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1.
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. 相似文献
2.
A Streptomyces sp. was isolated that produced novel thermoalkalotolerant cellulase activity after growth on crystalline cellulose at 50°C.
Three major components of the cellulases (CMCase, Avicelase and cellobiase) were produced with maximal activities (11.8, 7.8
and 3.9 IU/ml) and maximum specific activities 357, 276 and 118 IU/mg protein, respectively, after 120 h growth. Maximum CMCase
activity was between 50 and 60°C measured over 3 h. The enzyme also retained 88% of its maximum activity at 70°C and pH 5,
and 80% of the activity at pH 10 and 50°C when assayed after 1 h. After incubation at 40°C for 1 h with commercial detergent
(Tide) at pH 11, 95% activity was retained. The enzyme mixture produced glucose from crystalline cellulose. 相似文献
3.
Hajime Yoshioka Shinsaku Hayashida 《Bioscience, biotechnology, and biochemistry》2013,77(12):2817-2824
A thermophilic fungus, Mucor miehei YH-10, isolated from manure was selected to produce thermostable β-glucosidase among 207 isolates. When Mucor miehei YH-10 was grown on wheat bran medium, the maximal accumulation of thermostable β-glucosidase was obtained after 4 days at 50°C, The β-glucosidase had an optimal temperature of 60°C and retained 73% of original activity after heating at 95°C for 5 min. The β-glucosidase was fractionated by Sephadex G-100 chromatography into two components during the purification steps. These components were further purified by consecutive column chromatographies until they were homogeneous on disc electrophoresis. One retained 56% of original activity after heating at 95°C for 5 min, whereas the other was completely inactivated after heating at 80°C for 5 min. 相似文献
4.
Identification of Mycelium-Associated Cellulase from Streptomyces reticuli 总被引:2,自引:3,他引:2
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Gisela Wachinger Karin Bronnenmeier Walter L. Staudenbauer Hildgund Schrempf 《Applied microbiology》1989,55(10):2653-2657
Among 180 Streptomyces strains tested, 25 were capable of hydrolyzing microcrystalline cellulose (Avicel) at 30°C. Streptomyces reticuli was selected for further studies because of its ability to grow at between 30 and 50°C on Avicel. Enzymatic activities degrading Avicel, carboxymethyl cellulose, and cellobiose were found both in the culture supernatant and in association with the mycelium and crystalline substrate. The bound enzymes were efficiently solubilized by repeated washes with buffer of low ionic strength (50 mM Tris hydrochloride [pH 7.5]) and further purified by fast protein liquid chromatography. A high-molecular-weight Avicelase of >300 kilodaltons could be separated from carboxymethyl cellulase (CMCase) and β-glucosidase activities (molecular mass, 40 to 50 kilodaltons) by gel filtration on Superose 12. The CMCase fraction was resolved by Mono Q anion-exchange chromatography into two enzymes designated CMCase 1 and CMCase 2. The β-glucosidase activity was found to copurify with CMCase 2. The purified cellulase components showed optimal activity at around pH 7.0 and temperatures of between 45 and 50°C. Avicelase (but not CMCase) activity was stimulated significantly by the addition of CaCl2. 相似文献
5.
An extracellular alkaline carboxymethycellulase (CMCase) from Bacillus subtilis was purified by salt precipitation followed by anion-exchange chromatography using DEAE-Sepharose. The cell-free supernatant containing crude enzyme had a CMCase activity of 0.34 U/mg. The purified enzyme gave a specific activity of 3.33 U/mg, with 10-fold purification and an overall activity yield of 5.6%. The purified enzyme displayed a protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) with an apparent molecular size of 30 kDa, which was also confirmed by zymogram analysis. The enzyme displayed multisubstrate specificity, showing significantly higher activity with lichenan and β-glucan as compared to carboxymethylcellulose (CMC), laminarin, hydroxyethylcellulose, and steam-exploded bagasse, and negligible activity with crystalline substrate such as Avicel and filter paper. It was optimally active at pH 9.2 and temperature 45°C. The enzyme was stable in the pH range 6–10 and retained 70% activity at pH 12. Thermal stability analysis revealed that the enzyme was stable in temperature range of 20°C to 45°C and retained more than 50% activity at 60°C for 30 min. The enzyme had a Km of 0.13 mg/ml and Vmax of 3.38 U/mg using CMC as substrate. 相似文献
6.
Kavish Kumar Jain Tapati Bhanja Dey Sandeep Kumar Ramesh Chander Kuhad 《Bioprocess and biosystems engineering》2015,38(4):787-796
Thermophilic fungi are potential sources of thermostable enzymes and other value added products. Present study has focused on optimization of different physicochemical parameters for production of thermostable cellulases and xylanase by Thermoascus aurantiacus RCKK under SSF. Enzyme production was supported maximally on wheat bran fed with 20 % inoculum, at initial pH 5, temperature 45 °C and moisture ratio 1:3. The supplementation of wheat bran with yeast extract, Tween-80 and glycine further improved enzyme titres (CMCase 88 IU/g, FPase 15.8 IU/g, β-glucosidase 25.3 IU/g and xylanase 6,543 IU/g). The crude enzymes hydrolyzed phosphoric acid-swollen wheat straw, avicel and untreated xylan up to 74, 71 and 90 %, respectively. In addition, T. aurantiacus RCKK produced antioxidants as fermentation by-products with significant %DPPH? scavenging, FRAP and in vivo antioxidant capacity against H2O2-treated Saccharomyces cerevisiae. These capabilities show that it holds potential to exploit crop by-products for providing various commodities. 相似文献
7.
Aline Rodrigues Gama Carolina Candida Q. Brito-Cunha Ivan T. N. Campos Guilherme Rocha L. de Souza Lilian Carla Carneiro Luiz Artur Mendes Bataus 《Biotechnology progress》2020,36(2):e2934
Lignocellulosic wastes can be potentially converted into several bioproducts such as glucose, xylo-oligosaccharides, and bioethanol. Certain processes, such as enzymatic hydrolysis, are generally needed to convert biomass into bioproducts. The present study investigated the production of xylanases and cellulases by Streptomyces thermocerradoensis I3 under solid-state fermentation (SSF), using wheat bran as a low-cost medium. The activities of xylanase and carboxymethyl cellulase (CMCase) were evaluated until 96 hr of incubation. The highest enzyme activity was observed after 72 hr of incubation. The crude enzyme extract was sequentially filtered, first using a 50 kDa filter, followed by a 30 kDa filter. Fraction 3 (F3) exhibited activities of both xylanase and CMCase. Xylanase and CMCase showed optimum activity at 70°C and pH 6.0 and 55°C and pH 6.0, respectively. The zymogram analysis showed a single activity band with a molecular mass of approximately 17 kDa. These findings provide strong evidence that the enzyme is a bifunctional xylanase/endoglucanase. This enzyme improved the saccharification of sugarcane bagasse by 1.76 times that of commercial cellulase. This enzyme has potential applications in various biotechnological procedures. 相似文献
8.
A cellulase-producing bacterium strain was isolated from soil that produced novel thermoalkalotolerant cellulases after growth
on CMC-Na agar screening plate at 37°C. It was identified as Escherichia coli using the method of 16S rRNA and intergenic spacer gene analysis combined with morphological, physiological, and biochemical
tests. Three major components of the cellulases [carboxymethyl cellulase (CMCase), filter paper cellulase, and β-glucosidase]
were produced with maximal activities (0.23, 0.08, and 0.15 U/ml) and maximum specific activities 4.13, 0.56, and 0.50 U/mg
protein after 72, 96, and 120 h growth, respectively. Maximum CMCase activity was measured at 50°C and pH 6.0, respectively,
and it also retained more than 60% of its maximal activity for at least 20 min at 50–70°C and 10 min at 80°C, respectively,
and retained approximately 50% of its maximal activity after incubating at 90°C for 10 min. The enzyme could be applied in
bioconversion of lignocellulosic agricultural wastes. 相似文献
9.
Hye-Jin Kim You-Jung Lee Wa Gao Chung-Han Chung Chang-Woo Son Jin-Woo Lee 《Biotechnology and Bioprocess Engineering》2011,16(3):542-548
The optimal conditions for the production of cellulases by a marine bacterium, Psychrobacter aquimaris LBH-10, were established and their effects were compared using orthogonal array experiments based on the Taguchi method.
The optimal conditions of rice bran, peptone and initial pH for the production of avicelase and CMCase by P. aquimaris LBH-10 were 50.0, 3.0, and 8.0 g/L, respectively, whereas those for filter paperase (FPase) were 100.0, 3.0, and 8.0 g/L,
respectively. Rice bran was found to be the most important factor for the production of cellulases based on the calculated
percentage of participation P (%) from an analysis of the variance (ANOVA). The optimal temperature for the cell growth of P. aquimaris LBH-10 was 25°C, whereas that for the production of avicelase, CMCase and FPase was 30°C. The optimal agitation speed and
aeration rate for cell growth was 400 rpm and 1.5 vvm, respectively, whereas those for the production of CMCase were 300 rpm
and 1.0 vvm, respectively. Aeration was found to be more important for cell growth and CMCase production than agitation. The
maximum production of avicelase, CMCase and FPase in a 100 L bioreactor for 72 h under optimized conditions was 83.2, 388.7,
and 75.4 U/mL, respectively. 相似文献
10.
Production and characterization of thermostable cellulases from Streptomyces transformant T3-1 总被引:2,自引:0,他引:2
A total of 26 thermophilic isolates, selected from a compost of agricultural waste, which was mostly composed of vegetable, corncob and rice straw, were cultivated at 50 °C for further studies of thermostable cellulase production. The thermostable cellulase gene from the chromosomal DNA of actinomycetes isolate no. 10 was shotgun-cloned and transformed into Streptomyces sp. IAF 10-164. A transformant, T3-1, was found to be a good strain for the production of thermostable cellulases. Cultivation of T3-1 in modified Mandels–Reese broth containing 1% carboxymethylcellulose (CMC)-sodium salt and the optimal condition for microbial growth were studied. Batch cultivation in a flask revealed that CMCase and Avicelase production reached the maximum between the third to fifth day, whereas maximum -glucosidase production occurred on the ninth day. Microbial biomass increased from the first day to the fifth day and then decreased. The crude enzyme had the highest activity at 50 °C and at pH 6.5. The enzyme was shown to be a thermostable cellulase whose activities were stable at 50 °C for more than 7 days. 相似文献
11.
L. Benoit C. Cailliez A. Gehin J. Thirion G. Raval H. Petitdemange 《Current microbiology》1995,30(5):305-312
The extracellular cellulase enzyme system of Clostridium A11 was fractionated by affinity chromatography on Avicel: 80% of the initial carboxymethylcellulase (CMCase) activity was adhered. This cellulase system was a multicomponent aggregate. Several CMCase activities were detected, but the major protein P1 had no detectable activity. Adhered and unadhered cellulases showed CMCase activity with the highest specific activity in Avicel-adhered fraction. However, only afhered fractions could degrade Avicel. Thus, efficiency of the enzymatic hydrolysis of Avicel was related to the cellulase-adhesion capacity. Carboxymethylcellulase and Avicelase activities were studied with the extracellular enzyme system and cloned cellulases. Genomic libraries from Clostridium A11 were constructed with DNA from this Clostridium, and a new gene cel1 was isolated. The gene(s) product(s) from cel1 exhibited CMCase and p-nitrophenylcellobiosidase (pNPCbase) activities. This cloned cellulase adhered to cellulose. Synergism between adhered enzyme system and cloned endoglucanases was observed on Avicel degradation. Conversely, no synergism was observed on CMC hydrolysis. Addition of cloned endoglucanase to cellulase complex led to increase of the Vmax without significant K
m
variation. Cloned endoglucanases can be added to cellulase complexes to efficiently hydrolyze cellulose. 相似文献
12.
《Journal of Fermentation and Bioengineering》1994,77(1):109-111
Thermophilic Humicola sp. secreted thermostable xylanases when grown on wheat bran medium at 50°C. DEAE-Sephadex A-50 column chromatography of the crude xylanase separated three fractions of xylanase (I, II and III), xylanase I being homogeneous in polyacrylamide gel electrophoresis after CM-Sephadex column chromatography. The respective xylanases, including the crude xylanase, increased pulp brightness but xylanases II and III decreased the viscosity of the pulp due to CMCase activity. The crude xylanase contained lower CMCase activity than xylanases II and III. 相似文献
13.
Jean Carlos Rodrigues Silva Luis Henrique Souza Guimarães José Carlos Santos Salgado Rosa Prazeres Melo Furriel Maria Lourdes T. M. Polizeli José César Rosa João Atilio Jorge 《Folia microbiologica》2013,58(6):561-568
Two cellulases from Scytalidium thermophilum were purified and characterized, exhibiting tolerance to glucose and cellobiose. Characterization of purified cellulases I and II by mass spectrometry revealed primary structure similarities with an exoglucanase and an endoglucanase, respectively. Molecular masses were 51.2 and 45.6 kDa for cellulases I and II, respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Cellulases I and II exhibited isoelectric points of 6.2 and 6.9 and saccharide contents of 11 and 93 %, respectively. Optima of temperature and pH were 60–65 °C and 4.0 for purified cellulase I and 65 °C and 6.5 for purified cellulase II. Both cellulases maintained total CMCase activity after 60 min at 60 °C. Cysteine, Mn2+, dithiotreitol and ß-mercaptoethanol-stimulated cellulases I and II. The tolerance to cellulose hydrolysis products and the high thermal stabilities of Scytalidium cellulases suggest good potential for industrial applications. 相似文献
14.
Kuhad Ramesh Chander Kapoor Mukesh Rustagi Renuka 《World journal of microbiology & biotechnology》2004,20(3):257-263
An alkalophilic Streptomyces sp. RCK-SC, which produced a thermostable alkaline pectinase, was isolated from soil samples. Pectinase production at 45
°C in shaking conditions (200 rev min−1) was optimal (76,000 IU l−1) when a combination of glucose (0.25% w/v) and citrus pectin (0.25% w/v) was added along with urea (0.25% w/v) in the basal
medium devoid of yeast extract and peptone. All the tested amino acids and vitamins greatly induced pectinase production and
increased the specific productivity of pectinase up to 550%. In an immobilized cell system containing polyurethane foam (PUF),
the pectinase production was enhanced by 32% (101,000 IU l−1) compared to shake flask cultures. In solid-state cultivation (SSC) conditions, using wheat bran as solid substrate, pectinase
yield of 4857 IU g−1 dry substrate was obtained at substrate-to-moisture ratio of 1:5 after 72 h of incubation. The partially purified pectinase
was optimally active at 60 °C and retained 80% of its activity at 50 °C after 2 h of incubation. The half life of pectinase
was 3 h at 70 °C. Pectinase was stable at alkaline pH ranging from 6.0 to 9.0 for more than 8 h at room temperature retaining
more than 50% of its activity.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
15.
Bacillus subtilis AU-1 was found to produce carboxymethylcellulase (CMCase) and Avicelase activities in the culture supernatant when grown on a variety of carbohydrates as major carbon source. Maximum CMCase production was obtained in a liquid medium containing 0.2% D (+) raffinose as inducer, 0.5% each of yeast extract, casamino acids and proteose peptone at 50 °C and at an initial pH of 6.0. CMCase activity was detected at early log phase of growth, and reached the maximum level at early stationary phase of growth which occurred at the 10th hour of cultivation. The optimal temperature for CMCase activity was 65 °C, and the enzyme was highly stable up to 60 °C. CMCase synthesis was subjected to catabolite repression by glucose and cellobiose. 相似文献
16.
Bukhtojarov FE Ustinov BB Salanovich TN Antonov AI Gusakov AV Okunev ON Sinitsyn AP 《Biochemistry. Biokhimii?a》2004,69(5):542-551
Using different chromatographic techniques, eight cellulolytic enzymes were isolated from the culture broth of a mutant strain of Chrysosporium lucknowense: six endoglucanases (EG: 25 kD, pI 4.0; 28 kD, pI 5.7; 44 kD, pI 6.0; 47 kD, pI 5.7; 51 kD, pI 4.8; 60 kD, pI 3.7) and two cellobiohydrolases (CBH I, 65 kD, pI 4.5; CBH II, 42 kD, pI 4.2). Some of the isolated cellulases were classified into known families of glycoside hydrolases: Cel6A (CBH II), Cel7A (CBH I), Cel12A (EG28), Cel45A (EG25). It was shown that EG44 and EG51 are two different forms of one enzyme. EG44 seems to be a catalytic module of an intact EG51 without a cellulose-binding module. All the enzymes had pH optimum of activity in the acidic range (at pH 4.5-6.0), whereas EG25 and EG47 retained 55-60% of the maximum activity at pH 8.5. Substrate specificity of the purified cellulases against carboxymethylcellulose (CMC), beta-glucan, Avicel, xylan, xyloglucan, laminarin, and p-nitrophenyl-beta-D-cellobioside was studied. EG44 and EG51 were characterized by the highest CMCase activity (59 and 52 U/mg protein). EG28 had the lowest CMCase activity (11 U/mg) amongst the endoglucanases; however, this enzyme displayed the highest activity against beta-glucan (125 U/mg). Only EG51 and CBH I were characterized by high adsorption ability on Avicel cellulose (98-99%). Kinetics of Avicel hydrolysis by the isolated cellulases in the presence of purified beta-glucosidase from Aspergillus japonicus was studied. The hydrolytic efficiency of cellulases (estimated as glucose yield after a 7-day reaction) decreased in the following order: CBH I, EG60, CBH II, EG51, EG47, EG25, EG28, EG44. 相似文献
17.
《Journal of Fermentation and Bioengineering》1992,73(6):461-466
In order to investigate the interactive adsorption behaviors between each cellulase component purified from Trichoderma viride cellulase on microcrystalline cellulose, the adsorption of CMCase, Avicelase, and various compositions of CMCase and Avicelase was performed at 25–45°C. All adsorptions were found to apparently obey the Langmuir isotherm and the thermodynamic parameters, ΔHa, ΔSa, and ΔGa were calculated from the adsorption equilibrium constant, Kad. The adsorption process was found to be endothermic and an adsorption entropy-controlled reaction. The amount of adsorption of cellulase components decreased with increasing temperature and varied with a change in composition of the cellulase components. The maximum synergistic degradation occurred at the specific mass ratio of the cellulase components at which the maximum affinity of cellulase components occurred. 相似文献
18.
Hua Li Jing Chen Anna Li Duo-Chuan Li 《World journal of microbiology & biotechnology》2007,23(9):1297-1303
A thermostable extracellular β-1,3-glucanase from Chaetomium thermophilum was purified to homogeneity by fractional ammonium sulfate precipitation, Pheny1-Sepharose hydrophobic interaction chromatography,
ion exchange chromatography on DEAE-Sepharose and gel filtration on Sephacryl S-100. SDS-PAGE of the purified enzyme showed
a single protein band of molecular weight 76.3 kDa. The enzyme exhibited optimum catalytic activity at pH 6.0 and 60 °C. It
was thermostable at 50 °C, and retained 90% activity after 60 min at 60 °C. The half-life at 65 °C, 70 °C and 80 °C was 55 min,
21.5 min, and 5 min, respectively. The N-terminal amino acid sequence (8 residues) of the enzyme was HWLGDIPH. The HPLC analysis
showed that the only enzymatic product formed from laminarin by the purified β-1,3-glucanase was glucose, indicating that
the enzyme is an exo-β-1,3-glucanase (EC 3.2.1.58). 相似文献
19.
Kofi Abokitse Meiqun Wu Hélène Bergeron Stephan Grosse Peter C. K. Lau 《Applied microbiology and biotechnology》2010,87(1):195-203
A putative α/β hydrolase fold-encoding gene (locus tag TTE1809) from the genome of Thermoanaerobacter tengcongensis was cloned and expressed in Escherichia coli as a possible source of thermostable feruloyl esterase (FAE) for the production of antioxidant phenolic acids from biomass.
Designated as TtFAE, the 33-kDa protein was purified to apparent homogeneity. The lipase-like sequence characteristics of
TtFAE and its substrate specificity towards methyl ferulate, methyl sinapate, and methyl p-coumarate classify it as a new member of the type A FAEs. At 75°C, the enzyme retained at least 95% of its original activity
for over 80 min; at 80°C, its half-life was found to be 50 min, rendering TtFAE a highly thermostable protein. Under different
hydrolytic conditions, ferulic acid (FA) was shown to be released from feruloylated oligosaccharides prepared from triticale
bran. An estimated recovery of 68 mg FA/100 g triticale bran was demonstrated by a 30% release of the total FA from triticale
bran within a 5-h incubation period. Both the oxygen radical absorbing capacity values of the feruloylated oligosaccharides
and free FA were also determined. Overall, this work introduces a new bacterial member to the growing family of plant cell
wall degrading FAEs that at present is largely of fungal origin, and it benchmarks the bioproduction of FA from triticale
bran. 相似文献
20.
A strong fibrin-specific fibrinolytic enzyme was purified from the cell-free spent culture broth of a thermophilic organism,
Streptomyces megasporus SD5. The strain could produce 150 mg crude protein per litre of spent broth, with a specific activity of 80 IU (Plough units)
per milligram, within 18 h of incubation at 55 °C in glucose yeast/extract/peptone (GYP) medium, pH 8.0. For production of
the enzyme, the strain could utilize different carbon and nitrogen sources with a C:N ratio of ∼ 1:2. The enzyme was stable
at a broad range of pH ranging from 5 to 9, and highly thermostable with 50% activity after storage at 60 °C for 6 months.
The enzyme belonged to the serine endopeptidase group. In vitro clot lysis revealed that the enzyme was active at 37 °C.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献