Mixed Enzyme System Research Articles

Alginate Encapsulation Stabilizes Xylanase Toward the Laccase Mediator System.

Xylanase, a hydrolytic enzyme, is susceptible to inactivation by the oxidative conditions generated by the laccase mediator system (LMS). Given the impetus to develop a mixed enzyme system for application in biomass processing industries, xylanase was encapsulated with either Cu2+- or Ca2+-alginate and then exposed to the LMS with variations such as mediator type, mediator concentration, and treatment pH. Results demonstrate that alginate-encapsulated xylanase retains substantial activity (> 80%) when exposed to the LMS relative to non-encapsulated xylanase. Cu2+-alginate generally provided better protection than Ca2+-alginate for all mediators, and protection was observed even at a low pH, where the LMS is most potent. Despite encapsulation, xylanase was still capable of hydrolyzing its polymeric substrate xylan, given kcat/Km values within an order of magnitude of that for non-encapsulated xylanase. The alginate matrix does not impede the function of the oxidized mediator, since comparable Vmax values were observed for the conversion of veratryl alcohol to veratraldehyde by free and Cu2+-alginateencapsulated laccase. Overall, these results support development of a mixed enzyme system for biomass delignification and, more broadly, show potential for protecting protein function in an oxidative environment.

Research on complex enzyme system to extract Hibiscus Sabdariffa L reducing sugar

Hibiscus Sabdariffa L. has important nutritional and medicinal value. Currently, a safe process extraction of reducing sugar from Hibiscus Sabdariffa L. calyx by biological complex enzyme system is seldom investigated. In this experiment, the pectinase and cellulase complex enzyme system was used to optimize the enzymatic hydrolysis of Hibiscus Sabdariffa L. and Box behnken was used to design the experiment. The results showed that there was significant difference in enzymolysis effect of Hibiscus Sabdariffa L under different conditions (p < 0.05). When the enzymolysis temperature was 45 °C, pH 4.5, and the mass volume ratio of material to liquid was 5g:100ml, the optimum conditions were obtained by response surface methodology: the addition amount of pectinase and cellulase complex enzyme was 0.011 g/kg, the ratio of mixed enzyme was 1.36:1, the enzymolysis time was 2.09 h, and the reducing sugar content of Hibiscus Sabdariffa L extract was 3.56 mg/ml. The experimental results of reducing sugar content of Hibiscus Sabdariffa L (3.54 ± 0.1mg / ml) were in good agreement with the predicted value of the equation fitted by response surface. The mixed enzyme system of pectinase and cellulase has the advantages of environmental protection and energy saving, which can provide guidance for industrial production.

Current Status of Mining, Modification, and Application of Cellulases in Bioactive Substance Extraction.

Cellulases have been used to extract bioactive ingredients from medical plants; however, the poor enzymatic properties of current cellulases significantly limit their application. Two strategies are expected to address this concern: (1) new cellulase gene mining strategies have been promoted, optimized, and integrated, thanks to the improvement of gene sequencing, genomic data, and algorithm optimization, and (2) known cellulases are being modified, thanks to the development of protein engineering, crystal structure data, and computing power. Here, we focus on mining strategies and provide a systemic overview of two approaches based on sequencing and function. Strategies based on protein structure modification, such as introducing disulfide bonds, proline, salt bridges, N-glycosylation modification, and truncation of loop structures, have already been summarized. This review discusses four aspects of cellulase-assisted extraction. Initially, cellulase alone was used to extract bioactive substances, and later, mixed enzyme systems were developed. Physical methods such as ultrasound, microwave, and high hydrostatic pressure have assisted in improving extraction efficiency. Cellulase changes the structure of biomolecules during the extraction process to convert them into effective ingredients with better activity and bioavailability. The combination of cellulase with other enzymes and physical technologies is a promising strategy for future extraction applications.

Establishment of a highly efficient and low cost mixed cellulase system for bioconversion of corn stover by Trichoderma reesei and Aspergillus niger

The high cost of cellulase production is one of the main obstacles for the conversion of lignocellulose into high valued products. As a widely existing biomass resource, corn stover can be used to reduce the cost of cellulase production. In this study, Parameters of producing cellulase by Trichoderma reesei were optimized using Response Surface methodology, the optimum conditions were as follows: 3.84% solid acid pretreated corn stover, 1.19% yeast extract, initial pH of 5.01, 0.12% glycerol monostearate, 0.25% Na2HPO4, with 3% inoculum content for 12 days at 30 °C. Filter paper activity (FPA) of Trichoderma reesei was increased by 180% to 13.44 IU/mL. Enzymatic hydrolysis experiments were performed on different pretreated corn stovers with Trichoderma reesei cellulase produced under optimal conditions, and the highest cellulose conversion efficiency reached 62.39%. In addition, a blend of Trichoderma reesei cellulase at a concentration of 30 FPU/g cellulose and Aspergillus niger beta-glucosidase at a concentration of 50 FPU/g cellulose could effectively hydrolyse alkali pretreated corn stover, resulting in an improvement in the enzymatic hydrolysis efficiency by 17.26% compared to Trichoderma reesei cellulase alone. In parallel, the mixed enzymes achieved an equivalent enzymatic effect with that of commercial cellulase (Novozymes, Cellic Ctec2) at the same concentration. These data suggested that solid acid pretreated corn stover was a promising carbon source for fungal cellulase production for effective saccharification of corn stover biomass. Glycerol monostearate was first found to have a strong induction effect on enzyme production. The development of a high efficiency and low-cost mixed enzyme system had a potential application in future industrialization scale.

Establishing the oxidative tolerance of Thermomyces lanuginosus xylanase.

This work aims to determine the tolerance of xylanase towards enzyme-generated oxidative conditions, such as those produced by the peroxidase or laccase mediator systems (LMS). The activity of Thermomyces lanuginosus xylanase was measured after incubation with lignin peroxidase, manganese peroxidase or laccase plus various mediators. The laccase system, using mediators such as 1-hydroxybenzotriazole and violuric acid, resulted in complete loss of xylanase activity, accompanied by an increase in the solution potential. However, an increase in solution potential alone was not sufficient to inactivate xylanase, nor was loss of xylanase activity always accompanied by a significant increase in solution potential, as observed with N-hydroxyphthalimide as the mediator. Neither lignin peroxidase nor manganese peroxidase impacted xylanase activity; only extended treatment with elevated hydrogen peroxide concentration promoted modest xylanase activity loss. The mechanism of inactivation as determined by the tryptophan-modifying reagent N-bromosuccinimide (NBS) indicated that oxidation of just one of the eight tryptophan residues of T.lanuginosus xylanase would be sufficient to result in complete loss of xylanase activity, since xylanase is completely inactivated at 1:1 molar ratio of NBS to xylanase. While showing tolerance to peroxidase-based enzyme systems, T. lanuginosus xylanase is readily inactivated in the presence of the LMS. Based upon treatment with NBS as the oxidant, inactivation can be attributed to modification of a single tryptophan residue. The simultaneous application of mixed hydrolytic and oxidative enzyme systems is of importance to biomass processing industries. Understanding the tolerance of xylanase to oxidative conditions will facilitate the design of reaction conditions or enzyme variants to maximize the impact of mixed enzyme systems.

アミラーゼ・プロテアーゼ混合酵素による細胞壁物質の定量及び牧乾草・牧草サイレージのTDN含量推定法の検討

アミラーゼ・プロテアーゼ混合酵素による細胞壁物質の定量及び牧乾草・牧草サイレージのTDN含量推定法の検討

Effects of Acid/Alkali and Cellulase Enriched Mixed Enzyme Treatments on Properties of Jute-Cotton Union Furnishing Fabric

The effects of chemical pre-treatments using dilute acids [HCl, CH3COOH, HCOOH, (CH2COOH)2C(OH)COOH] and alkali (NaOH) on raw jute and jute-cotton(75:25) union fabric and the effects of specified mixed enzyme system (different proportion of mixture of cellulase, xylanase and pectinase) pretreatment on conventional 2% H2O2 bleached jute-cotton union fabric have been comparatively evaluated. Among different organic/ mineral acid pretreatments 1% HCl treatment shows higher surface brightening action than that obtained by formic acid, acetic acid and citric acid pre-treatment. All these acid pretreatment do not show desirable softening action but show varying degree of surface leaching action associated with measurable changes in K/S value, whiteness, yellowness and brightness indices. However, 10-18 % NaOH pretreatment of raw jute-cotton union fabric show appreciable softening action but is associated with higher mass loss, appreciable reduction in tenacity and surface darkening. While, 5% dilute NaOH pretreatment show somewhat less softening action, but exhibit lower mass loss, lower surface darkening and lower reduction in tenacity which may be practically useful in imparting desirable softness in jute-cotton union fabric. Treatment with specified cellulase enriched mixed enzyme system (175 unit/ml of cellulase, 96 unit/ml of xylanase and 136 unit/ml of pectinase) on either 1 or 5% NaOH pretreated or conventional 2%H2O2 bleached jute-cotton union fabric renders a moderate degree of softening action and surface smoothening with overall good balance in important textile related properties. However, higher degree of softening action may be achieved, if the said mixed enzyme treatment is carried out after both sequential pretreatments i.e. 5% NaOH pretreatment followed by conventional H2O2 bleaching at the cost of higher weight loss and reduction in tenacity. Studies on Dyeability of such chemically treated jute-cotton union fabric reveals that NaOH pretreatment show an increasing trend for acid dye uptake and reactive dye uptake, while all the dilute acid pretreatments show an increasing trend of basic dye uptake in general.

Mixed Enzyme Systems for Delignification of Lignocellulosic Biomass

The application of enzymes such as laccase and xylanase for the preparation of cellulose from lignocellulosic material is an option for those industries seeking to reduce the use of chlorine-containing bleach agents, thus minimizing the environmental impact of their processes. Mixed hydrolytic and oxidative enzyme systems have been well described in the context of biopulping, and thus provide good precedent regarding effectiveness, despite the susceptibility of xylanase to inactivation by laccase-generated oxidants. This paper examines the progress towards development of sequential and simultaneous mixed enzyme systems to accomplish delignification.

Pretreatment of cottage cheese to enhance biogas production.

This study evaluated the possibility of pretreating selected solid fraction of an anaerobic digester treating food waste to lower the hydraulic retention time and increase the methane production. The study investigated the effect of different pretreatments (thermal, chemical, thermochemical and enzymatic) for enhanced methane production from cottage cheese. The most effective pretreatments were thermal and enzymatic. Highest solubilisation of COD was observed in thermal pretreatment, followed by thermochemical. In single enzyme systems, lipase at low concentration gave significantly higher methane yield than for the experiments without enzyme additions. The highest lipase dosages decreased methane yield from cottage cheese. However, in case of protease enzyme an increase in concentration of the enzyme showed higher methane yield. In the case of mixed enzyme systems, pretreatment at 1 : 2 ratio of lipase : protease showed higher methane production in comparison with 1 : 1 and 2 : 1 ratios. Methane production potentials for different pretreatments were as follows: thermal 357 mL/g VS, chemical 293 mL/g VS, and thermochemical 441 mL/g VS. The average methane yield from single enzyme systems was 335 mL/g VS for lipase and 328 mL/g VS for protease. Methane potentials for mixed enzyme ratios were 330, 360, and 339 mL/g VS for 1 : 1, 1 : 2, and 2 : 1 lipase : protease, respectively.

Cellulose hydrolysis in evolving substrate morphologies III: Time‐scale analysis

We present a time-scale analysis for the enzymatic hydrolysis of solid cellulosic substrates, based on our recently developed kinetic model (Zhou et al., 2009a, Biotechnol Bioeng 104:261-274; Zhou et al., 2009b, Biotechnol Bioeng 104:275-289) which incorporates both enzymatic chain fragmentation and hydrolytic time evolution of the solid substrate morphology. Analytical order-of-magnitude estimates of the relevant single-layer chain depolymerization times are first discussed. These time-scale estimates for pure and mixed enzyme systems can be employed to calculate the degree of synergy between endo- and exo-acting enzymes in a mixed enzyme system. By the way of a quasi-steady-state approximation which allows for a greatly simplified analytical solution of the model, we also explain the origin and give order-of-magnitude estimates of the two characteristic hydrolysis time scales which arise in this model when the solid substrate morphology is taken into account. These analytically derived time-scale relations explain how the embedding of cellulose chains in a solid substrate acts as a crucial rate-limiting factor and results in a substantial slowing down of the hydrolytic conversion process, compared to a hypothetical substrate of immediately enzyme-accessible, isolated chains. The analytical time-scale results are verified by numerical simulations and compared to experimental observations.

Effect on physical characteristics of jute fibres with cellulase and specific mixed enzyme systems

This research investigated the effect of using cellulase enzyme and a mixture of enzymes (cellulase, xylanase and pectinase), under specific treatment conditions, on the physical characteristics of jute fibres. The physical characteristics such as tenacity, elongation to break, flexural rigidity and structural analysis using scanning electron microscopy and colour measurements using colour spectroscopy were studied. Tensile characteristics of these enzyme-treated jute fibres in terms of tenacity (cN/tex) were found to decrease by approximately 9.05%, 14.76% and 19.83% for cellulase enzyme treatment and 11.96%, 13.47% and 23.81% for mixed enzymes at 2%, 3% and 4% concentration levels, respectively. Significant improvements in physical characteristics such as elongation, whiteness index, brightness index and reflectance of these enzyme-treated jute fibres were noticed when compared to those of raw jute fibres. The significant reduction in flexural rigidity and yellowness index of these enzyme-treated jute fibres was also noticed. Scanning electron microscopy reveals the degradation of the jute fibre at surface level and along with the removal of the gummy substances present on the surface; this degradation depends on the various concentrations of the enzyme.

Process Optimization for Biosoftening of Lignocellulosic Fiber with White Rot Fungi and Specific Enzymatic Systems

Jute is a strong, stiff, natural fiber. Jute fibers are aggregates of single cells consisting of α-cellulose, which are cemented by lignin and hemicellulose. Lignin is responsible for the dark color, branching patterns, and harshness. The spinnability of the fiber is greatly reduced because of poor elongation and high flexural rigidity. This research investigated the effect of using white rot fungi (namely, Phanerochaete chrysosporium and Ceriporiopsis subvermispora), cellulase enzyme, and a mixture of enzymes (cellulase, xylanases, and pectinases) under specific treatment conditions on the physical characteristics of jute fibers. The physical characteristics such as tenacity, elongation to break, flexural rigidity, and structural analysis using scanning electron microscopy were studied. The results show that these fungi and enzymes are able to degrade the lignin effectively, culminating in the reduction of flexural rigidity and tenacity and improvement in elongation percentage of the fiber. Scanning electron microscopy reveals the degradation of the jute fiber at the surface level and also reveals the removal of the gummy substances present on the surface. The prediction of the desired quality of the enzymatic systems, both cellulase and mixed enzyme systems, using artificial neural network was reported. The results obtained from the network give an average training error of around 1% in the prediction of the process parameters for achieving the desired physical properties of the jute fibers.

Production of lactosucrose from sucrose and lactose by a levansucrase from Zymomonas mobilis.

Lactosucrose (4(G)-beta-D-galactosylsucrose) is an oligosaccharide consisting of galactose, glucose, and fructose. In this study, we prepared lactosucrose from lactose and sucrose using a levansucrase derived from Zymomonas mobilis. Optimum conditions for lactosucrose formation were 23 degrees C, pH 7.0, 18.0% (w/v) lactose monohydrate, and 18% (w/v) sucrose as substrates, and 1 unit of enzyme/ml of reaction mixture. Under these conditions, the lactosucrose conversion efficiency was 28.5%. The product was purified and confirmed to be O-beta-D-galactopyranosyl-(1-->4)-O-beta-D-glucopyranosyl- (1-->2)-beta-D-fructofuranoside, or lactosucrose. A mixed-enzyme system containing a levansucrase and a glucose oxidase was applied in order to increase the efficiency of lactose and sucrose conversion to lactosucrose, which rose to 43.2% as s result.

Effect of bio-friendly conditioning agents on jute fibre spinning

From early times, jute fibre has been generally conditioned for easy spinning by adding oil and water in the form of an emulsion. The commonly used oil consists of C 12–C 31 fractions of mineral oil that sometimes impart different intensities of oily (kerosene) or fishy smell to the end product. In the present work, efforts have been made to find a suitable sustainable substitute of mineral oil based conditioning agent for spinning of jute yarn and for this, three types of vegetable oil (rice bran oil, palmolein oil and castor oil), a silicone emulsion, a mixed enzyme system and glycerine have been used separately or in combinations as conditioning agents for jute fibre before its mechanical processing for making yarn in jute spinning machines. Considering comparable mechanical process performance for spinning of jute fibre ( viz., fibre loss as droppings during processing, moisture retention prior to spinning stage and spinning end breakage rate), tensile properties of yarn, and lower yarn hairiness, it may be suggested to use 2.5% castor oil alone, or 2% castor oil in combination with 0.1–0.5% glycerine in the form of oil-in-water emulsion as the most suitable alternatives to conventional mineral oil-based jute conditioning agent to spin ordinary jute yarn.

Production of High-content Galacto-oligosaccharide by Enzyme Catalysis and Fermentation with Kluyveromyces marxianus

Of three beta-galactosidases from Aspergillus oryzae, Kluyveromyces lactis and Bacillus sp., used for the production of low-content galacto- oligosaccharides (GOS) from lactose, the latter produced the highest yield of trisaccharides and tetrasaccharides. GOS production was enhanced by mixing beta-galactosidase glucose oxidase. The low-content GOS syrups, produced either by beta-galactosidase alone or by the mixed enzyme system, were subjected to the fermentation by Kluyveromyces marxianus, whereby glucose, galactose, lactose and other disaccharides were depleted, resulting in up to 97% and 98% on a dry weight basis of high-content GOS with the yields of 31% and 32%, respectively.

Recent trends in the microbial production, analysis and application of Fructooligosaccharides

This review focuses on the recent developments in the area of FOS research—its microbial production, functional properties and applications and an overview of the different analytical methods for the determination of the FTase. Different microbial sources of FTase reported in literature to produce FOS with different linkages to form 1-kestose, 6-kestose and neokestose in varying yields based on initial sucrose concentration is discussed. Different fermentative methods have been used for production of FOS. SSF has been used for the production of a value added product FOS utilizing various agroindustrial byproducts. The nutritional and culture parameters when optimized, the FOS yields and productivity could be improved. The use of immobilized enzymes and cells has led to the development of effective and economic methods for large-scale production of FOS. Forced flow Membrane reactor systems, biocatalyst system with a bioreactor equipped with a microfiltration systems, have been used for production of high content FOS by removing the released glucose and unreacted sucrose from the reaction mixture resulting in up to 98% FOS. The use of mixed enzyme system of Fructosyl Transferase and glucose oxidase or glucose dehydrogenase, could produce highly concentrated FOS up to 90–98%. Nano-filtration for removing glucose resulted in FOS of 90% concentration. The purified enzyme was found to produce kestose and nystose unlike the crude enzyme which produced GF5 and GF6 oligosaccharides Kinetic parameters (Vm, Km, and Ki) of the enzyme were determined from experimental data on the transfructosylation rate at various substrate concentrations with and without addition of glucose Techniques like HPLC, using polar-bonded phase and resin-based HPLC columns are commonly used for separation of oligosaccharides with refractive Index Detector or pulsed amperometric detector and annular size exclusion chromatography for large scale and continuous fractionation. Other techniques like gas liquid chromatography, thin layer chromatography, NMR and Mass Spectrometry have been used for structure analyses. The functional properties like use as prebiotics, dietary fiber, role in absorption and defense/Immunity, lipid metabolism control of diabetics have been discussed. A variety of applications in food formulations are also discussed.

High-performance hydrolysis of cellulose using mixed cellulase species and ultrasonication pretreatment

Effective approaches to a practical high-performance enzymatic hydrolysis of cellulose were investigated using a ultrasonic irradiation as a pretreatment and a combination of cellulases from Trichoderma viride and Aspergillus niger. Their efficacy on enhancement of reactivity was discussed based on the kinetic parameters, i.e. Michaelis constant K m, maximum reaction rate V max and initial reaction rate, correlating with ultrasonic conditions and enzyme combination, respectively. A relatively high-weight fraction of A. niger cellulase in the mixed-enzyme system afforded rapid initiation of the hydrolysis reaction, while a kinetic analysis revealed that a T. viride cellulase weight fraction of ca. 0.3 was optimal in enhancing V max without increasing K m. Pretreatment of the cellulose fibers with ultrasonic irradiation prior to initiating the enzyme reaction further improved the reaction rate. Enhancement of V max by ultrasonication was effective in greater weight fraction of T. viride used system. The correlation between the ultrasonic irradiation time required to obtain a desired initial reaction rate and the irradiation power employed was examined, and it was found that the time needed for effective degradation could be markedly reduced by increasing the irradiation power. This finding is considered to support the possibility of applying ultrasonication pretreatment in a practical cellulose saccharification process.

Production of fructooligosaccharides in high yield using a mixed enzyme system of β-fructofuranosidase and glucose oxidase

A mixed enzyme system, with β-fructofuranosidase (obtained from Aspergillus japonicus) and commercial glucose oxidase (Gluzyme, Novo Nordisk), produced fructooligosaccharides (FOS) in high yield from sucrose. The reaction was performed in an aerated stirred tank reactor controlled at pH 5.5 by a slurry of CaCO3. Glucose, an inhibitor of β-fructofuranosidase, produced in the reaction was converted by glucose oxidase to gluconic acid, which was then precipitated to calcium gluconate in solution. The system produced more than 90% (w/w) FOS on a dry weight basis, the remainder was glucose, sucrose and a small amount of calcium gluconate. Most of the FOS and sucrose was hydrolyzed to fructose in the mixed enzyme system with glucose oxidase and β-fructofuranosidase from Asp. niger.

Structural Changes in Hemp Fibers as a Result of Enzymatic Hydrolysis with Mixed Enzyme Systems

Hemp fabric is hydrolyzed with cellulase, hemicellulase/cellulase, and cellulase plus additional β-glucosidase. The effect of the admixture of hemicellulase and β-glucosi dase is evaluated in terms of hydrolysis rate and product properties. The influence of mechanical action during the treatment is also taken into account. Treated samples are characterized by weight loss per incubation time, tensile strength, crystallinity, acces sibility, and pore structure. The admixture of hemicellulase and,β-glucosidase produces only minor effects without mechanical action on the hydrolysis rate, but mechanical action applied during the treatment dramatically increases weight loss. While crystal linity changes upon hydrolysis are insignificant, the pore structure is strongly influenced by the choice of enzyme system. Hemicellulase seems to promote the formation of smaller pores, while cellobiase forms larger pores.

Enzymatic hydrolysis and saccharification optimisation of sago starch in a twin-screw extruder

The influence of processing conditions in a combined gelatinisation and liquefaction extrusion system on subsequent saccharification was examined. Extrusion conditions investigated were barrel temperature (70–130 °C), screw speed (70–190 rpm), enzyme concentration (0–1%) and feed moisture content (28.5–50.5%). Feed moisture, barrel temperature and enzyme concentration had significant effects on saccharification, which ranged from 32 to 98% (dextrose equivalent = 83–98) after 8 h reaction with 0.50 AGU/ml amyloglucosidase (AMG). Die pressure was negatively correlated with saccharification (r = −0.75), implying that products formed from a high viscous mass were poorly saccharified. Greater extent of saccharification was achieved with lower SME input. Saccharification in the extruder was initiated by adding AMG during the extrusion processing. Both initial and subsequent saccharification were dependent on the AMG concentration, moisture content and the stage of AMG addition. In this mixed-enzyme system, synergistic effects of both enzymes (α-amylase and amyloglucosidase) were evident. High-performance size-exclusion chromatography profiles showed that incorporating AMG led to loss of G6 and G2 in the oligosaccharide spectra but with formation of G5.