Crude Xylanase Preparation Research Articles

Production of Xylanase and <i>β</i>-Xylosidase Enzymes by <i>Pseudozyma hubeiensis</i> in Solid State Fermentation

Xylanase is an important enzyme with potential application in the degradation of xylan component in the lignocellulosic biomass. There are very few reports on the production of cellulase free xylanases especially by yeast strains which have great potential in paper and pulp industry in removing the hemicellulose from the treated or untreated pulp. In this study, P. hubeiensis NCIM 3574 isolated in our laboratory produced significant levels of extracellular cellulase free xylanase (2480 IU/g DSS) in solid state fermentation (SSF) using wheat bran and xylan. It also produced high levels of β-xylosidase (198 IU/g DSS) when grown in SSF using ground nut oil cake and xylan. These highest activities were obtained when fermented Koji was extracted with 1% NaCl supplemented with 0.5% of Triton X-100. These are the highest activities reported so far from yeast strains in the available literature. The crude xylanase preparation of P. hubeiensis produced xylooligosaccharides (XOS) without xylose proving its potential for XOS production with no further requirement of downstream processing. The XOS as prebiotic show beneficial effect on gut microflora such as Lactobacilli and Bifidobacteria which suppress the activity of pathogenic organisms. This xylanase also has a potential application as a bio-bleaching agent in paper and pulp industry.

Xylanase production by Aureobasidium pullulans on globe artichoke stem: Bioprocess optimization, enzyme characterization and application in saccharification of lignocellulosic biomass

ABSTRACTStatistical optimization of the factors affecting xylanase production by Aureobasidium pullulans NRRL Y-2311-1 on globe artichoke stem was performed for the first time. The optimization strategies used resulted in almost six-fold enhancement of xylanase production (66.48 U/ml). Biochemical and thermal characterization of the crude xylanase preparation was performed to elucidate its feasibility for different industrial applications. The optimum conditions for xylanase activity were pH 4.0 and 30–50°C. The enzyme was very stable over a wide pH range of 3.0–8.0. The thermal stability studies revealed an inactivation energy of 183 kJ/mol. Thermodynamic parameters (enthalpy, entropy, and Gibbs free energy) for thermal inactivation were also determined. Primary application of the crude xylanase preparation in saccharification of corn cob subjected to different pretreatment techniques has been evaluated. The crude xylanase preparation was very promising for saccharification of corn cob pretreated with aqueous ammonia. The maximum yield of reducing sugar was 357 mg/g dry substrate, which revealed that the crude xylanase from A. pullulans could be a very good alternative in saccharification of lignocellulosic biomass for biological fuel generation. This study also provides a basis for further exploitation of globe artichoke by-products in microbial production of several other industrially significant metabolites.

Hydrolysis of pretreated rice straw by an enzyme cocktail comprising acidic xylanase from Aspergillus sp. for bioethanol production

The most crucial enzyme involved in xylan hydrolysis is endoxylanase which cleaves the internal glycosidic bonds of xylan. The aim of this work was to study the production of extracellular xylanase by a locally isolated strain of Aspergillus sp. under solid-state fermentation (SSF) and to evaluate the potential of the enzyme in enzymatic hydrolysis of pretreated rice straw. Xylanase production reached maximum with incubation period (96 h), moisture level (80%), inoculum size (3 × 106 spores/mL), pH (4.8), temperature (25 °C), carbon source (wheat bran) and nitrogen source (yeast extract). Under optimized conditions, xylanase production reached to 5059 IU/gds. Crude xylanase was used for supplementing the enzyme cocktail comprising cellulases (Zytex, India), β-glucosidase (In-house) and xylanase (In-house) for the saccharification of alkali-pretreated rice straw to get the maximum reducing sugar production. The cocktail containing the three enzymes resulted a maximum of 574.8 mg/g of total reducing sugars in comparison to 430.2 mg/g sugars by the cocktail without xylanase. These results proved that the crude xylanase preparation from Aspergillus sp. could be a potent candidate for the enzyme cocktail preparation for biomass hydrolysis in lignocellulosic bioethanol program.

Purification, characterization, and molecular cloning of the xylanase from Streptomyces thermovulgaris TISTR1948 and its application to xylooligosaccharide production

A crude xylanase preparation from Streptomyces thermovulgaris TISTR1948 was able to hydrolyze KOH-treated corncob and to produce bioactive xylooligosaccharides (XOs). A thermostable cellulase-free endo-xylanase from strain TISTR1948 was purified 15.0-fold from the crude preparation, with a recovery yield of 13.0%. On SDS-PAGE, the purified enzyme had an apparent molecular mass of 46.2kDa. The N-terminal and internal amino acid sequences were determined and the cloned xylanase gene were sequenced. The 1434-bp gene encodes a protein with a predicted molecular mass of 46,976Da. The deduced amino acid sequence had a high degree of identity with the sequences of GH 10 xylanases from Streptomyces spp. The purified xylanase was highly stable within a pH range of 4.0–11.5 and was thermostable within a temperature range of 50–70°C. The activity of the enzyme reached a maximum at 65°C; the enzyme’s half-life was 90min at 70°C. Enzymatic activity was enhanced in the presence of metal ions, Ca2+, Co2+, and Mn2+ but almost completely inhibited by Hg2+, Pb2+, and SDS. The Km and Vmax values of the enzyme with beechwood xylan as the substrate were 37.6μM and 303U/mg, respectively. The crude, partially purified, and purified xylanases were assayed for XO production from KOH-treated corncob. The main component of the XO products was xylobiose, with very little xylose and arabinose. An in vitro evaluation of XOs from the purified xylanase showed that they enhanced the growth of probiotic Lactobacillus plantarum TISTR1465.

A Box–Behnken design approach for the production of xylanase by Aspergillus candidus under solid state fermentation and its application in saccharification of agro residues and Parthenium hysterophorus L.

Xylanase production by indigenous Aspergillus candidus using wheat bran as a substrate under solid state fermentation was studied. Inorganic nitrogen source ammonium nitrate was found best among all the nitrogen sources tested. Physical parameters like time of incubation, temperature and moisture content were optimized using Box–Behnken design and under optimized condition maximum xylanase activity found 770U/gds. Low amount of CMCase (77U/gds) and FPase (26.12U/gds) were produced concomitantly with xylanase. The crude xylanase preparation was used for saccharification of aqueous ammonia treated of various lignocellulosic materials like corn cob, wheat straw, sugar cane bagasse and Parthenium weed. Corn cob produced maximum sugar yield and further response surface method was employed to optimized saccharification yield. Under optimized condition maximum reducing sugar yield were found to be 438.47mg/g which was found significantly high considering earlier studies available in the literature.

Characterization of a novel xylanase from Armillaria gemina and its immobilization onto SiO2 nanoparticles

Enhanced catalytic activities of different lignocellulases were obtained from Armillaria gemina under statistically optimized parameters using a jar fermenter. This strain showed maximum xylanase, endoglucanase, cellobiohydrolase, and β-glucosidase activities of 1,270, 146, 34, and 15 U mL(-1), respectively. Purified A. gemina xylanase (AgXyl) has the highest catalytic efficiency (k (cat)/K (m) = 1,440mg mL(-1) s(-1)) ever reported for any fungal xylanase, highlighting the significance of the current study. We covalently immobilized the crude xylanase preparation onto functionalized silicon oxide nanoparticles, achieving 117% immobilization efficiency. Further immobilization caused a shift in the optimal pH and temperature, along with a fourfold improvement in the half-life of crude AgXyl. Immobilized AgXyl gave 37.8% higher production of xylooligosaccharides compared to free enzyme. After 17cycles, the immobilized enzyme retained 92% of the original activity, demonstrating its potential for the synthesis of xylooligosaccharides in industrial applications.

Immobilization of Pholiota adiposa xylanase onto SiO2 nanoparticles and its application for production of xylooligosaccharides

Enhanced yields of different lignocellulases were obtained under statistically-optimized parameters using Pholiota adiposa. The k (cat) value (4,261 s(-1)) of purified xylanase under standard assay conditions was the highest value ever reported. On covalent immobilization of the crude xylanase preparation onto functionalized silicon oxide nanoparticles, 66 % of the loaded enzyme was retained on the particle. Immobilized enzyme gave 45 % higher concentrations of xylooligosaccharides compared to the free enzyme. After 17 cycles, the immobilized enzyme retained 97 % of the original activity, demonstrating its prospects for the synthesis of xylooligosaccharides in industrial applications.

Production of Xylooligosaccharides from Xylans by Extracellular Xylanases fromThermobifida fusca

Xylooligosaccharides are produced for use as a valuable food sweetener or additive. They have many beneficial biomedical and health effects. In this study, a process for producing xylooligosaccharides from lignocellulolytic agricultural waste was developed. Bagasse, corncob, wheat bran, and peanut shell were used as carbon sources for production of xylanolytic enzymes from Thermobifida fusca NTU22. When using bagasse as the carbon source, the xylanolytic enzymes that simultaneously accumulated in the broth in a 500 mL Hinton flask after 72 h of cultivation at 50 degrees C were measured as xylanase (14.0 U/mL), beta-xylosidase (74.1 mU/mL), and acetyl esterase (29.1 mU/mL). The optimum pH and temperature for xylanases were 6.0-8.0 and 70 degrees C, respectively. Six proteins with xylanase activity were identified by zymogram analysis of isoelectric focusing gel. This was followed by heat treatment at 70 degrees C for 30 min that eliminated 90% of the beta-xylosidase activity. The xylanase and acetyl esterase activities were still 100%. Two percent of xylan extracted from the bagasse was then hydrolyzed by heat-treated crude xylanase preparation at 60 degrees C, pH 7.0, for 10 h. The xylooligosaccharides that accumulated in the broth were about 23.7%. After the purification process by activated charcoal chromatography, the purity of xylooligosaccharides was 71.4%.

Corn fiber hydrolysis by Thermobifida fusca extracellular enzymes.

Thermobifida fusca was grown on cellulose (Solka-Floc), xylan or corn fiber and the supernatant extracellular enzymes were concentrated. SDS gels showed markedly different protein patterns for the three different carbon sources. Activity assays on a variety of synthetic and natural substrates showed major differences in the concentrated extracellular enzyme activities. These crude enzyme preparations were used to hydrolyze corn fiber, a low-value biomass byproduct of the wet milling of corn. Approximately 180 mg of reducing sugar were produced per gram of untreated corn fiber. When corn fiber was pretreated with alkaline hydrogen peroxide, up to 429 mg of reducing sugars were released per gram of corn fiber. Saccharification was enhanced by the addition of beta-glucosidase or by the addition of a crude xylanase preparation from Aureobasidium sp.

Isolation and screening for alkaline thermostable xylanases

AbstractScreening of xylanase producing microorganisms from soil samples and hemicellulosic materials were carried out in three stages using wheat bran extract agar medium, xylan agar medium and liquid medium with xylan. One of the isolates, identified as Bacillus SSP‐34 produced 100 times more xylanase activity than the other isolates. Maximum enzyme production by this isolate was observed after 102 hours of growth. The optimum pH for the crude xylanase preparation range from 6–8 and optimum temperature was at 50 °C. The enzyme was stable up to 20 minutes at 50 °C and at pH range 4.5–9.0 for more than 2 hours.

Direct conversion of cellulosic materials to hydrogen by Clostridium sp. strain no. 2

We investigated hydrogen production by fermentation of enzymatic hydrolysates of Avicel and xylan, using Clostridium sp. strain no. 2. At 0.3% concentration in peptone yeast (PY) medium, pure xylose or glucose and crude hydrolysates of Avicel or xylan were converted to hydrogen at yields of 16.1 mmol or 14.6 mmol and 19.6 mmol or 18.6 mmol, per gram of substrate consumed, respectively. Alternatively, a batch culture of strain no. 2 grown in PY medium containing 1% of xylan or xylose with crude xylanase preparation and xylose alone as a reference, gave hydrogen production of 9.5 mmol or 9.6 mmol and 10.3 mmol, per gram of substrate supplemented, respectively.

Isolation of Xylanase Preparations for Biotechnological Applications fromAspergillus NigerA3

ABSTRACTThe crude xylanase preparation (P-1) for biotechnological applications was obtained from the culture supernatant of Aspergillus niger A3 by precipitation with ethanol. The pH optimum (5.0) and temperature optimum (40 °C) were estimated. This crude xylanase preparation was stable at pH 5.0 (80 % retained initial activity for 24 h) and temperature between 20 - 45 °C (75 % retained initial activity for over 24 h). At the optimal experimental conditions for enzyme hydrolysis (2.5 % substrate concentration, 0.1 % concentration of the crude xylanase preparation, 50 °C—temperature of hydrolysis, pH 5.0 and 72 h time of hydrolysis) the xylans of two substrates (xylan ex larch sawdust and pretreated com stalks with 1 % NaOH) were hydrolyzed to 55 and 65%, respectively. Six forms of xylanases were detected by molecular-sieve column chromatography of the P-1. The maximal active xylanase peaks (Fr-II and Fr-IV) were purified further by anion-exchange column chromatography. The two xylanase preparations P-2 (F...

Optimization of cellulase-free xylanase production by a novel yeast strain

A novel yeast strain, NCIM 3574, isolated from a decaying wood produced up to 570 IU ml−1 of xylanolytic enzymes when grown on medium containing 4% xylan. The yeast strain also produced xylanase activity (40–50 IU ml−1) in the presence of soluble carbon sources like xylose or arabinose. No xylanase activity was detected when the organism was grown on glucose. The crude xylanase preparation showed no activity towards cellulolytic substrates but low levels of β-xylosidase (0.1 IU ml−1) and α-l-arabinofuranosidase (0.05 IU ml−1) were detected. The temperature and pH optima for the crude xylanase preparation were 55°C and 4.5 respectively. The crude xylanase produced mainly xylose from xylan within 5 min. Prolonged hydrolysis of xylan produced xylobiose and arabinose, in addition to xylose, as the end products. The presence of arabinose as one of the end products in xylan hydrolysate could be due to the low levels of arabinofuranosidase enzyme present in the crude fermentation broth.

Thermophilic Alkaline Xylanase from Newly Isolated Alkaliphilic and Thermophilic Bacillus sp. Strain TAR-1.

Alkaliphilic and thermophilic Bacillus sp. strain TAR-1, isolated from soil, produced a xylanase extracellularly. The xylanase was most active over a pH range of 5.0 to 9.5 at 50°C. Optimum temperatures of the crude xylanase preparation were 75°C at pH 7.0 and 70°C at pH 9.0. Zymogram analyses of the culture supernatant showed that the molecular mass of the xylanase was 40 kDa and the isoelectric point was pH 4.1. The predominant products of xylan hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that the enzyme was an endoxylanase. Production of the thermophilic alkaline xylanase was induced by xylan and xylose, but was repressed in the presence of glucose.

Production of alkaline xylanase by a newly isolated alkaliphilic Bacillus sp. strain 41M-1

Alkaliphilic Bacillus sp. strain 41M-1, isolated from soil, produced xylan-degrading enzymes extracellularly. Optimum pH for the crude xylanase preparation was about pH 9, confirming the production of novel alkaline xylanase(s) by the isolate. Xylanases were induced by xylan, but were not produced in the presence of xylose, arabinose or glucose. Xylanase productivity was influenced by culture pH, and production at pH 10.5 was higher than that at pH 8.0. Zymogram analysis of the culture supernatant showed the alkaline xylanase with a molecular mass of 36 kDa.

Studies on xylan degrading enzymes: I. Purification and characterization of endo-1,4-β-xylanase from Aspergillus niger STR. 14

1. 1. From an extract of a 48-h culture of Aspergillus niger str. 14 a crude preparation degrading xylan to xylose was obtained by isopropanol precipitation. 2. 2. From 200 g of the crude xylanase preparation 21 mg of endo-1,4-β-xylanase (1,4-β- d-xylan xylanohydrolase, EC 3.2.1.8) were obtained by purification via 5 steps: ethanol precipitation, Sephadex G-50 gel filtration, chromatography and rechromatography on hydroxyapatite, and chromatography on CM-cellulose. The purification was 5000-fold with regard to the culture extract and 250-fold with regard to the crude enzyme preparation. 3. 3. Endo-1,4-β-xylanase was homogeneous when examined by polyacrylamide gel electrophoresis and ultracentrifugation. The sedimentation coefficient of the enzyme was 1.46 S. 4. 4. A pH optimum of 4.0 was found for the degradation of carboxymethylxylan by endo-1,4-β-xylanase. The temperature of 50°C was optimal for its activity. Endo-1,4-β-xylanase was stable at pH 3–8. 5. 5. The molecular weight of highly purified endo-1,4-β-xylanase from Asp. niger str. 14 was determined by four methods: gel filtration on Sephadex G-200, electrophoresis in polyacrylamide gel in the presence of sodium dodecyl sulphate, sedimentation ultracentrifugation and via amino acid and carbohydrate analysis. These methods gave molecular weights of 24 000; 33 000; 24 700; and 27 000 respectively. 6. 6. By isoelectric focusing the isoelectric point of endo-1,4-β-xylanase was found to be 4.2. 7. 7. Amino acid analysis showed that the endo-1,4-β-xylanase molecule consisted of 212 amino acid residues, including a large number of aspartic (30) and glutamic acid (17) residues. 8. 8. No free SH groups were detected in the experiments with para-chloromercuribenzene, which did not influence the enzyme activity. 9. 9. By the anthrone method 20% carbohydrates were found in the endo-1,4-β-xylanase molecule. After acid hydrolysis glucose and small quantities of galactose and glucosamine were detected in chromatograms.