Optimization and Characterization of an Ultra-Thermostable, Acidophilic, Cellulase-Free Xylanase from a New Obligate Thermophilic Geobacillus thermoleovorans AKNT10 and its Application in Saccharification of Wheat Bran.

Microbial xylanases are enzymes of great importance due to their wide industrial applications, especially in the degradation of lignocellulosic biomass into fermentable sugars. This study aimed to describe the production optimization and partial characterization of an ultra-thermostable, acidophilic, cellulase-free xylanase from an obligate thermophilic eubacterium Geobacillus thermoleovorans strain-AKNT10 (Ac.No. LT158229) isolated from a hot-spring of Puga Valley located at an altitude of 4419 m in Ladakh, India. The optimization of cultural conditions improved enzyme yield by 10.49-fold under submerged fermentation. The addition of 1% (w/v) xylose induced the enzyme synthesis by ~ 165 and 371% when supplemented in the fermentation medium containing wheat bran (WB) 1 and 3%, respectively. The supplementation of sucrose reduced the xylanase production by ~ 25%. Results of partial characterization exhibited that xylanase was optimally active at pH 6.0 and 100 °C. Enzyme retained > 75%, > 83%, and > 84% of activity at 4 °C for 28 days, 100 °C for 60 min, and pHs 3-8 for 60 min, respectively. An outstanding property of AKNT10-xylanase, was the retention of > 71% residual activity at extreme conditions (121 °C and 15 psi pressure) for 15 min. Enzymatic saccharification showed that enzyme was also capable to liberate maximum reducing sugars within 4-8 h under optimized conditions thus it could be a potential candidate for the bioconversion of lignocellulosic biomass as well as other industrial purposes. To the best of our knowledge, this is the first report on such an ultra-thermo-pressure-tolerant xylanase optimally active at pH 6 and 100 °C from the genus Geobacillus.

Comparison of lignocellulosic enzymes and CAZymes between ascomycetes (Trichoderma) and basidiomycetes (Ganoderma) species: a proteomic approach.

Wood decomposing ascomycetes and basidiomycetes group of fungi are the most valuable microbes on the earth's ecosystem that recycles the source of carbon; therefore, they are essential for the biorefinery industries. To understand the robustness of the enzymes and their metabolic pathways in the fungal system, label-free quantification of the total proteins was performed. The fungi showed a comparable quantity of protein abundance [Trichoderma citrinoviride (285), Thermoascus aurantiacus (206), Ganoderma lucidum MDU-7 (102), G. lucidum (242)]. Differentially regulated proteins of ascomycetes and basidiomycetes were analyzed, and their heatmap shows upregulated and downregulated proteins [25 differentially expressed proteins in T. citrinoviride (8.62 % up-regulated and 91.37 % down-regulated) and G. lucidum (5.74 % up-regulated and 94.25 % down-regulated)] by using the normalized peptide-spectrum match (PSMs) and log2fold change. These proteins were similarly matched to the carbohydrate active enzymes family (CAZymes) like glycoside hydrolase (GH family), carbohydrate-binding module (CBM family) with auxiliary activities, and also involved in the hydrolysis of carbohydrate, lignin, xylan, polysaccharides, peptides, and oxido-reductase activity that helps in antioxidant defense mechanism. The lignocellulolytic enzymes from two different divisions of fungi and proteomics studies gave a better understanding of carbon recycling and multi-product lignocellulosic biorefinery processes.

Valorization of onion peel waste: From trash to treasure.

Globally, fruits and vegetables are consumed as raw, processed, or as an additive, accounting for approximately 50% of total food wastage. Among the fruits and vegetables, onion is well known for its potential bioactive components; however, peels of onion are a major concern for the environmental health and food industries. Effective utilization methods for valorizing the onion peel should be needed to develop value-added products, which are more eco-friendly, cost-effective, and sustainable. Therefore, this review attempts to emphasize the conventional and emerging valorization techniques for onion peel waste to generate value-added products. Several vital applications including anticancerous, antiobesity, antimicrobial, and anti-inflammatory activities are thoroughly discussed. The findings showed that the use of advanced technologies like ultrasound-assisted extraction, microwave-assisted extraction, and enzymatic extraction, demonstrated improved extraction efficiency and higher yield of bioactive compounds, which showed the anticancerous, antiobesity, antimicrobial, and anti-inflammatory properties. However, in-depth studies are recommended to elucidate the mechanisms of action and potential synergistic effects of the bioactive compounds derived from onion peel waste, and to promote the sustainable utilization of onion peel waste in the long-term.

In-vitro antimicrobial and anti-inflammatory activity of modified solvent evaporated ethanolic extract of Calocybe indica: GCMS and HPLC characterization.

Recent interest in the utilization of mushroom-based bioactive compounds has increased due to their potential bioactivities and as alternatives in the reduction of high concentrations of chemical utilization. Therefore, we evaluated the physicochemical, functional, antimicrobial, and anti-inflammatory activity of the Calocybe indica. The nutritional composition of the mushroom was found to be a good source of proteins (12.48%) and fiber (6.87%). Polysaccharide and protein moiety showed both hydrophilic and hydrophobic domains and the sample showed higher water (3.01 g/g), oil binding (2.45 g/g) emulsifying (68.94), and foaming properties (59.39%). Structural characterization revealed the porous and small crystalline structure of the mushroom powder. Ethanolic extract was quantified for total phenolics and flavonoids and revealed 11.1534 ppm caffeic acid, 0.057 ppm syringic acid, 1.6385 ppm p-coumaric acid, and 0.3495 ppm rutin, respectively. Presence of ethyl tridecanoate, hexadecanoic acid ethyl ester, pentadecanoic acid ethyl ester, undecanoic acid ethyl ester, N, α, α'-trimethyl diphenethylamine, nicotinonitriles, phosphonic acid decyl-, 1-hexyl-2-nitrocyclohexane, diallyl divinylsilane, 3-phenyl-pyrrolo(2,3-ß) pyrazine was confirmed during GC-MS analysis. Furthermore, the mushroom extract showed effective antimicrobial against Gram-positive (23.67 mm) and negative bacteria (20.33 mm) in terms of zone of inhibition. Significantly comparable anti-inflammatory activity was observed for mushroom extract during protein denaturation (43.72-85.69%) and membrane stabilization. In conclusion, the mushroom extract has shown good functional properties and potential bioactivity, therefore, it can be scaled up as an effective food preservative, potential anti-inflammatory, and antimicrobial agent at the industrial level.

Advances and future prospects of pyrethroids: Toxicity and microbial degradation.

Pyrethroids are a class of insecticides structurally similar to that of natural pyrethrins. The application of pyrethrins in agriculture and pest control lead to many kinds of environmental pollution affecting human health and loss of soil microbial population that affect soil fertility and health. Natural pyrethrins have been used since ancient times as insect repellers, and their synthetic versions especially type 2 pyrethroids could be highly toxic to humans. PBO (Piperonyl butoxide) is known to enhance the toxicity of prallethrin in humans due to the resistance in its metabolic degradation. Pyrethroids are also known to cause plasma biochemical profile changes in humans and they also lead to the production of high levels of reactive oxygen species. Further they are also known to increase SGPT activity in humans. Due to the toxicity of pyrethrins in water bodies, soils, and food products, there is an urgent need to develop sustainable approaches to reduce their levels in the respective fields, which are eco-friendly, economically viable, and socially acceptable for on-site remediation. Keeping this in view, an attempt has been made to analyse the advances and prospects in using pyrethrins and possible technologies to control their harmful effects. The pyrethroid types, composition and biochemistry of necessary pyrethroid insecticides have been discussed in detail, in the research paper, along with their effect on insects and humans. It also covers the impact of pyrethroids on different plants and soil microbial flora. The second part deals with the microbial degradation of the pyrethroids through different modes, i.e., bioaugmentation and biostimulation. Many microbes such as Acremonium, Aspergillus, Microsphaeropsis, Westerdykella, Pseudomonas, Staphylococcus have been used in the individual form for the degradation of pyrethroids, while some of them such as Bacillus are even used in the form of consortia.

Arabinofuranosidases: Characteristics, microbial production, and potential in waste valorization and industrial applications.

Alpha-L-arabinofuranoside arabinofuranohydrolase (ARA), more commonly known as alpha-L-arabinofuranosidase (E.C. number 3.2.1.55), is a hydrolytic enzyme, catalyzing the cleavage of alpha-L-arabinose by acting on the non-reducing ends of alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)- and/or (1,5)-linked arabinoxylans and arabinogalactans. ARA functions as debranching enzyme removing arabinose substituents from arabinoxylan and arabinoxylooligomers, thereby, boosting the hydrolysis of arabinoxylan fraction of hemicellulose and improving bioconversion of lignocellulosic biomass. Previously, comprehensive information on this enzyme has not been reviewed thoroughly. Therefore, the main aim of this review is to highlight the important properties of this interesting enzyme, microorganisms used for its production, and enhanced production using genetic engineering approach. An account on synergism with other biomass hydrolyzing enzymes and various industrial applications of this enzyme has also been provided along with an outlook on further research and development.

Functional Expression of a Thermostable Endoglucanase from Thermoascus aurantiacus RCKK in Pichia pastoris X-33 and Its Characterization.

Thermostable cellulases offer several advantages like higher rates of substrate hydrolysis, lowered risk of contamination, and increased flexibility with respect to process design. In the present study, a thermostable native endoglucanase nEG (EC 3.2.1.4) was purified and characterized from T. aurantiacus RCKK. Further, it was cloned in P. pastoris X-33 and processed for over expression. Expression of recombinant endoglucanase (rEG) of molecular size ~ 33 kDa was confirmed by SDS-PAGE and western blotting followed by in gel activity determination by zymogram analysis. Similar to nEG, the purified rEG was characterized to harbor high thermostability while retaining 50% of its initial activity even after 6- and 10-h incubation at 80 and 70 °C, respectively, and exhibited considerable stability in pH range 3.0-7.0. CD spectroscopy revealed more than 20% ß-sheets in protein structure consistently when incubated upto 85 °C as a speculated reason for protein high thermostability. Interestingly, both nEG and rEG were found tolerant up to 10% of the presence of 1-ethyl-3-methylimidazolium acetate [C2mim][OAc]. Values of the catalytic constants Km and Vmax for rEG were recorded as 2.5 mg/ml and 303.4 µmol/mg/min, respectively. Thermostability, pH stability, and resistance to the presence of ionic liquid signify the potential applicability of present enzyme in cellulose hydrolysis and enzymatic deinking of recycled paper pulp.

Improved Production of Thermostable Cellulase from Thermoascus aurantiacus RCKK by Fermentation Bioprocessing and Its Application in the Hydrolysis of Office Waste Paper, Algal Pulp, and Biologically Treated Wheat Straw.

Thermostable cellulases have wide variety of applications and distinctive advantages, but their low titer becomes the hurdle in their commercialization. In the present work, an assessment of optimum levels of significant factors (temperature, moisture ratio, inoculum size, and ammonium sulfate) and the effect of their interactions on production of thermostable CMCase, FPase, and ß-glucosidase by Thermoascus aurantiacus RCKK under solid-state fermentation (SSF) was carried out using central composite design (CCD) of response surface methodology (RSM). The study revealed 33, 13, and 8 % improvement in FPase, CMCase, and ß-glucosidase production, respectively. Moreover, crude cellulase from T. aurantiacus RCKK efficiently hydrolyzed office waste paper, algal pulp (Gracillaria verulosa), and biologically treated wheat straw at 60 °C with sugar release of about 830 mg/ml, 285 mg/g, and 260 mg/g of the substrate, respectively. The thermostable enzyme from T. aurantiacus RCKK holds potential to be used in biofuel industry.

In-Vitro Refolding and Characterization of Recombinant Laccase (CotA) From Bacillus pumilus MK001 and Its Potential for Phenolics Degradation.

Among lignocellulolytic enzymes, laccases are the most versatile, broadly specific, and largely studied enzyme with a wide range of biotechnological potential. Putative laccase (CotA) from Bacillus pumilus MK001 was cloned and expressed in E. coli. In addition to soluble bioactive fraction, inactive inclusion body fraction was also harvested and refolded under optimized conditions resulting in 64 % of refolding efficiency. The enzyme was found to be thermostable exhibiting a half-life of 60 min at 80 °C. UV thermal CD spectra also supported the observation as about 9 % increase in ß-sheets was recorded after thermal induction. The 3D CotA structure was constructed through homology modeling and the best selected model was verified through PROCHECK, ERRAT, Verify 3D, and PROSA servers. Final 3D model showed potential binding affinities with ferulic acid, caffeic acid, and vanillin. Results of the docking studies were further validated by HPLC analysis which signified the efficient bioconversion ability of CotA.

Process development for the production of bioethanol from waste algal biomass of Gracilaria verrucosa.

The algal biomass of different species of Gracilaria were collected from coasts of Orissa and Tamil Nadu, India and characterized biochemically. Among various species, G. verrucosa was found to be better in terms of total carbohydrate content (56.65%) and hence selected for further studies. The agar was extracted from algal biomass and the residual pulp was enzymatically hydrolyzed. The optimization of algal pulp hydrolysis for various parameters revealed a maximum sugar release of 75.8mg/ml with 63% saccharification yield. The fermentation of enzymatic hydrolysate of algal pulp was optimized and 8% (v/v) inoculum size, 12h inoculum age, pH 5.0 were found to be optimum parameters for maximum ethanol concentration (27.2g/L) after 12h. The process of enzymatic hydrolysis and fermentation were successfully scaled up to 2L bioreactor scale.

Cost-effective production of cellulose hydrolysing enzymes from Trichoderma sp. RCK65 under SSF and its evaluation in saccharification of cellulosic substrates.

Trichoderma sp. is a potential cellulase producing mesophilic fungi which grow under mild acidic condition. In this study, growth and nutritional conditions were manipulated for the maximum and cost-effective production of cellulase using lab strain Trichoderma sp. RCK65 and checked for its efficiency in hydrolysis of Prosopis juliflora (a woody substrate). Preliminary studies suggested that when 48 h old secondary fungal culture (20 % v/w) was inoculated in wheat bran moistened with mineral salt solution (pH 4.5 and 1:3 solid to moisture ratio), incubated at 30 °C and after 72 h, it produced maximum cellulase (CMCase 145 U/gds, FPase 38 U/gds and ß-glucosidase 105 U/gds). However, using statistical approach a S:L ratio (1:1) was surprisingly found to be optimum that improved cellulase that is CMCase activity by 6.21 %, FPase activity by 23.68 % and ß-glucosidase activity by 37.28 %. The estimated cost of crude enzyme (Rs. 5.311/1000 FPase units) seems to be economically feasible which may be due to high enzyme titre, less cultivation time and low media cost. Moreover, when the crude enzyme was used to saccharify pretreated Prosopis juliflora (a woody substrate), it resulted up to 83 % (w/w) saccharification.

Microbiological Analyses of Traditional Alcoholic Beverage (Chhang) and its Starter (Balma) Prepared by Bhotiya Tribe of Uttarakhand, India.

Present article depicts microbiology of starter (Balma) used in traditional solid-state fermentation of alcoholic beverage (Chhang) by Bhotiya tribe of Uttarakhand. It also highlights the importance of herbs in Balma preparation and kinetics of lactic acid and ethanol fermentation under Chhang preparation using Balma. Balma contains 214 × 10(6) cfu/g yeasts, 2.54 × 10(6) cfu/g lactic acid bacteria (LAB) and 1.4 × 10(6) cfu/g other mesophilic bacteria. ITS sequence analysis revealed a rich diversity of yeast comprising of Saccharomyces cerevisiae, Saccharomycopsis fibuligera and Saccharomycopsis malanga in Balma. 16S rDNA sequence analysis revealed Lactobacillus pentosus and Pediococcus pentosaceus among LAB, while amylolytic Bacillus subtilis and B. aerophilus among other bacteria in Balma. Based on the results, it is speculated that herbs such as Inula cuspidata, Micromeria biflora, Origanum vulgare, Rubus sp. and Thymus linearis used earlier by Bhotiya in Balma preparation contribute as a source of yeasts, LAB and amylolytic bacilli. Study also demonstrates that Bhotiya tribe is rational in preparation of starter as they have circumvented the need of plants by using previous year Balma as inoculum and possibility of deficient quality of Balma due to weak colonization of phyllosphere and rhizosphere microbiota. Results suggest that simultaneous saccharification and lactic acid-ethanol fermentation take place in traditional cereal based Chhang fermentation system of Bhotiya.

Multiple Genes in a Single Host: Cost-Effective Production of Bacterial Laccase (cotA), Pectate Lyase (pel), and Endoxylanase (xyl) by Simultaneous Expression and Cloning in Single Vector in E. coli.

This study attempted to reduce the enzyme production cost for exploiting lignocellulosic materials by expression of multiple genes in a single host. Genes for bacterial laccase (CotA), pectate lyase (Pel) and endoxylanase (Xyl), which hold significance in lignocellulose degradation, were cloned in pETDuet-1 vector containing two independent cloning sites (MCS). CotA and xyl genes were cloned in MCS1 and MCS 2, respectively. Pel gene was cloned by inserting complete cassette (T7 promoter, ribosome binding site, pel gene, His tag and complete gene ORF) preceded by cotA open reading frame in the MCS1. IPTG induction of CPXpDuet-1 construct in E. coli BL21(DE3) resulted in expression of all three heterologous proteins of ~65 kDa (CotA), ~45 kDa (Pel) and ~25 kDa (Xyl), confirmed by SDS-PAGE and western blotting. Significant portions of the enzymes were also found in culture supernatant (~16, ~720 and ~370 IU/ml activities of CotA, Pel and Xyl, respectively). Culture media optimization resulted in 2, 3 and 7 fold increased secretion of recombinant CotA, Pel and Xyl, respectively. Bioreactor level optimization of the recombinant cocktail expression resulted in production of 19 g/L dry cell biomass at OD600nm 74 from 1 L induced culture after 15 h of cultivation, from which 9, 627 and 1090 IU/ml secretory enzyme activities of CotA, Xyl and Pel were obtained, respectively. The cocktail was also found to increase the saccharification of orange peel in comparison to the xylanase alone. Thus, simultaneous expression as well as extra cellular secretion of these enzymes as cocktail can reduce the enzyme production cost which increases their applicability specially for exploiting lignocellulosic materials for their conversion to value added products like alcohol and animal feed.

Reduced toxicity of malachite green decolorized by laccase produced from Ganoderma sp. rckk-02 under solid-state fermentation.

Statistical designs were applied for optimizing laccase production from a white-rot fungus, Ganoderma sp. rckk-02 under solid-state fermentation (SSF). Compared to unoptimized conditions [2,154 U/gds (Unit per gram of dry substrate)], the optimization process resulted in a 17.3-fold increase in laccase production (37,423 U/gds). The laccase produced was evaluated for its potential to decolorize a recalcitrant synthetic dye, malachite green. Laccase at dosage of 30 U/ml in presence of 1 mM of 1-hydroxybenzotriazole (HBT) almost completely decolorized 100 and 200 mg/l of malachite green in 16 and 20 h, respectively, at 30 °C, pH 5.5 and 150 rpm. While, higher dyes concentrations of 300, 400 and 500 mg/l were decolorized to 72, 62 and 55 % in 24, 28 and 32 h, respectively, under similar conditions. Furthermore, it was observed that the decolorized malachite green was less toxic towards the growth of five white-rot fungi tested viz. Crinipellis sp. RCK-1, Ganoderma sp. rckk-02, Coriolopsis Caperata RCK 2011, Phanerochaete chrysosporium K3 and Pycnoporous cinnabarinus PB. The present study demonstrates the potential of Ganoderma sp. rckk-02 to produce high titres of laccase under SSF, which can be exploited in conjunction with redox mediator for the decolorization of high concentrations of malachite green from water bodies.

Production of thermostable hydrolases (cellulases and xylanase) from Thermoascus aurantiacus RCKK: a potential fungus.

Thermophilic fungi are potential sources of thermostable enzymes and other value added products. Present study has focused on optimization of different physicochemical parameters for production of thermostable cellulases and xylanase by Thermoascus aurantiacus RCKK under SSF. Enzyme production was supported maximally on wheat bran fed with 20% inoculum, at initial pH 5, temperature 45 °C and moisture ratio 1:3. The supplementation of wheat bran with yeast extract, Tween-80 and glycine further improved enzyme titres (CMCase 88 IU/g, FPase 15.8 IU/g, ß-glucosidase 25.3 IU/g and xylanase 6,543 IU/g). The crude enzymes hydrolyzed phosphoric acid-swollen wheat straw, avicel and untreated xylan up to 74, 71 and 90%, respectively. In addition, T. aurantiacus RCKK produced antioxidants as fermentation by-products with significant %DPPH(∙) scavenging, FRAP and in vivo antioxidant capacity against H2O2-treated Saccharomyces cerevisiae. These capabilities show that it holds potential to exploit crop by-products for providing various commodities.

Characterization of recombinant pectate lyase refolded from inclusion bodies generated in E. coli BL21(DE3).

Pectate lyase (EC 4.2.2.2) gene from Bacillus subtilis RCK was cloned and expressed in Escherichia coli to maximize its production. In addition to soluble fraction, bioactive pectate lyase was also obtained from inclusion body aggregates by urea solubilization and refolding under in vitro conditions. Enzyme with specific activity ∼3194IU/mg and ∼1493IU/mg were obtained from soluble and inclusion bodies (IBs) fraction with recovery of 56% and 74% in terms of activity, respectively. The recombinant enzyme was moderately thermostable (t1/2 60min at 50°C) and optimally active in wider alkaline pH range (7.0-10.5). Interaction of protein with its cofactor CaCl2 was found to stimulate the change in tertiary structure as revealed by near UV CD spectra. Intrinsic tryptophan fluorescence spectra indicated that tryptophan is involved in substrate binding and there might be independent binding of Ca(2+) and polygalacturonic acid to the active site. The recombinant enzyme was found to be capable of degrading pectin and polygalacturonic acid. The work reports novel conditions for refolding to obtain active recombinant pectate lyase from inclusion bodies and elucidates the effect of ligand and substrate binding on protein conformation by circular dichroism (CD) and fluorescence spectrofluorometry.

Bioprocessing of wheat straw into nutritionally rich and digested cattle feed.

Wheat straw was fermented by Crinipellis sp. RCK-1, a lignin degrading fungus, under solid state fermentation conditions. The fungus degraded 18.38% lignin at the expense of 10.37% cellulose within 9 days. However, when wheat straw fermented for different duration was evaluated in vitro, the 5 day fungal fermented wheat straw called here "Biotech Feed" was found to possess 36.74% organic matter digestibility (OMD) and 5.38 (MJ/Kg Dry matter) metabolizable energy (ME). The Biotech Feed was also observed to be significantly enriched with essential amino acids and fungal protein by fungal fermentation, eventually increasing its nutritional value. The Biotech Feed upon in vitro analysis showed potential to replace 50% grain from concentrate mixture. Further, the calves fed on Biotech Feed based diets exhibited significantly higher (p<0.05) dry matter intake (DMI: 3.74 Kg/d), dry matter digestibility (DMD: 57.82%), total digestible nutrients (TDN: 54.76%) and comparatively gained 50 g more daily body weight.

Xylanase and laccase based enzymatic kraft pulp bleaching reduces adsorbable organic halogen (AOX) in bleach effluents: a pilot scale study.

In present study, xylanase and laccase were produced in a cost-effective manner up to 10 kg substrate level and evaluated in elemental chlorine free bleaching of Eucalyptus kraft pulp. Compared to the pulp pre-bleached with xylanase (15%) or laccase (25%) individually, the ClO2 savings were higher with sequential treatment of xylanase followed by laccase (35%) at laboratory scale. The sequential enzyme treatment when applied at pilot scale (50 kg pulp), resulted in improved pulp properties (50% reduced post color number, 15.71% increased tear index) and reduced AOX levels (34%) in bleach effluents. The decreased AOX level in effluents will help to meet AOX discharge limits, while improved pulp properties will be value addition to the paper.

Fungal pretreatment improves amenability of lignocellulosic material for its saccharification to sugars.

The sugarcane bagasse was biologically pretreated with three white-rot fungi; Pleurotus florida, Coriolopsis caperata RCK 2011 and Ganoderma sp. rckk-02, individually under solid-state fermentation. P. florida, C. caperata RCK 2011 and Ganoderma sp. rckk-02 degraded lignin up to 7.91, 5.48 and 5.58%, respectively. The lignocellulolytic enzymes produced by these fungi were also monitored during solid state fermentation of sugarcane bagasse. The fungal fermented sugarcane bagasse when hydrolyzed with crude cellulases from brown-rot fungus, Fomitopsis sp. RCK2010, released comparatively 1.5-2.4 fold higher sugars than in case of untreated sugarcane bagasse. The study demonstrated that white-rot fungal pretreatment improved the amenability of plant material for enzymatic hydrolysis.