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.

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.

Xylanase and cellulase activities during anaerobic decomposition of three aquatic acrophytes

Enzymatic activity during decomposition is extremely important to hydrolyze molecules that are assimilated by microorganisms. During aquatic macrophytes decomposition, enzymes act mainly in the breakdown of lignocellulolytic matrix fibers (i.e. cellulose, hemicellulose and lignin) that encompass the refractory fraction from organic matter. Considering the importance of enzymatic activities role in decomposition processes, this study aimed to describe the temporal changes of xylanase and cellulose activities during anaerobic decomposition of Ricciocarpus natans (freely-floating), Oxycaryum cubense (emergent) and Cabomba furcata (submersed). The aquatic macrophytes were collected in Óleo Lagoon, Luiz Antonio, São Paulo, Brazil and bioassays were accomplished. Decomposition chambers from each species (n = 10) were set up with dried macrophyte fragments and filtered Óleo Lagoon water. The chambers were incubated at 22.5ºC, in the dark and under anaerobic conditions. Enzymatic activities and remaining organic matter were measured periodically during 90 days. The temporal variation of enzymes showed that C. furcata presented the highest decay and the highest maximum enzyme production. Xylanase production was higher than cellulase production for the decomposition of the three aquatic macrophytes species.

One-step purification and characterization of cellulase-free xylanase produced by alkalophilic Bacillus subtilis ash

A purificação de uma etapa e caracterização de uma xilanase livre de celulase de uma linhagem recentemente isolada alcalofílicos e moderadamente termofílico de Bacillus subtilis ASH. Xilanase foi purificada à homogeneidade de 10,5 vezes, com ~ por cento de recuperação 43 através de cromatografia de troca iônica através de CM- Sephadex C -50. A enzima purificada revelou uma única banda no gel SDS-PAGE com uma massa molecular de 23 kDa. Ele mostrou um pH ótimo de 7,0 e manteve-se estável na faixa de pH 6,0-9,0 . A temperatura ótima para atividade da enzima foi 55 º C. A xilanase purificada não perder nenhuma atividade até 45 º C , no entanto, manteve 80 por cento e 51 por cento de sua atividade após pré-incubação a 55 º C e 60 º C , respectivamente. A enzima obedecido Michaelis- Menton cinética para xilano de madeira de bétula com aparente km 3,33 mg / ml e Vmax 100 UI / ml. A enzima foi fortemente inibida por Hg2 +, Cu2 + , enquanto reforçada por Co2 + e Mn2 +. A enzima purificada pode ser armazenado a 4 º C por seis semanas sem nenhuma perda de atividade catalítica. A purificação mais rápido e econômico da xilanase livre de celulase de B. subtilis ASH por um passo-a processo juntamente com a sua estabilidade sensível a alta temperatura e pH alcalino torna potencialmente eficazes para aplicações industriais.

Xylanase production with xylan rich lignocellulosic wastes by a local soil isolate of Trichoderma viride / Produção de xilanase com resíduos lignocelulósicos ricos em xilana por uma cepa local de Trichoderma viride isolada de solo

In the present study, cultural and nutritional conditions for enhanced production of xylanase by a local soil isolate of Trichoderma viride, using various lignocellulosic substrates in submerged culture fermentation have been optimized. Of the lignocellulosics used, maize straw was the best inducer followed by jowar straw for xylanase production. The highest activity achieved was between 14 to 17 days of fermentation. A continuous increase in xylanase production was observed with increasing level of lignocellulosics in the medium and highest activity was observed with maize straw at 5 percent level. Xylanase production with higher levels of lignocellulosics (3 to 5 percent) of maize, jowar and barseem was found to be higher as compared to that with commercial xylan as carbon source. Sodium nitrate was the best nitrogen source among the six sources used. Maximum xylanase production was achieved with initial medium pH of 3.5-4.0 and incubation temperature of 25ºC.The enzyme preparation was effective in bringing about saccharification of different lignocellulosics. The xylanase production could be further improved by using alkali treated straw as carbon source.

Neste estudo, otimizou-se as condições culturais e nutricionais para produção aumentada de xilanase por uma cepa local de Trichoderma viride isolada de solo, empregando-se vários substratos lignocelulósicos, em fermentação submersa. Entre os substratos utilizados, o melhor indutor de produção de xilanase foi palha de milho, seguido de palha de sorgo. A atividade mais alta foi obtida entre 14 e 17 dias de fermentação. Com palha de milho observou-se um aumento contínuo na produção de xilanase com o aumento da concentração dos substratos lignocelulósicos no meio, sendo que a melhor atividade foi obtida com 5 por cento de palha de milho. A produção de xilanase com níveis mais altos de (3 a 5 por cento) de milho, sorgo e forragem verde (barseem) foi mais levada do que com xilana comercial como fonte de carbono. Entre as fontes de nitrogênio testadas, a melhor foi nitrato de sódio. Produção máxima de xilanase foi obtida quando o pH inicial do meio foi 3,5 4,0 e a temperatura de incubação 25ºC. A enzima foi eficiente na sacarificação de diferentes substratos lignocelulósicos. A produção de xilanase poderia ser aumentada empregando-se álcali ao invés de palha tratada como fonte de carbono.