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1.
Kim SJ Lee CM Han BR Kim MY Yeo YS Yoon SH Koo BS Jun HK 《FEMS microbiology letters》2008,282(1):44-51
To detect cellulases encoded by uncultured microorganisms, we constructed metagenomic libraries from Korean soil DNAs. Screenings of the libraries revealed a clone pCM2 that uses carboxymethyl cellulose (CMC) as a sole carbon source. Further analysis of the insert showed two consecutive ORFs (celM2 and xynM2) encoding proteins of 226 and 662 amino acids, respectively. A multiple sequence analysis with the deduced amino acid sequences of celM2 showed 36% sequence identity with cellulase from the Synechococcus sp., while xynM2 had 59% identity to endo-1,4-beta-xylanase A from Cellulomonas pachnodae. The highest enzymatic CMC hydrolysis was observable at pH 4.0 and 45 degrees C with recombinant CelM2 protein. Although the enzyme CelM2 additionally hydrolyzed avicel and xylan, no substrate hydrolysis was observed on oligosaccharides such as cellobiose, pNP-beta-cellobioside, pNP-beta-glucoside, and pNP-beta-xyloside. These results showed that CelM2 is a novel endo-type cellulase. 相似文献
2.
J. C. Royer J. S. Novak J. P. Nakas 《Journal of industrial microbiology & biotechnology》1992,11(1):59-61
Summary Purified xylanase A ofTrichoderma longibrachiatum was active on one of two carboxymethyl cellulose (CMC) preparations used as cellulase assay substrates. The pattern of enzyme activity, and analysis of the substrate by acid hydrolysis and thin-layer chromatography (TLC) suggested that the enzyme had acted on xylan present in the CMC. 相似文献
3.
A rate equation has been derived to describe the hydrolysis of benzylpenicillin to 6-aminopenicillanic acid by penicillin amidase. The integrated from of the rate equation has been shown to predict satisfactorily the progress of the reaction in a batch reactor using either soluble or immobilized penicillin amidase. The rate equation was also used to predict the performance of a continuous feed stirred tank reactor containing immobilized enzyme. There was good agreement with experimental measurements. 相似文献
4.
Optimal pH, temperature, and concentration of enzyme conditions for the rate of hydrolysis of five isoflavone conjugates (daidzein, O-desmethylangolensin, equol, genistein, and glycitein) and two lignans (enterodiol and enterolactone) from two biological matrices (urine and plasma) were studied using beta-glucuronidase from Helix pomatia. In addition, the use of mixtures of beta-glucuronidase and sulfatase enzymes from different sources was investigated to find enzyme preparations that contained lower amounts of naturally present phytoestrogens. Quantification of aglycones spiked with (13)C(3)-labeled internal standards was carried out by LC-MS/MS. In urine, all of the phytoestrogen conjugates hydrolyzed within 2h under standard hydrolysis conditions (24mul H. pomatia, pH 5, 37 degrees C). Hydrolysis rates were improved at 45 degrees C and by doubling the enzyme concentration and may be used to further reduce hydrolysis times down to 100min. In plasma, a 16-h hydrolysis was required to ensure complete hydrolysis of all conjugates. As with urine, the use of increased temperature or increased enzyme concentration reduced hydrolysis times for most analytes. However, the rate of hydrolysis in plasma was significantly slower than that in urine for all analytes except enterodiol, for which the reverse was true. Neither increased temperature nor increased enzyme concentration increased the rate of hydrolysis of enterolactone. Hydrolysis at pH 6 proved to be detrimental to hydrolysis of phytoestrogen conjugates, especially those in plasma. Other enzyme preparations from different sources, such as beta-glucuronidase from Escherichia coli, were found to contain lower amounts of contaminating phytoestrogens and showed increased enzyme activity for isoflavones, but lower activity for lignans, when used with other sulfatase enzymes. In addition, this involved complicating the analytical procedure through using mixtures of enzymes. Therefore, the use of beta-glucuronidase from H. pomatia combined with an enzyme "blank" to correct for phytoestrogen contamination was shown to be a suitable method for hydrolysis of phytoestrogens. 相似文献
5.
A colored derivative of sphingomyelin was synthesized and used as substrate for several sphingomyelinases. The compound is N-omega-trinitrophenyl-aminolaurylsphingosylphosphorylcholine. The rate of hydrolysis of this substrate was compared to that of bovine brain sphingomyelin, labelled with tritium in the choline moiety. The following enzyme preparations were used: homogenate-less debris of brain, assayed at pH 5.0 or 7.4; a solubilized preparation derived from rat brain lysosomes, assayed at pH 5.0 and a purified enzyme of Staphylococcus aureus. With all preparations, the rates of hydrolysis of the yellow derivative were very similar to those of the brain sphingomyelin. Extracts of skin fibroblasts of normal and Niemann-Pick patients as well as amniotic cells were also used. Again, the rates of hydrolysis of the yellow derivative practically equalled those using brain sphingomyelin. 相似文献
6.
Characterization of the activation of rat liver beta-glucosidase by sialosylgangliotetraosylceramide
We show that sialosylgangliotetraosylceramide (GM1) is a potent activator of delipidated (sodium cholate- and 1-butanol-extracted) lysosomal rat liver glucocerebroside:beta-glucosidase. Stimulation of 4-methylumbelliferyl-beta-D-glucopyranoside hydrolysis by the beta-glucosidase was markedly dependent upon the concentration of GM1 in the assay medium. Estimations of critical micellar concentration (CMC) performed fluorometrically using the dye N-phenylnaphthylamine revealed two CMC values of GM1 above 18 degrees C; the CMC of the primary micelles (3.32 microM) was temperature-independent whereas that of the secondary micelles decreased with decreasing temperature (17.2 and 10.8 microM at 37 and 20 degrees C, respectively). In the temperature range of 18-39 degrees C, beta-glucosidase activity increased sharply when the GM1 concentration was above the CMC of the secondary micelles. Although a heat-stable factor, purified from the spleen of a patient with Gaucher's disease, had a profound effect on the activation of beta-glucosidase by GM1, it decreased the CMC only slightly (14.8 versus 17.2 microM at 37 degrees C). The heat-stable factor (8 micrograms/ml) changed the shape of the activation curve from sigmoidal to hyperbolic, suggesting that the heat-stable factor permits beta-glucosidase to be activated by primary micelles or monomers. The results of gel filtration chromatography and sucrose gradient centrifugation in H2O and D2O revealed that the activation of beta-glucosidase by GM1 was associated with an increase in the size of the enzyme from 45,800 to 178,500 daltons and an increase in the partial specific volume from 0.697 to 0.740 ml/g. The active, reconstituted beta-glucosidase appears to consist of 50% protein and 50% ganglioside (56 molecules/178,500 g). Concentrations of GM1 below the CMC of secondary micelles increased the rate of inactivation of the enzyme by the irreversible inhibitor conduritol B epoxide at 37 degrees C, indicating that GM1 monomers or primary micelles do interact with the enzyme, even though they do not increase the rate of hydrolysis of 4-methylumbelliferyl-beta-D-glucopyranoside by the enzyme. 相似文献
7.
Enzymatic hydrolysis of soluble cellulose derivatives as measured by changes in viscosity 总被引:5,自引:0,他引:5
Observation of changes in fluidity is presented as a method for following the enzymatic hydrolysis of soluble cellulose derivatives. The activity of different cell-free enzyme preparations may be compared by this method, providing certain precautions are observed. In general, results obtained by use of the fluidity method are similar to those obtained using the reducing sugar technique, indicating that the same enzyme system is measured by the two methods. Changes in the DP of the substratum may be followed within certain limits of molecular size. Results indicate that a random splitting of CMC occurs during enzymatic hydrolysis, with a concomitant decrease in intrinsic viscosity and an increase in reducing sugars. Certain inadequacies of the cellulose-cellobiose-glucose theory, together with more recent findings, have led to the postulation of an alternate explanation of the mechanism of cellulose hydrolysis. 相似文献
8.
The hydrolysis of maltodextrins (10 DE) by glucoamylase was studied in a batch reactor at temperatures between 40 and 80 degrees C and substrate concentration range from 17 to 300 kg/m(-3). The experimental data were fitted to a model including thermal deactivation of the enzyme. In the model, the reaction rate was correlated with an extended Michaelis-Menten equation including inhibition by product, and the thermal deactivation of glucoamylase was fitted with a first-order reaction. The dependence of rate parameters on temperature was correlated using the Arrhenius equation. The differential equation of the model was integrated and the optimal enzyme demand and temperature were determined for isothermal operation. 相似文献
9.
E. A. Kassim 《Folia microbiologica》1983,28(1):36-40
Three types of cellulase preparations were applied to different types of cellulose and cellulosic materials. The action of these types of cellulase on cellulose powder was increased with the increase of enzyme concentration. Both carboxymethyl cellulose (CMC) and sodium carboxymethyl cellulose (Na-CMC) released high amounts of reducing sugar as affected by cellulase application. Different types of paper pulp were moderately hydrolyzed, while agricultural wastes were slightly hydrolyzed. Vegetable and fruits cellulose were equally hydrolyzed but at low rate. Pretreatment of cellulose or cellulosic materials by grinding or by swelling with phosphoric acid gave rise to increased hydrolysis by the enzyme. Cellobiose was detected chromatographically as an intermediate product of hydrolysis of both cellulose and carboxymethyl cellulose with glucose. 相似文献
10.
The maximum product yield of endo-glucanase (650 IU g(-1) substrate) from Cellulomonas biazotea mutant 51 Sm(r) was 1.5- to 2.5-fold more than was produced by the wild type cells and was twice that reported by previous researchers. Mutation substantially improved the enthalpy (DeltaH (*)) and entropy of activation (DeltaS (*)) for product formation, turnover number, specificity constant activation energy, free energies for transition state formation and substrate binding for CMC hydrolysis respectively. 相似文献
11.
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. 相似文献
12.
A recombinant Trichoderma reesei cellulase was used for the ultrasound‐mediated hydrolysis of soluble carboxymethyl cellulose (CMC) and insoluble cellulose of various particle sizes. The hydrolysis was carried out at low intensity sonication (2.4–11.8 W cm?2 sonication power at the tip of the sonotrode) using 10, 20, and 40% duty cycles. [A duty cycle of 10%, for example, was obtained by sonicating for 1 s followed by a rest period (no sonication) of 9 s.] The reaction pH and temperature were always 4.8 and 50°C, respectively. In all cases, sonication enhanced the rate of hydrolysis relative to nonsonicated controls. The hydrolysis of CMC was characterized by Michaelis‐Menten kinetics. The Michaelis‐Menten parameter of the maximum reaction rate Vmax was enhanced by sonication relative to controls, but the value of the saturation constant Km was reduced. The optimal sonication conditions were found to be a 10% duty cycle and a power intensity of 11.8 W cm?2. Under these conditions, the maximum rate of hydrolysis of soluble CMC was nearly double relative to control. In the hydrolysis of cellulose, an increasing particle size reduced the rate of hydrolysis. At any fixed particle size, sonication at a 10% duty cycle and 11.8 W cm?2 power intensity improved the rate of hydrolysis relative to control. Under the above mentioned optimal sonication conditions, the enzyme lost about 20% of its initial activity in 20 min. Sonication was useful in accelerating the enzyme catalyzed saccharification of cellulose. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1448–1457, 2013 相似文献
13.
The effect of crystallinity of cellulose on the rate of reducing sugars production by heterogeneous enzymatic hydrolysis 总被引:4,自引:0,他引:4
Al-Zuhair S 《Bioresource technology》2008,99(10):4078-4085
A kinetic model is devised, from the reaction mechanism steps, to predict the rate of reducing sugar production by hydrolysis of two types of cellulose, namely, amorphous carboxymethylcellulose (CMC) and highly crystalline wood shavings, using Aspergillus niger cellulase. Experimental results in a stirred batch reactor at 40 degrees C show that the production of reducing sugar reduced at much shorter times for wood shavings in comparison to CMC at the same initial substrate concentration. The experimental results are used to determine the kinetic parameters of the model equations. The significance of crystallinity was determined using inert fraction coefficient, which is assumed to be constant and equals 0.05 and 0.98 for CMC and wood shavings, respectively. It is shown there is a good agreement between the experimental results and proposed kinetic model predictions. The effect of the inert fraction coefficient on the production of reducing sugar by the enzymatic hydrolysis of cellulose is also determined. It is found that the cellulase used extracted from A. niger is much more sensitive towards the substrate structure in comparison to that extracted from Trichoderma reesei. 相似文献
14.
Enzymatic hydrolysis of cellulose is often conducted in batch processes in which hydrolytic products tend to inhibit enzyme activity. In this study, we report a method for continuous hydrolysis of carboxymethyl cellulose (CMC) by using cross-linked cellulase aggregate (XCA) trapped inside a membrane. XCA particles prepared by using a millifluidic reactor have a uniform size distribution around 350 nm. Because of their large size, XCA particles in solutions can be filtered through a polyethersulfone membrane to collect 87.1 ± 0.9% of XCA particles. The membrane with impregnated XCA can be used as a catalyst for hydrolysis of CMC in a continuous mode. When the CMC concentration is 1.0 g/l and the flow rate is 2 μl/min, 53.9% of CMC is hydrolyzed to reducing sugars. The membrane with XCA is very stable under continuously flowing solutions. After 72 h of reaction, 97.5% of XCA remains inside the membrane. 相似文献
15.
For more than a century the simple single-substrate enzyme kinetics model and related Henri-Michaelis-Menten (HMM) rate equation have been thoroughly explored in various directions. In the present paper we are concerned with a possible generalization of this rate equation recently proposed by F. Kargi (BBRC 382 (2009) 157-159), which is assumed to be valid both in the case that the total substrate or enzyme is in excess and the quasi-steady-state is achieved. We demonstrate that this generalization is grossly inadequate and propose another generalization based on application of the quasi-steady-state condition and conservation equations for both enzyme and substrate. The standard HMM equation is derived by (a) assuming the quasi-steady-state condition, (b) applying the conservation equation only for the enzyme, and (c) assuming that the substrate concentration at quasi-steady-state can be approximated by the total substrate concentration [S](0). In our formula the rate is already expressed through [S](0), and we only assume that when quasi-steady-state is achieved the amount of product formed is negligible compared to [S](0). Numerical simulations show that our formula is generally more accurate than the HMM formula and also can provide a good approximation when the enzyme is in excess, which is not the case for the HMM formula. We show that the HMM formula can be derived from our expression by further assuming that the total enzyme concentration is negligible compared to [S](0). 相似文献
16.
Summary
Cellulomonas flavigena (strain NIAB 441) produced cellulase and hemicellulase activities when grown on Leptochloa fusca L. Kunth (Kallar grass), found to be the best inducer for enzyme production. The enzyme possessed the potential to saccharify bagasse, Kallar grass straw, wheat straw, carboxymethyl cellulose (CMC) and xylan to reducing sugars. 相似文献
17.
《Journal of biotechnology》1988,8(3):249-255
A genomic bank of Cellulomonas flavigena was constructed in E. coli using the pUC18 vector, and over 14000 clones screened for cellulolytic activity. Three different cellulolytic enzyme genes were cloned, one coding for an endo-β-glucanase (pJS10, CMC activity) and two coding for β-glucosidases, each with a distinct substrate specificity (pJS3, X-glu, and pJS4, X-glu and MUC activities). These three inserts have different restriction patterns to each other and the previously isolated cellulolytic enzyme genes from C. fimi and C. uda. 相似文献
18.
A cellulose-binding protein of 105 kDa (CBP105) from Cellulomonas flavigena was purified and its gene was cloned. CBP105 is a processive endoglucanase with maximum activity on carboxymethyl cellulose
(CMC) at pH 7.5 and 60°C. Limited proteolysis suggested that CBP105 is composed of one catalytic domain (CD) and two carbohydrate-binding
modules (CBM). The nucleotide sequence of the cbp105 gene (AY729806) indicates that CBP105 is a modular enzyme with a family 9 glycoside hydrolase CD linked to a family 3 CBM,
two fibronectin III-like domains and a family 2 CBM. This structural organization may be responsible for CBP105 processive
CMC degradation. 相似文献
19.
Cellulomonas flavigena CDBB-531 was found to secrete a bifunctional cellulase/xylanase with a molecular mass of 49 kDa and pI 4.3. This enzyme was active on Remazol brilliant blue-carboxymethylcellulose (RBB-CMC) and Remazol brilliant blue-xylan (RBB-X). Based on thin-layer chromatographic analysis of the degradation products, the cellulase activity produced glucose, cellobiose, cellotriose, and cellotetraose from CMC as the substrate. When xylan from birchwood was used, end products were xylose, arabinose, and xylobiose. The bifunctional enzyme showed a pH optimum of 6 for cellulase activity and 9 for xylanase activity, which pointed out that this enzyme had separate sites for each activity. In both cases, the apparent optimum temperature was 50 degrees C. The predicted amino acid sequence of purified protein showed similarity with the catalytic domain of several glycosyl hydrolases of family 10. 相似文献
20.
Direct 1H n.m.r. determination of the stereochemical course of hydrolyses catalysed by glucanase components of the cellulase complex 总被引:2,自引:0,他引:2
S G Withers D Dombroski L A Berven D G Kilburn R C Miller R A Warren N R Gilkes 《Biochemical and biophysical research communications》1986,139(2):487-494
The stereochemical courses of the hydrolyses catalysed by three glycosidases have been determined directly by 1H nmr. The anomeric configuration of the initially formed product was ascertained in each case by observation of the chemical shift and coupling constant of the anomeric proton at the new hemiacetal centre. Two of the enzymes investigated, an endo-glucanase and an exo-glucanase are components of the cellulase complex of Cellulomonas fimi. The third enzyme is the beta-glucosidase from almond emulsin. Two of these enzymes, the exo-glucanase and the almond beta-glucosidase catalysed hydrolysis with retention of anomeric configuration, in agreement with previous observations on the almond enzyme. The endo-glucanase catalysed hydrolysis with inversion of configuration, this result being confirmed by optical rotation measurements. This 1H nmr approach has several advantages over other techniques in that it is applicable to a wide variety of glycosidases and substrates and it is non-destructive, allowing recovery of the enzyme. 相似文献