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1.
Aims: The purification and biochemical properties of the 1,4‐β‐xylosidase of an oenological yeast were investigated. Methods and Results: An ethanol‐tolerant 1,4‐β‐xylosidase was purified from cultures of a strain of Pichia membranifaciens grown on xylan at 28°C. The enzyme was purified by sequential chromatography on DEAE cellulose and Sephadex G‐100. The relative molecular mass of the enzyme was determined to be 50 kDa by SDS‐PAGE. The activity of 1,4‐β‐xylosidase was optimum at pH 6·0 and at 35°C. The activity had a Km of 0·48 ± 0·06 mmol l ?1 and a Vmax of 7·4 ± 0·1 μmol min ?1 mg ?1 protein for p‐nitrophenyl‐β‐d ‐xylopyranoside. Conclusions: The enzyme characteristics (pH and thermal stability, low inhibition rate by glucose and ethanol tolerance) make this enzyme a good candidate to be used in enzymatic production of xylose and improvement of hemicellulose saccharification for production of bioethanol. Significance and Impact of the Study: This study may be useful for assessing the ability of the 1,4‐β‐xylosidase from P. membranifaciens to be used in the bioethanol production process. 相似文献
2.
The influence of dried cell powder and culture filtrates of endophytic fungi on production of inophyllum in cell suspension
cultures of leaf- and stem-derived callus of Calophyllum inophyllum was investigated. Two fungi, Nigrospora sphaerica and Phoma spp., endophytic to C. inophyllum, were isolated from leaf tissues, and were identified by both 18S rRNA gene amplification and sequencing. Elicitation of
suspension cultures of both callus types of C. inophyllum with dried cell powder and culture filtrates of both fungi consistently elicited production of inophyllum A, B, C, and P.
In comparison to stem-derived callus, suspension cultures of leaf-derived callus enhanced production of most inophyllum. Of
the four inophyllum studied, the highest production of inophyllum A, C, and P was achieved in elicited suspension cultures
of leaf-derived callus. Suspension cultures of stem-derived callus enhanced production only of inophyllum B. When suspension
cultures of leaf-derived callus were elicited with 40 mg dried cell powder of Phoma spp., a level of 751-fold (6.84 mg/100 g elicited biomass) of inophyllum A was produced, compared to control. Whereas, a
level of 414-fold (6.22 mg/100 g elicited biomass) of inophyllum B was produced when suspension cultures of stem-derived callus
were elicited with 20 mg dried cell powder of N. sphaerica. When compared to control, a 10% culture filtrate of N. sphaerica in suspension cultures of leaf-derived callus elicited inophyllum C and P production by 928-fold (7.43 mg/100 g elicited
biomass) and 750-fold (1.5 mg/100 g elicited biomass), respectively. 相似文献
3.
A commercial cellulase was first assessed to be effective in hydrolyzing glycosyl at the C-3 and C-26 positions in steroidal
saponins from yellow ginger ( Dioscorea zingiberensis C. H. Wright) to diosgenin, a very important chemical in the pharmaceutical industry. The effect of different parameters
on enzyme hydrolysis was further investigated by systematically varying them. The highest yield was achieved when the hydrolysis
ran at 55°C and pH 5.0 with an enzyme to substrate ratio of 15 × 10 3 U/g. The biotransformed products identified using TLC and HPLC confirmed that the cellulase was capable of releasing diosgenin
from steroidal saponins. Moreover, the biotransformation process was explored by LC-MS and LC-MS/MS analysis. Enzymatic hydrolysis
together with 40 % of the original sulphuric acid used increased the diosgenin yield by 15.4 ± 2.7% than traditional method.
Therefore, the commercial cellulase may serve as a promising tool for industrial diosgenin production and for further use
in saponin modification. 相似文献
4.
Batch suspension cultures of Digitalis lanata plant cell were performed to investigate the biotransformation of digitoxin. Digitalis lanata K3OHD plant cells were used to biotransform digitoxin into deacetyllanatoside C. A kinetic model was proposed to describe
cell growth, substrate consumption, depletion of digitoxin, formation and depletion of digoxin and purpureaglycoside A, and
formation of deacetyllanatoside C. The digoxin and purpureaglycoside A are intermediates of deacetyllanatoside C formation
from digitoxin. Interactions between extracellular and intracellular compounds were considered. The proposed model could accurately
predict cell growth, substrate consumption and product synthesis. And it can provide a useful framework for quantitative analysis
of biotransformation in a plant cell culture system. 相似文献
5.
Fermentation of xylooligosaccharides by 11 strains of Selenomonas ruminantium was examined. Xylooligosaccharides were prepared by the partial hydrolysis of oat spelt xylan in dilute phosphoric acid (50
mM, 121°C, 15 min) and were added to a complex, yeast extract-Trypticase-containing medium. Strains of S. ruminantium varied considerably in their capacity to ferment xylooligosaccharides. Strains GA192, GA31, H18, and D used arabinose, xylose,
and the oligosaccharides xylobiose through xylopentaose, as well as considerable quantities of larger, unidentified oligosaccharides.
Other strains of S. ruminantium (HD4, HD1, 20-21a, H6a, W-21, S23, 5-1) were able to use only the simple sugars present in the substrate mixture. The ability
of S. ruminantium strains to utilize xylooligosaccharides was correlated with the presence of xylosidase and arabinosidase activities. Both
enzyme activities were induced by growth on xylooligosaccharides, but no activity was detected in glucose- or arabinose-grown
cultures. Xylooligosaccharide-fermenting strains of S. ruminantium exhibited considerable variation in substrate utilization patterns, and the assimilation of individual carbohydrate species
also appeared to be regulated. Lactic, acetic, and propionic acids were the major fermentation end products detected.
Received: 2 August 1997 / Accepted: 18 September 1997 相似文献
6.
Strains of Selenomonas ruminantium vary considerably in their capacity to ferment xylooligosaccharides. This ability ranges from strain GA192, which completely
utilized xylose through xylotetraose and was able to ferment considerable quantities of larger oligosaccharides, to strain
HD4, which used only the simple sugars present in the hydrolysate. The ability of S. ruminantium GA192 to utilize xylooligosaccharides was correlated with the presence of xylosidase and arabinosidase activities. The production
of these activities appears to be regulated in response to carbon source used for growth. Both arabinosidase and xylosidase
were induced by growth on xylose or xylooligosaccharides, but no activity was detected in glucose-or arabinose-grown cultures.
A genetic locus from S. ruminantium GA192 was cloned into Escherichia coli JM83 that produced both xylosidase and arabinosidase activities. Analyses of crude extracts from the E. coli clone and S. ruminantium GA192 by using native polyacrylamide gel electrophoresis and methylumbelliferyl substrates indicated that a single protein
was responsible for both activities. The enzyme expressed in E. coli was capable of degrading xylooligosaccharides derived from xylan. DNA sequencing of the locus demonstrated the presence of
an open reading frame that encodes for a protein of 61,174 molecular weight.
Received: 12 January 2001 / Accepted: 5 March 2001 相似文献
7.
Summary In order to demonstrate enzyme activities playing a role in the biosynthesis of cardenolides and 2,6-dideoxysugars, 5H-pregnan-3ol-20-one and cardenolides (digitoxigenin, oleandrigenin/L-oleandrose, oleandrin, neriifolin, digitoxigeninmonodigitoxoside and strospeside) were fed to cell suspension cultures of Nerium oleander L.. It could be shown that cell suspension cultures of Nerium oleander L. are able to oxidize, isomerize and glucosylate 5H-steroidaglycones at C-3. The respective glucosides of the 5H-steroid-aglycones are the main biotransformation products. These cell cultures are an appropriate tool for the production of labelled 5H-steroidglucosides.Abbreviations 2,4-D
2,4-dichlorophenoxyacetic acid
- IAA
indole-3-acetic acid
- EtOAc
ethylacetate
- MeOH
methanol
- MS
Murashige & Skoog 相似文献
8.
Summary We have utilized strains of three actinomycete species, Actinomadura sp, Streptomyces cyaneus and Thermomonospora mesophila, to study the solubilisation of lignocellulose. The production of extracellular proteins, was measured for each of the organisms during 17 days growth using medium containing either glucose or ball-milled straw. Some of the extracellular proteins (as identified by SDS gel electrophoresis) were present under both growth conditions, but others were specific to the type of medium or the period of incubation. The levels of proteins were compared with the abilities of the extracellular protein preparations to solubilise a substrate of 14C-labelled lignocellulose. About 6% of the radioactive material were solubilised when the extracellular proteins from the cultures grown on glucose were incubated with the substrate, compared to 20–30% that were solubilised by the extracellular proteins from the cultures grown on ball-milled straw. Partial characterisation of an enzyme from S. cyaneus responsible for the solubilisation of lignocellulose was achieved by gel filtration of the extracellular proteins, using Superose 12. Material that eluted from the column with an apparent molecular weight of about 20 000 accounted for all of the solubilisation of 14C-labelled (i.e. lignin-derived) moieties. In contrast, when the eluate was tested for the presence of cellulases and xylanases most of the activities were found in fractions containing material with an apparent molecular weight of about 45 000. We conclude that in cultures of S. cyaneus grown on ball-milled straw, a single extracellular enzyme is responsible for the solubilisation of lignin in lignocellulose, and that this enzyme is unlikely to be a cellulase or a xylanase. 相似文献
9.
Microorganisms that colonize plants require a number of hydrolytic enzymes to help degrade the cell wall. The maize endophyte
Acremonium zeae was surveyed for production of extracellular enzymes that hydrolyze cellulose and hemicellulose. The most prominent enzyme
activity in cell-free culture medium from A. zeae NRRL 6415 was xylanase, with a specific activity of 60 U/mg from cultures grown on crude corn fiber. Zymogram analysis following
SDS-PAGE indicated six functional xylanase polypeptides of the following masses: 51, 44, 34, 29, 23, and 20 kDa. Xylosidase
(0.39 U/mg), arabinofuranosidase (1.2 U/mg), endoglucanase (2.3 U/mg), cellobiohydrolase (1.3 U/mg), and β-glucosidase (0.85 U/mg)
activities were also detected. Although apparently possessing a full complement of hemicellulolytic activities, cell-free
culture supernatants prepared from A. zeae required an exogenously added xylosidase to release more than 90% of the xylose and 80% of the arabinose from corn cob and
wheat arabinoxylans. The hydrolytic enzymes from A. zeae may be suitable for application in the bioconversion of lignocellulosic biomass into fermentable sugars.
Mention of a trade name or commercial products in this publication is solely for the purpose of providing specific information
and does not imply recommendation or endorsement by the U.S. Department of Agriculture. 相似文献
10.
紫杉醇是目前临床治疗癌症的一线化疗药物,资源紧张,价格昂贵。7-木糖-10-去乙酰基紫杉醇(7-XDT)在红豆杉中含量可达紫杉醇的10倍,脱除木糖基后生成的10-脱乙酰紫杉醇(10-DAT)经乙酰化可生成紫杉醇。通过木聚糖平板对不同菌株进行筛选,从52株供试微生物中,发现27株在木聚糖平板上生长良好。经转化实验筛选,发现一株天蓝色链霉菌(Streptomyces coelicolor YUCM 410115)具有转化7-XDT为10-脱乙酰紫杉醇的能力。菌体细胞经破碎离心后,沉淀及上清液均无转化反应出现,而发酵液的硫酸铵沉淀物则可以转化7-XDT生成10-DAT,表明该菌株能产生一种胞外紫杉醇-7-木糖苷酶,发酵液酶活为6 268U。首次发现天蓝色链霉菌能够产生紫杉醇-7-木糖苷酶,为7-XDT转化生产紫杉醇提供了新的酶源。 相似文献
11.
Three psychrotrophic and one mesophilic strains were isolated from winter water samples of different freshwater biotopes and identified as Cytophaga johnsonae (C-21), Cytophaga sp. (M-17), Pseudomonas fluorescens (KD), and Enterobacter cloacae (BS-2). Temperature shift-up experiments with emphasis on low temperatures were carried out with aerated pure batch cultures in glucose mineral medium. The effects of sudden temperature increases on growth rates and substrate conversion were investigated. All three psychrotrophic strains in the temperature increase experiments at low temperatures showed differing reactions within the linear zone of the Arrhenius plot. The C. johnsonae (C-21) shift-up cultures adjusted the growth rate immediately to the rate of the temperature adapted cultures, whereas Cytophaga sp. (M-17) shift-up cultures showed a lower and P. fluorescens (KD) a higher growth rate. The mesophilic E. cloacae (BS-2), like C. johnsonae (C-21), adjusted immediately to the new growth rate. Substrate conversion increased in all experiments immediately after the shift-up. The extracellular substrate conversion by P. fluorescens (KD) of glucose to gluconate and 2-ketogluconate was particularly affected by the sudden temperature increase. 相似文献
12.
Time‐course of biotransformation of racemic trans‐4‐(( E)‐4′,8′‐dimethylnona‐3′,7′‐dien‐1‐yl)‐5‐iodomethyl‐4‐methyldihydrofuran‐2‐one ( 1 ) in fungal and yeast cultures was investigated. In these conditions, the substrate 1 was enantioselectively dehalogenated yielding 4‐(( E)‐4′,8′‐dimethylnona‐3′,7′‐dien‐1‐yl)‐4‐methyl‐5‐methylenedihydrofuran‐2‐one ( 2 ) and its structure was established based on the spectroscopic data. The most effective biocatalyst used was Didymosphaeria igniaria, which catalyzed the process with highest rate and enantioselectivity ( ee of product = 76%). The antiproliferative activity of δ‐iodo‐ γ‐lactone 1 , product of its biotransformation 2 , and starting substrate (farnesol) were evaluated toward two cancer cell lines: A549 (human lung adenocarcinoma) and HL‐60 (human promyelocytic leukemia). 相似文献
13.
Extracellular enzyme‐producing yeasts might be involved in the supplementation of enzymes within the gastrointestinal tract of fish. The present study was intended to detect yeasts in the intestine of three Indian major carps ( Labeo rohita, Catla catla, Cirrhinus mrigala) , three exotic carps ( Hypophthalmichthys molitrix, Ctenopharyngodon idella, Cyprinus carpio), as well as Nile tilapia ( Oreochromis niloticus), and to identify the most promising extracellular enzyme‐producing (e.g. amylase, protease, lipase, cellulase, xylanase and phytase) yeast strains by 18S rDNA sequence analysis. Selected for qualitative enzyme assay were 121 yeast strains, from which 28 were further studied for quantitative enzyme assay. The strain CMH6A isolated from C. mrigala exhibited the best extracellular enzyme activities except for amylase and cellulase. The strain ONF19B isolated from O. niloticus was noted as the best extracellular enzyme producer among the strains that produced all of the extracellular enzymes studied. Sequencing of the 18S rDNA fragment followed by nucleotide blast in the National Centre for Biotechnology Information (NCBI) GenBank revealed that strains CMH6A and ONF19B were similar to Pichia kudriavzevii (Accession no. KF479403 ) and Candida rugosa (Accession no. KF479404 ), respectively. The test of antagonism ( in vitro) revealed that the isolated yeasts could not affect the growth of the autochthonous gut bacteria. This might indicate likely co‐existence of autochthonous yeasts and bacteria in the fish gut. Further research is necessary to explore the possibilities of utilizing the extracellular enzyme‐producing yeasts detected in the present study for commercial aquaculture. 相似文献
14.
A simple, rapid and reproducible procedure for the identification of extracellular cucumber ( Cucumis sativus L.) α‐galactosidase is described using callus cultures of seedlings from the tested plant, hairy roots of 2‐day‐old seedlings of cucumber germinating on agar plates as well as cell suspension cultures derived from callus cultures. For the determination of the intracellular and extracellular activities of α‐galactosidase, 6‐bromo‐2‐naphthyl‐ α‐ D‐galactopyranoside and p‐nitrophenyl‐ α‐ D‐galactopyranoside, respectively, were used as synthetic substrates. The extracellular α‐galactosidase activity was identified by evaluating the dye‐zones in agar medium. The enzyme from cucumber callus cultures and seedling roots, cultivated on agar plates supplemented with 6‐bromo‐2‐naphthyl‐ α‐ D‐galactopyranoside, hydrolyzed this substrate releasing 6‐bromo‐2‐naphthol. By simultaneous coupling with hexazonium p‐rosaniline the corresponding azodye was formed. Thus, the extracellular enzyme was detected by the presence of reddish‐brown zones on the agar plates around the plant material. The parallel extracellular and intracellular activities were determined in cell suspension cultures derived from callus cultures. The results show a 44.6% intracellular and 55.4% extracellular distribution of α‐galactosidase activity. The described agar plate method enables a rapid, simple and specific detection of plant producers of extracellular α‐galactosidase. 相似文献
15.
Enterobacter sp. CGMCC 2487, a bacterial strain isolated from the soil around a Taxus cuspidata Sieb. et Zucc. plant, was able to remove the xylosyl group from 7-xylosyltaxanes. The xylosidase of this strain was an inducible enzyme. In the bioconversion of 7-xylosyl-10-deacetyltaxol (7-XDT) to 10-deacetyltaxol (10-DT), for the purpose of enhancing the conversion efficiency, the effects of NH 4+, oat xylan, temperature, pH value, cell density and substrate concentration on the bioconversion have been systematically investigated. 3.0 mM NH 4+, 0.6% oat xylan in the media could enhance the yield of 10-DT; the optimum biocatalytic temperature was 26 °C and optimum pH value was 6.0. The highest conversion rate and yield of 10-DT from 7-XDT reached 92% and 764 mg/L, respectively. In addition, the biocatalytic capacity of the cell cultures remained 66.1% after continuous three batches. These results indicate that converting 7-XDT to 10-DT, a useful intermediate for the semisynthesis of paclitaxel or other taxane-based anticancer drugs by a novel bacterial strain, Enterobacter sp. CGMCC 2487, would be an alternative for the practical application in the future. 相似文献
16.
Cellulase, Tween 80, and β-glucosidase loading were studied and optimized by response surface methodology to improve saccharification.
Microwave alkali-pretreated rice straw used as substrate for onsite enzyme production by Aspergillus heteromorphus and Trichoderma reesei. The highest enzymatic hydrolysis (84%) was obtained from rice straw at crude enzyme loading of 10 FPU/gds of cellulase,
0.15% Tween 80, and 100 international unit/g dry solids of β-glucosidase activities. Enzymatic hydrolyzate of pretreated rice
straw was used for ethanol production by Saccharomyces cerevisiae, Scheffersomyces stipitis, and by co-culture of both. The yield of ethanol was 0.50, 0.47, and 0.48 g p/g s by S. cerevisiae, S. stipitis, and by co-culture, respectively, using pretreated rice straw hydrolyzate. The co-culture of S. cerevisiae and S. stipitis produced 25% more ethanol than S. cerevisiae alone and 31% more ethanol than S. stipitis alone. During anaerobic fermentation 65.08, 36.45, and 50.31 μmol/ml CO 2 released by S. cerevisiae, S. stipitis, and by co-culture, respectively. The data indicated that saccharification efficiency using optimized crude enzyme cocktail
was good, and enzymatic hydrolyzate could be fermented to produce ethanol. 相似文献
17.
A laboratory scale study to evaluate the potentiality of filamentous fungi for the production of cellulolytic enzymes using
palm oil mill effluent (POME) as a basal medium was initiated. A total of 25 filamentous fungi in which 16 filamentous fungi
were isolated and purified from oil palm industrial residues and 9 strains from laboratory stock were screened using POME
with 1% total suspended solids. Trichoderma reesei RUT C-30 was identified as a potential strain for cellulolytic enzyme production as compared to other genera of Aspergillus, Penicillum, Rhizopus, Phanerochaete, Trichoderma and basidiomycete groups. The results showed that T. reesei RUT C-30 gave the highest filter paper cellulase and carboxy methyl cellulase activity of 0.917 and 2.51 U/ml respectively
at day 5 of fermentation. Other parameters such as growth formation, pH, filterability and total biosolids were observed to
evaluate the bioconversion process. 相似文献
18.
The production of an extracellular -D-xylosidase (-D-xyloside xylohydrolase, EC 3.2.1.37) by four Aspergillus strains ( A. carbonarius, A. nidulans, A. niger and A. oryzae) grown on wheat bran medium was compared. The highest amount of the enzyme was found in the culture of A. carbonarius. The -D-xylosidase from A. carbonarius was purified to homogeneity by a rapid procedure, using hydrophobic interaction chromatography, chromatofocusing and affinity chromatography. The purified enzyme possessed not only -D-xylosidase activity, but also -L-arabinosidase activity. Mixed substrate experiments revealed that a single active centre was responsible for the splitting of the corresponding synthetic substrates. The molecular weight of the purified enzyme proved to be 100,000 Da, as estimated by SDS–PAGE. The isoelectric point was at pH 4.4. The pH and temperature optima were 4.0 and 60 °C, respectively. The enzyme remained stable over a pH range of 3.5–6.5 and up to 50 °C for 30 min. The Michaelis constant for p-nitrophenyl -D-xyloside was 0.198 mM. Kinetic studies demonstrated that the lack of the C-5 hydroxylmethyl group and the configuration of the C-4 hydroxyl group on the pyranoside ring play an important role in both substrate binding and splitting. 相似文献
19.
During prolonged cultivation of S. aureus strains 104 and NCTC 8178 in continuous culture, staphylocoagulase-negative mutants arose and accumulated progressively in increasing proportions. The resulting loss of production of staphylocoagulase was accompanied by a simultaneous loss of production of -haemolysin and PV-leucocidin. Characterization of the strains revealed no further differences in biotype, exoenzymes, phage pattern and plasmid content.Cultivation in batch cultures showed that the maximal specific growth rates and specific oxygen-consumption rates of the mutant strains were slightly higher than those of the parent strains, whereas the production of total extracellular protein of the mutant strains had decreased significantly.From competition experiments between parent and mutant strains in chemostat cultures at different dilution rates and cultivation temperatures, it was concluded that the underlying mechanism of accumulation of staphylocoagulase-negative mutants in the chemostat is based on differences in affinity for the limiting substrate(s) rather than on differences in the production rates of total extracellular proteins. The complete repression of three exoenzymes, a partial repression of the total extracellular protein production, and an increased affinity for the limiting substrate(s) suggested that a mutation in a regulatory gene is involved. The possible role of a transposon in this mutation is discussed. 相似文献
20.
The aim of this work is to characterize the potential probiotic properties of 14 antifungal Lactobacillus strains isolated from traditional fermenting Moroccan green olives. The molecular identification of strains indicated that they are composed of five Lactobacillus brevis, two Lactobacillus pentosus, and seven Lactobacillus plantarum. In combination with bile (0.3%), all the strains showed survival rates (SRs) of 83.19–56.51% at pH 3, while 10 strains showed SRs of 31.67–64.44% at pH 2.5. All the strains demonstrated high tolerance to phenol (0.6%) and produced exopolysaccharides. The autoaggregation, hydrophobicity, antioxidant activities, and surface tension value ranges of the strains were 10.29–41.34%, 15.07–34.67%, 43.11–52.99%, and 36.23–40.27 mN/m, respectively. Bacterial cultures exhibited high antifungal activity against Penicillium sp. The cell-free supernatant (CFS) of the cultures showed important inhibition zones against Candida pelliculosa (18.2–24.85 mm), as well as an antibacterial effect against some gram-positive and gram-negative bacteria (10.1–14.1 mm). The neutralized cell-free supernatant of the cultures displayed considerable inhibitory activity against C. pelliculosa (11.2–16.4 mm). None of the strains showed acquired or horizontally transferable antibiotic resistance or mucin degradation or DNase, hemolytic, or gelatinase activities. Lactobacillus brevis S82, Lactobacillus pentosus S75, and Lactobacillus plantarum S62 showed aminopeptidase, β-galactosidase, and β-glucosidase activities, while the other enzymes of API-ZYM were not detected. The results obtained revealed that the selected antifungal Lactobacillus strains are considered suitable candidates for use both as probiotic cultures for human consumption and for starters and as biopreservative cultures in agriculture, food, and pharmaceutical industries. 相似文献
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