cellulase and ferulic-acid

The aim of this work is to study a bifunctional endoglucanase/carboxylesterase in Sphingobacterium soilsilvae Em02 and express it in soluble form in engineered Escherichia coli. The molecular weight of the recombinant protein of the bifunctional enzyme was 41 KDa. This research also determined the enzymatic activities of the bifunctional enzymes using microcrystalline cellulose and p-nitrophenyl butyrate as substrates and found 40 °C as the optimum temperature for their enzymatic activities. The optimal pH in dual function was 6.0 for endoglucanase and 7.0 for carboxylesterase. The bifunctional enzyme also exhibited enzymatic activities on the natural biomass by generating up to 3.94 mg of glucose and 49.4 μg of ferulic acid from 20 mg of destarched wheat bran. This indicates the broad application prospects of the bifunctional enzyme in agriculture and industry.

Topics: Carboxylic Ester Hydrolases; Catalysis; Cellulase; Dietary Fiber; Glucose; Hydrogen-Ion Concentration

In this work, Monascus fermentation and cellulase hydrolysis (MCF) of oats (Avena sativa L.) to release and convert phenolic fraction was investigated. Results showed the fungus Monascus grew well with a biomass of 27.03 mg/g glucosamine equivalent in MCF, following the destruction of oat cellular structures. SDS-PAGE revealed lots of enzymes were regulated with the α-amylase and FPase activity achieved 139.25 U/g and 1.84 U/g in MCF, respectively. Compared with unfermented oats, content of the total phenolic fractions was increased by 19.2 times in MCF, suggesting a phenolic release process occurred during fermentation. Moreover, the soluble-free chlorogenic acid upregulated to 510.00 mg/kg whereas the insoluble-bound ferulic acid downregulated to 193.36 mg/kg in MCF, indicating a transformation process of chlorogenic acid from ferulic acid in oats was enhanced. Based on this, a possible pathway of phenolic release and conversion in oats during fermentation with Monascus spp. was revealed. This study was helpful to enrich the theory of microbial metabolism and transformation in grain materials.

Topics: Avena; Cellulase; Chlorogenic Acid; Edible Grain; Fermentation; Phenols

In the present work, the effects of combined enzymatic hydrolysis by cellulase and xylanase (CXEH), fed-batch enzymatic hydrolysis (FBEH) operation and kinetics on production of ferulic acid (FA) and p-coumaric acid (pCA) from pretreated corn straws were investigated. The results showed that CXEH could efficiently increase production of FA and pCA. When performed the FBEH operation by feeding 150 mL enzymatic hydrolysis solution (1.5 % enzyme concentration, 5:4 (v/v) ratio of cellulase to xylanase and 2.0 % substrate loading) to 250 mL batch enzymatic hydrolysis solution at 36 h, the maximum production (2178.58 and 2710.17 mg/L) and production rate (590.95 and 727.89 mg/L.h) of FA and pCA were respectively obtained. Moreover, the disruption of fiber tissues, enhancement of crystallinity and accelerated degradation of hemicelluloses and lignocelluloses caused by CXEH contributed to effectively improving production of FA and pCA in corn straws.

Topics: Cellulase; Hydrolysis; Zea mays

Solid-state fermentation (SSF) is the preferred method of enhancing the phenolic content of oats, while scientific optimization for improving specific phenolic compounds is limited. In this study, sequential targeting of phenolic conversion in simultaneous hydrolysis and fermentation (SHF) of oats was investigated. The results revealed that SHF with adding cellulase at 0, 6 and 12 days could increase the total phenolic content by 4.4%, 67.8% and 59.1%, respectively, over that of SSF. The α-amylase and CMCase activity were highly correlated with the soluble and insoluble phenolic contents in SHF (-6 and -12) systems (r > 0.8, p < 0.05). Interestingly, the content of phenolic fraction, such as ferulic acid, was up-regulated, whereas sinapic acid was down-regulated. These results indicated that the phenolic conversion occurred in SHF, resulting in variation in DPPH and ABTS

Topics: Amylases; Antioxidants; Avena; Cellulase; Coumaric Acids; Edible Grain; Fermentation; Hydrolysis; Phenols

Ammonia treatment of lignocellulose improves carbohydrate degradability, however, low ammonia dose treatment effects and mechanisms are hardly considered. This study describes low dose ammonia treatment of wheat straw in a statistical design of experiments (Taguchi design) to evaluate the effects of ammonia concentration, treatment time and the Solid:Liquid ratio on structure, composition and enzymatic degradability of the residual fractions. The results showed that low ammonia concentration (≤2 w/w % NH

Topics: Ammonia; Cell Wall; Cellulase; Coumaric Acids; Hydrolysis; Polysaccharides; Triticum

Evolution of Rhizopus oryzae and Trichoderma reesei biomass in rice bran, their enzyme activity, and the profile of phenolic compounds released from the lignocellulosic matrices were determined and correlated by principal component analysis (PCA). PCA analysis confirms that cultivation of rice bran affected the release of methanol-soluble phenolic compounds (MSPC), ethanol-soluble phenolic compounds (ESPC), and bound phenolic compounds (BPC) positively, due to their enzymatic activity. The release of MSPC was influenced by the activity of cellulase and endoglucanase, which increased 110.6% and 136.3%, respectively, for Rhizopus oryzae and Trichoderma reesei. Gallic acid was the main component in the MSPC and ESPC compound fractions. Ferulic and syringic acids were found in its bound (BPC) form in the biomass. This study showed that bioactive compounds be released from lignocellulosic materials by fungus action and this process can be conducted to obtain specific phenolic compounds. PRACTICAL APPLICATION: Due the demand by natural compounds with biological activity, such as phenolic compounds, it is interesting to purpose alternatives to enhance their yield, like for instance, by fungal fermentation of lignocellulosic material. Therefore, understanding the relations among different phenolic compounds released and the production of fungal hydrolases during growth of Rhizopus oryzae and Trichoderma reesei in solid state cultivation using rice bran as a substrate is fundamental to control the process. This knowledge gets viable scale up to apply the phenolic compounds as preservative in food chain, because this becomes possible directing the process to obtain specific bioactive compounds in less time of cultivation and with low cost.

Topics: Biomass; Cellulase; Cellulose; Coumaric Acids; Culture Media; Fermentation; Gallic Acid; Oryza; Phenols; Rhizopus; Trichoderma

Okara (soybean residue) is a highly perishable food processing by-product from soymilk and tofu manufacture. It contains a large proportion of insoluble dietary fibre (40-60% on a dry basis), as well as digestion-resistant proteins, trypsin inhibitors and phytic acid. These factors contribute lead to the under-utilisation of okara. To improve the overall nutritional quality of okara, sequential saccharification of okara by Celluclast® 1.5L (cellulase) or Viscozyme® L (cellulase and hemicellulase) and fermentation by the yeast Yarrowia lipolytica were performed. The changes in the antioxidant capacity, amino acids, phenolic acids, isoflavones, phytic acid and dietary fibre during biotransformation were studied. Carbohydrase pre-treatment increased the amounts of monosaccharides, trans-cinnamic acid and aglycone isoflavones in okara. After fermentation, the okara had higher antioxidant activity and greater amounts of total amino acids and ferulic acid. Some positive interactions between the carbohydrase and Y. lipolytica were hypothesised: the carbohydrase and Y. lipolytica proteases could have synergised with each other to break down the okara secondary cell wall more efficiently. After 52 h, Celluclast® 1.5 L and Viscozyme® L significantly reduced the insoluble dietary fibre content from 61.9 ± 0.6 to 45.6 ± 3.0% and 24.7 ± 0.3%, respectively (all w/w, dry basis), while increasing the soluble dietary fibre content by about onefold. Both carbohydrases also increased the amounts of monosaccharides, trans-cinnamic acid, and aglycone isoflavones in okara. The addition of Y. lipolytica led to a higher antioxidant capacity and greater amounts of total amino acids and ferulic acid in okara. The overall improvements in the digestibility and potential health benefits of okara highlight the promising applicability of biotransformation in okara valorisation.

Topics: Amino Acids; Antioxidants; Biotransformation; Cellulase; Coumaric Acids; Dietary Fiber; Fermentation; Food Handling; Food Microbiology; Glycine max; Glycoside Hydrolases; Hydroxybenzoates; Isoflavones; Phytic Acid; Yarrowia

The current study is focused on optimizing the parameters involved in enzymatic processing of red rice bran for maximizing total polyphenol (TP) and free radical scavenging activity (FRSA). The sequential optimization strategies using central composite design (CCD) and artificial neural network (ANN) modeling linked with genetic algorithm (GA) was performed to study the effect of incubation time (60-90 min), xylanase concentration (5-10 mg/g), cellulase concentration (5-10 mg/g) on the response, i.e., total polyphenol and FRSA. The result showed that incubation time has a negative effect on the response, while the square effect of xylanase and cellulase showed positive effect on the response. A maximum TP of 2,761 mg ferulic acid Eq/100 g bran and FRSA of 778.4 mg Catechin Eq/100 g bran was achieved with incubation time (min) = 60.491; xylanase (mg/g) = 5.4633; cellulase (mg/g) = 11.5825. Furthermore, ANN-GA-based optimization showed better predicting capabilities as compared to CCD.

Topics: Aspergillus niger; Biphenyl Compounds; Cellulase; Coumaric Acids; Endo-1,4-beta Xylanases; Free Radical Scavengers; Free Radicals; Neural Networks, Computer; Oryza; Picrates; Plant Extracts; Polyphenols

To characterize the hemicellulolytic and cellulolytic enzymes from novel fungi, and evaluate the potential of novel enzyme system in releasing ferulic acid (FA) from biomass resource.. A hemicellulolytic and cellulolytic enzyme-producing fungus 4-14 was isolated from soil by Congo red staining method, and identified as Eupenicillium parvum based on the morphologic and molecular phylogenetic analysis. The optimum temperature of fungal growth was 37°C. Hemicellulolytic and cellulolytic enzymes were produced by this fungus in solid-state fermentation (SSF), and their maximum activities were 554, 385, 218, 2·62 and 5·25 U g(-1) for CMCase, xylanase, β-glucosidase, FPase and FAE respectively. These enzymes displayed the best catalytic ability at low pH values (pH 4·5-5·0). The optimum temperatures were 70°C, 70°C, 75°C and 55°C for CMCase, β-glucosidase, xylanase and FAE respectively. CMCase, xylanase and FAE were stable at different pHs or high temperature (60°C). Enzymatic hydrolysis experiment indicated that the maximum (76·8 ± 4)% of total alkali-extractable FA was released from de-starched wheat bran by the fungal enzyme system.. High activities of thermotolerant CMCase, β-glucosidase, xylanase and FAE were produced by the newly isolated fungus E. parvum 4-14 in SSF. The fungal enzyme system displayed high efficiency at releasing FA from wheat bran.. This study provides a new fungal strain for researches of novel hemicellulolytic and cellulolytic enzymes and will improve the bioconversion and utilization of agricultural by-products.

Topics: beta-Glucosidase; Cellulase; Coumaric Acids; Dietary Fiber; Eupenicillium; Fermentation; Fungal Proteins; Hydrolysis; Phylogeny; Temperature

Fermentation of natural products is emerging as an important processing method and is attracting a lot of attention because it may have the advantage of having a new biological function. In this study, fruits of Opuntia ficus-indica were enzymatically hydrolyzed and then fermented with two species of yeast. We identified novel prominent markers in enzymatically hydrolyzed O. ficus-indica (EO) and fermented O. ficus-indica (FO) samples by using an ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry. We also evaluated the effect of EO and FO on photoaging of skin cells exposed to ultraviolet radiation. We identified the major fermented metabolite in the FO as ferulic acid. Our in vitro study indicated that FO significantly enhanced the concentration of pro-collagen type 1 than the EO, by increasing the TGF-β1 production.

Topics: Ascomycota; Cells, Cultured; Cellulase; Collagen Type I; Coumaric Acids; Drug Discovery; Fermentation; Freeze Drying; Fruit; Fungal Proteins; Humans; Hydrolysis; Opuntia; Pichia; Plant Extracts; Plant Preparations; Procollagen; Skin; Skin Aging; Sunscreening Agents; Trichoderma; Ultraviolet Rays

The production of ferulic acid esterase involved in the release of ferulic acid side groups from xylan was investigated in strains of Aspergillus tubingensis, Aspergillus carneus, Aspergillus niger and Rhizopus oryzae. The highest activity on triticale bran as sole carbon source was observed with the A. tubingensis T8.4 strain, which produced a type A ferulic acid esterase active against methyl p-coumarate, methyl ferulate and methyl sinapate. The activity of the A. tubingensis ferulic acid esterase (AtFAEA) was inhibited twofold by glucose and induced twofold in the presence of maize bran. An initial accumulation of endoglucanase was followed by the production of endoxylanase, suggesting a combined action with ferulic acid esterase on maize bran. A genomic copy of the A. tubingensis faeA gene was cloned and expressed in A. niger D15#26 under the control of the A. niger gpd promoter. The recombinant strain has reduced protease activity and does not acidify the media, therefore promoting high-level expression of recombinant enzymes. It produced 13.5 U/ml FAEA after 5 days on autoclaved maize bran as sole carbon source, which was threefold higher than for the A. tubingensis donor strain. The recombinant AtFAEA was able to extract 50 % of the available ferulic acid from non-pretreated maize bran, making this enzyme suitable for the biological production of ferulic acid from lignocellulosic plant material.

Topics: Aspergillus; Aspergillus niger; Carboxylic Ester Hydrolases; Cellulase; Coumaric Acids; Peptide Hydrolases; Zea mays

Sequential fractionation of extractive-free maize stems was carried out using two mild alkaline extractions (0.5 and 2 M NaOH, 20°C, 24h) before and after endoglucanase treatment. This procedure provided two lignin-carbohydrate fractions (LC1 and LC2) recovered after each alkali treatment. LC1 and LC2 contained 39% and 8% of the total lignin amount, respectively. These two fractions contained structurally distinct lignin molecules. While the content of resistant interunit bonds in lignin was 77% in LC1, it was increased up to 98% in LC2. Not unexpectedly, both alkali-soluble fractions contained substantial amount of p-coumaric and ferulic acids ether-linked to lignins. These results outline heterogeneity of maize stem lignins related to fractionation of grass materials.

Topics: Alkalies; Carbohydrates; Cellulase; Chemical Fractionation; Coumaric Acids; Lignin; Monosaccharides; Plant Stems; Propionates; Spectroscopy, Fourier Transform Infrared; Zea mays

In the cell walls of grasses, ferulic acid is esterified to arabinoxylans and undergoes oxidative reactions to form ferulates dimers, trimers and oligomers. Feruloylation of arabinoxylan is considered important not only because it leads to cross-linked xylans but also because ferulates may act as a nucleating site for the formation of lignin and hence link arabinoxylans to lignin by forming a lignin-ferulate-arabinoxylan complex. Such cross-linking is among the main factors inhibiting the release of fermentable carbohydrates from grasses either for ruminant nutrition or for biofuel production. We have found that significant reductions in the levels of monomeric and dimeric phenolics can be achieved in the growing cell walls during plant development in leaves of Festuca arundinacea by constitutive intracellular targeted expression of Aspergillus niger ferulic acid esterase (FAEA). We propose that this occurred by directly disrupting ester bonds linking phenolics to cell wall polysaccharides by apoplast targeting or by preventing excessive feruloylation of cell wall carbohydrates prior to their incorporation into the cell wall, by targeting to the Golgi membrane system. Plants with lower cell wall ferulate levels, which showed increased digestibility and increased rates of cellulase-mediated release of fermentable sugars, were identified. Targeting FAE to the Golgi was found to be more effective than targeting to the ER, which supports the current theories of the Golgi as the site of feruloylation of arabinoxylans. It is concluded that targeting FAEA expression to the Golgi or apoplast is likely to be an effective strategy for improving wall digestibility in grass species used for fodder or cellulosic ethanol production.

Topics: Aspergillus niger; Biodegradation, Environmental; Carboxylic Ester Hydrolases; Cell Wall; Cellulase; Coumaric Acids; Endo-1,4-beta Xylanases; Endoplasmic Reticulum; Festuca; Golgi Apparatus; Phenols; Plants, Genetically Modified; Transformation, Genetic; Xylans

Hydroxycinnamic acids, mainly ferulic and p-coumaric acids, are believed to be inhibitory to ruminal biodegradability of complex cell wall materials such as oat hulls. Previous studies have shown that a novel enzyme, Aspergillus ferulic acid esterase, and Trichoderma xylanase act synergistically to break the ester linkage between ferulic acid and the attached sugar of feruloyl polysaccharides, releasing ferulic acid from oat hulls. In this paper, we examined the enzymic release of reducing sugars from oat hulls by the actions of individual enzymes (Aspergillus ferulic acid esterase at 13 mU, 6.4 U, and 4678.4 U/assay; cellulase at 20 levels, ranging from 7.8 mU to 2772.7 U/assay; Trichoderma xylanase at 20 levels, ranging from 7.8 mU to 4096 U/assay) and by the combined action of cellulase at six levels (62.5 mU, 2 U, 16 U, 128 U, 1024 U, and 2772.7 U/assay), Aspergillus ferulic acid esterase at 13 mU/assay, and Trichoderma xylanase at two levels (1 U and 256 U/assay). The amount of total acid-extractable reducing sugars in the oat hulls used in this study was 793.8 +/- 8.0 microg/mg. The results show that after a 24-h incubation with Aspergillus ferulic acid esterase alone, no reducing sugars were observed to be released from oat hulls. With cellulase as the sole enzyme, as the concentration increased from 7.8 mU to 2772.7 U/assay, the release of reducing sugars increased (P < 0.01) from 0 to 39% of the total present, with the highest release at 512 U/assay. With Trichoderma xylanase alone, as the concentration increased from 7.8 mU to 4096 U/assay, the release of reducing sugars increased (P < 0.01) from 4.9 to 33%, with the highest release at 2048 U/assay. When incubated together with Trichoderma xylanase (1 U or 256 U/assay) and Aspergillus ferulic acid esterase (13 mU/assay), cellulase at all six levels (62.5 mU, 2 U, 16 U, 128 U, 1024 U and 2772.7 U/assay) significantly increased the release of reducing sugars (P < 0.01) from 8 to 69%. These results indicate that the synergistic interaction between Aspergillus ferulic acid esterase and Trichoderma xylanase on the release of ferulic acid from feruloyl polysaccharides of oat hulls makes the remainder of the polysaccharides open for further hydrolytic attack and facilitates the accessibility of the main chain of polysaccharides to cellulase. This action extends the cell wall hydrolysis, thus releasing a higher yield of reducing sugars. Such enzymic pretreatment of oat hulls may provide a unique advanta

Topics: Aspergillus; Avena; Carbohydrate Metabolism; Carbohydrates; Carboxylic Ester Hydrolases; Cellulase; Coumaric Acids; Propionates; Regression Analysis; Seeds; Trichoderma; Xylan Endo-1,3-beta-Xylosidase; Xylosidases

Most studies published thus far on the four brown midrib (bm) mutants (bm1, bm2, bm3, and bm4) in maize (Zea mays L.) have focused on one or two individual mutants, and comparisons between studies have been difficult because of variation in genetic backgrounds, maturity, and source of tissue. Detailed analyses of the stalks of the four bm single mutants and a bm1-bm2 double mutant in a common genetic background (inbred A619) revealed structural and compositional changes in their isolated cell walls and lignins compared to the wild-type inbred. 2D-NMR revealed a significant presence of benzodioxane units in the bm3 isolated lignin. 1D (13)C NMR revealed increased aldehyde levels in the bm1 and bm1-bm2 mutants compared to the wild-type inbred. The bm3 and bm1-bm2 mutants contained less Klason lignin in the isolated cell walls. The bm1, bm3, and bm1-bm2 mutants contained approximately 50% less esterified p-coumaric acid with noticeably elevated levels of ferulate in the bm3 mutant. A difference among bm mutants in the solubility of p-coumaric acid-lignin complexes during cellulase enzyme treatment was also discovered, suggesting that the bm mutations might also differ in the structural organization of lignin.

Topics: Aldehydes; Cell Wall; Cellulase; Coumaric Acids; Esterification; Lignin; Magnetic Resonance Spectroscopy; Mutation; Propionates; Zea mays

trans-p-Coumaric acid inhibited the growth of Bacteroides ruminicola on both cellobiose and glucose, while trans-ferulic acid and vanillin retarded growth. The phenolic monomers varied in their potential to inhibit the Bacteroides succinogenes beta-glucosidase, carboxymethylcellulase, and xylanase, with p-coumaric acid being the most inhibitory. The B. ruminicola beta-glucosidase was inhibited less than 10% by all three compounds.

Topics: Bacteroides; Benzaldehydes; beta-Glucosidase; Cellulase; Coumaric Acids; Glycoside Hydrolases; Phenols; Propionates; Xylan Endo-1,3-beta-Xylosidase