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.

Xylanase gene mutation by error-prone pcr and expression in Pichia pastoris / Mutação do gene da xilanase por pcr propensa a erros e expressão em Pichia pastoris

Xylanase can hydrolyze xylan for reducing its anti-nutritional impact and improving nutrient availability, so obtaining suitable xylanase to degrade xylan is essential. Error-prone PCR and gene transformation were used in this study to obtain the ideal xylanase for degrading xylan effectively. The result showed that one mutant xylanase gene with high xylanase expression was obtained. After the mutant xylanase gene was connected with pGAPZA and transformed into Pichia pastoris (P. pastoris), the recombinant P. pastoris with mutant gene was found to produce higher xylanase activity (0.1480 U/mL) than that with the native xylanase gene (0.1360 U/mL) after 12 h incubation (p<0.05). The optimal temperature and pH of xylanase expressed by native and mutant genes were the same, i.e. 40°C and 5.50 (p<0.05). In addition, adding 0.2% Tween 80 during recombinant P. pastoris incubation could significantly increase xylanase yield by about 30-35% (p<0.05). The mutant xylanase could significantly increase xylose yield from wheat meal more than the native xylanase (p<0.05).

A xilanase pode hidrolisar o xilano para reduzir seu impacto antinutricional e melhorar a disponibilidade de nutrientes, portanto, obter xilanase adequada para degradar o xilano é essencial. A PCR propensa a erros e a transformação genética foram utilizadas neste estudo para obter a xilanase ideal para degradar eficazmente a xilana. O resultado mostrou que um gene mutante de xilanase com alta expressão de xilanase foi obtido. Depois que o gene mutante da xilanase foi conectado ao pGAPZA e transformado em Pichia pastoris (P. pastoris), o recombinante P. pastoris com o gene mutante produziu maior atividade de xilanase (0,1480 U / mL) do que com o gene nativo da xilanase (0,1360 U / mL) após 12 h de incubação (p <0,05). A temperatura e o pH ótimos da xilanase expressa pelos genes nativos e mutantes foram os mesmos, ou seja, 40 ºC e 5,50 (p <0,05). Além disso, a adição de Tween 80 a 0,2% durante a incubação de P. pastoris recombinante poderia aumentar significativamente o rendimento de xilanase em cerca de 30-35% (p <0,05). A xilanase mutante poderia aumentar significativamente o rendimento de xilose da farinha de trigo mais do que a xilanase nativa (p <0,05).

Produção de xilanases por fermentação em estado sólido de farelo de trigo utilizando consórcios fúngicos / Production of xylanases by solid state fermentation of wheat bran using fungal consortia

A fim de se agregar valor ao resíduo farelo de trigo gerado em indústrias do setor alimentício avaliou-se, no presente trabalho, o potencial deste subproduto como substrato para produção de enzima xilanase no cultivo em estado sólido, utilizando consórcios fúngicos bem como os fungos Aspergillus oryzae CCT nº 0975 (ATCC9362) eTrichoderma reesei CCT nº 2768 - QM 9414. Para tanto utilizou-se o farelo de trigo, não lavado e não autoclavado, como fonte de carbono e energia na fermentação em estado sólido pelo fungo Aspergillus oryzae que apresentou maior produção do percentual de proteína nas 72 horas de cultivo. Depois de realizado um Delineamento Composto Central Rotacional (DCCR) - planejamento fatorial 23,com três repetições no ponto central e seis pontos axiais - partiu-se para otimização dos fatores que foram considerados significativos no processo: umidade, pH e granulometria. Os fatores foram considerados significativos pela A NOVA com o nível de 95% de confiança e com o resultado otimizado de atividade enzimática de (1.84 ± 0.01) UI/mL utilizando pH 3,3, granulometria de 900,0 µm e umidade de 40%. O caldo enzimático obtido foi considerado eficiente na modificação de tipificação de farinhas de trigo pelo estudo dos parâmetros reológicos do falling number e alveografia sendo estável por cerca de 3 meses

This study aimed to find alternatives for wheat bran disposal destination generated in food industry sector,thus contributing to the reduction of the resultant impact of residue depo-sition in the environment. The poten-tial of the wheat bran as a substrate for xylanase production by solid-state fermentation using fungal con-sortiums as well as Aspergillus ory-zae (ATCC9362) and Trichoderma reesei (2768) was valued. The use of non-washed and non-autoclaved wheat bran as carbon and energy source in solid-state fermentation by A. oryzae fungus showed greater per-centage of produced protein after 72 h of cultivation. The use of a central composite rotatable design(CCRD), 23 factorial planning with three rep-etitions at the central point as well as six axial points, coupled with Sur-face Response Methodology (SRM) allowed to assay the influence of hu-midity, pH, and grain size (indepen-dent variables or factors) on the xy-lanase activity(dependent variable or response) as well as to optimize the best conditions for the enzyme production. The results showed that all factors and their combinations were significant at 95% confidence level. The optimized xylanase activi-ty was (1.84 ± 0.01) UI/mL, obtained at 40% humidity and pH 3.3 with a grain size of 900.0 µm. The produced broth was stable for 3 months and approximately had 50% of the initial xylanase activity at 4°C. SDS-PAGE assay showed that xylanase has 30 kDa molar mass. The obtained en-zymatic broth was efficient to modify wheat flours as shown by the falling number rheologic parameters and alveography assay

Improving the thermostability of Trichoderma reesei xylanase 2 by introducing disulfide bonds

Background: Xylanases are considered one of the most important enzymes in many industries. However, their low thermostability hampers their applications in feed pelleting, pulp bleaching, and so on. The main aim of this work was to improve the thermostability of Trichoderma ressei xylanase 2 (Xyn2) by introducing disulfide bonds between the N-terminal and α-helix and the ß-sheet core. Results: In this work, two disulfide bonds were separately introduced in the Xyn2 to connect the N-terminal and α-helix to the ß-sheet core of Xyn2. The two disulfide bonds were introduced by site-directed mutagenesis of the corresponding residues. The half-life of the mutants Xyn2C14­52 (disulfide bond between ß-sheets B2 and B3) and Xyn2C59­149 (disulfide bond between ß-sheets A5 and A6) at 60°C was improved by approximately 2.5- and 1.8-fold compared to that of the wild type Xyn2. In addition, the enzyme's resistance to alkali and acid was enhanced. Conclusion: Our results indicated that the connection of the N-terminal and α-helix to the ß-sheet core is due to the stable structure of the entire protein.

Xylooligosaccharides: an economical prebiotic from agroresidues and their health benefits.

Oligosaccharides and dietary fibres are non-digestible food ingredients that preferentially stimulate the growth of prebiotic Bifidobacterium and other lactic acid bacteria in the gastro-intestinal tract. Xylooligosaccharides (XOS) provide a plethora of health benefits and can be incorporated into several functional foods. In the recent times, there has been an over emphasis on the microbial conversion of agroresidues into various value added products. Xylan, the major hemicellulosic component of lignocellulosic materials (LCMs), represents an important structural component of plant biomass in agricultural residues and could be a potent bioresource for XOS. On an industrial scale, XOS can be produced by chemical, enzymatic or chemo-enzymatic hydrolysis of LCMs. Chemical methods generate XOS with a broad degree of polymerization (DP), while enzymatic processes will be beneficial for the manufacture of food grade and pharmaceutically important XOS. Xylooligomers exert several health benefits, and therefore, have been considered to provide relief from several ailments. This review provides a brief on production, purification and structural characterization of XOS and their health benefits.

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.

Thermostability of xylanolytic enzymes produced by Lentinula edodes UFV70

Xylanolytic enzymes produced by Lentinula edodes UFV70, cultivated in eucalyptus sawdust/rice bran medium, were stable at 50, 60 and 65ºC for 21 hours, losing only 15-25% activity. Fungus incubation at 50ºC for 12 hours and at 65ºC for 24 hours increased the amount of xylose produced.

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.

Xylanolytic enzymes production by Aspergillus niger GS1 from solid-state fermentation on corn stover and their effect on ruminal digestibility

Hemicellulosic agricultural by-products such as corn stover (CS) are highly available materials which represent an opportunity to develop value added products. Native Aspergillus niger GS1 was used for solid-state fermentation (SSF) on alkali pre-treated CS (ACS) aimed to optimize xylanolytic enzymes production, and their effect on in vitro ruminal and true digestibility of ACS. Enzyme production was empirically modelled using a fractional factorial design 2(9-5), and the resulting significant factors were glucose, yeast extract and two mineral salts, which were arranged in a Draper-Lin optimization design at two levels. Predicted optimum xylanolytic activity of 33.6 U (mg protein)-1 was achieved at 48 hrs of SSF, and was validated by confirmatory experiments. ACS was incubated with a semipurified enzymatic extract (EE) showing a xylanolytic activity of 1600 U kg-1 dry ACS for 12 hrs before exposure to cow's ruminal liquid for 72 hrs, which led to 5 percent and 10 percent increase of in vitro ruminal and true digestibility, respectively. CS is a readily available by-product in different regions which after alkaline treatment and partial hydrolysis with the EE, may be advantageously used as supplement for ruminant feed.

Hyperexpression of two Aspergillus niger xylanase genes in Escherichia coli and characterization of the gene products

The analysis of individual gene product should enable to clarify the role of a particular enzyme in a complex xylanase system of A. niger. The two genes encoding precursors of co-produced endo-1,4-¥â-D-xylanases, xynA1 and xynB, were isolated from Aspergillus niger SCTCC 400264 (SCTCC, China) by using RT-PCR technique and then successfully expressed in Escherichia coli BL21. The nucleotide sequences of the xynA1 and xynB genes revealed that they were only 52.5 percent homology to each other. Characterization of the recombinant enzymes revealed the different properties: the specific activity of recombinant XYNA1 was 16.58 U/mg compared to 1201.7 U/mg for recombinant XYNB; The optimum temperature and pH of the recombinant XYNA1 were 35 ¨¬C and 3.0, respectively, whereas the corresponding values for the recombinant XYNB were 55 ¨¬C and 5.0, respectively; The recombinant XYNB showed much more thermostability than recombinant XYNA1; The recombinant XYNB showed 94 percent of maximal activity after incubating in water for 60 min at 60 ¨¬C compared to no activity for recombinant XYNA1. Various metal ions had different effects on activity between the two recombinant xylanases.

Molecular cloning, purification and characterization of thermostable -1,3-1,4 glucanase from Bacillus subtilis A8-8.

A gene encoding a -1,3-1,4-glucanase (CelA) belonging to family 5 of glycoside hydrolases was cloned and sequenced from the Bacillus subtilis A8-8. The open-reading-frame of celA comprised 1499 base pairs and the enzyme was composed of 500 amino acids with a molecular mass of 55 kDa. The recombinant -1,3-1,4 glucanase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 8.0 and 60oC, respectively. The enzyme was stable within pH 6.0-9.0. It was stable up to 60oC and retained 30% of its original activity at 70oC for 60 min. It hydrolyzed lichenan, CMC, xylan, laminarin, avicel and pNPC, but was inactive towards cellobiose. The enzyme activity was markedly activated by Co2+ and Mn2+, but was strongly inactivated by Fe3+. The truncated gene, devoid of cellulose-binding domain (CBD) showed 60% of activity and bound to avicel.