Simultaneous production and induction of cellulolytic and xylanolytic enzymes in aStreptomyces sp.

Extracellular cellulolytic and xylanolytic enzymes ofStreptomyces sp. EC22 were produced during submerged fermentation. The cell-free culture supernatant of the streptomycete grown on microcrystalline cellulose contained enzymes able to depolymerize both crystalline and soluble celluloses and xylans. Higher cellulase and xylanase activities were found in the cell-free culture supernatant of the strain when grown on microcrystalline cellulose than when grown on xylan. Total cellulase and endoglucanase [carboxymethyl-cellulase (CMCase)] activities reached maxima after 72 h and xylanase activity was maximal after 60h. Temperature and pH optima were 55°C and 5.0 for CMCase activity and 60°C and 5.5 for total crystalline cellulase and xylanase activities. At 80°C, approximate half-lives of the enzymes were 37, 81 and 51 min for CMCase, crystalline cellulose depolymerization and xylanase, respectively.     

Adsorption characteristics of cellulolytic enzymes from the anaerobic fungus Piromyces sp. strain E2 on microcrystalline cellulose.

Characteristics of the cellulolytic system of the anaerobic fungus Piromyces sp. strain E2 with respect to adsorption onto microcrystalline cellulose were examined. Cellulolytic enzymes were separated by gel filtration chromatography into a high-molecular-mass complex with an apparent mass of approximately 1,200 to 1,400 kDa and proteins of lower molecular weights. Adsorption of cellulolytic enzymes was not only very fast (within 2 min, equilibrium was attained) but also very effective: Avicelase, endoglucanase, and beta-glucosidase activities from the high-molecular-mass complex were almost completely removed by Avicel. Adsorption of these enzyme activities was proportional and appeared to obey the Langmuir isotherm. For Avicelase, endoglucanase, and beta-glucosidase activities, the maximum amounts adsorbed (Amax) and apparent adsorption constants (Kad) were 16.8, 600, and 33.5 IU/g and 284, 6.93 and 126 ml/IU, respectively. The results of this study strongly support the existence of a multiprotein enzyme complex. This complex was found not to be specifically associated with cell wall fragments as judged by chitin determination.     

Saccharification of cellolulose by the cellulolytic enzyme system of Thermonospora sp. I. Stability of cellulolytic activities with respect to time,temperature, and pH

The stabilities and optima with respect to temperature and pH of the β-glucosidase, Avicelase, and carboxymethylcellulase (CMCase) activity of Thermomonospora sp., in the culture filtrate, culture whole broth, and filtrate after sonication of culture solids, are reported. The β-glucosidase is cell associated and has an optimal activity at about pH 6.5 and 55°C. In the whole culture broth, it has a half-life of about 8 hr at 55°C and less than 1 hr at 60°C, while the half-life of the activity in the sonicated, cell-free filtrate is less than 1 hr at 55°C. The Avicelase and CMCase activities occur in the extracellular culture fluid and have optima at about pH 7.0 and 5.9, and 65 and 70°C, respectively. The CMCase activity is stable over 24 hr at 60°C, but declines by 50% in the same period at 65°C. The Avicelase activity declines by 15% over 24 hr at 55°C, and by 50% at 60°C. The highest pH studied (pH 7.3) was the most destabilizing for all three activities. The thermostable characteristics of the cellulases from Themomonospora appear to make them suitable for commercial saccharification processes operating at elevated temperatures.     

Pectinolytic Enzymes from Actinomycetes for the Degumming of Ramie Bast Fibers

Actinomycetes isolated from 10 different soil and compost samples were screened for production of pectinolytic enzyme activities when grown on pectin-containing solid and liquid media. Pectinolytic enzymes, detected by using plate diffusion tests with a medium containing ramie (Boehmeria nivea) plant material as the sole carbon source, were mainly pectate lyases, but low activities of pectinesterases were also observed. Polygalacturonases and polymethylgalacturonases were not produced. Multiple forms of pectate lyases were detected in the culture supernatants of some of the strains by using the zymogram technique of isoelectric focusing gels. Xylanolytic and cellulolytic activities were always found to be associated with pectinolytic activities. None of the pectinolytic enzymes were produced in a medium with glucose as the sole carbon source. Treatment of ramie bast fibers with crude enzyme preparations from a selection of strains showed a good correlation between the pectate lyase activity applied and the degumming effect, resulting in good separation of the bast fibers.     

Production of cellulase enzymes during the solid-state fermentation of empty palm fruit bunch fiber

Penicillium verruculosum COKE4E is a fungal strain isolated from bituminous coal. The microorganism cultivated in a minimal medium supplemented with Avicel, carboxymethylcellulose, and oat spelt xylan produced cellulase enzymes as exhibiting carboxymethylcellulase (CMCase), Avicelase, xylanase, and cellobiosidase activities. In this study, the productivity of the extracellular enzymes in the strain was evaluated by using empty palm fruit bunch fiber (EPFBF), a lignocellulosic biomass, as a substrate for solid-state bioconversion. The highest cellulase activities were observed after 6 days of fermentation at pH 6.0 and 30 °C. The enzymes were secreted as cellulosomes for the degradation of EPFBF as a sole carbon source. Focused ion beam analysis showed that P. verruculosum COKE4E produced cellulolytic enzymes that were able to effectively biodegrade EPFBF during solid-state fermentation. In this process, 6.5 U of CMCase, 6.8 U of Avicelase, and 8.8 U of xylanase per gram of dry solid EPFBF were produced. These results demonstrate that EPFBF may be a potential raw material in solid-state fermentation for the production of cellulase enzymes to be used for biofuel production.     

Xylanase Activity of Phanerochaete chrysosporium

Xylan-degrading enzymes were induced when Phanerochaete chrysosporium was grown at 30°C in shake flask media containing xylan, Avicel PH 102, or ground corn stalks. The highest xylanase activity was produced in the corn stalk medium, while the xylan-based fermentation resulted in the lowest induction. Analytical and preparative isoelectric focusing were used to characterize xylanase multienzyme components. Preparative focusing was performed only with the cultures grown on Avicel and corn stalk. Of over 30 protein bands separated by analytical focusing from the Avicel and corn stalk media, three main groups (I, II, and III) of about five isoenzymes each showed xylanase activity when a zymogram technique with a xylan overlay was used. Enzyme assays revealed the presence of 1,4-β-endoxylanase and arabinofuranosidase activities in all three isoenzyme groups separated by preparative isoelectric focusing. β-Xylosidase activity appeared in the first peak and also as an independent peak between peaks II and III. Denatured molecular masses for the three isoenzyme groups were found to be between 18 and 90 kDa, and pI values were in the range of 4.2 to 6.0. β-Xylosidase has an apparent molecular mass of 20, 30, and 90 kDa (peak I) and 18 and 45 kDa (independent peak), indicating a trimer and dimer structure, respectively, with pI values of 4.2 and 5.78, respectively. Three more minor xylanase groups were produced on corn stalk medium: a double peak in the acidic range (pI 6.25 to 6.65 and 6.65 to 7.12) and two minor peaks in the alkaline range (pI 8.09 to 8.29 and 9.28 to 9.48, respectively). The profile of xylanases separated by isoelectric focusing (zymogram) of culture filtrate from cells grown on corn stalk media was more complex than that of culture supernatants from cells grown on cellulose. The pH optima of the three major xylanase groups are in the range of pH 4 to 5.5.     

Biochemical and Electron Microscopic Studies of the Streptomyces reticuli Cellulase (Avicelase) in Its Mycelium-Associated and Extracellular Forms

Streptomyces reticuli is able to grow efficiently with crystalline cellulose (Avicel) as the sole carbon source. Cultivation in the presence of the nonionic detergent Tween 80 at a concentration of 0.1% led to a 10-fold increase in extracellular cellulolytic activity. Under these conditions, one single 82-kDa cellulase (Avicelase) capable of degrading crystalline and soluble cellulose as well as cellodextrins and p-nitrophenylcellobioside was purified to apparent homogeneity by a procedure which consisted of two consecutive anion-exchange chromatographies followed by chromatofocusing. Aggregation, which was a major problem during protein purification, could be avoided by including Triton X-100 at a concentration of 0.1% in every chromatographic step. The Avicelase was identified in extracellular and mycelium-associated forms, the latter of which could be released efficiently by nonionic detergents. In addition, a 42-kDa truncated form retaining cellulolytic activity was identified which had been generated from the 82-kDa enzyme by a protease. Antibodies raised against the mycelium-associated Avicelase reacted with the 42-kDa derivative and the extracellular form. The mycelial association of the enzyme was confirmed by immunofluorescence and immunoelectron microscopies.     

Tamarind kernel powder co-induces xylanase and cellulase production during submerged fermentation of <Emphasis Type="Italic">Termitomyces clypeatus</Emphasis>

Tamarind kernel powder (TKP), a soluble agro-residue, was used to examine the production of both cellulolytic and xylanolytic enzymes in a submerged culture of Termitomyces clypeatus, an edible mushroom. Soluble TKP containing xyloglucan as the major polysaccharide induced all cellulolytic and xylanolytic enzymes, and enzyme production increased up to 3% (w/v) TKP with culture filtrate consisting of xylanase and CMCase at a ratio of 4: 1 app. Strong catabolic repression of enzyme production was also observed with the soluble substrate, although fed-batch addition of soluble substrate at late growth phase modified the enzyme kinetics by improving the yield by 30%. The results indicate that inducers were possibly released from TKP by cellulose and xylan fractions of the lignocellulosic polymer. Therefore, the present study reports the successful economic utilization of TKP, an abundantly available soluble agro-residue, for the production of both cellulolytic and xylanolytic enzymes in a single fermentation method.     

Growth of Cellulomonas sp. ATCC 21399 on different polysaccharides as sole carbon source Induction of extracellular enzymes

Summary Growth and extracellular enzyme production of Cellulomonas sp. ATCC 21399 on carboxymethylcellulose (CMC), microcrystalline cellulose (Avicel), xylan, galactomannan and starch were compared. The bacteria grew poorly on CMC, whereas high cell densities were obtained on the other substrates. Growth on Avicel resulted in extracellular enzyme activities against CMC, Avicel, xylan, galactomannan and amylose. By contrast, growth on xylan, galactomannan and starch induced only the enzymes neccessary for the degradation of the growth substrate. Extracellular proteinase activity could be measured during growth on all substrates but CMC, and the possibility of proteolytic inactivation of some of the unstable enzymes (i.e. Avicelase and amylase) in discussed.     

Production of cellulolytic enzymes from theXylaria andHypoxylon species of xylariaceae

Eighteen strains of xylariaceous fungi have been screened for higher activities of cellulolytic enzymes,Trichoderma reesei QM 9414 was also examined for comparison. Strains ofXylaria anisopleura andX. regalis had higher endocellulase (CMCase) and exocellulase (Avicelase) activities after 2 weeks' incubation.Hypoxylon stygium produced the highest activity of -glucosidase 3 days after inoculation. The optimum pH for these cellulolytic enzymes was approx. 5.0 and the optimum temperatures ranged from 37 to 50°C. A mixed culture process usingT. reesei QM 9414 andH. stygium was developed to obtain enhanced synthesis of cellulase. -Glucosidase activities in the mixed culture increased within 48h whenH. stygium was introduced after 24h.     

Recovery and fractionation of the extracellular degradative enzymes from Lentinula edodes cultures cultivated on a solid lignocellulosic substrate

The white-rot basidiomycete Lentinula edodes produces shiitake, a commercial edible mushroom grown on wood. Large-scale cultivation of this fungus on lignocellulose particles provides the opportunity to recover its extracellular enzymes in quantity from spent commercial cultures. Here we show that anion exchange chromatography is a particularly useful step in the initial purification and identification of the range of enzymes present in a crude culture filtrate made from a commercial wood-containing medium. We report the level of major degradative enzyme activities detected both in crude filtrates and in fractions resulting from their fractionation by a single representative chromatography run. The enzymes included cellulases, hemicellulases, fungal cell wall-degrading enzymes, oxidative enzymes (including potential ligninases), acid phosphatases, and acid proteinases. Screening for activity in fractions with multiple substrates was a powerful method both to determine the range of different polysaccharidase activities present and to pinpoint enzymes that either were nonspecific or that required further purification.     

Production of extracellular enzymes and degradation of biopolymers by saprotrophic microfungi from the upper layers of forest soil

Production of extracellular enzymes participating in the degradation of biopolymers was studied in 29 strains of nonbasidiomycetous microfungi isolated from Quercus petraea forest soil based on the frequency of occurrence. Most of the isolates were ascomycetes and belonged to the genera Acremonium, Alternaria, Cladosporium, Geomyces, Hypocrea, Myrothecium, Ochrocladosporium, and Penicillium (18 isolates), and two isolates were zygomycetes. Only six isolates showed phenol oxidation activity which was low and none of the strains were able to degrade humic acids. Approximately half of the strains were able to degrade cellulose and all but six degraded chitin. Most strains produced significant amounts of the cellulolytic enzymes cellobiohydrolase and ??-glucosidase and the chitinolytic enzymes chitinase, chitobiosidase, and N-acetylglucosaminidase. The highest cellulase activities were found in Penicillium strains, and the highest activity of chitinolytic enzymes was found in Acremonium sp. The production of the hemicellulose-degrading enzymes ??-galactosidase, ??-galactosidase, and ??-mannosidase was mostly low. The microfungal strains were able to produce significant growth on a range of 41?C87, out of 95 simple C-containing substrates tested in a Biolog? assay, monosaccharides being for all strains the most rapidly metabolized C-sources. Comparison with saprotrophic basidiomycetes from the same environment showed that microfungi have similar cellulolytic capabilities and higher chitinase activities which testifies for their active role in the decomposition of both lignocellulose and dead fungal biomass, important pools of soil carbon.     

Lignocarbohydrate Solubilization from Straw by Actinomycetes

Actinomycetes grown on wheat straw solubilized a lignocarbohydrate fraction which could be recovered by acid precipitation. Further characterization of this product (APPL) during growth of Streptomyces sp. strain EC1 revealed an increase in carboxylic acid and phenolic hydroxyl content, suggesting progressive modification. This was also observed in dioxane-extracted lignin fractions of degraded straw, and some similarity was further suggested by comparative infrared spectroscopy. However, the molecular weight profile of APPL was relatively constant during growth of Streptomyces sp. strain EC1 on straw, while analysis of the dioxane-extracted lignin fractions appeared to show fragmentation followed by repolymerization. Lignocarbohydrate solubilization could be monitored in all cultures by routine assay of APPL-associated protein, which accounted for up to 20% of the extracellular culture protein in some cases. Interestingly, this protein fraction was found to include active hydrolytic and oxidative enzymes involved in the degradation of lignocellulose, and specific enzyme activities were often increased in the acid-insoluble fractions of culture supernatants. This was particularly important for peroxidase and veratryl oxidase activities, which could be readily detected in the acid-precipitable lignocarbohydrate complex but were virtually undetectable in untreated culture supernatants.     

Occurrence of a procellulase in the culture filtrates of Penicillium janthinellum

The endo-1,4-β-d-glucanase [cellulase, 1,4-(1,3:1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] activity of two-day old culture filtrates of Penicillium janthinellum has been enhanced four-fold by incubating with a 10-day old culture filtrate of Penicillium funiculosum grown on the same medium. An inactive protein isolated by fractionation of two-day old culture filtrate of P. janthinellum using preparative isoelectric focusing, showed 30- to 50-fold enhancement of endo-1,4-β-d-glucanase activity. This fraction has been designated the ‘procellulase’ in the present paper. The purity of the procellulase was confirmed by analytical isoelectric focusing and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. It had a molecular weight of 68 000 and an isoelectric point of pH 3.7.     

Isolation by isoelectric focusing of a mitogenic substance released by stimulated human lymphocytes

Supernatants of human lymphocytes incubated for 4 hr with extracellular products of group A streptococci (streptococcal filtrate) and then washed and reincubated had a mitogenic activity for homologous lymphocytes. Fractionation by isoelectric focusing of the lymphocyte supernatants and of the streptococcal filtrates showed mitogenic activity in fractions with different isoelectric points. Moreover, human lymphocytes stimulated either with a mitogenic fraction of the lymphocyte supernatant or with a mitogenic fraction of the streptococcal filtrate, after treatment with bromodeoxyuridine (BUdR) and reciprocal restimulation, were responsive only to the heterologous stimulant.     

Induction of cellulose- and xylan-degrading enzyme systems in Aspergillus terreus by homo- and heterodisaccharides composed of glucose and xylose

Synthetic heterodisaccharides composed of glucose and xylose were tested as inducers of cellulose- and xylan-degrading enzymes in Aspergillus terreus, and the inducing abilities were compared with those of sophorose and xylobiose or their positional isomers. Measurement of secreted and cell-associated enzyme activities revealed that the heterodisaccharides induced the synthesis of the cellulolytic and xylanolytic enzymes, 2-O-beta-D-glucopyranosyl D-xylose (Glcbeta 1-2Xyl) being the most powerful inducer. Sophorose and 2-O-beta-D-xylopyranosyl D-Xylose (Xylbeta 1-2Xyl), or their positional isomers, selectively induced the synthesis of cellulases and beta-xylanases, respectively. An analysis of the extracellular enzymes (which were separated by isoelectric focusing followed by detection using chromogenic and fluorogenic substrates) showed that Glcbeta 1-2Xyl initiated the synthesis of specific endo-1,4-beta-glucanases and specific endo-1,4-beta-xylanases identical to those produced separately in response to sophorose or Xylbeta 1-2Xyl. Glcbeta 1-2Xyl also induced specific endo-1,4-beta-glucanases that hydrolysed 4-methylumbelliferyl beta-lactoside at the agluconic bond. The results strengthen the concept of separate regulatory control of the synthesis of cullulases and beta-xylanases. The results also suggest that mixed disaccharides, composed of glucose and xylose moieties, which may occur in nature, could play an important role in regulating the synthesis of wood-degrading enzymes.     

Biosynthesis of cellulolytic enzymes in semi-continuous cultures

The aim of the research was to check the possibility of the semicontinuous cultivation of two Fusarium sp. strains producing cellulolytic enzymes. The cultures were grown in 5 1 fermentor on the mineral medium with addition of 1% cellulose cotton. In these cultures lasting 9 to 16 days the total activity was about 13 mg of reducing sugar/ml culture filtrate/hour. The comparison of the results of semi-continuous and periodic cultures pointed to the productivity of cellulolytic enzymes which was 1.2 to 2 times higher during one hour in semi-continuous cultures.     

Growth of bacteria on chitin, fungal cell walls and fungal biomass, and the effect of extracellular enzymes produced by these cultures on the antifungal activity of amphotericin B

Vibrio alginolyticus, Streptomyces griseus, Arthrobacter G12, Bacillus sp. and Cytophaga sp. were grown on solid and liquid media containing soluble and insoluble carbon sources. Arthrobacter G12, Bacillus sp. and Cytophaga sp. grew well on media which contained fungal cell walls or fungal biomass as the main carbon source. All bacteria produced extracellular proteases and all bacteria except Arthrobacter G12 produced extracellular chitinases. Growth of Cytophaga sp. on colloidal chitin was paralleled by the accumulated chitinase activity in the culture filtrate, and growth of Cytophaga sp. and Arthrobacter G12 on cell walls of Geotrichum candidum and cell walls of Candida pseudotropicalis was paralleled by the accumulation of laminarinase activity in the culture filtrate, but little or no extracellular chitinase activity was observed in these cultures. Mycolases purified from the culture filtrates of Cytophaga sp. grown on colloidal chitin on cell walls of C. pseudotropicalis potentiated the antifungal activity of amphotericin B.     

Biochemical Characterization of a Protease Involved in the Processing of a Streptomyces reticuli Cellulase (Avicelase)

A 36-kDa protease from Streptomyces reticuli had recently been shown to be responsible for the in vivo and in vitro processing of the 82-kDa cellulase (Avicelase) Cel-1 from S. reticuli to a 42-kDa truncated enzyme. It was induced only in the presence of Avicel, hydroxyethylcellulose, and xylan. The addition of the nonionic detergent Tween 80 to the culture medium containing Avicel as the carbon source led to a 10-fold increase in extracellular proteolytic activity. The protease, which has an isoelectric point of 3.9, was purified to homogeneity from the culture filtrate by a combination of anion-exchange and hydrophobic-interaction chromatographies and was characterized biochemically. The enzyme hydrolyzed gelatin and the chromogenic substrates Azocoll, Azocasein, and Azoalbumin. Its highest activity was determined between pH 7.0 and 7.7 and at 55°C. The proteolytic activity was inhibited by 1,10-phenanthroline and EDTA; however, no metal ions were detected to be associated with the protein. The protease was stable in the presence of 1 M urea and 0.01 M sodium dodecyl sulfate. The inhibitory effect of alpha-2-macroglobulin indicated an endo-mode of proteolytic cleavage. Studies with lectins and sugar analysis by mass spectroscopy indicated that the cellulase (Avicelase) Cel-1 was neither N nor O glycosylated. Its processing by the protease occurred at temperatures ranging from 30 to 55°C, pH 7.5, in the presence of 2 mM dithiothreitol.     

Amylolytic lactic acid bacteria in fish silage

An ∝aL-amylase activity has been observed in lactic acid bacteria occurring initially in fermented fish silage. The organisms belong to the genus Leuconostoc . The main fraction of the amylolytic enzyme produced by one of the isolated bacteria is cell-bound and is released into the medium at a late stage of growth. Treating cells with ultrasound or Triton X-100 increases enzyme activity in the culture filtrate. The pH range for enzyme activity is 5.0–7.0, with an optimum at pH 6.0. The enzyme is extremely labile at pH 8.0 and is inactivated at temperatures above 50°C at pH 5.8. Two enzyme fractions were found by isoelectric focusing, the main one at pH 5.00 and another at pH 4.5. Chromatography on DEAE cellulose gave two active peaks.