Immobilization of mutated xylanase from Neocallimastix patriciarum in E coli and application for kraft pulp biobleaching / Imobilização de xilanase mutada de Neocallimastix patriciarum em E coli e aplicação para polpa kraft

As an important enzyme, xylanase is widely used in the food, pulp, and textile industry. Different applications of xylanase warrant specific conditions including temperature and pH. This study aimed to carry out sodium alginate beads as carrier to immobilize previous reported mutated xylanase from Neocallimastix patriciarum which expressed in E. coli, the activity of immobilization of mutated xylanase was elevated about 4% at pH 6 and 13% at 62 °C. Moreover, the immobilized mutated xylanase retained a greater proportion of its activity than the wide type in thermostability. These properties suggested that the immobilization of mutated xylanase has potential to apply in biobleaching industry.

Como importante enzima, a xilanase é amplamente utilizada na indústria alimentícia, de celulose e têxtil. Diferentes aplicações de xilanase garantem condições específicas, incluindo temperatura e pH. Este estudo teve como objetivo realizar grânulos de alginato de sódio como carreador para imobilizar xilanase mutada relatada anteriormente de Neocallimastix patriciarum que expressa em E. coli, a atividade de imobilização da xilanase mutada foi elevada em cerca de 4% em pH 6 e 13% a 62 °C. Além disso, a xilanase mutada imobilizada reteve uma proporção maior de sua atividade do que o tipo amplo em termoestabilidade. Essas propriedades sugerem que a imobilização da xilanase mutada tem potencial para aplicação na indústria de biobranqueamento.

Modulating the production of xylanase by Bacillus pumilus BS131 through optimization using waste fiber sludge / Modulando a produção de xilanase por Bacillus pumilus BS131 por meio da otimização usando lodo de fibra residual

In recent days, cheapest alternative carbon source for fermentation purpose is desirable to minimize production cost. Xylanases have become attractive enzymes as their potential in bio-bleaching of pulp and paper industry. The objective of the present study was to identify the potential ability on the xylanase production by locally isolated Bacillus pumilus BS131 by using waste fiber sludge and wheat bran media under submerged fermentation. Culture growth conditions were optimized to obtain significant amount of xylanase. Maximum xylanase production was recorded after 72 hours of incubation at 30 °C and 7 pH with 4.0% substrate concentration. In the nutshell, the production of xylanase using inexpensive waste fiber sludge and wheat-bran as an alternative in place of expensive xylan substrate was more cost effective and environment friendly.

Nos últimos dias, a fonte alternativa de carbono mais barata para fins de fermentação é desejável para minimizar o custo de produção. As xilanases têm se tornado enzimas atraentes como seu potencial no biobranqueamento da indústria de papel e celulose. O objetivo do presente estudo foi identificar a capacidade potencial na produção de xilanase por Bacillus pumilus BS131 isolado localmente usando lodo de fibra residual e farelo de trigo em meio de fermentação submersa. As condições de crescimento da cultura foram otimizadas para obter uma quantidade significativa de xilanase. A produção máxima de xilanase foi registrada após 72 horas de incubação a 30 °C e pH 7 com concentração de substrato de 4,0%. Resumindo, a produção de xilanase usando lodo de fibra residual de baixo custo e farelo de trigo como uma alternativa no lugar do substrato de xilano caro foi mais econômica e ecológica.

Bamboo shavings derived O-acetylated xylan alleviates loperamide-induced constipation in mice.

BSH-1 is an O-acetylated xylan obtained from bamboo shavings. This study determined the protective effects of BSH-1 against loperamide (Lop)-induced constipation in mice. Mice received BSH-1 by gavage daily for 14 days. In constipated mice, BSH-1 significantly shortened the defecation time and raised the gastrointestinal (GI) transit rate, stool production, and cecal concentration of short-chain fatty acids (SCFAs). BSH-1 regulated the serum levels of gut hormones and neurotransmitters. BSH-1 also significantly altered the cecal microbiota of the constipated mice by increasing the abundance of potentially beneficial bacteria (e.g., Lactobacillus, Roseburia, and Bacteroidales_S24-7) and decreasing potentially pathogenic bacteria (e.g., Alloprevotella and Staphylococcus). Furthermore, colonic transcriptome analysis revealed that BSH-1 significantly reversed the expression changes of genes related to intestinal motility, water and ion transport, inflammation and cancer in constipated mice. Our findings indicated that BSH-1 effectively relieved Lop-induced constipation in mice and could be potentially used for constipation treatment.

Wheat-derived arabinoxylans reduced M2-macrophage functional activity, but enhanced monocyte-recruitment capacity.

The immunomodulatory properties of non-digestible polysaccharides (NDPs) have been recognized in in vitro and in vivo studies. The latter mostly demonstrated altered frequencies and inflammatory status of immune cells as clinical parameters. Most of the NDP activity will be exerted in the intestine where they can directly interact with macrophages. The predominant macrophage phenotype in the intestine is M2-like, with M1-like macrophages arising during inflammation. Here, we investigated transcriptional and functional impact on these macrophage phenotypes by NDP-treatment (i.e. yeast-derived soluble ß-glucan (yeast-ßG), apple-derived RG-I (apple-RGI), shiitake-derived ß-glucan (shiitake-ßG) or wheat-derived arabinoxylan (wheat-AX)). Wheat-AX, and to a lesser extent shiitake-ßG and apple-RGI but not yeast-ßG, reduced endocytosis and antigen processing capacity of M1- and M2-like macrophages. Moreover, the NDPs, and most notably wheat-AX, strongly induced transcription and secretion of a unique set of cytokines and chemokines. Conditioned medium from wheat-AX-treated M2-like macrophages subsequently demonstrated strongly increased monocyte recruitment capacity. These findings are in line with clinically observed immunomodulatory aspects of NDPs making it tempting to speculate that clinical activity of some NDPs is mediated through enhanced chemoattraction and modifying activity of intestinal immune cells.

Male grower pigs fed cereal soluble dietary fibres display biphasic glucose response and delayed glycaemic response after an oral glucose tolerance test.

Acute and sustained soluble dietary fibre (SDF) consumption are both associated with improved glucose tolerance in humans and animal models (e.g. porcine). However, the effects on glucose tolerance in grower pigs, adapted to diets with a combination of SDF have not been studied previously. In this experiment, cereal SDF wheat arabinoxylan (AX) and oat ß-glucan (BG) were fed individually and in combination to determine the effect on glucose tolerance in jugular vein catheterized grower pigs. Five groups of Large White male grower pigs were fed highly digestible diets containing either 10% AX, 10% BG, 5% AX with 5% BG, a model cereal whole wheat flour (WWF), or a control wheat starch diet (WS) with no SDF. Blood was collected via jugular vein catheters over 240 minutes following a feed challenge and an oral glucose tolerance test (OGTT) on two separate days. Postprandial blood samples were used to determine plasma glucose, insulin, non-esterified fatty acids (NEFA), glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), peptide tyrosine tyrosine (PYY), ghrelin, glucagon and cortisol concentrations. No dietary effects on glycaemic response were observed following the feed challenge or the OGTT as determined by the area under the curve (AUC). A biphasic glucose and insulin response was detected for all pigs following the OGTT. The current study showed male grower pigs have tight glycaemic control and glucose tolerance regardless of diet. In addition, pigs fed the combined SDF had a reduced GIP response and delayed insulin peak following the feed challenge. Incretin (GLP-1 and GIP) secretion appeared asynchronous reflecting their different enteroendocrine cell locations and response to nutrient absorption.

Phenolic compounds and antioxidant properties of arabinoxylan hydrolysates from defatted rice bran.

BACKGROUND: The water unextractable arabinoxylans (WUAX) contain beneficial phenolic compounds that can be used for food rather than for animal feed. The antioxidant activities of defatted rice bran obtained by xylanase-aided extraction is reported herein. The chemical and molecular characteristics of extracted fractions were investigated. RESULTS: The WUAX hydrolysate precipitated by 0-60% ethanol (F60), 60-90% ethanol (F6090), and more than 90% ethanol (F90) had decreased molar masses with increasing ethanol concentration. The fractions of interest, F60 and F6090, contained 75% arabinoxylans with ferulic acid as the major bound phenolic acid, followed by p-coumaric acid. According to chemical-based antioxidant assays F60 and F6090 exhibited higher diphenyl-1-picrylhydrazyl (DPPH) radical scavenging and ferric iron reducing ability than F90 which contained minor contents of small sugars and free phenolic acids. In cell-based antioxidant assays, using the fluorescent 2',7'-dichlorofluorescein diacetate probe, all three fractions were potent intracellular scavengers. CONCLUSION: The high molar mass of WUAX hydrolysates with high amount of bound phenolics contributes to the chemical-based antioxidant activity. All fractions of WUAX hydrolysates showed high potent intracellular scavenging activity regardless of molar mass, content and the component of bound phenolics. © 2017 Society of Chemical Industry.

The contribution of cell wall composition in the expansion of Camellia sinensis seedlings roots in response to aluminum.

MAIN CONCLUSION: Treatment with aluminum triggers a unique response in tea seedlings resulting in biochemical modification of the cell wall, regulation of the activity of the loosening agents, and elongation of root. Unlike most terrestrial plants, tea (Camellia sinensis L.) responds to aluminum (Al) through the promotion of its root elongation; but the real mechanism(s) behind this phenomenon is not well understood. A plausible relationship between the modifications of the cell wall and the promotion of root elongation was examined in tea seedlings treated for 8 days with 400 µM Al. The mechanical properties of the cell wall, the composition of its polysaccharides and their capacity to absorb Al, the expression of genes, and the activities of the wall-modifying proteins were studied. With 6 h of the treatment, about 40% of the absorbed Al was bound to the cell wall; however, the amount did not increase thereafter. Meanwhile, the activity of pectin methylesterase, the level of pectin demethylation, the amounts and the average molecular mass of xyloglucan in the root apices significantly decreased upon exposure to Al, resulting in the reduction of Al binding sites. On the other hand, the activity and the gene expression of peroxidase decreased, whereas the activity and gene expression of xyloglucan-degrading enzymes, the expression of expansin A and the H +-ATPase4 genes increased in the Al-treated plants. Interestingly, it was accompanied by the increase of elastic and viscous extensibility of the root apices. From the results, it can be suggested that the biochemical modification of the cell walls reduces sites of Al binding to roots and triggers the activity of the loosening agents, thereby increasing the length of tea roots.

Arabinoxylan hydrolyzates as immunomodulators in lipopolysaccharide-induced RAW264.7 macrophages.

Inflammation is an important healthy immune response of the body during lesions and infection. However, uncontrolled excessive inflammation can be damaging to the cells. The specific objective of this research was to evaluate the effect of structural details of enzymatically derived wheat arabinoxylan hydrolyzates (AXH) on their immunomodulatory properties. Out of the 30 AXH, six AXH showed statistically significant reduction in NO production compared to the control, causing an approximately 24 to 12% reduction in NO production. Five AXH exhibited statistically significant pro-inflammatory properties in the LPS induced cells, causing an approximately 10 to 14% increase compared to the control. A negative correlation was seen between NO production and total arabinoxylan (AX) × amount of 1,4-linked xylose with arabinose substituted at the O-3 position. Thus, AXH with higher AX and substitution at the O-3 position are favorable candidates to reduce the lipopolysaccharide induced inflammation. These results suggest that there may be a structure-function relationship for these AXH as immunomodulators.

Use of Empty Fruit Bunches from the oil palm for bioethanol production: a thorough comparison between dilute acid and dilute alkali pretreatment.

In the present work, two pretreatment techniques using either dilute acid (H2SO4) or dilute alkali (NaOH) have been compared for producing bioethanol from Empty Fruit Bunches (EFBs) from oil palm tree, a relevant feedstock for tropical countries. Treatments' performances under different conditions have been assessed and statistically optimized with respect to the response upon standardized enzymatic saccharification. The dilute acid treatment performed at optimal conditions (161.5°C, 9.44 min and 1.51% acid loading) gave 85.5% glucose yield, comparable to those of other commonly investigated feedstocks. Besides, the possibility of using fibers instead of finely ground biomass may be of economic interest. Oppositely, treatment with dilute alkali has shown lower performances under the conditions explored, most likely given the relatively significant lignin content, suggesting that the use of stronger alkali regime (with the associated drawbacks) is unavoidable to improve the performance of this treatment.

In vitro grown pollen tubes of Nicotiana alata actively synthesise a fucosylated xyloglucan.

Nicotiana alata pollen tubes are a widely used model for studies of polarized tip growth and cell wall synthesis in plants. To better understand these processes, RNA-Seq and de novo assembly methods were used to produce a transcriptome of N. alata pollen grains. Notable in the reconstructed transcriptome were sequences encoding proteins that are involved in the synthesis and remodelling of xyloglucan, a cell wall polysaccharide previously not thought to be deposited in Nicotiana pollen tube walls. Expression of several xyloglucan-related genes in actively growing pollen tubes was confirmed and xyloglucan epitopes were detected in the wall with carbohydrate-specific antibodies: the major xyloglucan oligosaccharides found in N. alata pollen grains and tubes were fucosylated, an unusual structure for the Solanaceae, the family to which Nicotiana belongs. Finally, carbohydrate linkages consistent with xyloglucan were identified chemically in the walls of N. alata pollen grains and pollen tubes grown in culture. The presence of a fucosylated xyloglucan in Nicotiana pollen tube walls was thus confirmed. The consequences of this discovery to models of pollen tube growth dynamics and more generally to polarised tip-growing cells in plants are discussed.

XTH31, encoding an in vitro XEH/XET-active enzyme, regulates aluminum sensitivity by modulating in vivo XET action, cell wall xyloglucan content, and aluminum binding capacity in Arabidopsis.

Xyloglucan endohydrolase (XEH) and xyloglucan endotransglucosylase (XET) activities, encoded by xyloglucan endotransglucosylase-hydrolase (XTH) genes, are involved in cell wall extension by cutting or cutting and rejoining xyloglucan chains, respectively. However, the physiological significance of this biochemical activity remains incompletely understood. Here, we find that an XTH31 T-DNA insertion mutant, xth31, is more Al resistant than the wild type. XTH31 is bound to the plasma membrane and the encoding gene is expressed in the root elongation zone and in nascent leaves, suggesting a role in cell expansion. XTH31 transcript accumulation is strongly downregulated by Al treatment. XTH31 expression in yeast yields a protein with an in vitro XEH:XET activity ratio of >5000:1. xth31 accumulates significantly less Al in the root apex and cell wall, shows remarkably lower in vivo XET action and extractable XET activity, has a lower xyloglucan content, and exhibits slower elongation. An exogenous supply of xyloglucan significantly ameliorates Al toxicity by reducing Al accumulation in the roots, owing to the formation of an Al-xyloglucan complex in the medium, as verified by an obvious change in chemical shift of (27)Al-NMR. Taken together, the data indicate that XTH31 affects Al sensitivity by modulating cell wall xyloglucan content and Al binding capacity.

Lignocellulolytic enzyme production of Pleurotus ostreatus growth in agroindustrial wastes

The mushroom Pleurotus ostreatus has nutritional and medicinal characteristics that depend on the growth substrate. In nature, this fungus grows on dead wood, but it can be artificially cultivated on agricultural wastes (coffee husks, eucalyptus sawdust, corncobs and sugar cane bagasse). The degradation of agricultural wastes involves some enzyme complexes made up of oxidative (laccase, manganese peroxidase and lignin peroxidase) and hydrolytic enzymes (cellulases, xylanases and tanases). Understanding how these enzymes work will help to improve the productivity of mushroom cultures and decrease the potential pollution that can be caused by inadequate discharge of the agroindustrial residues. The objective of this work was to assess the activity of the lignocellulolytic enzymes produced by two P. ostreatus strains (PLO 2 and PLO 6). These strains were used to inoculate samples of coffee husks, eucalyptus sawdust or eucalyptus bark add with or without 20 % rice bran. Every five days after substrate inoculation, the enzyme activity and soluble protein concentration were evaluated. The maximum activity of oxidative enzymes was observed at day 10 after inoculation, and the activity of the hydrolytic enzymes increased during the entire period of the experiment. The results show that substrate composition and colonization time influenced the activity of the lignocellulolytic enzymes.

Endoglucanase production with the newly isolated Myceliophtora sp. i-1d3b in a packed bed solid state fermentor

This work is aimed to produce endoglucanase through solid state fermentation in a packed bed bioreactor with the use of the fungus Myceliophtora sp. I-1D3busing a mixture of wheat bran (WB) and sugar cane bagasse (SCB) as culture medium. Preliminary tests were performed in polypropylene plastic bags, controlling the variables temperature (40, 45, and 50ºC), initial moisture content (75, 80, and 85%, w.b.), and weight proportion SCB/WB (1:1, 7:3, and 9:1). The highest enzyme activities in plastic bags were obtained using the substrate proportion of 7:3, 50ºC temperature, and 80% initial moisture content (878 U/grams of dry solid). High activities of filter-paper cellulase and xylanase were also obtained in plastic bags and some results are reported. For the packed bed experiments, the temperature (45 and 50ºC) and the air flow rate (80, 100 and 120L/h) were the controlled variables. Activity of endoglucanase was similar to plastic bag tests. A longitudinal gradient of moisture content, was observed increasing from the bottom to the top of the reactor, even though the longitudinal enzyme activity profile was flat for almost the whole bed. Air flow rate did not affect enzyme activity, while experiments carried out at 50ºC showed higher enzyme activities. The maximum temperature peak observed was at about 6ºC above the process temperature.

Utilization of Agro-industrial Wastes for the Simultaneous Production of Amylase and Xylanase by Thermophilic Actinomycetes

Agro-industrial wastes such as sugarcane bagasse, wheat bran, rice bran, corn cob and wheat straw are cheapest and abundantly available natural carbon sources. The present study was aimed to production of amylase and xylanase simultaneously using agro-industrial waste as the sole carbon source. Seven thermophilic strains of actinomycete were isolated from the mushroom compost. Among of these, strain designated MSC702 having high potential to utilize agro-industrial wastes for the production of amylase and xylanase. Strain MSC702 was identified as novel species of Streptomyces through morphological characterization and 16S rRNA gene sequence. Enzyme production was determined using 1% (w/v) of various agro-industrial waste in production medium containing (g/100mL): K2HPO4(0.1), (NH4)2SO4(0.1), NaCl (0.1), MgSO4(0.1) at pH 7.0 after incubation of 48 h at 50°C. The amylase activity (373.89 IU/mL) and xylanase activity (30.15 IU/mL) was maximum in rice bran. The decreasing order of amylase and xylanase activity in different type of agro-industrial wastes were found rice bran (RB) > corn cob (CC) > wheat bran (WB) > wheat straw (WS) > sugarcane bagasse (SB) and rice bran (RB) > wheat bran (WB) > wheat straw (WS) > sugarcane bagasse (SB) > corn cob (CC), respectively. Mixed effect of different agro-industrial wastes was examined in different ratios. Enzyme yield of amylase and xylanase was ~1.3 and ~2.0 fold higher with RB: WB in 1:2 ratio.

Selection of thermophilic and thermotolerant fungi for the production of cellulases and xylanases under solid-state fermentation

Twenty-seven thermophilic and thermotolerant fungal strains were isolated from soil, decaying organic matter and sugarcane piles based on their ability to grow at 45ºC on medium containing corn straw and cardboard as carbon sources. These fungi were identified in the genera Aspergillus, Thermomyces, Myceliophthora, Thermomucor and Candida. The majority of the isolated strains produced xylanase and cellulases under solid state fermentation (SSF). The highest cellulase and xylanase productions were obtained by the cultivation of the strains identified as Aspergillus fumigatus M.7.1 and Myceliophthora thermophila M.7.7. The enzymes from these strains exhibited maximum activity at pH 5.0 and at 60 and 70ºC. The endo-glucanase from A. fumigatus was stable from 40ºC to 65ºC and both endo-glucanase and xylanase from M. thermophila were stable in this temperature range when in absence of substrate. The enzymes were stable from pH 4.0 to 9.0.

A biodegradation study of forest biomass by Aspergillus niger F7: correlation between enzymatic activity, hydrolytic percentage and biodegradation index

Aspergillus niger F7 isolated from soil was found to be the potent producer of cellulase and xylanase. The residue of forest species Toona ciliata, Celtris australis, Cedrus deodara and Pinus roxburghii was selected as substrate for biodegradation study due to its easy availability and wide use in industry. It was subjected to alkali (sodium hydroxide) treatment for enhancing its degradation. Biodegradation of forest waste by hydrolytic enzymes (cellulase and xylanase) secreted by A. niger under solid state fermentation (SSF) was explored. SSF of pretreated forest biomass was found to be superior over untreated forest biomass. Highest extracellular enzyme activity of 2201±23.91 U/g by A. niger was shown in pretreated C. australis wood resulting in 6.72±0.20 percent hydrolysis and 6.99±0.23 biodegradation index (BI). The lowest BI of 1.40±0.08 was observed in untreated saw dust of C. deodara having the least enzyme activity of 238±1.36 U/g of dry matter. Biodegradation of forest biomass under SSF was increased many folds when moistening agent i.e. tap water had been replaced with modified basal salt media (BSM). In BSM mediated degradation of forest waste with A. niger, extracellular enzyme activity was increased up to 4089±67.11 U/g of dry matter in turn resulting in higher BI of 15.4±0.41 and percent hydrolysis of 19.38±0.81 in pretreated C. australis wood. A. niger exhibited higher enzyme activity on pretreated biomass when moistened with modified BSM in this study. Statistically a positive correlation has been drawn between these three factors i.e. enzyme activity, BI and percent hydrolysis of forest biomass thus proving their direct relationship with each other.

The hydrolysis of agro-industrial residues by holocellulose-degrading enzymes

Holocellulose structures from agro-industrial residues rely on main and side chain attacking enzymes with different specificities for complete hydrolysis. Combinations of crude enzymatic extracts from different fungal species, including Aspergillus terreus, Aspergillus oryzae, Aspergillus niger and Trichoderma longibrachiatum, were applied to sugar cane bagasse, banana stem and dirty cotton residue to investigate the hydrolysis of holocellulose structures. A. terreus and A. oryzae were the best producers of FPase and xylanase activities. A combination of A. terreus and A. oryzae extracts in a 50% proportion provided optimal hydrolysis of dirty cotton residue and banana stem. For the hydrolysis of sugar cane bagasse, the best results were obtained with samples only containing A. terreus crude extract.

Biosynthesis, purification and characterization of endoglucanase from a xylanase producing strain Aspergillus niger B03

An extracellular endoglucanase was isolated from the culture liquid of xylanase producing strain Aspergillus niger B03. The enzyme was purified to a homogenous form, using consecutive ultrafiltration, anion exchange chromatography, and gel filtration. Endoglucanase was a monomer protein with a molecular weight of 26,900 Da determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and 28,800 Da determined by gel filtration. The optimal pH and temperature values for the enzyme action were 3.5 and 65ºC respectively. Endoglucanase was stable at 40ºC, pH 3.0 for 210 min. The substrate specificity of the enzyme was determined with carboxymethyl cellulose, filter paper, and different glycosides. Endoglucanase displayed maximum activity in the case of carboxymethyl cellulose, with a Km value of 21.01 mg/mL. The substrate specificity and the pattern of substrate degradation suggested that the enzyme is an endoglucanase. Endoglucanase showed a synergism with endoxylanase in corn cobs hydrolysis.

A simple and efficient diffusion technique for assay of endo b-1, 4-xylanase activity

Endo-β-1, 4-xylanases is thought to be of great significance for several industries namely paper, pharmaceuticals, food, feed etc. in addition to better utilization of lignocellulosic biomass. The present investigation was aimed to develop an easy, simple and efficient assay technique for endo-β-1, 4-xylanases secreted by the aerobic fungi. Under the proposed protocol, 9 g/L xylan containing agar was prepared in 100 mM phosphate buffer at different pH (4.5, 5.5 and 6.5). The sterilized xylan agar was dispensed in 90 mm petri dishes. 100 µl of culture supernatant of 12 fungal isolates was added to the wells and left overnight at 31±10C. The petri dishes were observed for zone of clearance by naked eye and diameter was measured. Congo red solution (1 g/L) was applied over the petri dishes as per the established protocol and thereafter plates were flooded with 1M Sodium chloride solution for the appearance of zone of clearance. The diameter for zone of clearance by the proposed method and the established protocol was almost identical and ranged from 21 to 42 mm at different pH depending upon the activity of endo-β-1, 4-xylanases. Change of pH towards alkaline side enabled similar or marginal decrease of diameter for the zone of clearance in most of the fungal isolates. The specific activities of these fungal isolates varied from 1.85 to 11.47 IU/mg protein. The present investigation revealed that the proposed simple diffusion technique gave similar results as compared to the established Congo red assay for endo-β-1, 4-xylanases. Moreover, the present technique avoided the cumbersome steps of staining by Congo red and de-staining by sodium chloride.

Xylanase production by Aspergillus awamori under solid state fermentation conditions on tomato pomace

In this work, tomato pomace, a waste abundantly available in the Mediterranean and other temperate climates agro-food industries, has been used as raw material for the production of some hydrolytic enzymes, including xylanase, exo-polygalacturonase (exo-PG), cellulase (CMCase) and ¥á-amylase. The principal step of the process is the solid state fermentation (SSF) of this residue by Aspergillus awamori. In several laboratory experiments, maximum xylanase and exo-PG activities were measured during the first days of culture, reaching values around 100 and 80 IU/gds (international units of enzyme activity per gram of dried solid), respectively. For CMCase and ¥á-amylase production remained almost constant along fermentation, with average values of 19 and 21.5 IU/gds, respectively. Experiments carried out in a plate-type bioreactor at lab scale showed a clear positive effect of aeration on xylanase and CMCase, while the opposite was observed for exo-PG and ¥á-amylase. In general, xylanase was the enzyme produced in higher levels, thus the optimum conditions for the determination of the enzyme activity was characterized. The xylanase activity shows an optimum pH of 5 and an optimum temperature of 50 ¨¬C. The enzyme is activated by Mg2+, but strongly inhibited by Hg2+ and Cu2+. The enzymatic activity remains quite high if the extract is preserved in a range of pH from 3 to 10 and a temperature between 30 ¨¬C to 40 ¨¬C.