Rapid production of thermostable cellulase-free xylanase by a strain of Bacillus subtilis and its properties

A Bacillus subtilis strain isolated from a hot-spring was shown to produce xylanolytic enzymes. Their associative/synergistic effect was studied using a culture medium with oat spelts xylan as xylanase inducer. Optimal xylanase production of about 12 U ml⁻¹ was achieved at pH 6.0 and 50°C, within 18 h fermentation. At 50°C, xylanase productivity obtained after 11 h in shake-flasks, 96,000 U l⁻¹ h⁻¹, and in reactor, 104,000 U l⁻¹ h⁻¹ was similar. Increasing temperature to 55°C a higher productivity was obtained in the batch reactor 45,000 U l⁻¹ h⁻¹, compared to shake-flask fermentations, 12,000 U l⁻¹ h⁻¹. Optimal xylanolytic activity was reached at 60°C on phosphate buffer, at pH 6.0. The xylanase is thermostable, presenting full stability at 60°C during 3 h. Further increase in the temperature caused a correspondent decrease in the residual activity. At 90°C, 20% relative activity remains after 14 min. Under optimised fermentation conditions, no cellulolytic activity was detected on the extract. Protein disulphide reducing agents, such as DTT, enhanced xylanolytic activity about 2.5-fold. When is used xylan as substrate, xylanase production decreased as function of time in contrast, with trehalose as carbon source, xylanase production in maintained constant for at least 80 h fermentation.     

Production of thermostable pectinase and xylanase for their potential application in bleaching of kraft pulp

A very high level of alkalophilic and thermostable pectinase and xylanase has been produced from newly isolated strains of Bacillus subtilis and Bacillus pumilus respectively. Enzyme production for pectinase was carried out under SSF using combinations of cheap agricultural residues while xylanase was produced under submerged fermentation using wheat bran as substrate to minimize the cost of production of these enzymes Among the various substrates tested, the highest yield of pectinase production was observed by using combination of WB + CW (6592 U/g of dry substrate) supplemented with 4% yeast extract when incubated at 37 °C for 72 h using deionized water of pH 7.0 as moistening agent. The biobleaching effect of these cellulase free enzymes on kraft pulp was determined. Both xylanase and pectinase showed stability over a broad range of pH from 6 to 10 and temperature from 55 to 70 °C. The bleaching efficiency of the pectinase and xylanase on kraft pulp was maximum after 150 min at 60 °C using enzyme dosage of 5 IU/ml of each enzyme at 10% pulp consistency with about 16% reduction in kappa number and 84% reduction in permanganate number. Enzyme treated pulp when subjected to CDED₁D₂ steps, 25% reduction in chlorine consumption and upto 19% reduction in consumption of chlorine dioxide was observed for obtaining the same %ISO brightness. Also an increase of 22 and 84% in whiteness and fluorescence respectively and a decrease of approximately 19% in the yellowness of the biotreated pulp were observed by pretreatment of the pulp with our enzymatic mixture.     

Enhanced production of cellulase-free thermostable xylanase by Bacillus pumilus ASH and its potential application in paper industry

A very high level of cellulase-free, thermostable xylanase has been produced from newly isolated strain of Bacillus pumilus under submerged fermentation in a basal medium supplemented with wheat bran (2%, w/v) pH 8.0 and at 37 °C. After optimization of various production parameters, an increase of nearly 13-fold in xylanase production (5407 IU/ml) was achieved. The produced xylanase is stable in neutral to alkaline pH region at 70 °C. The suitability of this xylanase for use in the bioleaching of eucalyptus Kraft pulp was investigated. A xylanase dose of 5 IU/g of oven dried pulp of 10% consistency exhibited the optimum bleach boosting of the pulp at pH 7.0 and 60 °C after 180 min of treatment. An increase of 5% in brightness along with an increase of 21% and 28% in whiteness and fluorescence respectively, whereas 18% decrease in the yellowness of the biotreated pulp was observed. Enzyme treated pulp when subjected to chemical bleaching, resulted in 20% reduction in chlorine consumption and up to 10% reduction in consumption of chlorine dioxide. Also a reduction of about 16% in kappa number and 83% in permanganate number, along with a reduction in COD value and significant improvement in various pulp properties, viz. viscosity, tensile strength, breaking length, burst factor, burstness, tear factor and tearness were observed in comparison to the conventional chemical bleaching.     

Purification and properties of thermostable lipase from a thermophilic Bacillus stearothermophilus MC 7

Extracellular thermostable lipase produced by the thermophilic Bacillus stearothermophilus MC 7 was purified to 19.25-fold with 10.2% recovery. The molecular weight of the purified enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was shown to be 62 500 Da. The purified enzyme expressed maximum activity at 75–80 °C and its half life was 30 min at 70 °C. The K_m and V_max were calculated to be, respectively, 0.33 mM and 188 μM min⁻¹ mg⁻¹ with p-nitrophenyl palmitate (pNPP) as a substrate. Enzyme activity was inhibited by divalent ions of heavy metals, thiol and serine inhibitors, whereas calcium ion stimulated its activity. The most advantageous method for immobilization was found to be ionic binding to DEAE Cellulose. The enzyme was able to hydrolyze both soluble and insoluble emulsified substrates and was classified as a lipase, expressing some esterase activity as well.     

Purification and characterization of a novel cellulase-free xylanase from Acrophialophora nainiana

A beta-xylanase (XynIII) of Acrophialophora nainiana was purified to homogeneity from the culture supernatant by ultrafiltration and a combination of ion exchange and gel filtration chromatographic methods. It was optimally active at 55 degrees C and pH 6.5. XynIII had molecular masses of 27.5 and 54 kDa, as estimated by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. The purified enzyme hydrolyzed preferentially xylan as the substrate. The half-lives of XynIII at 50 and 60 degrees C were 96 and 1 h, respectively. It was activated by L-tryptophan, dithiothreitol, 5,5-dithio-bis(2-nitrobenzoic acid, L-cysteine and beta-mercaptoethanol and strongly inhibited by N-bromosuccinimide. The presence of carbohydrate was detected in the pure XynIII.     

Surfactants from xylan: Production of n-octyl xylosides using a highly thermostable xylanase from Thermotoga neapolitana

A highly thermostable recombinant xylanase from Thermotoga neapolitana was used as a catalyst for single-step synthesis of n-octyl xylobioside and n-octyl xylotrioside from xylan and n-octanol. Effect of xylan concentration, enzyme dose, reaction water content, reaction temperature and initial pH on the yield of these surfactants has been studied. The optimal conditions for n-octyl xylobioside and n-octyl xylotrioside synthesis are found to be different. The maximum n-octyl xylobioside and n-octyl xylotrioside yield were 120 and 38 mg/g of xylan, respectively. The n-octyl xylobioside yield achieved in this study was better than the yield achieved in all other enzymatic synthesis studies reported so far except what is achieved with the use of supercritical fluoroform under high pressure. The n-octyl xylotrioside yield is the highest ever achieved through enzymatic synthesis. An integrated system of production and recovery of n-octyl xylosides has improved the yield of n-octyl xylobioside and n-octyl xylotrioside by a factor of 1.7 and 2.4, respectively.     

Production of a high level of cellulase-free xylanase by the thermophilic fungus Thermomyces lanuginosus in laboratory and pilot scales using lignocellulosic materials

Thermomyces lanuginosus, isolated from self-heated jute stacks in Bangladesh, was able to produce a very high level of cellulase-free xylanase in shake cultures using inexpensive lignocellulosic biomass. Of the nine lignocellulosic substrates tested, corn cobs were found to be the best inducer of xylanase activity. The laboratory results of xylanase production have been successfully scaled up to VABIO (Voest-Alpine Biomass Technology Center) scale using a 15-m³ fermentor for industrial production and application of xylanase. In addition, some properties of the enzyme in crude culture filtrate produced on corn cobs are presented. The enzyme exhibited very satisfactory storage stability at 4–30°C either as crude culture filtrate or as spray- or freeze-dried powder. The crude enzyme was active over a broad range of pH and had activity optima at pH 6.5 and 70–75°C. The enzyme was almost thermostable (91–92%) at pH 6.5 and 9.0 after 41 h preincubation at 55°C and lost only 20–33% activity after 188 h. In contrast, it was much less thermostable at pH 5.0 and 11.0. Xylanases produced on different lignocellulosic substrates exhibited differences in thermostability at 55°C and pH 6.5. Correspondence to: J. Gomes     

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

Summary 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 -xylosidase (0.1 IU ml^–1) and -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.     

Production of cellulase and xylanase by a wild strain of Trichoderma viride

Summary The production of cellulase and xylanase was investigated with a newly isolated strain of Trichoderma viride BT 2169. The medium composition was optimized on a shake-flask scale using the Graeco-Latin square technique. The temperature and time for optimal growth and production of the enzymes in shake cultures were optimized using a central composite design. The temperature optima for maximal production of filter paper cellulase (FPase), xylanase and -gluosidase were 32.8°, 34.7° and 31.1° C, respectively, and the optimum times for production of these enzymes were found to be 144, 158 and 170 h, respectively. The optimized culture medium and conditions (33° C) gave 0.55 unit of FPase, 188.1 units of xylanase and 3.37 units of -glucosidase per milliliter of culture filtrate at 144 h of shake culture. Among different carbon sources tested, the maximum enzyme activities were produced with sulphite pulp and all three enzymes were produced irrespective of the carbon sources used. Batch fermentation in a laboratory fermentor using 2% sulphite pulp allowed the production of 0.61 unit of FPase, 145.0 units of xylanase and 2.72 units of -glucosidase. In a fed-batch fermentation on 6% final Avicel concentration FPase and -glucosidase were 3.0 and 2.4 times higher respectively than those in batch fermentation on 2% Avicel. The pH and temperature optima as well as pH and temperature stabilities of T. viride enzymes were found to be comparable to T. reesei and some other fungal enzymes.     

Production of xylooligosaccharides from enzymatic hydrolysis of xylan by the white-rot fungi Pleurotus

The white-rot fungi basidiomycetes Pleurotus sp. BCCB068 and Pleurotus tailandia were used to degrade oat-spelt xylan under submerged fermentation over a period of 40 days. Activities of endo-1,4-β-xylanase and β-xylosidase and xylan degradation products were determined. Xylan degradation by Pleurotus sp. BCCB068 and P. tailandia reached 75.1% and 73.4%, respectively. The formation of xylooligosaccharides and the simple sugars xylose, arabinose, cellobiose, mannose, and maltose were observed for both strains. The xylan degradation exhibited by these Pleurotus strains indicates they have potential for use in biotechnological processes related to degradation of hemicellulose sources.     

Production and purification of high levels of cellulase-free bacterial xylanase by Bacillus sp. SV-34S using agro-residue

Xylanase produced from the isolated bacterial strain Bacillus sp. SV-34S showed a 8.74-fold increase in enzyme activity under optimized submerged fermentation conditions. Cultivation using wheat bran as the carbon source and beef extract and (NH₄)H₂PO₄ as the nitrogen source resulted in productivity of 3,454.01 IU/mL xylanase. Xylanase was purified by 12.94-fold, with a recovery of 13.4 % and a specific activity of 3417.2 IU/mg protein, employing ammonium sulphate fractionation followed by cation-exchange chromatography using CM-Sephadex C-50 column chromatography, with a product of 27 kDa. The purified xylanase showed an optimum temperature and pH of 50 °C and 6.5, respectively although it was active even at pH 11.0. The thermostability study revealed that Bacillus sp. SV-34S was thermotolerant, being stable up to 50 °C; the residual activity at 55 and 60 °C was 96 and 93 %, respectively. The enzyme was stable between pH 6.0 and 8.0, although it retained >100 % activity at pH 8.0 and 9.0, respectively, following pre-incubation for 24 h. Xylanase activity was inhibited by various metal ions added to the assay mixture, with maximum inhibition observed in the presence of HgCl₂. The K_m and V_max values of the purified xylanase using birch wood xylan as substrate were 3.7 mg/mL and 133.33 IU/mL, respectively. The isolated bacterial strain produced high levels of extremophilic cellulase-free xylanase. The fact that it can be used in crude form and that it can be produced cheaply with renewable carbon sources make the process economically feasible. The characteristics of the purified enzyme suggest its potential application in industries such as the paper and pulp industry.     

Fusion of family VI cellulose binding domains to Bacillus halodurans xylanase increases its catalytic activity and substrate-binding capacity to insoluble xylan

A tandem repeat of the family VI cellulose binding domain (CBD) from Clostridium stercorarium xylanase (XylA) was fused at the carboxyl-terminus of Bacillus halodurans xylanase (XylA). B. halodurans XylA is an enzyme which is active in the alkaline region of pH and lacks a CBD. The constructed chimera was expressed in Escherichia coli, purified to homogeneity, and then subjected to detailed characterization. The chimeric enzyme displayed pH activity and stability profiles similar to those of the parental enzyme. The optimal temperature of the chimera was observed at 60 °C and the enzyme was stable up to 50 °C. Binding studies with insoluble polysaccharides indicated that the chimera had acquired an increased affinity for oat spelt xylan and acid-swollen cellulose. The bound chimeric enzyme was desorbed from insoluble substrates with sugars and soluble polysaccharides, indicating that the CBDs also possess an affinity for soluble sugars. Overall, the chimera displayed a higher level of hydrolytic activity toward insoluble oat spelt xylan than its parental enzyme and a similar level of activity toward soluble xylan.     

Phosphorylation of a 25 kDa protein is induced by thermostable direct hemolysin of Vibrio parahaemolyticus

Thermostable direct hemolysin (TDH) is a possible virulence factor produced by Vibrio parahaemolyticus. Although TDH has a variety of biological activities, including hemolytic activity, the biochemical mechanism of action remains uncertain. Here we analysed biochemical events, especially phosphorylation, caused by TDH in erythrocytes, and found that TDH caused significant phosphorylations of proteins on erythrocyte membrane. Phosphorylation of proteins was studied using γ-³²P ATP and SDS-PAGE. A number of protein kinase inhibitors were tested, to determine which types of kinases were involved in the phosphorylation events. TDH induced the phosphorylation of two proteins on membranes of human erythrocyte that are sensitive to TDH. The estimated molecular weight of these proteins was 25 and 22.5 kDa. Interestingly, the 22.5 kDa, but not the 25 kDa protein, was phosphorylated on the membrane of TDH-insensitive (resistant) horse erythrocytes. Moreover, a mutant TDH (R7), which retained binding ability but lost hemolytic activity, also phosphorylated only the 22.5 kDa protein on human erythrocyte membranes. Among the protein kinase inhibitors used the protein kinase C inhibitors, (staurosporine and calphostin C) showed marked inhibition of phosphorylation of 25 kDa protein. In addition to phosphorylation, these protein kinase C inhibitors suppresssed hemolysis by TDH. These results indicate that the phosphorylation of the 25 kDa protein seems to be essential for the hemolysis by TDH after it binds to erythrocyte membranes.     

Production and potential applications of a xylanase from a new strain of Streptomyces cuspidosporus

Enzyme production by a new mesophilic Streptomyces isolate was investigated which grew optimally on 1% (w/v) xylan and 10% (w/v) wheat bran at pH 7 and 37 °C. Xylan induced only CMCase (0.29 U/ml) besides xylanase (22–35 U/ml, 40–49 U/mg protein). Wheat bran induced xylanase (105 U/ml, 17.5 U/mg protein), CMCase (0.74 U/ml), -xylosidase (0.009 U/ml), -glucosidase (0.026 U/ml), -L-arabinofuranosidase (0.049 U/ml), amylase (1.6 U/ml) and phytase (0.432 U/ml). The isolate was amenable to solid state cultivation and produced increased levels of xylanase (146 U/ml, 28 U/mg protein). The pH and temperature optima of the crude xylanase activity were 5.5 and 65 °C respectively. The pI was 6.0 as determined by PEG precipitation. The crude enzyme was applied in treatment of paper pulp and predigestion of poultry feed and was found to be effective in releasing sugars from both and soluble phosphorus from the latter.     

Production and biochemical characterization of xylanase from an alkalitolerant novel species Aspergillus niveus RS2

The novel fungus Aspergillus niveus RS2 isolated from rice straw showed relatively high xylanase production after 5 days of fermentation. Of the different xylan-containing agricultural by-products tested, rice husk was the best substrate; however, maximum xylanase production occurred when the organism was cultured on purified xylan. Yeast extract was found to be the best nitrogen source for xylanase production, followed by ammonium sulfate and peptone. The optimum pH for maximum enzyme production was 8 (18.2 U/ml); however, an appreciable level of activity was obtained at pH 7 (10.9 U/ml). Temperature and pH optima for xylanase were 50°C and 7.0, respectively; however the enzyme retained considerably high activity under high temperature (12.1 U/ml at 60°C) and high alkaline conditions (17.2 U/ml at pH 8 and 13.9 U/ml at pH 9). The enzyme was strongly inhibited by Hg²⁺, while Mn²⁺ was slight activator. The half-life of the enzyme was 48 min at 50°C. The enzyme was purified by 5.08-fold using carboxymethyl-sephadex chromatography. Zymogram analysis suggested the presence of a single candidate xylanase in the purified preparation. SDS-PAGE revealed a molecular weight of approximately 22.5 kDa. The enzyme had K _m and V _max values of 2.5 and 26 μmol/mg per minute, respectively.     

疏绵状嗜热丝孢菌热稳定几丁质酶的纯化及其性质研究

采用硫酸铵沉淀、DEAE SepharoseFastFlow阴离子层析、Phenyl Sepharose疏水层析等步骤获得了凝胶电泳均一的疏绵状嗜热丝孢菌 (Thermomyceslanuginosus)几丁质酶。经SDS PAGE和凝胶过滤层析测得纯酶蛋白的分子量在 4 8～ 4 9 .8kD之间。该酶反应的最适温度和最适pH分别为 5 5℃和 4 5 ,在pH4 5条件下 ,该酶在 5 0℃以下稳定 ;6 5℃的半衰期为 2 5min ;70℃保温 2 0min后 ,仍保留 2 4 %的酶活性。其N 端氨基酸序列为AQGYLSVQYFVNWAI。金属离子对几丁质酶的活性影响较大 ,Ca2 、Na 、K 、Ba2 对酶有激活作用 ;Ag 、Fe2 、Cu2 、Hg2 对酶有显著的抑制作用 ;以胶体几丁质为底物的Km 和Vmax值分别为 9 .5 6mg mL和 2 2 . 12 μmol min。抗菌活性显示 ,该酶对供试病原菌有不同程度的抑制作用。     

Purification and characterization of a thermophilic alkaline xylanase from thermoalkaliphilic Bacillus sp. strain TAR-1

Thermoalkaliphilic Bacillus sp. strain TAR-1 isolated from soil produced an extracellular xylanase. The enzyme (xylanase R) was purified to homogeneity by ammonium sulfate fractionation and anion-exchange chromatography. The molecular mass of xylanase R was 40 kDa and the isoelectric point was 4.1. The enzyme was most active over the range of pH 5.0 to 10.0 at 50°C. The optimum temperatures for activity were 75°C at pH 7.0 and 70°C at pH 9.0. Xylanase R was stable up to 65°C at pH 9.0 for 30 min in the presence of xylan. Mercury(ll) ion at 1 mM concentration abolished all the xylanase activity. The predominant products of xylan-hydrolysate were xylobiose, xylotriose, and higher oligosaccharides, indicating that xylanase R was an endo-acting enzyme. Xylanase R had a K_m of 0.82 mg/ml and a V_max of 280 μmol min⁻¹ mg⁻¹ for xylan at 50°C and pH 9.0.     

Production of a cellulase-free xylanase from agricultural waste materials by a thermotolerant Streptomyces sp.

1444 microorganisms were isolated from soil samples from the northern Thai and screened at 55 °C by using basal medium supplemented with 1% carboxymethyl cellulose as a sole carbon source. One isolate, Streptomyces Ab106, had a high activity of a cellulase-free xylanase also without mannanase activity. The maximum cellulase-free xylanase activities of 3.5, 3.3, 3.1 and 2.7 IU were after growth of the organism with 1% (w/v) corn hull, corncob, bagasse and oat spelt xylan, respectively, at 55 °C for 6 days, respectively. The activity was more than 5 times higher than that at 35 °C.     

Production of cellulase-free thermostable xylanases by an isolated strain of Aspergillus niger PPI, utilizing various lignocellulosic wastes

A strain of Aspergillus niger PPI having prolific xylanolytic potential was isolated and the optimum conditions for maximum xylanase production was studied, resulting in the following: 4% substrate concentration, 10% v/v inoculum size, 72 h of incubation and pH 3.5–4.5 at 28 °C. The production profile of xylanase was examined with various lignocellulosics and maximum yield was achieved with oat. The hemicellulose content of wastes was also determined and oatmeal was found to have maximum hemicellulose content followed by wheat straw, sugarcane bagasse, rice husk and gram residue respectively. The enzyme showed maximum activity at pH 4 and temperature 60 °C. However, maximum stability was achieved at pH 3.5 and temperature 55 °C. Cellulase activity was found altogether absent in the enzyme broth.     

Chitobiose production by using a novel thermostable chitinase from Bacillus licheniformis strain JS isolated from a mushroom bed

The thermophilic Bacillus licheniformis strain JS was isolated from a bed of mushrooms, Pleurotus sajor-caju. The organism could produce a novel, single-component, thermostable chitinase that was purified by ion-exchange chromatography using DEAE-cellulose in 7.64% yield and in an 8.1-fold enhancement in purity. Its molecular weight is 22 kDa. The enzyme is a chitobiosidase, since the chitin hydrolysate is N^I,N^II-diacetylchitobiose. The optimum temperature for enzyme activity is 55 °C, and the optimum pH is 8.0. It was completely inhibited by Hg²⁺ ions whereas Co²⁺ ions served as an activator. The thermostability of this enzyme is important in the bioconversion of chitinous waste and for the production of chitooligosaccharides.