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1.
l-arabinose isomerase (EC5.3.1.4. AI) mediates the isomerization of d-galactose into d-tagatose as well as the conversion of l-arabinose into l-ribulose. The AI from Lactobacillus plantarum SK-2 was purified to an apparent homogeneity giving a single band on SDS–PAGE with a molecular mass of 59.6 kDa. Optimum
activity was observed at 50°C and pH 7.0. The enzyme was stable at 50°C for 2 h and held between pH 4.5 and 8.5 for 1 h. AI
activity was stimulated by Mn2+, Fe3+, Fe2+, Ca2+ and inhibited by Cu2+, Ag+, Hg2+, Pb2+. d-galactose and l-arabinose as substrates were isomerized with high activity. l-arabitol was the strongest competitive inhibitor of AI. The apparent Michaelis–Menten constant (K
m), for galactose, was 119 mM. The first ten N-terminal amino acids of the enzyme were determined as MLSVPDYEFW, which is identical to L. plantarum (Q88S84). Using the purified AI, 390 mg tagatose could be converted from 1,000 mg galactose in 96 h, and this production
corresponds to a 39% equilibrium. 相似文献
2.
Prabhu P Tiwari MK Jeya M Gunasekaran P Kim IW Lee JK 《Applied microbiology and biotechnology》2008,81(2):283-290
Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues
with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni–NTA chromatography. The molecular mass of the purified enzyme
was estimated to be ~53 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography,
suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn2+or Co2+, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°C, respectively, with a k
cat of 12,455 min−1 and a k
cat/K
m of 34 min−1 mM−1 for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far. 相似文献
3.
M. Helanto K. Kiviharju T. Granström M. Leisola A. Nyyssölä 《Applied microbiology and biotechnology》2009,83(1):77-83
l-Ribose is a rare and expensive sugar that can be used as a precursor for the production of l-nucleoside analogues, which are used as antiviral drugs. In this work, we describe a novel way of producing l-ribose from the readily available raw material l-arabinose. This was achieved by introducing l-ribose isomerase activity into l-ribulokinase-deficient Escherichia coli UP1110 and Lactobacillus plantarum BPT197 strains. The process for l-ribose production by resting cells was investigated. The initial l-ribose production rates at 39°C and pH 8 were 0.46 ± 0.01 g g−1 h−1 (1.84 ± 0.03 g l−1 h−1) and 0.27 ± 0.01 g g−1 h−1 (1.91 ± 0.1 g l−1 h−1) for E. coli and for L. plantarum, respectively. Conversions were around 20% at their highest in the experiments. Also partially purified protein precipitates
having both l-arabinose isomerase and l-ribose isomerase activity were successfully used for converting l-arabinose to l-ribose. 相似文献
4.
Bacillus coagulans has been of great commercial interest over the past decade owing to its strong ability of producing optical pure l-lactic acid from both hexose and pentose sugars including l-arabinose with high yield, titer and productivity under thermophilic conditions. The l-arabinose isomerase (L-AI) from Bacillus coagulans was heterologously over-expressed in Escherichia coli. The open reading frame of the L-AI has 1,422 nucleotides encoding a protein with 474 amino acid residues. The recombinant
L-AI was purified to homogeneity by one-step His-tag affinity chromatography. The molecular mass of the enzyme was estimated
to be 56 kDa by SDS-PAGE. The enzyme was most active at 70°C and pH 7.0. The metal ion Mn2+ was shown to be the best activator for enzymatic activity and thermostability. The enzyme showed higher activity at acidic
pH than at alkaline pH. The kinetic studies showed that the K
m, V
max and k
cat/K
m for the conversion of l-arabinose were 106 mM, 84 U/mg and 34.5 mM−1min−1, respectively. The equilibrium ratio of l-arabinose to l-ribulose was 78:22 under optimal conditions. l-ribulose (97 g/L) was obtained from 500 g/l of l-arabinose catalyzed by the enzyme (8.3 U/mL) under the optimal conditions within 1.5 h, giving at a substrate conversion
of 19.4% and a production rate of 65 g L−1 h−1. 相似文献
5.
Xiong H Turunen O Pastinen O Leisola M von Weymarn N 《Applied microbiology and biotechnology》2004,64(3):353-358
Trichoderma reesei Rut C-30 was grown on eight different natural or rare aldopentoses as the main carbon source and on mixtures of an aldopentose with d-glucose or lactose. The fungal cells consumed all aldopentoses tested, except l-xylose and l-ribose. The highest total xylanase and cellulase activities were achieved when cells were grown on l-arabinose as the main carbon source. The total xylanase activity produced by cells grown on l-arabinose was even higher than that produced by cells grown on an equal amount of lactose. In co-metabolism of d-glucose (15 g l–1) and l-arabinose (5 g l–1), the total volumetric and specific xylanase productivities were improved (derepressed) approximately 23- and 18-fold, respectively, compared to a cultivation on only d-glucose (20 g l–1). In a similar experiment, in which cells were grown on a mixture of lactose and l-arabinose, the xylanase productivity was approximately doubled, compared to a cultivation on only lactose. The cellulase productivities, however, were not improved by the addition of l-arabinose. Compared with a typical industrial fungal enzyme production process with lactose as the main carbon source, better volumetric and specific xylanase productivities were achieved both on a lactose/arabinose mixture and on a glucose/arabinose mixture. 相似文献
6.
Chang-Su Park Soo-Jin Yeom Yu-Ri Lim Yeong-Su Kim Deok-Kun Oh 《World journal of microbiology & biotechnology》2011,27(4):743-750
A putative ribose-5-phosphate isomerase (RpiB) from Streptococcus pneumoniae was purified with a specific activity of 26.7 U mg−1 by Hi-Trap Q HP anion exchange and Sephacryl S-300 HR 16/60 gel filtration chromatographies. The native enzyme existed as
a 96-kDa tetramer with activity maxima at pH 7.5 and 35°C. The RpiB exhibited isomerization activity with l-lyxose, l-talose, d-gulose, d-ribose, l-mannose, d-allose, l-xylulose, l-tagatose, d-sorbose, d-ribulose, l-fructose, and d-psicose and exhibited particularly high activity with l-form monosaccharides such as l-lyxose, l-xylulose, l-talose, and l-tagatose. With l-xylulose (500 g l−1) and l-talose (500 g l−1) substrates, the optimum concentrations of RpiB were 300 and 600 U ml−1, respectively. The enzyme converted 500 g l−1
l-xylulose to 350 g l−1
l-lyxose after 3 h, and yielded 450 g l−1
l-tagatose from 500 g l−1
l-talose after 5 h. These results suggest that RpiB from S. pneumoniae can be employed as a potential producer of l-form monosaccharides. 相似文献
7.
l-Arabinose is the second most abundant pentose beside d-xylose and is found in the plant polysaccharides, hemicellulose and pectin. The need to find renewable carbon and energy
sources has accelerated research to investigate the potential of l-arabinose for the development and production of biofuels and other bioproducts. Fungi produce a number of extracellular arabinanases,
including α-l-arabinofuranosidases and endo-arabinanases, to specifically release l-arabinose from the plant polymers. Following uptake of l-arabinose, its intracellular catabolism follows a four-step alternating reduction and oxidation path, which is concluded
by a phosphorylation, resulting in d-xylulose 5-phosphate, an intermediate of the pentose phosphate pathway. The genes and encoding enzymes l-arabinose reductase, l-arabinitol dehydrogenase, l-xylulose reductase, xylitol dehydrogenase, and xylulokinase of this pathway were mainly characterized in the two biotechnological
important fungi Aspergillus niger and Trichoderma reesei. Analysis of the components of the l-arabinose pathway revealed a number of specific adaptations in the enzymatic and regulatory machinery towards the utilization
of l-arabinose. Further genetic and biochemical analysis provided evidence that l-arabinose and the interconnected d-xylose pathway are also involved in the oxidoreductive degradation of the hexose d-galactose. 相似文献
8.
Kawaguchi H Sasaki M Vertès AA Inui M Yukawa H 《Applied microbiology and biotechnology》2008,77(5):1053-1062
Corynebacterium glutamicum was metabolically engineered to broaden its substrate utilization range to include the pentose sugar l-arabinose, a product of the degradation of lignocellulosic biomass. The resultant CRA1 recombinant strain expressed the Escherichia coli genes araA, araB, and araD encoding l-arabinose isomerase, l-ribulokinase, and l-ribulose-5-phosphate 4-epimerase, respectively, under the control of a constitutive promoter. Unlike the wild-type strain,
CRA1 was able to grow on mineral salts medium containing l-arabinose as the sole carbon and energy source. The three cloned genes were expressed to the same levels whether cells were
cultured in the presence of d-glucose or l-arabinose. Under oxygen deprivation and with l-arabinose as the sole carbon and energy source, strain CRA1 carbon flow was redirected to produce up to 40, 37, and 11%,
respectively, of the theoretical yields of succinic, lactic, and acetic acids. Using a sugar mixture containing 5% d-glucose and 1% l-arabinose under oxygen deprivation, CRA1 cells metabolized l-arabinose at a constant rate, resulting in combined organic acids yield based on the amount of sugar mixture consumed after
d-glucose depletion (83%) that was comparable to that before d-glucose depletion (89%). Strain CRA1 is, therefore, able to utilize l-arabinose as a substrate for organic acid production even in the presence of d-glucose. 相似文献
9.
The rpiB gene, encoding ribose-5-phosphate isomerase (RpiB) from Clostridium thermocellum, was cloned and expressed in Escherichia coli. RpiB converted d-psicose into d-allose but it did not convert d-xylose, l-rhamnose, d-altrose or d-galactose. The production of d-allose by RpiB was maximal at pH 7.5 and 65°C for 30 min. The half-lives of the enzyme at 50°C and 65°C were 96 h and 4.7 h,
respectively. Under stable conditions of pH 7.5 and 50°C, 165 g d-allose l−1
was produced without by-products from 500 g d-psicose l−1 after 6 h. 相似文献
10.
In the skin of fire-bellied toads (Bombina species), an aminoacyl-l/d-isomerase activity is present which catalyses the post-translational isomerization of the l- to the d-form of the second residue of its substrate peptides. Previously, this new type of enzyme was studied in some detail and
genes potentially coding for similar polypeptides were found to exist in several vertebrate species including man. Here, we
present our studies to the substrate specificity of this isomerase using fluorescence-labeled variants of the natural substrate
bombinin H with different amino acids at positions 1, 2 or 3. Surprisingly, this enzyme has a rather low selectivity for residues
at position 2 where the change of chirality at the alpha-carbon takes place. In contrast, a hydrophobic amino acid at position
1 and a small one at position 3 of the substrate are essential. Interestingly, some peptides containing a Phe at position
3 also were substrates. Furthermore, we investigated the role of the amino-terminus for substrate recognition. In view of
the rather broad specificity of the frog isomerase, we made a databank search for potential substrates of such an enzyme.
Indeed, numerous peptides of amphibia and mammals were found which fulfill the requirements determined in this study. Expression
of isomerases with similar characteristics in other species can therefore be expected to catalyze the formation of peptides
containing d-amino acids. 相似文献
11.
Zhu L Tao R Wang Y Jiang Y Lin X Yang Y Zheng H Jiang W Yang S 《Applied microbiology and biotechnology》2011,90(3):903-910
l-2-Aminobutyric acid can be synthesized in a transamination reaction from l-threonine and l-aspartic acid as substrates by the action of threonine deaminase and aromatic aminotransferase, but the by-product l-alanine was produced simultaneously. A small amount of l-alanine increased the complexity of the l-2-aminobutyric acid recovery process because of their extreme similarity in physical and chemical properties. Acetolactate
synthase has been introduced to remove the pyruvate intermediate for reducing the l-alanine concentration partially. To eliminate the remnant l-alanine, alanine racemase of Bacillus subtilis in combination with d-amino acid oxidase of Rhodotorula gracilis or Trigonopsis variabilis respectively was introduced into the reaction system for the l-2-aminobutyric acid synthesis. l-Alanine could be completely removed by the action of alanine racemase of B. subtilis and d-amino acid oxidase of R. gracilis; thereby, high-purity l-2-aminobutyric acid was achieved. The results revealed that alanine racemase could discriminate effectively between l-alanine and l-2-aminobutyric acid, and selectively catalyzed l-alanine to d-alanine reversibly. d-Amino acid oxidase then catalyzed d-alanine to pyruvate stereoselectively. Furthermore, this method was also successfully used to remove the by-product l-alanine in the production of other neutral amino acids such as l-tertiary leucine and l-valine, suggesting that multienzymatic whole-cell catalysis can be employed to provide high purity products. 相似文献
12.
Li Y Kawakami N Ogola HJ Ashida H Ishikawa T Shibata H Sawa Y 《Applied microbiology and biotechnology》2011,90(6):1953-1962
l-aspartate dehydrogenase (EC 1.4.1.21; l-AspDH) is a rare member of amino acid dehydrogenase superfamily and so far, two thermophilic enzymes have been reported.
In our study, an ORF PA3505 encoding for a putative l-AspDH in the mesophilic bacterium Pseudomonas aeruginosa PAO1 was identified, cloned, and overexpressed in Escherichia coli. The homogeneously purified enzyme (PaeAspDH) was a dimeric protein with a molecular mass of about 28 kDa exhibiting a very
high specific activity for l-aspartate (l-Asp) and oxaloacetate (OAA) of 127 and 147 U mg−1, respectively. The enzyme was capable of utilizing both nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine
dinucleotide phosphate (NADP) as coenzyme. PaeAspDH showed a T
m value of 48°C for 20 min that was improved to approximately 60°C by the addition of 0.4 M NaCl or 30% glycerol. The apparent
K
m values for OAA, NADH, and ammonia were 2.12, 0.045, and 10.1 mM, respectively; comparable results were observed with NADPH.
The l-Asp production system B consisting of PaeAspDH, Bacillus subtilis malate dehydrogenase and E. coli fumarase, achieved a high level of l-Asp production (625 mM) from fumarate in fed-batch process with a molar conversion yield of 89.4%. Furthermore, the fermentative
production system C released 33 mM of l-Asp after 50 h by using succinate as carbon source. This study represented an extensive characterization of the mesophilic
AspDH and its potential applicability for efficient and attractive production of l-Asp. Our novel production systems are also hopeful for developing the new processes for other compounds production. 相似文献
13.
Summary. Hydrogensquarates of dipeptide l-threonyl-l-serine (H-Thr-Ser-OH) and l-serine (HSq × Ser) have been synthesized, isolated and spectroscopic characterized by solid-state linear-polarized IR-spectroscopy, 1H- and 13C-NMR, ESI-MS and HPLC with tandem masspectrometry (MS-MS) methods. The structures of the salts and neutral dipeptide have
been predicted theoretically by ab initio calculations. In the case of H-Thr-Ser-OH the theoretical data are supported by IR-LD ones. The hydrogensquarates consist in positive charged dipeptide or amino acid
moiety and negative hydrogensquarate anion (HSq) stabilizing by strong intermolecular hydrogen bonds. The data about the l-serine hydrogensquarate are compared with known crystallographic data thus indicating a good correlation between the theoretical
predicted structures and experimentally obtained by single crystal X-ray diffraction. 相似文献
14.
Deok-Kun Oh Nam-Hee Kim Hye-Jung Kim Chang-Su Park Seon Won Kim Minsu Ko Bueng Wan Park Min Ho Jung Ki-Hong Yoon 《World journal of microbiology & biotechnology》2007,23(4):559-563
Sinorhizobium sp., which can convert d-fructose into d-psicose, was isolated from soil. The optimal pH, temperature, and cell concentration for d-psicose production with the isolated strain were 8.5, 40°C, and 60 mg/ml, respectively. The toluene-treated cells showed
2.5- and 4.8-fold increases in the d-psicose concentration and productivity compared with untreated washed cells. Under the optimal conditions, the toluene-treated
cells produced 37 g d-psicose/l from 70% (w/v) (3.9 M) d-fructose after 15 h. 相似文献
15.
Ma C Gao C Qiu J Hao J Liu W Wang A Zhang Y Wang M Xu P 《Applied microbiology and biotechnology》2007,77(1):91-98
Pseudomonas stutzeri SDM was newly isolated from soil, and two stereospecific NAD-independent lactate dehydrogenase (iLDH) activities were detected
in membrane of the cells cultured in a medium containing dl-lactate as the sole carbon source. Neither enzyme activities was constitutive, but both of them might be induced by either
enantiomer of lactate. P. stutzeri SDM preferred to utilize lactate to growth, when both l-lactate and glucose were available, and the consumption of glucose was observed only after lactate had been exhausted. The
Michaelis–Menten constant for l-lactate was higher than that for d-lactate. The l-iLDH activity was more stable at 55°C, while the d-iLDH activity was lost. Both enzymes exhibited different solubilization with different detergents and different oxidation
rates with different electron acceptors. Combining activity staining and previous proteomic analysis, the results suggest
that there are two separate enzymes in P. stutzeri SDM, which play an important role in converting lactate to pyruvate.
Ma and Gao contributed equally to this work. 相似文献
16.
Kim P 《Applied microbiology and biotechnology》2004,65(3):243-249
d-Tagatose is a hexoketose monosaccharide sweetener, which is an isomer of d-galactose and is rarely found in nature. Recently, there has been industrial interest in d-tagatose as a low-calorie sugar-substituting sweetener. This article describes the properties and metabolism of tagatose as well as its commercial importance. The comparison between the biological tagatose production and the chemical production was reviewed based on the example of the glucose isomerization into fructose. The industrial problems facing its commercial application is described and evolving potential solutions are suggested. 相似文献
17.
A non-characterized gene, previously proposed as the d-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with d-fructose and decreased for other substrates in the order: d-tagatose, d-psicose, d-ribulose, d-xylulose and d-sorbose. Its activity was maximal at pH 9 and 40°C while being enhanced by Mn2+. At pH 9 and 40°C, 118 g d-psicose l−1 was produced from 700 g d-fructose l−1 after 3 h.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
18.
Sebastián Sánchez Vicente Bravo Juan Francisco García Nicolás Cruz Manuel Cuevas 《World journal of microbiology & biotechnology》2008,24(5):709-716
The fermentation of d-glucose and d-xylose mixtures by the yeast Candida tropicalis NBRC 0618 has been studied under the most favourable operation conditions for the culture, determining the most adequate
initial proportion in these sugars for xylitol production. In all the experiments a synthetic culture medium was used, with
an initial total substrate concentration of 25 g L−1, a constant pH of 5.0 and a temperature of 30 °C. From the experimental results, it was deduced that the highest values of
specific rates of production and of overall yield in xylitol were achieved for the mixtures with the highest percentage of
d-xylose, specifically in the culture with the initial d-glucose and d-xylose concentrations of 1 and 24 g L−1, respectively, with an overall xylitol yield of 0.28 g g−1. In addition, the specific rates of xylitol production declined over the time course of the culture and the formation of
this bioproduct was favoured by the presence of small quantities of d-glucose. The sum of the overall yield values in xylitol and ethanol for all the experiments ranged from 0.26 to 0.56 g bioproduct/g
total substrate. 相似文献
19.
Immobilization of Bacillus licheniformis
l-arabinose isomerase (BLAI) on aminopropyl glass modified with glutaraldehyde (4 mg protein g support−1) was found to enhance the enzyme activity. The immobilization yield of BLAI was proportional to the quantity of amino groups
on the surface of support. Reducing particle size increased the adsorption capacity (q
m) and affinity (k
a). The pH and temperature for immobilization were optimized to be pH 7.1 and 33°C using response surface methodology (RSM).
The immobilized enzyme was characterized and compared to the free enzyme. There is no change in optimal pH and temperature
before and after immobilization. However, the immobilized BLAI enzyme achieved 145% of the activity of the free enzyme. Correspondingly,
the catalytic efficiency (k
cat/K
m) was improved 1.47-fold after immobilization compared to the free enzyme. The thermal stability was improved 138-fold (t
1/2 increased from 2 to 275 h) at 50°C following immobilization. 相似文献
20.
Achromobacter xylosoxidans is known to utilize d-glucose via the modified Entner-Doudoroff pathway. Although d-gluconate dehydratase produced from this bacterium was purified and partially characterized previously, a gene that encodes
this enzyme has not yet been identified. To obtain protein information on bacterial d-gluconate dehydratase, we partially purified d-gluconate dehydratase in A. xylosoxidans and investigated its biochemical properties. Two degenerate primers were designed based on the N-terminal amino acid sequence
of the partially purified d-gluconate dehydratase. Through PCR performed using degenerate primers, a 1,782-bp DNA sequence encoding the A. xylosoxidans
d-gluconate dehydratase (gnaD) was obtained. The deduced amino acid sequence of A. xylosoxidans gnaD showed strong similarity with that of proteins belonging to the dihydroxy-acid dehydratase/phosphogluconate dehydratase family
(COG0129). This is in contrast to the archaeal d-gluconate dehydratase, which belongs to the enolase superfamily (COG4948). The phylogenetic tree showed that A. xylosoxidans
d-gluconate dehydratase is closer to the 6-phosphogluconate dehydratase than the dihydroxy-acid dehydratase. Interestingly,
a clade containing A. xylosoxidans enzyme was clustered with proteins annotated as a second and a third dihydroxy-acid dehydratase in the genomes of Clostridium acetobutylicum (Cac_ilvD2) and Streptomyces ceolicolor (Sco_ilvD2, Sco_ilvD3), indicating that the function of these enzymes is the dehydration of d-gluconate. 相似文献