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1.
Summary Cells of Escherichia intermedia were immobilized by entrapment in a polyacrylamide gel and used for the enzymatic production of l-tyrosine from phenol, pyruvate, and ammonia. A preparation containing 50 mg of cells/g of gel retained 60% of its original activity. The effect of temperature, pH and substrate concentration on the activity of free cells was almost identical with the effect on immobilized cells. Phenol showed inhibition and inactivation of the catalyst at high concentration. Synthesis of l-tyrosine (up to 10 g/l) was demonstrated in batch reactors with high conversion yields (95–100%) and a maximal productivity of 2 g/l/h. In continuous reactor the catalyst showed a very high operational stability (more than 54 days without losses). 相似文献
2.
von Weymarn N Kiviharju K Leisola M 《Journal of industrial microbiology & biotechnology》2002,29(1):44-49
Ten heterofermentative lactic acid bacteria were compared in their ability to produce D-mannitol from D-fructose in a resting state. The best strain, Leuconostoc mesenteroides ATCC-9135, was examined in high cell density membrane cell-recycle cultures. High volumetric mannitol productivity (26.2
g l−1 h−1) and mannitol yield (97 mol%) were achieved. Using the same initial biomass, a stable high-level production of mannitol was
maintained for 14 successive bioconversion batches. Applying response surface methodology, the temperature and pH were studied
with respect to specific mannitol productivity and yield. Moreover, increasing the initial fructose concentration from 100
to 120 and 140 g l−1 resulted in decreased productivities due to both substrate and end-product inhibition of the key enzyme, mannitol dehydrogenase
(MDH). Nitrogen gas flushing of the bioconversion media was unnecessary, since it did not change the essential process parameters.
Journal of Industrial Microbiology & Biotechnology (2002) 29, 44–49 doi:10.1038/sj.jim.7000262
Received 12 November 2001/ Accepted in revised form 30 March 2002 相似文献
3.
Vasu V Kumaresan J Babu MG Meenakshisundaram S 《Applied microbiology and biotechnology》2012,93(6):2377-2386
Cis-epoxysuccinate hydrolase (CESH, EC 3.3.2.3) from Nocardia tartaricans is known to catalyze the opening of an epoxide ring of cis-epoxysuccinate (CES), thereby converting it to corresponding vicinal diol, l(+)-tartaric acid. An attempt has been made to build a 3D homology model of CESH to investigate the structure–function relationship,
and also to understand the mechanism of the enzymatic reaction. Using a combination of molecular-docking simulation and multiple
sequence alignment, a set of putative residues that are involved in the CESH catalysis has been identified. Functional roles
of these putative active-site residues were further evaluated by site-directed mutagenesis. Interestingly, the mutants D18A,
D18E, Q20E, T22A, R55E, N134D, K164A, H190A, H190N, H190Q, D193A, and D193E resulted in complete loss of activity, whereas
the mutants Y58F, T133A, S189A, and Y192D retained partial enzyme activity. Furthermore, the active-site residues responsible
for the opening of CES were analyzed, and the mechanism underlying the catalytic triad involved in l(+)-tartaric acid biosynthesis was proposed. 相似文献
4.
l-2-Amino-Δ2-thiazoline-4-carboxylic acid hydrolase (ATC hydrolase) was purified and characterized from the crude extract of Escherichia coli, in which the gene for ATC hydrolase of Pseudomonas sp. strain ON-4a was expressed. The results of SDS–polyacrylamide gel electrophoresis and gel filtration on Sephacryl S-200 suggested that the ATC hydrolase was a tetrameric enzyme consisted of identical 25-kDa subunits. The optimum pH and temperature of the enzyme activity were pH 7.0 and 30–35°C, respectively. The enzyme did not require divalent cations for the expression of the activity, and Cu2+ and Mn2+ ions strongly inhibited the enzyme activity. An inhibition experiment by diethylpyrocarbonic acid, 2-hydroxy-5-nitrobenzyl bromide, and N-bromosuccinimide suggested that tryptophan, cysteine, or/and histidine residues may be involved in the catalytic site of this enzyme. The enzyme was strictly specific for the l-form of d,l-ATC and exhibited high activity for the hydrolysis of l-ATC with the values of K
m (0.35 mM) and V
max (69.0 U/mg protein). This enzyme could not cleave the ring structure of derivatives of thiazole, thiazoline, and thiazolidine tested, except for d,l- and l-ATC. These results show that the ATC hydrolase is a novel enzyme cleaving the carbon–sulfur bond in a ring structure of l-ATC to produce N-carbamoyl-l-cysteine. 相似文献
5.
l-threo-3,4-Dihydroxyphenylserine (DOPS) is a chiral unnatural β-hydroxy amino acid used for the treatment of Parkinson disease.
We developed a continuous bioconversion system for DOPS production that uses whole-cell biocatalyst of recombinant Escherichia coli expressing l-threonine aldolase (l-TA) genes cloned from Streptomyces avelmitilis MA-4680. Maximum conversion rates were observed at 2 M glycine, 145 mM 3,4-dihydroxybenzaldehyde, 0.75% Triton-X, 5 g E. coli cells/l, pH 6.5 and 10°C. In the optimized condition, overall productivity was 8 g/l, which represents 40 times the synthesis
yield possible with no optimization of conditions. 相似文献
6.
An alternative microbiological method for the production of malate from fumarate is presented. The yeast Dipodascus magnusii was used for this bioconversion. The optimum cell growth temperature was 28°C and the working volume 120 ml. The highest level of fumarase activity during bioconversion was achieved at a pH of 7.5 and a temperature of 37°C. These conditions were determined as optimal. Using sodium fumarate (1M), the maximum specific productivity of malic acid obtained was 1.72 g/(gDCW × h) for intact cells. In the case of ammonium fumarate, it was 2.25 g/(gDCW × h). 相似文献
7.
I-Son Ng Chen-Wei Li Yi-Fang Yeh Po Ting Chen Jiun-Ly Chir Chin-Hua Ma Su-May Yu Tuan-hua David Ho Chii-Gong Tong 《Extremophiles : life under extreme conditions》2009,13(3):425-435
A thermophilic Geobacillus bacterium secreting high activity of endo-glucanase (EC 3.2.1.4) was isolated from rice straw compost supplemented with pig
manure. A full-length gene of 1,104 bp, celA, encoding this glycosyl hydrolase family 5 endo-glucanase of 368 amino acids was isolated. No related gene from Geobacillus has been reported previously. The recombinant CelA expressed in Escherichia coli had an optimal activity at 65°C and pH 5.0, and it exhibited tenfold greater specific activity than the commercially available
Trichoderma reesei endo-glucanase. CelA displayed activity over a broad temperature range from 45 to 75°C and was a thermostable enzyme with
90% activity retained after heating at 65°C for 6 h. Interestingly, CelA activity could be enhanced by 100% in the presence
of 2 mM MnSO4. CelA had high specific activity over β-d-glucan from barley and Lichenan, making it a potentially useful enzyme in biofuel and food industries. 相似文献
8.
Novel stabilization of phenylalanine ammonia-lyase catalyst during bioconversion of trans-cinnamic acid to l-phenylalanine 总被引:4,自引:0,他引:4
Christopher T. Evans Dayle Conrad Kim Hanna Wendy Peterson Christin Choma Masanaru Misawa 《Applied microbiology and biotechnology》1987,25(5):399-405
Summary Production of l-phenylalanine from trans-cinnamic acid using isolate SPA10 cells was reduced to 26% of that observed initially when cells were reacted a second time with fresh substrate mixture. The stability (reuseability) of Phenylalanine Ammonia-Lyase (PAL) containing cells was significantly influenced by both the trans-cinnamate concentration and initial reaction pH. Using 2% t-cinnamate, l-phenylalanine production was 7-fold greater after 3 successive runs at pH 9.0 than at the optimum of pH 10.2. Cells reacted in the presence of 5% t-cinnamate were relatively unstable. Permeabilising agents, such as toluene and xylene, stimulated l-phenylalanine production but also enhanced instability of the catalyst. Several effectors were shown to stimulate the initial rate of the PAL bioconversion, but only sorbitol, alginate, glutaraldehyde, polyethylene glycol and glycerol conferred any significant degree of stability. Sparging of cultures and bioreactors with various gases revealed that oxygen enhanced PAL inactivation, CO2 had little effect and nitrogen conferred remarkable stability on PAL activity for several weeks in culture medium. The presence of chloride ions (from HCl) and aeration of substrate mixtures resulted in poor reuseability of catalyst. A combination of H2SO4 substitution for HCl and N2-sparging resulted in excellent initial conversions and good catalyst stability at 26°C but less at 30°C. The inclusion of 1.5 M sorbitol in reaction mixtures maintained PAL stability over several successive incubations. 相似文献
9.
Characterization of salt-tolerant glutaminase from Stenotrophomonas maltophilia NYW-81 and its application in Japanese soy sauce fermentation 总被引:1,自引:0,他引:1
Wakayama M Yamagata T Kamemura A Bootim N Yano S Tachiki T Yoshimune K Moriguchi M 《Journal of industrial microbiology & biotechnology》2005,32(9):383-390
Glutaminase from Stenotrophomonas maltophilia NYW-81 was purified to homogeneity with a final specific activity of 325 U/mg. The molecular mass of the native enzyme was
estimated to be 41 kDa by gel filtration. A subunit molecular mass of 36 kDa was measured with SDS-PAGE, thus indicating that
the native enzyme is a monomer. The N-terminal amino acid sequence of the enzyme was determined to be KEAETQQKLANVVILATGGTIA.
Besides l-glutamine, which was hydrolyzed with the highest specific activity (100%), l-asparagine (74%), d-glutamine (75%), and d-asparagine (67%) were also hydrolyzed. The pH and temperature optima were 9.0 and approximately 60°C, respectively. The enzyme
was most stable at pH 8.0 and was highly stable (relative activities from 60 to 80%) over a wide pH range (5.0–10.0). About
70 and 50% of enzyme activity was retained even after treatment at 60 and 70°C, respectively, for 10 min. The enzyme showed
high activity (86% of the original activity) in the presence of 16% NaCl. These results indicate that this enzyme has a higher
salt tolerance and thermal stability than bacterial glutaminases that have been reported so far. In a model reaction of Japanese
soy sauce fermentation, glutaminase from S. maltophilia exhibited high ability in the production of glutamic acid compared with glutaminases from Aspergillus oryzae, Escherichia coli, Pseudomonas citronellolis, and Micrococcus luteus, indicating that this enzyme is suitable for application in Japanese soy sauce fermentation. 相似文献
10.
Summary A hydantoin-hydrolyzing enzyme has been purified from an newly isolatedAgrobacterium sp. by procedures including ammonium sulfate fractionation and ion-exchange chromatography. Kinetic studies have demonstrated that this enzyme, which is strictlyd-selective and has a broad substrate specificity exhibits remarkable stability. Microbial bioconversion at 60°C and pH 10.0, allowed complete conversion of 30 g/L ofd,l 5-benzylhydantoin into thed
N-carbamyl derivative of phenylalanine (molar yield of 96%) in less than 10 h. The hydantoinase is activated by Ni2+ ions. 相似文献
11.
An epoxide hydrolase gene of about 0.8 kb was cloned from Rhodococcus opacus ML-0004, and the open reading frame (ORF) sequence predicted a protein of 253 amino acids with a molecular mass of about
28 kDa. An expression plasmid carrying the gene under the control of the tac promotor was introduced into Escherichia coli, and the epoxide hydrolase gene was successfully expressed in the recombinant strains. Some characteristics of purified recombinant
epoxide hydrolase were also studied. Epoxide hydrolase showed a high stereospecificity for l(+)-tartaric acid, but not for d(+)-tartaric acid. The epoxide hydrolase activity could be assayed at the pH ranging from 3.5 to 10.0, and its maximum activity
was obtained between pH 7.0 and 7.5. The enzyme was sensitive to heat, decreasing slowly between 30°C and 40°C, and significantly
at 45°C. The enzyme activity was activated by Ca2+ and Fe2+, while strongly inhibited by Ag+ and Hg+, and slightly inhibited by Cu2+, Zn2+, Ba2+, Ni+, EDTA–Na2 and fumarate. 相似文献
12.
Eun-Seon Seo Yu-Ri Lim Yeong-Su Kim Chang-Su Park Deok-Kun Oh 《Biotechnology and Bioprocess Engineering》2010,15(4):590-594
The bacterium Bacillus licheniformis, which exhibits high hydrolytic activity toward arabinan, was isolated from soil, and its gene encoding endo-1,5-α-l-arabinanase was cloned and sequenced. The gene has an open reading frame that encodes 328 amino acids, including a signal
peptide of 37 amino acids. Endo-1,5-α-l-arabinanase, a member of glycosyl hydrolase family 43, was expressed in Escherichia coli and purified as a 34-kD monomer with a specific activity of 27 U/mg. Optimal activity toward debranched arabinan (linear
1,5-α-l-arabinan) occurred at pH 6.0 and 35°C, with a k
cat of 160/sec and a K
m of 19 mg/mL. 相似文献
13.
Hiroyuki Horitsu Yasushi Takahashi Junkō Tsuda Keiichi Kawai Yoshio Kawano 《Applied microbiology and biotechnology》1983,18(6):358-360
Summary Living Aspergillus terreus cells were entrapped in polyacrylamide gels and employed in both replacement batch and continuous column reactors to produce itaconic acid from glucose.With the replacement batch reactor, maximum itaconic acid productivity was observed under the following conditions: pH 2.50, temperature at 35°C, addition of NH4H2PO4 and MgSO4·7H2O. Using the continuous reactor, the maximum itaconic acid yield was 60 mg/h/40 g of gel. The biocatalyst activity or half-life was about 10 days. 相似文献
14.
Summary The cell-associated and exocellular hemicellulolytic polysaccharide depolymerase and glycoside hydrolase activity ofBacillus macerans NCDO 1764 was monitored over a range of anaerobic growth conditions in batch and continuous culture. The enzymes were detectable throughout the complete growth cycle in batch culture reaching and maintaining maximum levels in the stationary phase. In continuous culture enzyme activity was largely independent of growth rate (D=0.025–0.1 h-1) although the activity was reduced at higher dilution rates (0.125–0.15 h-1). Although activity was detectable over a wide pH range (pH 5.5–7.5) it was pH dependent, and maximum activities of both the cell-associated and exocellular enzymes were measured in cultures maintained at pH 6.5–7.0±0.1.The principal metabolites formed anaerobically from xylose byB. macerans in batch and continuous culture were acetic acid, formic acid and ethanol which represented 95–99% of the products formed. Smaller amounts of acetone,d,l-lactic acid and succinic acid were formed together with traces of butyric acid (<5 nmol/ml) and isovaleric acid (<25 nmol/ml). The proportions of the metabolites produced varied with growth conditions and were influenced by the pH of the culture and the rate and stage of growth of the microorganism. 相似文献
15.
Citrobacter freundii DSM 30040 immobilized on modified polyurethane carrier particles PUR 90/16 was used for continuous glycerol fermentation in an anaerobic fixed bed reactor with effluent recycle and pH control (fixed bed loop reactor). The fermentor was run with buffered mineral medium under growth conditions resulting in the permanent renewal of active biomass. The effects of glycerol concentration in the feed, dilution rate (D), pH and temperature (T) were investigated to optimize the process. With 400 mm glycerol in the feed, pH 6.9, T = 36°C and D = 0.5 h–1 the maximum productivity could be determined as 8.2 g/l per hour of 1,3-propanediol. 相似文献
16.
Makhongela HS Glowacka AE Agarkar VB Sewell BT Weber B Cameron RA Cowan DA Burton SG 《Applied microbiology and biotechnology》2007,75(4):801-811
An amidase (EC 3.5.1.4) in branch 2 of the nitrilase superfamily, from the thermophilic strain Geobacillus pallidus RAPc8, was produced at high expression levels (20 U/mg) in small-scale fermentations of Escherichia coli. The enzyme was purified to 90% homogeneity with specific activity of 1,800 U/mg in just two steps, namely, heat-treatment
and gel permeation chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electron microscopic
(EM) analysis of the homogenous enzyme showed the native enzyme to be a homohexamer of 38 kDa subunits. Analysis of the biochemical
properties of the amidase showed that the optimal temperature and pH for activity were 50 and 7.0°C, respectively. The amidase
exhibited high thermal stability at 50 and 60°C, with half-lives greater than 5 h at both temperatures. At 70 and 80°C, the
half-life values were 43 and 10 min, respectively. The amidase catalyzed the hydrolysis of low molecular weight aliphatic
amides, with d-selectivity towards lactamide. Inhibition studies showed activation/inhibition data consistent with the presence of a catalytically
active thiol group. Acyl transfer reactions were demonstrated with acetamide, propionamide, isobutyramide, and acrylamide
as substrates and hydroxylamine as the acyl acceptor; the highest reaction rate being with isobutyramide. Immobilization by
entrapment in polyacrylamide gels, covalent binding on Eupergit C beads at 4°C and on Amberlite-XAD57 resulted in low protein
binding and low activity, but immobilization on Eupergit C beads at 25°C with cross-linking resulted in high protein binding
yield and high immobilized specific activity (80% of non-immobilized activity). Characterization of Eupergit C-immobilized
preparations showed that the optimum reaction temperature was unchanged, the pH range was somewhat broadened, and stability
was enhanced giving half-lives of 52 min at 70°C and 30 min at 80°C. The amidase has potential for application under high
temperature conditions as a biocatalyst for d-selective amide hydrolysis producing enantiomerically pure carboxylic acids and for production of novel amides by acyl transfer. 相似文献
17.
Ye-Wang Zhang Marimuthu Jeya Jung-Kul Lee 《Applied microbiology and biotechnology》2010,87(6):1993-1999
Recombinant Escherichia coli harboring the l-arabinose isomerase (BLAI) from Bacillus licheniformis was used as a biocatalyst to produce l-ribulose in the presence of borate. Effects of substrate concentration, the borate to l-arabinose ratio, pH, and temperature on the conversion of l-arabinose to l-ribulose were investigated. l-Ribulose production was efficient when pH was higher than 9 and temperature was higher than 50 °C. Borate addition to the
reaction mixture was essential for high conversion of l-arabinose to l-ribulose as it resulted in an equilibrium shift in favor of the product. Under the optimal conditions determined by response
surface methodology, the E. coli harboring BLAI produced 375 g l−1 L-ribulose from 500 g l−1
l-arabinose at a reaction time of 60 min, corresponding to a conversion yield of 75% and productivity of 375 g l−1 h−1. When the resting recombinant E. coli cells were recycled, 85% of the yield was obtained even after seven cycles of reuse. The productivity and final concentration
of l-ribulose obtained in the present study were the highest yet reported. 相似文献
18.
Anne Usvalampi Kristiina Kiviharju Matti Leisola Antti Nyyssölä 《Journal of industrial microbiology & biotechnology》2009,36(10):1323-1330
Factors affecting the production of the rare sugar l-xylulose from xylitol using resting cells were investigated. An E. coli BPT228 strain that recombinantly expresses a gene for xylitol dehydrogenase was used in the experiments. The ratio of xylitol
to l-xylulose was three times lower in the cytoplasm than in the medium. The effects of pH, temperature, shaking speed, and initial
xylitol concentration on l-xylulose production were investigated in shaking flasks using statistical experimental design methods. The highest production
rates were found at high shaking speed and at high temperature (over 44°C). The optimal pH for both productivity and conversion
was between 7.5 and 8.0, and the optimal xylitol concentration was in the range 250–350 g l−1. A specific productivity of 1.09 ± 0.10 g g−1 h−1 was achieved in a bioreactor. The response surface model based on the data from the shake flask experiments predicted the
operation of the process in a bioreactor with reasonable accuracy. 相似文献
19.
Koseki T Mese Y Nishibori N Masaki K Fujii T Handa T Yamane Y Shiono Y Murayama T Iefuji H 《Applied microbiology and biotechnology》2008,80(6):1007-1013
An α-l-rhamnosidase was purified by fractionating a culture filtrate of Aspergillus kawachii grown on l-rhamnose as the sole carbon source. The α-l-rhamnosidase had a molecular mass of 90 kDa and a high degree of N-glycosylation of approximately 22%. The enzyme exhibited optimal activity at pH 4.0 and temperature of 50 °C. Further, it
was observed to be thermostable, and it retained more than 80% of its original activity following incubation at 60 °C for
1 h. Its T
50 value was determined to be 72 °C. The enzyme was able to hydrolyze α-1,2- and α-1,6-glycosidic bonds. The specific activity
of the enzyme was higher toward naringin than toward hesperidin. The A. kawachii α-l-rhamnosidase-encoding gene (Ak-rhaA) codes for a 655-amino-acid protein. Based on the amino acid sequence deduced from the cDNA, the protein possessed 13 potential
N-glycosylation recognition sites and exhibited a high degree of sequence identity (up to 75%) with the α-l-rhamnosidases belonging to the glycoside hydrolase family 78 from Aspergillus aculeatus and with hypothetical Aspergillus oryzae and Aspergillus fumigatus proteins.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
20.
Canakci S Kacagan M Inan K Belduz AO Saha BC 《Applied microbiology and biotechnology》2008,81(1):61-68
The gene, AbfAC26Sari, encoding an α-l-arabinofuranosidase from Anoxybacillus kestanbolensis AC26Sari, was isolated, cloned, sequenced, and characterizated. On the basis of amino acid sequence similarities, this 57-kDa
enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. Characterization of the purified recombinant
α-l-arabinofuranosidase produced in Escherichia coli BL21 revealed that it is active at a broad pH range (pH 4.5 to 9.0) and at a broad temperature range (45–85°C) and it has
an optimum pH of 5.5 and an optimum temperature of 65°C. Kinetic experiment at 65°C with p-nitrophenyl α-l-arabinofuranoside as a substrate gave a V
max and K
m values of 1,019 U/mg and 0.139 mM, respectively. The enzyme had no apparent requirement of metal ions for activity, and its
activity was strongly inhibited by 1 mM Cu2+ and Hg2+. The recombinant arabinofuranosidase released l-arabinose from arabinan, arabinoxylan, oat spelt xylan, arabinobiose, arabinotriose, arabinotetraose, and arabinopentaose.
Endoarabinanase activity was not detected. These findings suggest that AbfAC26Sari is an exo-acting enzyme. 相似文献