Isolation of a Novel <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-lactosamine Specific Lectin from <Emphasis Type="Italic">Alocasia cucullata</Emphasis> (Schott.)

An N-acetyl-d-lactosamine (LacNAc) specific lectin from tubers of Alocasia cucullata was purified by affinity chromatography on asialofetuin-linked amino activated silica. The pure lectin showed a single band in SDS-PAGE at pH 8.8 and was a homotetramer with a subunit molecular mass of 13.5 kDa and native molecular mass of 53 kDa. It was heat stable up to 55 °C for 15 min and showed optimum hemagglutination activity from pH 2 to 11. The lectin was affected by denaturing agents such as urea (2 m), thiourea (2 m) and guanidine–HCl (0.5 m) and did not require Ca²⁺ and Mn²⁺ for its activity. It was a potent mitogen at 10 μg/ml towards human peripheral blood mononuclear cells with 50% growth inhibitory potential towards SiHa (human cervix ) cancer cell line at 100 μg/ml.     

Characterization of <Emphasis Type="SmallCaps">d</Emphasis>-tagatose-3-epimerase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> that converts <Emphasis Type="SmallCaps">d</Emphasis>-fructose into <Emphasis Type="SmallCaps">d-</Emphasis>psicose

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.     

<Emphasis Type="SmallCaps">d</Emphasis>-Psicose production from <Emphasis Type="SmallCaps">d</Emphasis>-fructose using an isolated strain, <Emphasis Type="Italic">Sinorhizobium</Emphasis> sp.

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.     

Characterization of ribose-5-phosphate isomerase of <Emphasis Type="Italic">Clostridium thermocellum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-allose from <Emphasis Type="SmallCaps">d</Emphasis>-psicose

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.     

Purification and characterization of <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> producing <Emphasis Type="SmallCaps">d</Emphasis>-tagatose

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.     

Fermentation of <Emphasis Type="SmallCaps">d</Emphasis>-glucose and <Emphasis Type="SmallCaps">d</Emphasis>-xylose mixtures by <Emphasis Type="Italic">Candida tropicalis</Emphasis> NBRC 0618 for xylitol production

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.     

Substrate specificity of a peptidyl-aminoacyl-<Emphasis Type="SmallCaps">l</Emphasis>/<Emphasis Type="SmallCaps">d</Emphasis>-isomerase from frog skin

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.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-alanine from <Emphasis Type="SmallCaps">dl</Emphasis>-alanine using immobilized cells of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> HLZ-68

Immobilized cells of Bacillus subtilis HLZ-68 were used to produce d-alanine from dl-alanine by asymmetric degradation. Different compounds such as polyvinyl alcohol and calcium alginate were employed for immobilizing the B. subtilis HLZ-68 cells, and the results showed that cells immobilized using a mixture of these two compounds presented higher l-alanine degradation activity, when compared with free cells. Subsequently, the effects of different concentrations of polyvinyl alcohol and calcium alginate on l-alanine consumption were examined. Maximum l-alanine degradation was exhibited by cells immobilized with 8% (w/v) polyvinyl alcohol and 2% (w/v) calcium alginate. Addition of 400 g of dl-alanine (200 g at the beginning of the reaction and 200 g after 30 h of incubation) into the reaction solution at 30 °C, pH 6.0, aeration of 1.0 vvm, and agitation of 400 rpm resulted in complete l-alanine degradation within 60 h, leaving 185 g of d-alanine in the reaction solution. The immobilized cells were applied for more than 15 cycles of degradation and a maximum utilization rate was achieved at the third cycle. d-alanine was easily extracted from the reaction solution using cation-exchange resin, and the chemical and optical purity of the extracted d-alanine was 99.1 and 99.6%, respectively.     

Cloning and expression of <Emphasis Type="SmallCaps">d</Emphasis> -lactonohydrolase cDNA from <Emphasis Type="Italic">Fusarium moniliforme</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A d -lactonohydrolase gene of about 1.1 kb was cloned from Fusarium moniliforme. The ORF sequence predicted a protein of 382 amino acids with a molecular mass of about 40 kDa. An expression plasmid carrying the gene under the control of the triose phosphate isomerase gene promotor was introduced into Saccharomyces cerevisiae, and the d -lactonohydrolase gene was successfully expressed in the recombinant strains.Revisions requested 10 September 2004; Revisions received 15 October 2004The nucleotide sequence data reported in this paper has been assigned accession number AY728018 in the GeneBank database.     

Identification and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Anoxybacillus flavithermus</Emphasis> useful in <Emphasis Type="SmallCaps">d</Emphasis>-tagatose production

d-Tagatose is a highly functional rare ketohexose and many attempts have been made to convert d-galactose into the valuable d-tagatose using l-arabinose isomerase (l-AI). In this study, a thermophilic strain possessing l-AI gene was isolated from hot spring sludge and identified as Anoxybacillus flavithermus based on its physio-biochemical characterization and phylogenetic analysis of its 16s rRNA gene. Furthermore, the gene encoding l-AI from A. flavithermus (AFAI) was cloned and expressed at a high level in E. coli BL21(DE3). l-AI had a molecular weight of 55,876 Da, an optimum pH of 10.5 and temperature of 95°C. The results showed that the conversion equilibrium shifted to more d-tagatose from d-galactose by raising the reaction temperatures and adding borate. A 60% conversion of d-galactose to d-tagatose was observed at an isomerization temperature of 95°C with borate. The catalytic efficiency (k _cat /K _m) for d-galactose with borate was 9.47 mM⁻¹ min⁻¹, twice as much as that without borate. Our results indicate that AFAI is a novel hyperthermophilic and alkaliphilic isomerase with a higher catalytic efficiency for d-galactose, suggesting its great potential for producing d-tagatose.     

Membrane-bound <Emphasis Type="SmallCaps">l</Emphasis>- and <Emphasis Type="SmallCaps">d</Emphasis>-lactate dehydrogenase activities of a newly isolated <Emphasis Type="Italic">Pseudomonas stutzeri</Emphasis> strain

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.     

Production of <Emphasis Type="SmallCaps">d</Emphasis>-arabitol by a newly isolated <Emphasis Type="Italic">Zygosaccharomyces rouxii</Emphasis>

A newly isolated Zygosaccharomyces rouxii NRRL 27,624 produced d-arabitol as the main metabolic product from glucose. In addition, it also produced ethanol and glycerol. The optimal conditions were temperature 30°C, pH 5.0, 350 rpm, and 5% inoculum. The yeast produced 83.4 ± 1.1 g d-arabitol from 175 ± 1.1 g glucose per liter at pH 5.0, 30°C, and 350 rpm in 240 h with a yield of 0.48 g/g glucose. It also produced d-arabitol from fructose, galactose, and mannose. The yeast produced d-arabitol and xylitol from xylose and also from a mixture of xylose and xylulose. Resting yeast cells produced 63.6 ± 1.9 g d-arabitol from 175 ± 1.8 g glucose per liter in 210 h at pH 5.0, 30°C and 350 rpm with a yield of 0.36 g/g glucose. The yeast has potential to be used for production of xylitol from glucose via d-arabitol route. Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. department of Agriculture.     

Phosphoenolpyruvate-supply module in <Emphasis Type="Italic">Escherichia coli</Emphasis> improves <Emphasis Type="Italic">N</Emphasis>-acetyl-<Emphasis Type="SmallCaps">d</Emphasis>-neuraminic acid biocatalysis

Objectives

N-Acetyl-d-neuraminic acid (Neu5Ac) is often synthesized from exogenous N-acetylglucosamine (GlcNAc) and excess pyruvate. We have previously constructed a recombinant Escherichia coli strain for Neu5Ac production using GlcNAc and intracellular phosphoenolpyruvate (PEP) as substrates (Zhu et al. Biotechnol Lett 38:1–9, 2016).

Results

PEP synthesis-related genes, pck and ppsA, were overexpressed within different modes to construct PEP-supply modules, and their effects on Neu5Ac production were investigated. All the PEP-supply modules enhanced Neu5Ac production. For the best module, pCDF-pck-ppsA increased Neu5Ac production to 8.6 ± 0.15 g l^?1, compared with 3.6 ± 0.15 g l^?1 of the original strain. Neu5Ac production was further increased to 15 ± 0.33 g l^?1 in a 1 l fermenter.

Conclusions

The PEP-supply module can improve the intracellular PEP supply and enhance Neu5Ac production, which benefited industrial Neu5Ac production.

     

Continuous <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid production by a novelthermotolerant <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis> QU 41

We isolated and characterized a d-lactic acid-producing lactic acid bacterium (d-LAB), identified as Lactobacillus delbrueckii subsp. lactis QU 41. When compared to Lactobacillus coryniformis subsp. torquens JCM 1166 ^T and L. delbrueckii subsp. lactis JCM 1248 ^T, which are also known as d-LAB, the QU 41 strain exhibited a high thermotolerance and produced d-lactic acid at temperatures of 50 °C and higher. In order to optimize the culture conditions of the QU 41 strain, we examined the effects of pH control, temperature, neutralizing reagent, and initial glucose concentration on d-lactic acid production in batch cultures. It was found that the optimal production of 20.1 g/l d-lactic acid was acquired with high optical purity (>99.9% of d-lactic acid) in a pH 6.0-controlled batch culture, by adding ammonium hydroxide as a neutralizing reagent, at 43 °C in MRS medium containing 20 g/l glucose. As a result of product inhibition and low cell density, continuous cultures were investigated using a microfiltration membrane module to recycle flow-through cells in order to improve d-lactic acid productivity. At a dilution rate of 0.87 h⁻¹, the high cell density continuous culture exhibited the highest d-lactic acid productivity of 18.0 g/l/h with a high yield (ca. 1.0 g/g consumed glucose) and a low residual glucose (<0.1 g/l) in comparison with systems published to date.     

Strain improvement of <Emphasis Type="Italic">Lactobacillus lactis</Emphasis> for <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid production

Three mutants, isolated by repeated UV mutagenesis of Lactobacillus lactis NCIM 2368, produced increased d-lactic acid concentrations. These mutants were compared with the wild type using 100 g hydrolyzed cane sugar/l in the fermentation medium. One mutant, RM2-24, produced 81 g lactic acid/l which was over three times that of the wild type. The highest d-lactic acid (110 g/l) in batch fermentation was obtained with 150 g cane sugar/l with a 73% lactic acid yield. The mutant utilizes cellobiose efficiently, converting it into d-lactic acid suggesting the presence of cellobiase. Thus, this strain could be used to obtain d-lactic acid from cellulosic materials that are pre-hydrolyzed with cellulase.     

Uptake and conversion of <Emphasis Type="SmallCaps">d</Emphasis>-amino acids in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

The d-enantiomers of proteinogenic amino acids fulfill essential functions in bacteria, fungi and animals. Just in the plant kingdom, the metabolism and role of d-amino acids (d-AAs) still remains unclear, although plants have to cope with significant amounts of these compounds from microbial decay in the rhizosphere. To fill this gap of knowledge, we tested the inhibitory effects of d-AAs on plant growth and established a method to quantitate 16 out of 19 proteinogenic amino acids and their d-enantiomers in plant tissue extracts. Therefore, the amino acids in the extracts were derivatized with Marfey’s reagent and separated by HPLC–MS. We used two ecotypes (Col-0 and C24) and a mutant (lht1) of the model plant Arabidopsis thaliana to determine the influence and fate of exogenously applied d-AAs. All of them were found in high concentrations in the plant extracts after application, even in lht1, which points to additional transporters facilitating the import of d-AAs. The addition of particular amino acids (d-Trp, d-Phe, d-Met and d-His) led to the accumulation of the corresponding l-amino acid. In almost all cases, the application of a d-AA resulted in the accumulation of d-Ala and d-Glu. The presented results indicate that soil borne d-AAs can actively be taken up and metabolized via central metabolic routes.     

Engineering an ATP-dependent <Emphasis Type="SmallCaps">d</Emphasis>-Ala:<Emphasis Type="SmallCaps">d</Emphasis>-Ala ligase for synthesizing amino acid amides from amino acids

We successfully engineered a new enzyme that catalyzes the formation of d-Ala amide (d-AlaNH₂) from d-Ala by modifying ATP-dependent d-Ala:d-Ala ligase (EC 6.3.2.4) from Thermus thermophilus, which catalyzes the formation of d-Ala-d-Ala from two molecules of d-Ala. The new enzyme was created by the replacement of the Ser293 residue with acidic amino acids, as it was speculated to bind to the second d-Ala of d-Ala-d-Ala. In addition, a replacement of the position with Glu performed better than that with Asp with regards to specificity for d-AlaNH₂ production. The S293E variant, which was selected as the best enzyme for d-AlaNH₂ production, exhibited an optimal activity at pH 9.0 and 40 °C for d-AlaNH₂ production. The apparent K _m values of this variant for d-Ala and NH₃ were 7.35 mM and 1.58 M, respectively. The S293E variant could catalyze the synthesis of 9.3 and 35.7 mM of d-AlaNH₂ from 10 and 50 mM d-Ala and 3 M NH₄Cl with conversion yields of 93 and 71.4 %, respectively. This is the first report showing the enzymatic formation of amino acid amides from amino acids.     

Biotransformation of glycerol to <Emphasis Type="SmallCaps">d</Emphasis>-glyceric acid by <Emphasis Type="Italic">Acetobacter tropicalis</Emphasis>

Bacterial strains capable of converting glycerol to glyceric acid (GA) were screened among the genera Acetobacter and Gluconacetobacter. Most of the tested Acetobacter and Gluconacetobacter strains could produce 1.8 to 9.3 g/l GA from 10% (v/v) glycerol when intact cells were used as the enzyme source. Acetobacter tropicalis NBRC16470 was the best GA producer and was therefore further investigated. Based on the results of high-performance liquid chromatography analysis and specific rotation, the enantiomeric composition of the produced GA was d-glyceric acid (d-GA). The productivity of d-GA was enhanced with the addition of both 15% (v/v) glycerol and 20 g/l yeast extract. Under these optimized conditions, A. tropicalis NBRC16470 produced 22.7 g/l d-GA from 200 g/l glycerol during 4 days of incubation in a jar fermentor.     

A broader role for AmyR in <Emphasis Type="Italic">Aspergillus niger</Emphasis>: regulation of the utilisation of <Emphasis Type="SmallCaps">d</Emphasis>-glucose or <Emphasis Type="SmallCaps">d</Emphasis>-galactose containing oligo- and polysaccharides

AmyR is commonly considered a regulator of starch degradation whose activity is induced by the presence of maltose, the disaccharide building block of starch. In this study, we demonstrate that the role of AmyR extends beyond starch degradation. Enzyme activity assays, genes expression analysis and growth profiling on d-glucose- and d-galactose-containing oligo- and polysaccharides showed that AmyR regulates the expression of some of the Aspergillus niger genes encoding α- and β-glucosidases, α- and β- galactosidases, as well as genes encoding α-amlyases and glucoamylases. In addition, we provide evidence that d-glucose or a metabolic product thereof may be the inducer of the AmyR system in A. niger and not maltose, as is commonly assumed.