A novel strain of Streptomyces malaysiensis isolated from Brazilian soil produces high endo-β-1,4-xylanase titres

One hundred and sixty two actinomycete strains isolated from Brazilian soils were screened for xylanase activity, according to the size of the hydrolysis zones observed in oat spelts xylan agar plates. The strain AMT-3, later identified as Streptomyces malaysiensis, was selected as the best producer. In subsequent shake flasks fermentations using growth media contanning 1% (w/v) of either birchwood, or oat spelts xylan, plus organic nitrogen and salts, high endo--1,4-xylanase titres (EC 3.2.1.8) (116 U ml^–1) were observed in the larchwood medium within 6 days. This is the first report concerning xylanase production by streptomyces malaysiensis, which has been recently described as a new species.     

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.     

A novel cold-tolerant Clostridium strain PXYL1 isolated from a psychrophilic cattle manure digester that secretes thermolabile xylanase and cellulase

A Clostridium strain PXYL1 was isolated from a cold-adapted cattle manure biogas digester at 15 degrees C. It could grow at temperatures as low as 5 degrees C up to 50 degrees C with highest specific growth rate at 20 degrees C and is a psychrotroph. It produced extracellular hydrolytic enzymes namely xylanase, endoglucanase, beta-xylosidase, beta-glucosidase and filter paper cellulase, all of which had maximal activity at 20 degrees C. The induction of xylanase was highest on birch wood xylan (37 IU(mg protein)(-1)) compared with xylose (1.11 IU(mg protein)(-1)), cellobiose (1.43 IU(mg protein)(-1)) and glucose (no activity). The xylanase was thermolabile with a half-life of 30 min at 40 degrees C and 8 min at 50 degrees C but stable for over 2 h at 20 degrees C. The crude enzyme released reducing sugars (1.25 g l(-1)) from finger millet flour at 20 degrees C, while commercial food-grade xylanases showed no hydrolysis at this temperature. This is the first report of a Clostridium strain growing at 20 degrees C and producing an array of xylanolytic and cellulolytic enzymes, possessing low temperature optima of 20 degrees C, which may facilitate degradation of plant fibre under low-temperature conditions.     

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.     

Keratinase Production by Newly Isolated Antarctic Actinomycete Strains

Summary The ability of actinomycete strains newly isolated from Antarctic soils to produce keratinolytic enzymes during growth on sheep wool waste was investigated. The strains which displayed highest keratinase activity and identified as Streptomyces flavis 2BG (mesophilic) and Microbispora aerata IMBAS-11A (thermophilic) were selected for a more detailed analysis. The addition of starch to the growth medium affected keratinase secretion by both strains. After 5 days of cultivation, a 6-fold increase in keratinase activity of strain 11A was observed in the presence of 11 g starch/l and a 9-fold increase in keratinase activity of the strain 2BG in the presence of 5 g starch/l. The results obtained showed that both newly isolated strains are very promising for effective processing of native keratinous wastes. To our knowledge, this is the first report of Antarctic actinomycete strains that were able to grow on keratin-containing wastes by producing keratinolytic enzymes.     

Xylanase-induced reduction of chlorine dioxide consumption during elemental chlorine-free bleaching of different pulp types

The potential of crude xylanase from Thermomyces lanuginosus and Xylanase P (a commercial xylanase) was evaluated in bleaching of various paper pulp types. Xylanases released chromophores and reducing sugars and decreased kappa number of pulps. Chlorine-bleached, alkali-extracted bagasse and post-oxygen kraft pulps, pretreated with enzymes, gained over 5 brightness points over controls. Biobleaching of soda-aq pulp with Xylanase P produced chlorine dioxide savings of up to 30% or 4.5 kg chlorine dioxide t^–1 pulp.     

Cellulose and xylan degrading enzymes of Fusarium avenaceum

It was found that crude preparation obtained from the culture medium of Fusarium avenaceum degraded cellulose and xylan. After chromatography on CM-Sepharose CL-6B of this preparation six fraction were obtained. The eluted fractions II and V showed xylanase activity, fraction IV — cellulase activity and fraction III — xylanase and cellulase activity. The end products of xylan hydrolysis by all xylanase fractions (II, III, V) were xylobiose, xylose, xylotriose and xylotetrose. The end products of cellulose hydrolysis by fractions III and IV was cellobiose, glucose and cellotriose. The data from gel filtration on Sephacryl S-200 indicated a molecular weight of more than 250,000 for both cellulase IV and xylanase V. After gel filtration in the presence of urea disaggregation of those high molecular xylanase and cellulase particles was observed. Xylanase II in difference from the other fractions contained higher amount of sugar. Digestion of fraction II with cellulase-hemicellulase preparation from Phoma hibernica decreased the content of sugar from 17% to 8%, but did not change its enzymatic properties. Cellulase IV as well as xylanase V were inactivated by N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide and tetranitromethane, hence it is suggested that tryptophan and tyrosine are the essential for the activity of these enzymes.     

Enzymatic hydrolysis of xylans I. A high xylanase and β-xylosidase producing strain of aspergillus niger

Summary Aspergillus niger, strain 110.42 (CBS), has been selected as a producer of high xylanolytic activities. The time course of xylanase and -xylosidase production as well as the effect of pH and temperature on the activity of these enzymes were studied. HPLC analysis of the enzymatic degradation of arabinoxylan showed a nearly complete conversion to pentose sugars. Aspects of using crude xylanase preparations for enzymatic saccharification of xylans are discussed.     

Production of alkali-tolerant cellulase-free xylanase by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. WLUN024 with wheat bran as the main substrate

An alkali-tolerant cellulase-free xylanase producer, WLI-11, was screened from soil samples collected from a pulp and paper mill in China. It was subsequently identified as a Pseudomonas sp. A mutant, WLUN024, was selected by consecutive mutagenesis by u.v. irradiation and NTG treatment using Pseudomonas sp. WLI-11 as parent strain. Pseudomonas sp. WLUN024 produced xylanase when grown on xylosidic materials, such as hemicellulose, xylan, xylose, and wheat bran. Effects of various nutritional factors on xylanase production by Pseudomonas sp. WLUN024 with wheat bran as the main substrate were investigated. A batch culture of Pseudomonas sp. WLUN024 was conducted under suitable fermentation conditions, where the maximum activity of xylanase reached 1245 U ml⁻¹ after incubating at 37 °C for 24 h. Xylanase produced by Pseudomonas sp. WLUN024 was purified and the molecular weight was estimated as 25.4 kDa. Primary studies on the characteristics of the purified xylanase revealed that this xylanase was alkali-tolerant (optimum pH 7.2–8.0) and cellulase-free. In addition, the xylanase was also capable of producing high quality xylo-oligosaccharides, which indicated its application potential in not only pulp bio-bleaching processes but also in the nutraceutical industry.     

Biological pretreatment of rice straw with Streptomyces griseorubens JSD-1 and its optimized production of cellulase and xylanase for improved enzymatic saccharification efficiency

Biological pretreatment of rice straw and production of reducing sugars by hydrolysis of bio-pretreated material with Streptomyces griseorubens JSD-1 was investigated. After 10 days of incubation, various chemical compositions of inoculated rice straw were degraded and used for further enzymatic hydrolysis studies. The production of cellulolytic enzyme by S. griseorubens JSD-1 favored the conversion of cellulose to reducing sugars. The culture medium for cellulolytic enzyme production by using agro-industrial wastes was optimized through response surface methodology. According to the response surface analysis, the concentrations of 11.13, 20.34, 4.61, and 2.85 g L^?1 for rice straw, wheat bran, peptone, and CaCO₃, respectively, were found to be optimum for cellulase and xylanase production. Then the hydrolyzed spent Streptomyces cells were used as a nitrogen source and the maximum filter paper cellulase, carboxymethylcellulase, and xylanase activities of 25.79, 78.91, and 269.53 U mL^?1 were achieved. The crude cellulase produced by S. griseorubens JSD-1 was subsequently used for the hydrolysis of bio-pretreated rice straw, and the optimum saccharification efficiency of 88.13% was obtained, indicating that the crude enzyme might be used instead of commercial cellulase during a saccharification process. These results give a basis for further study of bioethanol production from agricultural cellulosic waste.     

Isolation and characterization of fatty acid methyl ester (FAME)‐producing Streptomyces sp. S161 from sheep (Ovis aries) faeces

An actinomycete producing oil‐like mixtures was isolated and characterized. The strain was isolated from sheep faeces and identified as Streptomyces sp. S161 based on 16S rRNA gene sequence analysis. The strain showed cellulase and xylanase activities. The ¹H nuclear magnetic resonance (NMR) spectra of the mixtures showed that the mixtures were composed of fatty acid methyl esters (52·5), triglycerides (13·7) and monoglycerides (9·1) (mol.%). Based on the gas chromatography–mass spectrometry (GC‐MS) analysis, the fatty acid methyl esters were mainly composed of C14‐C16 long‐chain fatty acids. The results indicated that Streptomyces sp. S161 could produce fatty acid methyl esters (FAME) directly from starch. To our knowledge, this is the first isolated strain that can produce biodiesel (FAME) directly from starch.

Significance and Impact of the Study

Nowadays, production of biodiesel is based on plant oils, animal fats, algal oils and microbial oils. Lipid mostly consists of triacylglycerols (TAG), and conversion of these lipids into fatty acid short‐chain alcohol esters (methanol or ethanol) is the final step in biodiesel production. In this study, an oil‐producing Streptomyces strain was isolated from sheep faeces. The oil was composed of C₁₄‐C₁₆ long‐chain fatty acid methyl esters, triglycerides and monoglycerides. This is the first isolated strain‐producing biodiesel (FAME) directly from starch. Due to showing cellulase and xylanase activities, the strain would be helpful for converting renewable lignocellulose into biodiesel directly.     

Partial Purification and Some Properties of Mitomycin C Inactivating Factors of Pseudomonas convexa 4–87

Strains producing higher levels of cellulolytic enzymes were selected from among 520 strains of plant pathogenic fungi, Fusarium species, and F. oxysporum strain SUF850 was found to be the best producer. When strain SUF850 was cultured using one of three polysaccharides, Avicel, carboxy- methyl cellulose (CMC) or xylan, as a carbon source, the culture filtrate contained degrading activi- ties toward all three substrates, i.e., irrespective of the carbon source used. From the culture filtrate of Avicel-grown cells, four distinct enzymes were purified to homogeneity, as judged on SDS-PAGE. They were designated as CMCase I, CMCase II, /Miitrophenyl-β-d-cellobiosidase and xylanase, and the characteristics of the individual enzymes were examined and compared.     

Xylanases of thermophilic bacteria from Icelandic hot springs

Thermophilic, aerobic bacteria isolated from Icelandic hot springs were screened for xylanase activity. Of 97 strains tested, 14 were found to be xylanase positive. Xylanase activities up to 12 nkat/ml were produced by these strains in shake flasks on xylan medium. The xylanases of the two strains producing the highest activities (ITI 36 and ITI 283) were similar with respect to temperature and pH optima (80°C and pH 8.0). Xylanase production of strain ITI 36 was found to be induced by xylan and xylose. Xylanase activity of 24 nkat/ml was obtained with this strain in a laboratory-scale-fermentor cultivation on xylose medium. -Xylosidase activity was also detected in the culture filtrate. The thermal half-life of ITI 36 xylanase was 24 h at 70°C. The highest production of sugars from hydrolysis of beech xylan was obtained at 70°C, although xylan depolymerization was detected even up to 90°C. Correspondence to: M. Rättö     

Production of xylanases by mangrove fungi from the Philippines and their application in enzymatic pretreatment of recycled paper pulps

Mangrove fungi are vastly unexplored for enzymes with industrial application. This study aimed to assess the biocatalytic activity of mangrove fungal xylanases on recycled paper pulp. Forty-four mangrove fungal (MF) isolates were initially screened for xylanolytic activity in minimal medium with corn cob xylan as the sole carbon source. Eight MF were further cultivated under submerged fermentation for the production of crude xylanases. These crude enzymes were then characterized and tested for the pretreatment of recycled paper pulps. Results showed that 93 % of the tested MF isolates exhibited xylanolytic activity in solid medium. In submerged fermentation, salinity improved the growth of the fungal isolates but did not influence xylanase production. The crude xylanases were mostly optimally active at 50 °C and pH 7. Changes in pH had a greater effect on xylanase stability than temperature. More than half of the activity was lost at pH 9 for majority of the crude enzymes. However, two thermophilic xylanases from Fusarium sp. KAWIT-A and Aureobasidium sp. 2LIPA-M and one alkaliphilic xylanase from Phomopsis sp. MACA-J were also produced. All crude enzymes exhibited cellulase activities ranging from 4 to 21 U/ml. Enzymatic pretreatment of recycled paper pulps with 5 % consistency produced 70–650 mg of reducing sugars per gram of pulp at 50 °C after 60 min. The release of high amounts of reducing sugars showed the potential of mangrove fungal crude xylanases in the local paper and pulp industry. The diverse properties shown by the tested crude enzymes also indicate its potential applications to other enzyme-requiring industries.     

A comparison of two xylanases from the thermophilic fungi Thielavia terrestris and Thermoascus crustaceus

Two thermophilic xylanases (xylanase II from Thielavia terrestris 255B and the 32-kDa xylanase from Thermoascus crustaceus 235E) were studied to determine if they had different and complementary modes of action when they hydrolysed various types of xylans. Partial amino acid sequencing showed that these two enzymes belonged to different families of -1,4-glycanases. Xylanase II achieved faster solubilization of insoluble xylan whereas the 32-kDa xylanase was more effective in producing xylose and short xylooligomers. An assessment of the combined hydrolytic action of the two xylanases did not reveal any co-operative action. The sugars released when the two thermophilic xylanases were used together were almost identical to those released when the 32-kDa xylanase acted alone. The two xylanases were able to remove about 12% of the xylan remaining in an aspen kraft pulp. This indicated that either one of these thermophilic enzymes may be useful for enhancing the bleaching of kraft pulps. Correspondence to: J. N. Saddler     

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 of cellulases and xylanases under catabolic repression conditions from mutant PR-22 of Cellulomonas flavigena

Derepressed mutant PR-22 was obtained by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) mutagenic treatment of Cellulomonas flavigena PN-120. This mutant improved its xylanolytic activity from 26.9 to 40 U mg⁻¹ and cellulolytic activity from 1.9 to 4 U mg⁻¹; this represented rates almost 2 and 1.5 times higher, respectively, compared to its parent strain growing in sugarcane bagasse. Either glucose or cellobiose was added to cultures of C. flavigena PN-120 and mutant PR-22 induced with sugarcane bagasse in batch culture. The inhibitory effect of glucose on xylanase activity was more noticeable for parent strain PN-120 than for mutant PR-22. When 20 mM glucose was added, the xylanolytic activity decreased 41% compared to the culture grown without glucose in mutant PR-22, whereas in the PN-120 strain the xylanolytic activity decreased by 49% at the same conditions compared to its own control. Addition of 10 and 15 mM of glucose did not adversely affect CMCase activity in PR-22, but glucose at 20 mM inhibited the enzymatic activity by 28%. The CMCase activity of the PN-120 strain was more sensitive to glucose than PR-22, with a reduction of CMCase activity in the range of 20–32%. Cellobiose had a more significant effect on xylanase and CMCase activities than glucose did in the mutant PR-22 and parent strain. Nevertheless, the activities under both conditions were always higher in the mutant PR-22 than in the PN-120 strain. Enzymatic saccharification experiments showed that it is possible to accumulate up to 10 g l⁻¹ of total soluble sugars from pretreated sugarcane bagasse with the concentrated enzymatic crude extract from mutant PR-22.     

Integrated Strategies to Enhance Cellulolytic Enzyme Production Using an Instrumented Bioreactor for Solid-State Fermentation of Sugarcane Bagasse

The conversion of agro-industrial residues, such as sugarcane bagasse, into high-value products and renewable energy, within the biorefinery concept, is a potential alternative towards the sustainable management of these resources. This work evaluates the production of cellulolytic enzymes by a selected strain of Aspergillus niger cultivated in sugarcane bagasse under solid-state fermentation using an instrumented lab-scale bioreactor. The effects of environmental factors including the type of substrate and medium composition, as well as the operational conditions (air flow rate, inlet air relative humidity, and initial substrate moisture content) on the production of the enzymatic complex were evaluated using statistical design tools. Significant increases in FPase, endoglucanase, and xylanase activities were achieved under the optimized conditions predicted by the models, with values of 0.88, 21.77, and 143.85 IU/g of dry solid substrate, respectively, representing around ten-, four-, and twofold increases compared to the activities obtained under the initial growth conditions. This demonstrates the importance of evaluating environmental and operational criteria in order to achieve efficient enzyme production. The crude enzymatic extract obtained under optimized conditions was employed for enzymatic hydrolysis of pretreated sugarcane bagasse. Approximately 13 % of total reducing sugars, and a glucose concentration of 2.54 g/L, were obtained after 22 h of hydrolysis of steam exploded sugarcane bagasse, indicating that the enzymatic cocktail produced has good potential for use in the conversion of biomass.     

Comparison of different strains ofCellulomonas for production of cellulolytic and xylanolytic enzymes from biomass produced on saline lands

A comparison of the rate of carboxymethyl cellulase (CMCase), avicelase, xylanase, -glucosidase and -xylosidase production and rates of growth by 4 different strains ofCellulomonas revealed a wide range of behaviour; with some strains producing more CMCase, avicelase, xylanase, -glucosidase and -xylosidase from complex lignocellulosic (LC) biomass (from saline land) and CMC while some others producing small amounts of these enzymes. One strain,C. biazotea, was better with respect to enzyme production potential and growth behaviour than most of the other strains and has been chosen as a starting strain for genetic improvement for producing enzymes of the cellulase complex.     

Enhanced cellulase production through random mutagenesis of Talaromyces pinophilus OPC4-1 and fermentation optimization

Reducing cellulase cost remains a major challenge for lignocellulose to fuel and chemical industries. In this study, mutants of a novel wild-type cellulolytic fungal strain Talaromyces pinophilus OPC4-1 were developed by consecutive UV irradiation, N-methyl-N`-nitro-N-nitrosoguanidine (NTG) and ethylmethane sulfonate (EMS) treatment. A potential mutant EMM was obtained and displayed enhanced cellulase production. Using Solka Floc cellulose as the substrate, through fed-batch fermentation, mutant strain T. pinophilus EMM generated crude enzymes with an FPase activity of 27.0 IU/mL and yield of 900 IU/g substrate. When corncob powder was used, strain EMM produced crude enzymes with an FPase activity of 7.3 IU/mL and yield of 243.3 IU/g substrate. In addition, EMM crude enzymes contained 29.2 and 16.3 IU/mL β-glucosidase on Solka Floc cellulose and corncob power, respectively. The crude enzymes consequently displayed strong biomass hydrolysis performance. For corncob hydrolysis, without supplement of any commercial enzymes, glucose yields of 591.7 and 548.6 mg/g biomass were obtained using enzymes produced from Solka Floc cellulose and corncob powder, respectively. It was 553.9 mg/g biomass using the commercial enzyme mixture of Celluclast 1.5 L and Novozyme 188. Strain T. pinophilus EMM was therefore a potential fungus for on-site enzyme production in biorefinery processes.