Influence of temperature on the properties of the xylanolytic enzymes of the thermotolerant fungus<Emphasis Type="Italic"> Aspergillus phoenicis</Emphasis>

This study reports on the effects of growth temperature on the secretion and some properties of the xylanase and -xylosidase activities produced by a thermotolerant Aspergillus phoenicis. Marked differences were observed when the organism was grown on xylan-supplemented medium at 25 °C or 42 °C. Production of xylanolytic enzymes reached maximum levels after 72 h of growth at 42 °C; and levels were three- to five-fold higher than at 25 °C. Secretion of xylanase and -xylosidase was also strongly stimulated at the higher temperature. The optimal temperature was 85 °C for extracellular and 90 °C for intracellular -xylosidase activity, independent of the growth temperature. The optimum temperature for extracellular xylanase increased from 50 °C to 55 °C when the fungus was cultivated at 42 °C. At the higher temperature, the xylanolytic enzymes produced by A. phoenicis showed increased thermostability, with changes in the profiles of pH optima. The chromatographic profiles were distinct when samples obtained from cultures grown at different temperatures were eluted from DEAE–cellulose and Biogel P-60 columns.     

Production of thermostable α-galactosidase from thermophilic fungusHumicola sp

The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular -galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu²⁺, Ni²⁺ and Hg²⁺ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml^–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg²⁺ strongly inhibited enzyme activity. TheK _m andV _max forp-nitrophenyl--d-galactopyranoside were 60M and 33.6 mol min^–1 mg^–1, respectively, while for raffinose those values were 10.52 mM and 1.8 mol min^–1 mg^–1, respectively.     

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.     

Purification and characterization of an extracellular β-D-xylosidase from Aspergillus carbonarius

The production of an extracellular -D-xylosidase (-D-xyloside xylohydrolase, EC 3.2.1.37) by four Aspergillus strains (A. carbonarius, A. nidulans, A. niger and A. oryzae) grown on wheat bran medium was compared. The highest amount of the enzyme was found in the culture of A. carbonarius. The -D-xylosidase from A. carbonarius was purified to homogeneity by a rapid procedure, using hydrophobic interaction chromatography, chromatofocusing and affinity chromatography. The purified enzyme possessed not only -D-xylosidase activity, but also -L-arabinosidase activity. Mixed substrate experiments revealed that a single active centre was responsible for the splitting of the corresponding synthetic substrates. The molecular weight of the purified enzyme proved to be 100,000 Da, as estimated by SDS–PAGE. The isoelectric point was at pH 4.4. The pH and temperature optima were 4.0 and 60 °C, respectively. The enzyme remained stable over a pH range of 3.5–6.5 and up to 50 °C for 30 min. The Michaelis constant for p-nitrophenyl -D-xyloside was 0.198 mM. Kinetic studies demonstrated that the lack of the C-5 hydroxylmethyl group and the configuration of the C-4 hydroxyl group on the pyranoside ring play an important role in both substrate binding and splitting.     

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.     

Simultaneous production of α-arabinofuranosidase and xylanase by Termitomyces clypeatus

Termitomyces clypeatus produced xylanase and -L-arabinofuranosidase simultaneously in various media. The arabinofuranosidase had pH and temperature optima of 5.5 and 50°C, respectively, and was stable at 50°C for 30 min and at pH values from 2 to 5. The partially purified enzyme was distinct from xylanase present in the same medium.The authors are with the Department of Applied Biochemistry, Indian Institute of Chemical Biology, 4 Raja S.C. Mullick Road, Calcutta 700 032, India     

Cloning of a gene encoding a thermo-stable endo-beta-1,4-glucanase from Thermoascus aurantiacus and its expression in yeast

A gene encoding a thermo-stable endo--1,4-glucanase was isolated from the thermophilic fungus, Thermoascus aurantiacusIFO9748, and designated as eg1. Induction of this gene expression at 50°C was stronger than at 30°C. The deduced amino acid sequence encoded by eg1 showed that it belongs to the glycoside hydrolase family 5. The cloned gene was expressed in Saccharomyces cerevisiae and the gene product was purified and characterized. No significant activity loss was detected over 2 h at 70°C and the product was stable from pH 3–10. The enzyme was optimally active at 70°C over 20 min and the optimal pH was 6.     

Induction and partial characterization of intracellular <Emphasis Type="Italic">β</Emphasis> from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> and its application in the synthesis of 6-kestose

Production of -fructofuranosidase from the thermophilic fungus Thermoascus aurantiacuswas enhanced by optimization of the type of nitrogen source as well as the type and concentration of carbon source. Submerged batch cultivation in a laboratory bioreactor (7 l) using the optimized medium allowed the production of 85 mU/ml of -fructofuranosidase. The enzyme showed both transfructosylating and hydrolytic activities and was optimally active at 60 °C and pH 5.0. The enzyme showed the ability to catalyse the synthesis of 1-kestose and the reaction was maximized at 30% (w/v) initial sucrose concentration.     

Production and characterization of xylanases of a Bacillus strain isolated from soil

Xylanase production was performed by growing a Bacillus isolate on agricultural by-products, wheat straw, wheat bran, corn cobs and cotton bagasse. A maximum xylanase activity of 180 U/ml was obtained together with a cellulase activity of 0.03 U/ml on 4 (w/v) corn cobs. Electrophoretic analysis showed the presence of three endo--1, 4-xylanases having molecular weights of about 22, 23 and 40 kDa. Xylanolytic activity was stable up to 50 °C in the pH range of 4.5–10 and the highest activity was observed at 70 °C and pH 6.5.     

Cellulase-free xylanase production from an alkalophilicBacillus species

An alkalophilicBacillus (NCL-87-6-10, NCIM 2128), with a high productivity for extracellular xylanase (EC 3.2.1.8) and free of cellulase, was isolated from soil containing coconut fibre detritus. When grown on a wheat bran/yeast extract medium in submerged culture for 48 h, it produced 100 to 120 IU of enzyme activity per ml. The crude enzyme consists of two fractions of apparent mol sizes of approx 10.4 and 29 kDa in the proportion of 90:10, as determined by native gel exclusion chromatography. Optimum activity of the xylanase was at 60°C and pH 8.0. A two-fold increase in enzyme activity was obtained when reducing agents, thioethanol and dithiothreitol, were included in the assay.NCL Communication No. 5381.     

Alkaline-active xylanase produced by an alkaliphilicBacillus sp isolated from kraft pulp

ABacillus sp (V1-4) was isolated from hardwood kraft pulp. It was capable of growing in diluted kraft black liquor at pH 11.5 and produced 49 IU (mol xylose min^–1 ml^–1) of xylanase when cultivated in alkaline medium at pH 9. Maximal enzyme activity was obtained by cultivation in a defined alkaline medium with 2% birchwood xylan and 1% corn steep liquor at pH 9, but high enzyme production was also obtained on wheat bran. The apparent pH optimum of the enzyme varied with the pH used for cultivation and the buffer system employed for enzyme assay. With cultivation at pH 10 and assays performed in glycine buffer, maximal activity was observed at pH 8.5; with phosphate buffer, maximal activity was between pH 6 and 7. The xylanase temperature optimum (at pH 7.0) was 55°C. In the absence of substrate, at pH 9.0, the enzyme was stable at 50°C for at least 30 min. Elecrophoretic analysis of the crude preparation showed one predominant xylanase with an alkaline pl. Biobleaching studies showed that the enzyme would brighten both hardwood and softwood kraft pulp and release chromophores at pH 7 and 9. Because kraft pulps are alkaline, this enzyme could be used for prebleaching with minimal pH adjustment.     

Purification and properties of an extracellular endo-1,4-β-glucanase fromLenzites trabea

An extracellular endo-1,4--glucanase (EC 3.2.1.4) has been isolated and purified from the culture solution of the basidiomyceteLenzites trabea grown on glucose and cellulose. Besides-glucosidase activity (EC 3.2.1.21) no evidence for C₁-activity (EC 3.2.1.91) in the culture solution was found.The endoglucanase has been purified in a four-step procedure including chromatography on Sepharose 6-B and DEAE-Sephadex A-50, adsorption on hydroxylapatite and gel filtration on Bio-Gel P-100. The enzyme showed maximum activity at pH 4.4 and 70°C. A molecular weight of 29000 Daltons was estimated by calibration on Bio-Gel P-100. The enzyme hydrolyses carboxymethyl cellulose (CMC) as well as xylan.List of Abbreviations CMC carboxymethyl cellulose - D.S. degree of substitution - D.P. degree of polymerisation - MW molecular weight     

Culture conditions for enhanced cellulase production by a native strain of Penicillium purpurogenum

A cellulolytic wild-type strain of Penicillium purpurogenum was isolated from a soil sample in southern Chile. It grew best at 28°C from an inoculum of 4×10⁷ spores/100 ml medium. Highest endoglucanase activity was with Sigmacell as carbon source and corn steep liquor as nitrogen source. Wheat bran enhanced the production of endoglucanase and -glucosidase. The enzymes in the crude supernatants were stable up to 50°C and between pH 4.4 and 5.6 for 48 h.J.Steiner and C. Socha are with the Facultad de Ciencias Quimicas y Farmacéuticas, Universidad de Chile, Casilla 233, Santiago 1, Chile; J. Eyzaguirre is with the Laboratorio de Bioquímica, Pontificia Universidad Católica de Chile, Casilla 114-D, Santiago, Chile.     

Cellodextrin utilization and β-glucosidase production by Bacteroides polypragmatus

Bacteroides polypragmatus, a mesophilic obligate anaerobe, was shown to simultaneously ferment glucose and cellobiose giving ethanol as a major metabolic end-product. A mixture of higher cellodextrins was also utilized. The bacterium produced a -glucosidase with a pI value of 4.2 and a molecular weight of approximately 100000 daltons. The enzyme was intracellular and functioned optimally at pH 7. The K _m values obtained with p-nitrophenyl--d-glucoside and cellobiose as substrates were 0.73 mM and 100 mM, respectively. The enzyme was quite stable at elevated temperatures; in the presence of 10% glycerol (v/v), it had a half-life of 4 h at 55°C. It was also stable during long-term storage at either 4°C or-20°C, provided that 10% (v/v) glycerol was added to preparations maintained at-20°C.Abbreviations HPLC high-performance liquid chromatography - IEF isoelectric focusing - pNPG p-nitrophenyl--d-glucoside NRCC No. 25676     

Production and properties of carboxymethylcellulase (endo-1,4-β-glucanase) fromCurvularia lunata

An extracellular carboxymethylcellulase (endo-1,4--glucanase) fromCurvularia lunata, grown at 30°C with an initial pH of 6.0, had optimal activity at pH 4.8 and 50°C. The enzyme was unstable above 50°C. The enzyme had aK _m for carboxymethylcellulose of 0.97 g/l and aV _max of 5.4 IU/ml.     

Purification and characterization of β-mannanase from Bacillus licheniformis for industrial use

An easily scaled-up technique has been designed to purify -mannanase from Bacillus licheniformis. Using flocculation, ultrafiltration and ion-exchange chromatography, the enzyme was purified 33-fold with a final recovery of 47% and a specific activity of 4341 U mg^–1protein. The enzyme had maximum activity at 60 °C and pH 7.0. It was stable at 50 °C and pH 6.0 for 6 h, but lost all of its activity when held at 70 °C and pH 6.0 for 1 h.     

Characterization of cellulase and xylanase activities of Streptomyces lividans

Summary The production of cellulases and of xylanase by Streptomyces lividans 1326 was studied under different growth conditions. The strain grew between 18°C and 46°C and is therefore thermotolerant. Submerged cultures of the microorganism, when grown on a defined salt medium containing xylan as main carbon source, exhibited an overall cellulolytic activity as determined by the filter paper test. S. lividans produced optimal levels of extracellular -1,4-glucan-glucanohydrolase (1 IU/ml) and large amounts of -1,4-xylanxylanohydrolase (50 IU/ml) at 40°C. A better production of both enzymes was observed when xylan instead of cellulose was used as substrate.The stability of the enzyme was found to be significantly greater than those of the cellulases and xylanases produced by other streptomycetes. The optimal incubation temperatures for the enzyme assays were 55°C and 60°C for CM-cellulase and xylanase respectively and optimal pH values were found in the range of pH 6–7.     

Liquefaction of dulse (Palmaria palmata (L.) Kuntze) by a commercial enzyme preparation and a purified endo ,β-1,4-D-xylanase

Liquefaction of dry and freshPalmaria palmata by food grade enzyme preparations and a purified endo--1,4-D-xylanase was studied.The endo--1,4-D-xylanase (EC 3.2.1.8) was purified to homogeneity from a commercial food grade enzyme prepared fromAspergillus niger. It has a molecular weight of 22 500, a pI of 3.5, is inactive toward corn arabinoxylan,p-nitrophenyl--D-xylose, carboxymethyl cellulose but shows a weak activity toward microcrystalline cellulose. It hydrolyzes oat and dulse xylan equally well in seawater and deionized water essentially into xylose and xylobiose. It is stable between pH 5.5 to 9.0 and 0 to 30 °C and its activity is optimal at pH 4.5–5.5 and 40–60 °C. It has a K_m of 2.2 and 2.8 mg ml^-1 and V_max of 3600 and 3900 nkat mg^-1 of protein on oat and dulse xylan, respectively.Acetate buffer, deionized water and seawater alone extracted 62.6 to 64.5 % of the dry weight of dry dulse, but the use of commercial food grade enzyme preparations or the purified xylanase improved liquefaction to 81.2–87.1 %. Xylose and galactose were the only sugars present in the soluble extracts. Deionized and seawater extracted 58.8–52.7 and 39.1–42.2% of the dry weight of the fresh algae collected in fall and summer, respectively. Only galactose was found in the seawater extract, while some xylose with galactose were measured in the deionized water extract of the fresh autumn algal sample. Purified and crude xylanase improved liquefaction of fresh algae to 79.8–81.4 and 71.9–77.9% of the fresh dry weight (fall and summer, respectively) in deionized and seawater, respectively, and increased the xylose content of the soluble fractions. Polysaccharides in the soluble residues were composed of 1,3/1,4-linked xylose, 1-linked galactose (floridoside) and 1,4-linked glucose (cellulose) and contained essentially 1,4-linked xylose and 1,4-linked glucose in insoluble fractions obtained after enzymatic treatment.The use of xylanase-containing food grade enzyme preparations improves liquefaction ofPalmaria palmata, particularly from fresh alga. This study indicates that processing such as drying may modify markedly the solubility ofP. palmata cell wall polysaccharides, which would imply the existence of some organization and/or other components in the fresh cell wall that lower xylan solubility in seawater.     

Production and properties of xylanases from thermophilic actinomycetes

30 strains of xylanolytic thermophilic actinomycetes were isolated from composted grass and cattle manure and identified as members of the generaThermomonospora, Saccharomonospora, Microbispora, Streptomyces andActinomadura. Screening of these strains for extracellular xylanase indicated that strains ofSaccharomonospora andMicrobispora generally were poor xylanase producers (0.5–1.5 U/ml) whereas relatively high activities were observed in cultures ofStreptomyces andActionomadura (4–12 U/ml).A preliminary characterization of the enzymes of strains of the latter genera suggested that xylanases of all the strains ofActinomadura exhibited higher thermostabilities than those ofStreptomyces. To evaluate the potential of thermophilicActinomadura for industrial applications, xylanases of three strains were studied in more detail. The highest activity levels for xylanases were observed in cultures grown on xylan and wheat bran. The optimal pH and temperature for xylanase activities ranged from 6.0 to 7.0 and 70 to 80°C. The enzymes exhibited considerable thermostability at their optimum temperature. The half-lives at 75°C were in the range from 6.5 to 17h. Hydrolysis of xylan by extracellular xylanases yielded xylobiose, xylose and arabinose as principal products. Estimated by the amount of reducing sugars liberated the degree of hydrolysis was 55 to 65%. Complete utilization of xylan is presumably achieved by -xylosidase activities which could be shown to be largely cell-associated in the 3Actinomadura strains.     

Selective preparation of N-acetyl-D-glucosamine and N,N'-diacetylchitobiose from chitin using a crude enzyme preparation from Aeromonas sp

A bacterium, Aeromonas sp. GJ-18, having strong chitinolytic activity was isolated from coastal soil and used for crude enzyme preparations. This enzyme preparation contained N-acetyl-D-glucosaminidase and N,N-diacetylchitobiohydrolase. N-Acetyl-D-glucosaminidase was inactive above 50 °C, but N,N-diacetylchitobiohydrolase was stable at this temperature. Utilizing the temperature sensitivities of the chitin degradation enzymes in crude enzyme preparation, N-acetyl-D-glucosamine (GlcNAc) and N,N-diacetylchitobiose [(GlcNAc)₂] were selectively produced from chitin. At 45 °C, GlcNAc was produced as a major hydrolytic product (94% composition) with a yield of 74% in 5 d, meanwhile at 55 °C (GlcNAc)₂ was the major product (86%) with a yield of 35% within 5 d.Revisions requested 29 September 2004; Revisions received 1 November 2004