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1.
The induction of synthesis of the secreted enzymes endo-1,4--xylanase (EC 3.2.1.8) and -galactosidase (EC 3.2.1.23) in original and recombinant Penicillium canescens strains has been studied. In all producer strains, the synthesis of these enzymes was induced by arabinose and its metabolite arabitol. The two enzymes differed in the concentration of arabinose required for induction: the synthesis of -galactosidase was most pronounced at 1 mM, whereas maximum synthesis of endo-1,4--xylanase was observed at 5–10 mM. An increase in the number of endo-1,4--xylanase copies in the high-copy-number strain of the fungus suppressed the synthesis of -galactosidase; the synthesis of endo-1,4--xylanase in the high-copy-number recombinant producing -galactosidase was affected to a lesser extent. The amount of enzymes synthesized did not depend on the saccharide used as the sole source of carbon for growing the mycelium prior to its transfer to the inducer-containing medium. 相似文献
2.
Vavilova EA Antonova SV Barsukov ED Vinetskiĭ IuP 《Prikladnaia biokhimiia i mikrobiologiia》2003,39(3):284-292
The fungus Penicillium canescens strain F178 (VKPM) and its niaD- mutant exhibited an increased capability of synthesizing extracellular enzymes beta-galactosidase (70-80 U/ml) and xylanase (100 U/ml). The synthesis was induced by arabinose and its catabolite, arabitol. A deficiency in arabitol dehydrogenase, leading to arabitol accumulation in the cell, was detected in the chain of reactions of arabinose catabolism. The increased synthesis of beta-galactosidase and xylanase in P. canescens is accounted for by (1) cellular accumulation of the inducer (arabitol) at low concentrations of arabinose in the medium and (2) prevalence of induction over repression. 相似文献
3.
Krisana A Rutchadaporn S Jarupan G Lily E Sutipa T Kanyawim K 《Journal of biochemistry and molecular biology》2005,38(1):17-23
During the screening of xylanolytic enzymes from locally isolated fungi, one strain BCC14405, exhibited high enzyme activity with thermostability. This fugal strain was identified as Aspergillus cf. niger based on its morphological characteristics and internal transcribed spacer (ITS) sequences. An enzyme with xylanolytic activity from BCC14405 was later purified and characterized. It was found to have a molecular mass of ca. 21 kDa, an optimal pH of 5.0, and an optimal temperature of 55 degrees C. When tested using xylan from birchwood, it showed K(m) and V(max) values of 8.9 mg/ml and 11,100 U/mg, respectively. The enzyme was inhibited by CuSO(4) EDTA, and by FeSO(4) The homology of the 20-residue N-terminal protein sequence showed that the enzyme was an endo-1,4-beta-xylanase. The full-length gene encoding endo-1,4-beta-xylanase from BCC14405 was obtained by PCR amplification of its cDNA. The gene contained an open reading frame of 678 bp, encoding a 225 amino acid protein, which was identical to the endo-1,4-a-xylanase B previously identified in A. niger. 相似文献
4.
Activities of cellulolytic and hemicellulolytic enzymes endo-1,4-beta-glucanase, exo-1,4-beta-glucanase, 1,4-beta-glucosidase, endo-1,4-beta-xylanase, 1,4-beta-xylosidase and 1,4-beta-mannosidase and ligninolytic enzymes Mn-peroxidase and laccase were detected during the growth of the white-rot fungus Pleurotus ostreatus on wheat straw in the presence and absence of cadmium. The loss of substrate dry weight and Mn-peroxidase activity decreased with increasing Cd concentration, whereas the activities of endo-1,4-beta-glucanase, 1,4-beta-glucosidase and laccase were highly increased in the presence of metal. The onset of hemicellulose-degrading enzyme activity was delayed in the presence of cadmium. The degradation of a model synthetic dye Poly B-411 did not correspond to the activities of ligninolytic enzymes. This is the first report about 1,4-beta-mannosidase in P. ostreatus. 相似文献
5.
Two endo-1,4-beta-zylanases (m. w. 24,000 and 41,000) and six exo-1,4-beta-xylosidases, differing in their molecular weights and isoelectric points, were found in a xylanase preparation from Aspergillus niger, using different methods of fractionation. An electrophoretically homogeneous exo-1,4-beta-xylosidase (m. w. 30,000) purified 120-fold, with pI 4.6, having optimal effect on methyl-beta-D-xyloside at pH 3.0 was obtained. Exo-1,4-beta-xylosidase splits off xylose from the ends of the xylan chains at xylotriose, xylobiose and methyl-beta-D-xyloside and is characterized by a high transglycosilase activity. An electrophoretically homogeneous endo-1,4-beta-xylanase (m. w. 24,000) purified 250-fold, with pI 4.2 and optimal effect on carboxymethylxylan at pH 4.2 was isolated. Endo-1,4-beta-xylanase splits arabinoglucuronoxylan to form xylooligosaccharides; however, it does not hydrolyze xylobiose. 相似文献
6.
7.
Synthesis and hydrolysis of 1,3-beta-xylosidic linkages by endo-1,4-beta-xylanase of Cryptococcus albidus 总被引:1,自引:0,他引:1
Purified extracellular endo-1,4-beta-xylanase (EC 3.2.1.8) of the yeast Cryptococcus albidus was found to catalyze not only the known 1,4-beta-transfer, but an alternative transglycosylation reaction leading to the formation of 1,3-beta-glycosidic linkages. From a mixture of products of beta-xylanase degradation of phenyl beta-D-xylopyranoside three xylooligosaccharide fractions, differing chromatographically from the 1,4-beta-linked products, were isolated by preparative paper chromatography. Their structure was elucidated by mass spectrometry, 13C-NMR spectroscopy and enzymic hydrolysis by beta-xylanase and beta-xylosidase. The isomeric xylotriose was identified as 3-O-beta-D-xylopyranosyl-4-O-beta-D-xylopyranosyl-D-xylose. The fraction of isomeric tetrasaccharides was found to be represented mainly by 4-O-beta-D-xylopyranosyl-3-O-beta-D-xylopyranosyl-4-O-beta-D-xylopyranosyl- D-xylose. The xylooligosaccharides containing one 1,3-beta-linkage were also produced on the enzyme treatment of 1,4-beta-xylotriose and 1,4-beta-xylan. When treated with the enzyme responsible for their synthesis, the isomeric xylooligosaccharides were hydrolyzed at the 1,3-beta-linkage, despite the fact the enzyme does not attack 1,3-beta-xylan. The results are interpreted in the relation to the characterized four-subsite substrate-binding site of the enzyme. 相似文献
8.
Intracellular concentration of cAMP regulates the synthesis of enzymes sensitive to catabolite repression. The relationship between the single and multiple induction of beta-galactosidase (EC 3.2.1.23), L-tryptophanase (EC 4.1.99.1), D-serine deaminase (EC 4.2.1.14), L-asparaginase (EC 3.5.1.1) and L-malate dehydrogenase (EC 1.1.1.37) was studied and the effect of cAMP level on the induction in Escherichia coli Crookes (ATCC 8739) was investigated. A varying degree of catabolite repression was observed during induction of individual enzymes induced separately on different energy sources. The synthesis of l-tryptophanase was most sensitive, whereas l-asparaginase was not influenced at all. Exogenous cAMP was found to overcome partially the catabolite repression of beta-galactosidase and D-serine deaminase, both during single induction. The synthesis of l-malate dehydrogenase was negatively influenced by the multiple induction even in the presence of cAMP; on the other hand, the synthesis of l-tryptophanase was stimulated, independently of the level of the exogenous cAMP. Similarly, the activity of L-asparaginase slightly but significantly increased during the multiple induction of all five enzymes; here too the activity increase did not depend on exogenous cAMP. 相似文献
9.
The substrate binding site of an acidic endo-1,4-beta-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) of Aspergillus niger was investigated using 1,4-beta-xylooligosaccharides (1-3H)-labelled at the reducing end. Bond cleavage frequencies and V/Km parameters of the oligosaccharides were determined under conditions of unimolecular hydrolysis and, according to the method of Suganuma et al. (J. Biochem. (Tokyo) (1978) 84, 293-316), used for evaluation of subsite affinities. The substrate binding site of the enzyme was found to consist of seven subsites, numbered -IV, -III, -II, -I, I, II and III, towards the subsite binding the reducing end unit of xyloheptaose. The catalytic groups were localized between subsites -I and I, the affinities of which have not been determined. All other subsites showed positive values of affinities for binding xylosyl residues. The values decrease from subsites -II and II, similarly in both directions. As a consequence of such an almost symmetric distribution of affinities around the catalytic groups, the enzyme cleaves preferentially the bonds in the oligosaccharides which are most distant from both terminals. Thus, the acidic A. niger beta-xylanase appears to be an endo-1,4-beta-xylanase attacking polymeric substrates in a random fashion. This conclusion was supported by viscosimetric measurements with carboxymethylxylan as a substrate. 相似文献
10.
Thermoactinomyces thalophilus produced cellulase free extracellular endo-1,4-beta-xylanase (EC 3.2.1.8) at 50 degrees C and pH 8.5. Maximum xylanase production was achieved in fermentation medium using birchwood xylan as substrate after 96 h of growth at 50 degrees C. Other agricultural substrates such as wheat bran, wheat straw, sugarcane bagasse and cornstover produced less xylanase. The crude enzyme preparation from mutant T. thalophilus P2 grown under optimised fermentation conditions showed no cellulase contamination and maximum xylanase activity of 42 U/ml at 65%deg;C and pH 8.5-9.0. This enzyme with initial xylanase activity of 42 U/ml was found thermostable up to 65 degrees C and retaining 50% of its activity after its incubation for 125 min at 65 degrees C. 相似文献
11.
Low-molecular-weight xylanase from Trichoderma viride 总被引:1,自引:0,他引:1
An endo-1,4-beta-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) has been isolated from a commercial preparation of Trichoderma viride. The molecular weight was 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the pI value was 9.3. The xylanase was a true xylanase without cellulase activity. When the N-terminal amino acid sequence of the first 50 residues was compared with that of a xylanase from Schizophyllum commune, strong evidence for homology was found, with more than 50% amino acid identity. T. viride xylanase also possessed extensive identity with a proposed amino-terminal consensus sequence of xylanases from bacteria. 相似文献
12.
An endo-1,4-beta-xylanase (1,4-beta-D-xylan xylanohydrolase, EC 3.2.1.8) has been isolated from a commercial preparation of Trichoderma viride. The molecular weight was 22,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the pI value was 9.3. The xylanase was a true xylanase without cellulase activity. When the N-terminal amino acid sequence of the first 50 residues was compared with that of a xylanase from Schizophyllum commune, strong evidence for homology was found, with more than 50% amino acid identity. T. viride xylanase also possessed extensive identity with a proposed amino-terminal consensus sequence of xylanases from bacteria. 相似文献
13.
The mechanism of induction of secreted beta-galactosidase was studied in the filamentous fungus Penicillium canescens. L-Arabinose and its metabolite L-arabitol induce the synthesis of the enzyme. Apart from beta-galactosidase, L-arabinose induces the synthesis of other extracellular carbohydrolases including alpha-L-arabinofuranosidase. Increasing L-arabinose concentration above 1 mM or addition of other carbon sources results in carbon catabolite repression of the synthesis of the secreted enzymes. The data suggest that arabinofuranosidase can regulate the synthesis of secreted enzymes in P. canescens, thus controlling the level of free L-arabinose. 相似文献
14.
提取了台湾家白蚁总RNA并反转录获得eDNA,PCR扩增出白蚁内切葡聚糖酶的基因,并将目的基因分别克隆到大肠杆菌和酿酒酵母载体中,构建了产内切-β-1,4-葡聚糖酶的基因工程菌。由于大肠杆菌会有少量的泄漏表达,而所用的酿酒酵母表达载体是本实验室构建带有INU信号肽的表达载体,故都可采用刚果红平板染色法筛选具有羧甲基纤维素酶(CMCase)活性的重组转化子。利用金属镍亲和层析对大肠杆菌表达的内切-β-1,4-葡聚糖酶进行纯化,CMC酶活检测显示纯化酶的最适温度和最适pH值分别为42℃、6.5;内切-β-1,4-葡聚糖酶的Vmax为0.071mg/mL·min,Km值为80.2712mg/mL。 相似文献
15.
A Fusarium graminearum-based enzyme blend for the efficient hydrolysis of hemicellulose, a crucial step for competitive bioethanol production, is described. The heretofore-uncharacterized endo-1,4-beta-xylanase (XylD), 1,4-beta-xylosidase (XyloA), and bifunctional xylosidase/arabinofuranosidase (Xylo/ArabA) were produced at high levels in Escherichia coli (10-38 mg/l). They displayed compatible pH and temperature-dependences, allowing their utilization for simultaneous substrate digestions. Monosaccharide analysis indicated a strong positive synergism between the enzymes during the degradation of oat spelt xylan. Two units of each protein catalyzed the release of 61% and 15% of the total amount of available d-xylose and l-arabinose, respectively, in only 4 h. The detailed cooperative mechanism of the three hydrolases was elucidated by polysaccharide analysis using carbohydrate gel electrophoresis (PACE) and the enzymes were shown to be suitable for the partial hydrolysis of pretreated crude plant biomass. 相似文献
16.
The extracellular enzymes of seven fungal strains isolated from koala faeces have been comprehensively characterised for the
first time, revealing potential for biotechnological applications. The fungal isolates were grown in a hydrolase-inducing
liquid medium and the supernatants were analysed using enzyme assays and zymogram gels. Temperature and pH profiles were established
for xylanase (EC 3.2.1.8 endo-1,4-β-xylanase), mannanase (EC 3.2.1.78 mannan endo-1,4-β-mannosidase), endoglucanase (EC 3.2.1.4
cellulase), β-glucosidase (EC 3.2.1.21 β-glucosidase), amylase (EC 3.2.1.1 α-amylase), lipase (EC 3.1.1.3 triacylglycerol
lipase) and protease (EC 3.4 peptidase) activities. Comparisons were made to the high-secreting hypercellulolytic mutant strain
Trichoderma reesei RUT-C30 and the wild-type T. reesei QM6a. The isolates from koala faeces Gelasinospora cratophora A10 and Trichoderma atroviride A2 were good secretors of total protein and heat-tolerant enzymes. Doratomyces stemonitis C8 secreted hemicellulase(s), endoglucanase(s) and β-glucosidase(s) with neutral to alkaline pH optimums. A cold-tolerant
lipase was secreted by Mariannaea camptospora A11. The characteristics displayed by the enzymes are highly sought after for industrial processes such as the manufacture
of paper, detergents and food products. Furthermore, the enzymes were produced at good starting levels that could be increased
further by strain improvement programs. 相似文献
17.
The Clostridium cellulovorans xynA gene encodes the cellulosomal endo-1,4-beta-xylanase XynA, which consists of a family 11 glycoside hydrolase catalytic domain (CD), a dockerin domain, and a NodB domain. The recombinant acetyl xylan esterase (rNodB) encoded by the NodB domain exhibited broad substrate specificity and released acetate not only from acetylated xylan but also from other acetylated substrates. rNodB acted synergistically with the xylanase CD of XynA for hydrolysis of acetylated xylan. Immunological analyses revealed that XynA corresponds to a major xylanase in the cellulosomal fraction. These results indicate that XynA is a key enzymatic subunit for xylan degradation in C. cellulovorans. 相似文献
18.
【目的】在毕赤酵母中表达特异腐质霉Humicola insolens的中性内切葡聚糖酶Ⅱ,并对其性质加以研究。【方法】利用RT-PCR的方法,以特异腐质霉(Humicola insolens)NC3总RNA为模板,克隆到中性内切葡聚糖酶Ⅱ基因(egⅡ)的cDNA。将其插入表达载体pPIC9K,重组质粒经线性化后电击转化毕赤酵母(Pichia pastoris)菌株GS115。【结果】SDS-PAGE和酶活的检测结果均表明:egⅡ基因在毕赤酵母中成功表达。重组酶的部分酶学性质研究表明,该酶的最适反应温度为70°C,且在65°C以下具有较好的热稳定性。最适反应pH为6.5,在pH 6.0?7.0之间有较好的稳定性。【结论】用重组毕赤酵母可高效表达外源中性内切葡聚糖酶,为其今后在工业应用奠定了基础。 相似文献
19.
This study describes the identification of the key enzyme activities required in a "minimal" enzyme cocktail able to catalyze hydrolysis of water-soluble and water-insoluble wheat arabinoxylan and whole vinasse, a fermentation effluent resulting from industrial ethanol manufacture from wheat. The optimal arabinose-releasing and xylan-depolymerizing enzyme activities were identified from data obtained when selected, recombinant enzymes were systematically supplemented to the different arabinoxylan substrates in mixtures; this examination revealed three novel alpha-l-arabinofuranosidase activities: (i) one GH51 enzyme from Meripilus giganteus and (ii) one GH51 enzyme from Humicola insolens, both able to catalyze arabinose release from singly substituted xylose; and (iii) one GH43 enzyme from H. insolens able to catalyze the release of arabinose from doubly substituted xylose. Treatment of water-soluble and water-insoluble wheat arabinoxylan with an enzyme cocktail containing a 20%:20%:20%:40% mixture and a 25%:25%:25%:25% mixture, respectively, of the GH43 alpha-l-arabinofuranosidase from H. insolens (Abf II), the GH51 alpha-l-arabinofuranosidase from M. giganteus (Abf III), a GH10 endo-1,4-beta-xylanase from H. insolens (Xyl III), and a GH3 beta-xylosidase from Trichoderma reesei (beta-xyl) released 322 mg of arabinose and 512 mg of xylose per gram of water-soluble wheat arabinoxylan dry matter and 150 mg of arabinose and 266 mg of xylose per gram of water-insoluble wheat arabinoxylan dry matter after 24 h at pH 5, 50 degrees C. A 10%:40%:50% mixture of Abf II, Abf III, and beta-xyl released 56 mg of arabinose and 91 mg of xylose per gram of vinasse dry matter after 24 h at pH 5, 50 degrees C. The optimal dosages of the "minimal" enzyme cocktails were determined to be 0.4, 0.3, and 0.2 g enzyme protein per kilogram of substrate dry matter for the water-soluble wheat arabinoxylan, the water-insoluble wheat arabinoxylan, and the vinasse, respectively. These enzyme protein dosage levels were approximately 14, approximately 18, and approximately 27 times lower than the dosages used previously, when the same wheat arabinoxylan substrates were hydrolyzed with a combination of Ultraflo L and Celluclast 1.5 L, two commercially available enzyme preparations produced by H. insolens and T. reesei. 相似文献
20.
Beliën T Hertveldt K Van den Brande K Robben J Van Campenhout S Volckaert G 《Journal of biotechnology》2005,115(3):249-260
Two family 11 endoxylanases (EC 3.2.1.8) were functionally displayed on the surface of bacteriophage M13. The genes encoding endo-1,4-xylanase I from Aspergillus niger (ExlA) and endo-1,4-xylanase A from Bacillus subtilis (XynA) were fused to the gene encoding the minor coat protein g3p in phagemid vector pHOS31. Phage rescue resulted in functional monovalent display of the enzymes as was demonstrated by three independent tests. Firstly, purified recombinant phage particles showed a clear hydrolytic activity in an activity assay based on insoluble, chromagenic arabinoxylan substrate. Secondly, specific binding of endoxylanase displaying phages to immobilized endoxylanase inhibitors was demonstrated by interaction ELISA. Finally, two rounds of selection and amplification in a biopanning procedure against immobilized endoxylanase inhibitor were performed. Phages displaying endoxylanases were strongly enriched from background phages displaying unrelated proteins. These results open perspectives to use phage display for analysing protein-protein interactions at the interface between endoxylanases and their inhibitors. In addition, this technology should enable engineering of endoxylanases into novel variants with altered binding properties towards endoxylanase inhibitors. 相似文献