiles of enzyme. Enzyme concentration was achieved 7.20g/l after 96h of batch fermentation with specific activity levels of 42 FPU/ ml and CMCase 75U/ml. Enzyme concentration was further improved to 9.58g/l with activity levels of 54 FPU/ml and CMCase 93U/ml by adopting sequential feeding of coconut mesocarp in fed-batch fermentation mode. The presence of pure cellulase in the sample was confirmed by FTIR analysis.

Isolation, growth, enzyme assay and identification via 16S rRNA full sequencing of cellulolytic microbes from Nepal for biofuel production

Abstract With its rich climatic and geographical variance, Nepal harbors a habitat for a diverse set of microbes. This research aims to identify and study the potency of native cellulolytic microbes. Cellulolytic microbes are being used to produce biofuels. The microorganisms were isolated from varied sources and grown in three valid media: Tryptone Soy Broth (TSB), Nutrient Broth (NB) and Gelatin. The findings from the growth quality observations, hydrolysis capacity calculations, and total cellulase assay were used to assess the cellulase enzyme potency of the microbes. Although all three media can be used to study the growth and isolation of the cellulolytic microbes, and different media showed different application potential, Gelatin media may be preferred for screening a large sample size. Hydrolysis capacity in TSB, NB and Gelatin media ranged from 4.0–5.2, 3.8–5.4 and 4.0–5.4 for the bacterial samples and 8.8, 9.0 and 9.2 for 0.1% pure cellulase enzyme, respectively. The results from 16S rRNA full-sequence and cladogram analysis indicate the possibility of a new strain of cellulolytic bacteria. Among the isolated microorganisms, four microbes were discovered close to the Bacillus genus, and one microbe was close to Paenibacillus. The selection of media for isolation and culture of the cellulolytic microbes depends on the researcher’s decision and the availability of the resources. As one of the first studies to identify the native cellulolytic strains of Nepali origin, the study pushes other national and international researchers to consider the divergent climatic conditions of Nepal for potential cellulolytic strains.

English

The ionicliquid [MMIM][DMP] was synthesized from the reactants methyl imidazole [MIM] and trimethylphosphate [TMP] and verified using 1HNMR and FTIR. Coconut coir dust was pretreated with a 1% alkaline solution.Its crystalline structure increased significantly due to the dissolution of lignin and hemicelluloses under alkaline conditions, exposing the cellulose. After NaOH and IL were employed, the XRD showed that peak (002) decreased significantly and peak (101) almost vanished. This significant decrease in crystallinity was related to the alteration of the substrate from the cellulose I structure to the cellulose II structure. The pretreated substrates were hydrolyzed to convert them to reducing sugars by pure cellulase and xylanase,and the reaction was conducted at 60°C, pH 3, for 12 or 48 hours. The yields of sugar hydrolyzed from untreated and NaOH-pretreated substrates were 0.07 and 0.12 g sugar/g lignocellulose, respectively. Pretreatment with IL or the combination of NaOH+IL resulted in yields of reducing sugars of 0.11 and 0.13 g/g, respectively. These findings showed that IL pretreatment of the high-lignin lignocellulose is a new prospect for the economical manufacture of reducing sugars and bioethanol in the coming years.

Preparation of Reducing Sugar Hydrolyzed from High-Lignin Coconut Coir Dust Pretreated by the Recycled Ionic Liquid [mmim][dmp] and Combination with Alkaline

This study aims to produce reducing sugar hydrolyzed from substrate, coconut coir dust pretreated by recycled ionic liquid and its combination with alkaline. The 1H NMR and FTIR were performed to ver-ify the synthesized ionic liquid methylmethylimidazolium dimethyl phosphate ([mmim][dmp]). The structure of pretreated substrates was analyzed by XRD measurement. The used ionic liquid was recy-cled twice to re-employ for substrate pretreatment. The treated- and untreated-coconut coir dust were hydrolyzed into sugars using pure cellulase. The reaction, which called an enzymatic hydrolysis, was conducted at 60 °C, pH 3, for 48 h. The yields of sugar hydrolyzed from fresh IL-pretreated, 1R*IL-pretreated and 2R*IL-pretreated substrates were of 0.19, 0.15 and 0.15 g sugar / g cellu-lose+hemicellulose, respectively. Pretreatment with NaOH or the combination of NaOH+IL resulted in yields of reducing sugars of 0.25, 0.28 g/g, respectively. When alkaline combined with the recycled ionic liquids, NaOH+1R*IL, NaOH+2R*IL in the pretreatment, the yields of sugar were relatively similar to those obtained using alkaline followed by fresh ionic liquid. If the mixture enzymes, cellu-lase+xylanase, used to liberate sugars from fresh IL-pretreated, or recycled IL-pretreated substrates, the amount of sugar (concentration or yield) increased slightly compared to that employing a single cel-lulase. These findings showed that recycled IL pretreatment of the high-lignin lignocellulose, coconut coir dust, is a new prospect for the economical manufacture of fermentable sugars and biofuel in the coming years. © 2015 by Authors, Published by BCREC Group. This is an open access article under the CC BY-SA License (https://creativecommons.org/licenses/by-sa/4.0)

Effect of Enzyme Homogenization on the Physical Properties of Carrot Cell Wall Suspensions

Plant cell wall suspensions are widely present in daily food, such as soups, dressings, and sauces. Cell walls of edible plants are made up of an intricate biopolymer network of mainly cellulose microfibrils, pectins, and hemicelluloses. The current study aims to obtain a better insight in cell wall biopolymer interactions and their relation to physical functionalities of cell wall suspensions. In addition to standard thermal and mechanical treatments to effectively disrupt the cell wall, enzymes were added to hydrolyze specific classes of biopolymers. Incubation with pure cellulases, pectinases, or hemicellulases resulted in suspensions with different physical properties. The effect of the enzymes were measured on the rheology over an 8 h period, and the microstructure was investigated by cryo-scanning electron microscopy. Homogenization of the enzyme-treated system was further investigated. It was observed that there was an increase in the storage modulus after homogenizing the enzyme-treated samples. For example, after homogenizing the 8 h pectinase-treated sample, the storage modulus went from 30 to 70 Pa. This was due to an increase in the particle size and, hence, the volume fraction. The enzyme treatment weakened the cell wall; the additional homogenizing step caused the cell wall to break along the defects caused by the enzyme. This resulted in distinctly different cell wall microstructures based on the type and extent of the treatment.

Chemical modification of β-endoglucanase from Trichoderma viridin by methanol and determination of the catalytic functional groups

β -Endoglucanase from Trichoderma viride was modified by methanol to explore the catalytic functional groups of cellulase. Crude cellulase was produced, and the conditions of saturation and pH by salting out with ammonium sulfate were optimized. Under optimal conditions, crude cellulase was isolated and purified. The pure cellulase was modified by excess methanol, and it was found that the carboxyl in the side-chain radical of cellulase proved to be the catalytic functional group by analysis of the infrared spectrum of modification of cellulase. Modification of cellulase with different concentrations of methanol was carried out and results show that the modified side-chain radical lies at the active site of cellulase or on essential groups. Sodium carboxymethyl cellulose (CMC-Na) which protected the cellulase from inactivation by methanol indicated that the carboxyl modified by methanol is not only a structural radical but also lies at the active site. The inactivation degree of cellulase modified by methanol could be decreased by glucose and it showed that the modification occurred in the active site of cellulase. The kinetic analysis according to Keech and Farrant equation demonstrated that one carboxyl was an essential group for cellulase activity. Keywords: Chemical modification, cellulase, methanol, carboxyl

Enhanced stability and activity of cellulase in an ionic liquid and the effect of pretreatment on cellulose hydrolysis

We discuss the hydrolysis of cellulose using a pure cellulase: endo-1,4-β-D-glucanase (EG) from the fungus, Aspergillus niger, in buffer, the pure ionic liquid (IL), tris-(2-hydroxyethyl)-methylammonium methylsulfate (HEMA), and various mixtures of the two at different temperatures. Steady-state fluorescence and absorbance studies were performed to monitor the stability and activity of EG using cellulose azure as the substrate. EG attains its highest activity at 45°C in buffer and denatures at ∼55°C. On the other hand, HEMA imparts substantial stability to the enzyme, permitting the activity to peak at 75°C. The relative roles of temperature, viscosity, pH, polarity, and the constituent ions of the ILs on the hydrolysis reaction are examined. It is demonstrated that pretreatment of cellulose with ILs such as BMIM Cl, MIM Cl, and HEMA results in more rapid conversion to glucose than hydrolysis with cellulose that is not pretreated. The percent conversion to glucose from pretreated cellulose is increased when the temperature is increased from 45 to 60°C. Two different ILs are used to increase the efficiency of cellulose conversion to glucose. Cellulose is pretreated with BMIM Cl. Subsequent hydrolysis of the pretreated cellulose in 10-20% solutions of HEMA in buffer provides higher yields of glucose at 60°C. Finally, to our knowledge, this is the first study dealing with a pure endoglucanase from commercial A. niger. This enzyme not only shows higher tolerance to ILs, such as HEMA, but also has enhanced thermostability in the presence of the IL.

Synergistic action of xylanase and mannanase improves the total hydrolysis of softwood

The impact of xylan and glucomannan hydrolysis on cellulose hydrolysis was studied on five pretreated softwood substrates with different xylan and glucomannan contents, both varying from 0.2% to 6.9%, using mixtures of purified enzymes.The supplementation of pure cellulase mixture with non-specific endoglucanase TrCel7B and xylanase TrXyn11 enhanced the hydrolysis of all substrates, except the steam pretreated spruce, by more than 50%. The addition of endo-β-mannanase increased the overall hydrolysis yield by 20–25%, liberating significantly more glucose than theoretically present in glucomannan.When supplemented together, xylanolytic and mannanolytic enzymes acted synergistically with cellulases. Moreover, a linear correlation was observed between the hydrolysis of polysaccharides, irrespective of the composition, indicating that glucomannan and xylan form a complex network of polysaccharides around the cellulosic fibres extending throughout the lignocellulosic matrix. Both hemicellulolytic enzymes are crucial as accessory enzymes when designing efficient mixtures for the total hydrolysis of lignocellulosic substrates containing both hemicelluloses.

Preparation and enzymatic behavior of surfactant-enveloped enzymes for glycosynthesis in nonaqueous aprotic media

Abstract Surfactant-enveloped enzymes (SEEs) were prepared from pure cellulases, cellobiohydrolase I and endoglucanase I (Cel7A and Cel7B, respectively), via simply freeze-drying water-in-oil emulsions, wherein the aqueous phase containing each cellulase was stabilized with the nonionic surfactant, dioleyl- N - d -glucona- l -glutamate. The enzymatic tolerance of SEEs to various nonaqueous solvents was investigated, aiming at a novel synthetic approach in biocatalytic glycoengineering. SEE-Cel7A preserved ca . 67% of the original activity after 3 h incubation in lithium chloride (LiCl)/dimethylacetamide (DMAc) that is a good solvent for carbohydrates but completely deactivates intact enzymes. This excellent enzymatic durability depended on the preparation conditions of SEEs, e.g. pH and salt species of the aqueous phase during SEE preparation. SEE-Cel7A or SEE-Cel7B was applied as a biocatalyst to synthesize cellulose, a sugar polymer which is insoluble in common solvents but dissolves in LiCl/DMAc. Both SEEs could catalyze the direct dehydration of cellobiose without any activation of the anomeric carbon, a property that is indispensable for conventional chemo-enzymatic synthesis. The SEE-Cel7A provided short-chain cellulose with the degree of polymerization (DP) ca . 20, and longer-chain cellulose with DP ca . 60 was preferentially obtained by the SEE-Cel7B, possibly through preferential reverse hydrolysis instead of inherent hydrolysis. Nonaqueous SEE-mediated biocatalysis using inexpensive glycohydrolases and sugars that do not need to be chemically modified beforehand would have potentially wide applications in glycoengineering.

The Physical Action of Cellulases Revealed by a Quartz Crystal Microbalance Study Using Ultrathin Cellulose Films and Pure Cellulases

The effects of fungal cellulases on model cellulose films were studied using a high-resolution quartz crystal microbalance (QCM) sensitive to minute changes of the nanometer thick model cellulose films. It was found that endoglucanases not only produce new end groups but also cause a swelling of the cellulose film. The cellobiohydrolases degraded the films quickly, which was detected as a rapid decrease in the remaining amount of cellulose on the QCM crystal. However, changing viscoelastic properties of the films also indicated a softening of the film during the degradation. A defined mixture of selected cellulases caused a significantly higher rate of degradation than only cellobiohydrolases. Cellulase synergism is discussed with the endoglucanase swelling effects and film softening added.

Effects of exogenous cellulase supplementation on microbial growth and ruminal fermentation of a high-forage diet in Rusitec fermenters1

Two incubation runs were carried out with a Rusitec system to investigate the effects of 2 exogenous pure cellulases on ruminal microbial growth and fermentation of a 70:30 grass hay:concentrate (DM basis) substrate. The substrate was sprayed with buffer (control; pH = 6.5), a cellulase from Trichoderma longibrachiatum (TRI), a cellulase from Aspergillus niger (ASP), or a 1:1 mixture of both cellulases (MIX) 24 h before being placed in the fermenters. Enzymes were applied at a rate of 30 endoglucanase units/g of substrate DM. Treating the substrate with enzymes reduced substrate NDF and ADF content (P < 0.001 to P = 0.002) and increased DM, NDF, and ADF disappearance after 6 and 24 h of incubation (P < 0.001 to P = 0.004) but not after 48 h of incubation. Daily VFA production was increased (P = 0.004) by 15, 9, and 15% for TRI, ASP, and MIX, respectively, with half of the increase being due to production of acetate. All enzyme treatments augmented (P = 0.009) methane production, but none of them altered the methane:VFA ratio (P = 0.70). There were no differences (P = 0.80) among treatments in the daily flow of solid-associated microorganisms, as measured using 15N as a microbial marker. Although the TRI and MIX treatments increased (P < 0.05) the daily flow of liquid-associated microorganisms and the proportion of microbial N in the solid residue after 48 h of incubation, no effects were observed (P = 0.92 and P = 0.95, respectively) for the ASP treatment. The results show that the TRI and MIX treatments enhanced in vitro fermentation by increasing substrate fiber degradation, VFA production, and ruminal microbial growth. The lack of differences between TRI and MIX in most of the measured variables indicates that treating the substrate with a mixture of both cellulases did not further improve the effects of the TRI treatment.

Effect of digestion by pure cellulases on crystallinity and average chain length for bacterial and microcrystalline celluloses

In this study we employed Size Exclusion Chromatography (SEC) and X-ray diffraction to monitor the molecular weight and crystallinity of bacterial cellulose I and II (BC-I, BC-II) and microcrystalline cellulose (MCC) digested with three “pure” Thermobifida fusca cellulases (Cel6A, Cel6B, and Cel9A ). For each enzyme, cellulose crystallinity was found to increase modestly with treatment time. The digestion rate of BC-II was higher than that of BC-I for Cel6A and Cel9A, both endocellulases. SEC results show that the endocellulases create a very rapid decrease in cellulose molecular weight while a slower molecular weight loss was observed with Cel6B, an exocellulase. This work suggests that conversion of native cellulose I to cellulose II by mercerization may beneficially impact the rate of sugar release by cellulases from biomass. In general, lower conversion rates are observed for MCC compared to BC, possibly due to a higher initial crystallinity for MCC. Surface area effects may also be important.

A mixture of pure cellulase, hemicellulase and pectinase improves broiler performance

1. The effects of a mixture of pure enzymes (cellulase, hemicellulase and pectinase) and a commercial enzyme, Energex, were examined on performance and metabolisabilities in broiler chicks given a maize–soybean meal diet. Composition of the mixed enzyme was similar to Energex except that protease was not present. 2. Chicks were divided into three groups: control, mixed enzyme and Energex with 7 replicates per group. Male broiler chicks were raised at 25°C in wire-floored cages for 12 d from 15 d of age. Feed and water were offered ad libitum. 3. The Energex group gained significantly more weight and the mixed enzyme group tended to gain more than the control. Feed intakes were similar and thus the feed conversion ratio of Energex was significantly improved while it tended to be improved by the mixed enzyme. 4. The mixed enzyme group showed significant improvement in carcase and muscle weight when compared with the control group. The mixed enzyme group also showed significant improvement in organic matter and crude protein metabolisabilities. In the groups given enzyme, abdominal fat weight tended to decrease. 5. It is concluded that a combination of cellulase, hemicellulase and pectinase is effective in improving organic matter and crude protein metabolisabilities and carcase yield of broilers on a maize–soybean meal diet.