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The discharge of industrial dyes and their breakdown products are often environmentally harmful. Here, we describe a biodegradation method using Burkholderia multivorans CCA53, which exhibits a capacity to degrade azo dyes, particularly ethyl red. Under the optimized culture conditions, 100 μM ethyl red was degraded more than 99% after incubation for 8 h. Real-time PCR analysis of azoR1 and azoR2, encoding two azoreductases, revealed that transcription level of these genes is enhanced at early phase under the optimized conditions. For a more practical approach, hydrolysates were prepared from eucalyptus or Japanese cedar chips or rice straw, and rice straw hydrolysate was used as the best medium for ethyl red biodegradation. Under those conditions, ethyl red was also degraded with high efficiency (>91%). We have thus constructed a potentially economical method for the biodegradation of ethyl red.

Construction of acid-tolerant strains of Saccharomyces cerevisiae is required for various bioproduction processes. We previously isolated the gene IoGAS1 from multiple stress-tolerant Issatchenkia orientalis as a gene conferring sulfuric acid resistance in S. cerevisiae, but its acid tolerance was only investigated using sulfuric acid. Here, we evaluated the growth and ethanol fermentation ability of the IoGAS1-expressing S. cerevisiae strain, B4-IoGAS1, by using various acidic reagents. B4-IoGAS1 exhibited faster growth than the control strain, B4-CON, when cultured aerobically with sulfuric, hydrochloric, formic, acetic, and lactic acids at pH below 2.4. However, the growth of B4-IoGAS1 was suppressed at pH above 2.48, irrespective of the type of acid reagents. Furthermore, B4-IoGAS1 exhibited higher performance of ethanol fermentation than B4-CON under 250 mM lactic acid condition at pH 2.37. These results demonstrate that IoGAS1 could facilitate the aerobic growth and anaerobic ethanol production under different acidic stressed conditions.

Simultaneous saccharification and fermentation (SSF) is capable of performing enzymatic saccharification and fermentation for biofuel production in a single vessel. Thus, SSF has several advantages such as simplifying the manufacturing process, operating easily, and reducing energy input. Here, we describe the application of Pichia kudriavzevii NBRC1279 and NBRC1664 to SSF for bioethanol production. When each strain was incubated for 144 h at 35 °C with Japanese cedar particles, the highest ethanol concentrations were reached 21.9 ± 0.50 g/L and 23.8 ± 3.9 g/L, respectively. In addition, 21.6 ± 0.29 g/L and 21.3 ± 0.21 g/L of bioethanol were produced from Japanese eucalyptus particles when each strain was incubated for 144 h at 30 °C. Although previous methods require pretreatment of the source material, our method does not require pretreatment, which is an advantage for industrial use. To elucidate the different characteristics of the strains, we performed genome sequencing and genome comparison. Based on the results of the eggNOG categories and the resulting Venn diagram, the functional abilities of both strains were similar. However, strain NBRC1279 showed five retrotransposon protein genes in the draft genome sequence, which indicated that the stress tolerance of both strains is slightly different.

meso‐Diaminopimelate dehydrogenase (meso‐DAPDH) catalyzes the reversible NADP+‐dependent oxidative deamination of meso‐2,6‐diaminopimelate (meso‐DAP) to produce l‐2‐amino‐6‐oxopimelate. Moreover, d‐amino acid dehydrogenase (d‐AADHs) derived from protein‐engineered meso‐DAPDH is useful for one‐step synthesis of d‐amino acids with high optical purity. Here, we report the identification and functional characterization of a novel NAD(P)+‐dependent meso‐DAPDH from Numidum massiliense (NmDAPDH). After the gene encoding the putative NmDAPDH was expressed in recombinant Escherichia coli cells, the enzyme was purified 4.0‐fold to homogeneity from the crude extract through five purification steps. Although the previously known meso‐DAPDHs use only NADP+ as a coenzyme, NmDAPDH was able to use both NADP+ and NAD+ as coenzymes. When NADP+ was used as a coenzyme, NmDAPDH exhibited an approximately 2 times higher kcat/Km value toward meso‐DAP than that of meso‐DAPDH from Symbiobacterium thermophilum (StDAPDH). NmDAPDH also catalyzed the reductive amination of corresponding 2‐oxo acids to produce acidic d‐amino acids such as d‐aspartate and d‐glutamate. The optimum pH and temperature for the oxidative deamination of meso‐DAP were about 10.5 and 75°C, respectively. Like StDAPDH, NmDAPDH exhibited high stability: it retained more than 75% of its activity after 30 min at 60°C (pH 7.2) or at pHs ranging from 5.5 to 13.0 (50°C). Alignment of the amino acid sequences of NmDAPDH and the known meso‐DAPDHs suggested NmDAPDH has a hexameric structure. Given its specificity for both NADP+ and NAD+, high stability, and a broad range of reductive amination activity toward 2‐oxo acids, NmDAPDH appears to offer advantages for engineering a more effective d‐AADH.

Pyruvate, a potential precursor of various chemicals, is one of the fundamental chemicals produced by the fermentation process. We previously reported a pyruvate-producing Escherichia coli strain LAFCPCPt-accBC-aceE (PYR) that has the potential to be applied to the industrial production of pyruvate. In this study, the availability of the PYR strain for the production of pyruvate-derivative chemicals was evaluated using a d-lactate-producing strain (LAC) based on the PYR strain. The LAC strain expresses a d-lactate dehydrogenase-encoding gene from Lactobacillus bulgaricus under the control of a T7 expression system. The d-lactate productivity of the LAC strain was further improved by limiting aeration and changing the induction period for the expression of d-lactate dehydrogenase-encoding gene expression. Under combined conditions, the LAC strain produced d-lactate at 21.7 &plusmn, 1.4 g&middot, L&minus, 1, which was compatible with the pyruvate production by the PYR strain (26.1 &plusmn, 0.9 g&middot, 1). These results suggest that we have succeeded in the effective conversion of pyruvate to d-lactate in the LAC strain, demonstrating the wide versatility of the parental PYR strain as basal strain for various chemicals production.

To generate an organism capable of producing d-lactate, NAD+-dependent d-lactate dehydrogenase was expressed in our pyruvate-producing strain, Escherichia coli strain LAFCPCPt-accBC-aceE. After determining the optimal culture conditions for d-lactate production, 18.4 mM d-lactate was produced from biomass-based medium without supplemental mineral or nitrogen sources. Our results show that d-lactate can be produced in simple batch fermentation processes.

Malaysia is the second largest palm oil producer and exporter globally. When crude palm oil is produced in both plantations and oil processing mills, a large amount of oil palm empty fruit bunch (OPEFB) is simultaneously produced as a waste product. Here, we describe the preparation of hydrolysate from OPEFB. After OPEFB was hydrothermally treated at 180–200 °C, the resultant liquid phase was subjected to high-performance liquid chromatography analysis, while the solid phase was used for acidic and enzymatic hydrolysis. Hemicellulose yield from the acid-treated solid phase decreased from 153 mg/g-OPEFB to 27.5 mg/g-OPEFB by increasing the hydrothermal treatment temperature from 180 to 200 °C. Glucose yield from the enzyme-treated solid phase obtained after hydrothermal treatment at 200 °C was the highest (234 ± 1.90 mg/g-OPEFB, 61.7% production efficiency). In contrast, xylose, mannose, galactose, and arabinose yields in the hydrolysate prepared from the solid phase hydrothermally treated at 200 °C were the lowest. Thus, we concluded that the optimum temperature for hydrothermal pretreatment was 200 °C, which was caused by the low hemicellulose yield. Based on these results, we have established an effective method for preparing OPEFB hydrolysates with high glucose content.

We previously reported the crystal structure of an acetyl esterase (TcAE206) belonging to carbohydrate esterase family 3 from Talaromyces cellulolyticus. In this study, we solved the crystal structure of an S10A mutant of TcAE206 complexed with an acetate ion. The acetate ion was stabilized by three hydrogen bonds in the oxyanion hole instead of a water molecule as in the structure of wild-type TcAE206. Furthermore, the catalytic triad residue His182 moved 0.8 Å toward the acetate ion upon substrate entering the active site, suggesting that this movement is necessary for completion of the catalytic reaction., Highlights • The crystal structure of TcAE206_S10A with acetate ion was solved at 1.4 Å resolution. • The complex structure revealed the catalytic mechanism. • His182, which moves 0.8 Å toward the acetate ion, is necessary for enzymatic activity. • This study provides insights into the substrate specificity of fungal CE3 enzymes.

meso-Diaminopimelate dehydrogenase (meso-DAPDH) catalyzes the reversible NADP+-dependent oxidative deamination of meso-2,6-diaminopimelate (meso-DAP) to produce l -2-amino-6-oxopimelate. meso-DAPDH is divided into two major clusters, types I and II, based on substrate specificity and structural characteristic. Here, we describe a novel type II meso-DAPDH from Thermosyntropha lipolytica (TlDAPDH). The gene encoding a putative TlDAPDH was expressed in Escherichia coli cells, and then the enzyme was purified 7.3-fold to homogeneity from the crude cell extract. The molecule of TlDAPDH seemed to form a hexamer, which is the typical structural characteristic of type II meso-DAPDHs. The purified enzyme exhibited oxidative deamination activity toward meso-DAP with both NADP+ and NAD+ as coenzymes. TlDAPDH exhibited reductive amination activity of corresponding 2-oxo acid to produce d -amino acid. In particular, the productivities for d -aspartate and d -glutamate have not been reported in the type II enzymes. The optimum pH and temperature for oxidative deamination of meso-DAP were 10.5 and 55°C, respectively. TlDAPDH retained more than 80% of its activity after incubation for 30 min at temperatures between 50°C and 65°C and in the pH range of 4.5–9.5. Moreover, the coenzyme and substrate recognition mechanisms of TlDAPDH were elucidated based on a multiple sequence alignment and the homology model. The results of these analyses suggested that the molecular mechanisms for coenzyme and substrate recognition of TlDAPDH were similar to those of meso-DAPDH from S. thermophilum, which is the representative type II enzyme. Based on the kinetic characteristics and structural comparison, TlDAPDH was considered to be a novel type II meso-DAPDH.

Many kinds of NAD(P)+-dependent L-amino acid dehydrogenases have been so far found and effectively used for synthesis of L-amino acids and their analogs, and for their sensing. By contrast, similar biotechnological use of D-amino acid dehydrogenase (D-AADH) has not been achieved because useful D-AADH has not been found from natural resources. Recently, using protein engineering methods, an NADP+-dependent D-AADH was created from meso-diaminopimelate dehydrogenase (meso-DAPDH). The artificially created D-AADH catalyzed the reversible NADP+-dependent oxidative deamination of D-amino acids to 2-oxo acids. The enzyme, especially thermostable one from thermophiles, was efficiently applicable to synthesis of D-branched-chain amino acids (D-BCAAs), with high yields and optical purity, and was useful for the practical synthesis of 13C- and/or 15N-labeled D-BCAAs. The enzyme also made it possible to assay D-isoleucine selectively in a mixture of isoleucine isomers. Analyses of the three-dimensional structures of meso-DAPDH and D-AADH, and designed mutations based on the information obtained made it possible to markedly enhance enzyme activity and to create D-AADH homologs with desired reactivity profiles. The methods described here may be an effective approach to artificial creation of biotechnologically useful enzymes.

Isobutanol is attracting attention as a potential biofuel because it has higher energy density and lower hygroscopicity than ethanol. To date, several effective methods for microbial production of isobutanol have been developed, but they require expensive reagents to maintain expression plasmids and induce expression, which is not suitable for practical production. Here, we describe a simple and efficient method for isobutanol production in Escherichia coli. It is noteworthy that no expression plasmids or inducers were used during the production. Instead, heterologous genes necessary for isobutanol production were all knocked into the genome, and the expression of those genes was induced by xylose, which is present in most biomass feedstocks. The constructed strain (mlcXT7-LAFC-AAKCD) contains Bacillus subtilis alsS, E. coli ilvCD, Lactococcus lactis adhA, and L. lactis kivd genes in its genome and efficiently produced isobutanol from glucose and xylose in flask batch cultures. Under conditions in which the temperature and pH of the medium and the aeration in the culture were all optimized, the final isobutanol concentration reached 8.4 g L(-1) after 48 h. Isobutanol was also produced using hydrolysate from Japanese cedar as the carbon source without supplemented glucose, xylose, or yeast extract. Under those conditions, isobutanol (3.7 g L(-1)) was produced in 96 h. Taken together, these results indicate that the developed strain is potentially useful for industrial isobutanol production.

Acetoin is used in the biochemical, chemical and pharmaceutical industries. Several effective methods for acetoin production from petroleum-based substrates have been developed, but they all have an environmental impact and do not meet sustainability criteria. Here we describe a simple and efficient method for acetoin production from oil palm mesocarp fiber hydrolysate using engineered Escherichia coli. An optimization of culture conditions for acetoin production was carried out using reagent-grade chemicals. The final concentration reached 29.9gL-1 with a theoretical yield of 79%. The optimal pretreatment conditions for preparing hydrolysate with higher sugar yields were then determined. When acetoin was produced using hydrolysate fortified with yeast extract, the theoretical yield reached 97% with an acetoin concentration of 15.5gL-1. The acetoin productivity was 10-fold higher than that obtained using reagent-grade sugars. This approach makes use of a compromise strategy for effective utilization of oil palm biomass towards industrial application.

Objective A lignin-degrading bacterium, Burkholderia sp. CCA53, was previously isolated from leaf soil. The purpose of this study was to determine phenotypic and biochemical features of Burkholderia sp. CCA53. Results Multilocus sequence typing (MLST) analysis based on fragments of the atpD, gltD, gyrB, lepA, recA and trpB gene sequences was performed to identify Burkholderia sp. CCA53. The MLST analysis revealed that Burkholderia sp. CCA53 was tightly clustered with B. multivorans ATCC BAA-247T. The quinone and cellular fatty acid profiles, carbon source utilization, growth temperature and pH were consistent with the characteristics of B. multivorans species. Burkholderia sp. CCA53 was therefore identified as B. multivorans CCA53.

A stable NADP + -dependent d -amino acid dehydrogenase (DAADH) was recently created from Ureibacillus thermosphaericus meso -diaminopimelate dehydrogenase through site-directed mutagenesis. To produce a novel DAADH mutant with different substrate specificity, the crystal structure of apo-DAADH was determined at a resolution of 1.78 Å, and the amino acid residues responsible for the substrate specificity were evaluated using additional site-directed mutagenesis. By introducing a single D94A mutation, the enzyme's substrate specificity was dramatically altered; the mutant utilized d -phenylalanine as the most preferable substrate for oxidative deamination and had a specific activity of 5.33 μmol/min/mg at 50°C, which was 54-fold higher than that of the parent DAADH. In addition, the specific activities of the mutant toward d -leucine, d -norleucine, d -methionine, d -isoleucine, and d -tryptophan were much higher (6 to 25 times) than those of the parent enzyme. For reductive amination, the D94A mutant exhibited extremely high specific activity with phenylpyruvate (16.1 μmol/min/mg at 50°C). The structures of the D94A-Y224F double mutant in complex with NADP + and in complex with both NADPH and 2-keto-6-aminocapronic acid (lysine oxo-analogue) were then determined at resolutions of 1.59 Å and 1.74 Å, respectively. The phenylpyruvate-binding model suggests that the D94A mutation prevents the substrate phenyl group from sterically clashing with the side chain of Asp94. A structural comparison suggests that both the enlarged substrate-binding pocket and enhanced hydrophobicity of the pocket are mainly responsible for the high reactivity of the D94A mutant toward the hydrophobic d -amino acids with bulky side chains. IMPORTANCE In recent years, the potential uses for d -amino acids as source materials for the industrial production of medicines, seasonings, and agrochemicals have been growing. To date, several methods have been used for the production of d -amino acids, but all include tedious steps. The use of NAD(P) + -dependent d -amino acid dehydrogenase (DAADH) makes single-step production of d -amino acids from oxo-acid analogs and ammonia possible. We recently succeeded in creating a stable DAADH and demonstrated that it is applicable for one-step synthesis of d -amino acids, such as d -leucine and d -isoleucine. As the next step, the creation of an enzyme exhibiting different substrate specificity and higher catalytic efficiency is a key to the further development of d -amino acid production. In this study, we succeeded in creating a novel mutant exhibiting extremely high catalytic activity for phenylpyruvate amination. Structural insight into the mutant will be useful for further improvement of DAADHs.

In this study, we describe a novel method for producing valuable chemicals from glucose and xylose in Escherichia coli. The notable features in our method are avoidance of plasmids and expensive inducers for foreign gene expression to reduce production costs; foreign genes are knocked into the chromosome, and their expression is induced with xylose that is present in most biomass feedstock. As loci for the gene knock-in, lacZYA and some pseudogenes are chosen to minimize unexpected effects of the knock-in on cell physiology. The promoter of xylF is inducible with xylose and is combined with the T7 RNA polymerase-T7 promoter system to ensure strong gene expression. This expression system was named BICES (biomass-inducible chromosome-based expression system). As examples of BICES application, 2,3-butanediol and acetoin were successfully produced from glucose and xylose, and the maximal concentrations reached 54gL(-1) [99.6% in (R,S)-form] and 31gL(-1), respectively. 2,3-Butanediol and acetoin are industrially important chemicals that are, at present, produced primarily through petrochemical processes. To demonstrate usability of BICES in practical situations, we produced these chemicals from a saccharified cedar solution. From these results, we can conclude that BICES is suitable for practical production of valuable chemicals from biomass.

D-isoleucine (D-Ile) can be assayed using chiral chromatography but the availability of that method is limited by the necessity for special expertise and expensive equipment. We therefore developed a simple and specific colorimetric assay system for D-Ile determination using an artificially created NADP(+)-dependent D-amino acid dehydrogenase (DAADH). The system consists of two reaction steps: the first is the quantitative conversion of D-Ile to (3R)-2-oxo-3-methyl valerate by DAADH in which NADP(+) is converted to NADPH, while the second is chemical conversion of NADPH to reduced water-soluble Tetrazolium-3 via a redox mediator. D-Ile was determined from 1 to 50 µM, and the assay was unaffected by the presence of any of three other isomers (100 µM), alcohol and organic acids.

D-Branched-chain amino acids (D-BCAAs) such as D-leucine, D-isoleucine, and D-valine are known to be peptide antibiotic intermediates and to exhibit a variety of bioactivities. Consequently, much effort is going into achieving simple stereospecific synthesis of D-BCAAs, especially analogs labeled with stable isotopes. Up to now, however, no effective method has been reported. Here, we report the establishment of an efficient system for enantioselective synthesis of D-BCAAs and production of D-BCAAs labeled with stable isotopes. This system is based on two thermostable enzymes: D-amino acid dehydrogenase, catalyzing NADPH-dependent enantioselective amination of 2-oxo acids to produce the corresponding D-amino acids, and glucose dehydrogenase, catalyzing NADPH regeneration from NADP(+) and D-glucose. After incubation with the enzymes for 2 h at 65°C and pH 10.5, 2-oxo-4-methylvaleric acid was converted to D-leucine with an excellent yield (99 %) and optical purity (99 %). Using this system, we produced five different D-BCAAs labeled with stable isotopes: D-[1-(13)C,(15)N]leucine, D-[1-(13)C]leucine, D-[(15)N]leucine, D-[(15)N]isoleucine, and D-[(15)N]valine. The structure of each labeled D-amino acid was confirmed using time-of-flight mass spectrometry and nuclear magnetic resonance analysis. These analyses confirmed that the developed system was highly useful for production of D-BCAAs labeled with stable isotopes, making this the first reported enzymatic production of D-BCAAs labeled with stable isotopes. Our findings facilitate tracer studies investigating D-BCAAs and their derivatives.

Differences in sludge reduction effect due to chlorinated sites of dichlorophenol (DCP) isomers used in an uncoupling method were investigated. The study involved two different experiments. A batch experiment conducted to examine the effect of DCPs on SOUR clearly indicated that 3,5-DCP exhibited the strongest uncoupling activity. In the second experiment, in which six different sequence batch reactors were amended with 100 mM DCPs and operated for 2 months, 3,5-DCP also exhibited the strongest sludge reducing effect. The operations examined in this research maintained the concentration of DCPs at 100 mM, and reactors added with 3,5-DCP produced a COD removal rate of >80%, with almost no excess sludge generated in 2 months of continual operation. These results were not obtained with other DCPs. This is the first study to report differences in the sludge reduction effect associated with the chlorination site in the DCP isomer. The results indicate that 3,5-DCP is the most effective option for use in uncoupling methods involving DCPs.

Pyruvate plays an essential role in the central carbon metabolism of multiple organisms and is used as a raw material in the chemical, biochemical and pharmaceutical industries. To meet demand, large amounts of pyruvate are produced through fermentation processes. Here we describe a simple and efficient method for producing pyruvate in Escherichia coli. To stop carbon flux from pyruvate to fatty acids, the accBC genes, which encode the enzyme that catalyzes the first step of fatty acid biosynthesis and is essential for vegetative growth, were manipulated within the genome; its native promoter was replaced with the tetracycline (or doxycycline)-regulated promoter and the corresponding transcriptional regulator genes. The resulting strain grew normally in the presence of doxycycline, but showed poor growth upon withdrawal of doxycycline. Using this strain, we developed a high pyruvate producing strain (strain LAFCPCPt-accBC-aceE), in which the tetracycline-regulated promoter was also introduced upstream of aceE, and the ackA-pta, adhE, cra, ldhA, pflB and poxB genes were deleted. After determining the optimal culture conditions for this strain, the final pyruvate concentration reached 26.1 g L−1 after 72 h with a theoretical yield of 55.6 %. These levels are high enough to indicate that the developed strain has the potential for application to industrial production of pyruvate.

A thermostable, NADP(+)-dependent D: -amino acid dehydrogenase (DAADH) was created from the meso-diaminopimelate dehydrogenase of Ureibacillus thermosphaericus strain A1 by introducing five point mutations into amino acid residues located in the active site. The recombinant protein, expressed in Escherichia coli, was purified to homogeneity using a two-step separation procedure and then characterized. In the presence of NADP(+), the protein catalyzed the oxidative deamination of several D: -amino acids, including D: -cyclohexylalanine, D: -isoleucine and D: -2-aminooctanoate, but not meso-diaminopimelate, confirming the creation of a NADP(+)-dependent DAADH. For the reverse reaction, the corresponding 2-oxo acids were aminated in the presence of NADPH and ammonia. In addition, the D: -amino acid dehydrogenase showed no loss of activity at 65 °C, indicating the mutant enzyme was more thermostable than its parental meso-diaminopimelate dehydrogenase.

Microbial degradation of lignin releases fermentable sugars, effective utilization of which could support biofuel production from lignocellulosic biomass. In the present study, a lignin-degrading bacterium was isolated from leaf soil and identified as Burkholderia sp. based on 16S rRNA gene sequencing. This strain was named CCA53, and its lignin-degrading capability was assessed by observing its growth on medium containing alkali lignin or lignin-associated aromatic monomers as the sole carbon source. Alkali lignin and at least eight lignin-associated aromatic monomers supported growth of this strain, and the most effective utilization was observed for p-hydroxybenzene monomers. These findings indicate that Burkholderia sp. strain CCA53 has fragmentary activity for lignin degradation.

The open reading frame YGL157w in the genome of the yeast Kluyveromyces marxianus strain DMB1 encodes a putative uncharacterized oxidoreductase. However, this protein shows 46% identity with the Saccharomyces cerevisiae S288c NADPH-dependent methylglyoxal reductase, which exhibits broad substrate specificity for aldehydes. In the present study, the YGL157w gene product (KmGRE2) was purified to homogeneity from overexpressing Escherichia coli cells and found to be a monomer. The enzyme was strictly specific for NADPH and was active with a wide variety of substrates, including aliphatic (branched-chain and linear) and aromatic aldehydes. The optimal pH for methylglyoxal reduction was 5.5. With methylglyoxal as a substrate, the optimal temperature for enzyme activity at pH 5.5 was 45°C. The enzyme retained more than 70% of its activity after incubation for 30 min at temperatures below 35°C or at pHs between 5.5 and 9.0. In addition, the KmGRE2-overexpressing E. coli showed improved growth when cultivated in cedar hydrolysate, as compared to cells not expressing the enzyme. Taken together, these results indicate that KmGRE2 is potentially useful as an inhibit decomposer in E. coli cells.

Crystal structures of the thermostablemeso-diaminopimelate dehydrogenase (DAPDH) fromUreibacillus thermosphaericuswere determined for the enzyme in the apo form and in complex with NADP+andN-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid. The main-chain coordinates of the enzyme showed notable similarity to those ofSymbiobacterium thermophilumDAPDH. However, the subunit arrangement ofU. thermosphaericusDAPDH (a dimer) was totally different from that of theS. thermophilumenzyme (a hexamer). Structural comparison with the dimeric enzyme from the mesophileCorynebacterium glutamicumrevealed that the presence of large numbers of intrasubunit and intersubunit hydrophobic interactions, as well as the extensive formation of intersubunit ion-pair networks, were likely to be the main factors contributing to the higher thermostability ofU. thermosphaericusDAPDH. This differs fromS. thermophilumDAPDH, within which the unique hexameric assembly is likely to be responsible for its high thermostability. Analysis of the active site ofU. thermosphaericusDAPDH revealed the key factors responsible for the marked difference in substrate specificity between DAPDH and the D-amino acid dehydrogenase recently created from DAPDH by introducing five point mutations [Akitaet al.(2012).Biotechnol. Lett.34, 1693–1699; 1701–1702].

We screened various thermophiles for meso-diaminopimelate dehydrogenase (meso-DAPDH, EC 1.4.1.16), which catalyzes the NAD(P)-dependent oxidative deamination of meso-diaminopimelate, and found the enzyme in a thermophilic bacterium isolated from compost in Japan. The bacterium grew well aerobically at around 55°C and was identified as Ureibacillus thermosphaericus strain A1. We purified the enzyme about 47-fold to homogeneity from crude cell extract using five successive purification steps. The molecular mass of the purified protein was about 80 kDa, and the molecule consists of a homodimer with the subunit molecular mass of about 40 kDa. The optimum pH and temperature for the catalytic activity of the enzyme are about 10.5 and 65°C, respectively. The enzyme is highly selective for meso-diaminopimelate as the electron donor, and NADP but not NAD can serve as the electron acceptor. The K m values for meso-diaminopimelate and NADP at 50°C and pH 10.5 are 1.6 mM and 0.13 mM, respectively. The nucleotide sequence of this meso-DAPDH gene encodes a 326-amino acid peptide. When the gene was cloned and overexpressed in Escherichia coli Rosetta (DE3), the specific activity in the crude extract of the recombinant cells was about 18.0-fold higher than in the extract from U. thermosphaericus strain A1. This made more rapid and simpler purification of the enzyme possible.

Two genes from Kluyveromyces marxianus strain DMB1, YGL039w1 and YGL039w2, encode putative uncharacterized oxidoreductases that respectively share 42 and 44% identity with the Saccharomyces cerevisiae S288c NADPH-dependent methylglyoxal reductase (EC 1.1.1.283). To determine the enzymatic characteristics of their products, the two genes were expressed in recombinant Escherichia coli cells, after which the YGL039w1 and YGL039w2 proteins were purified to homogeneity. In the presence of NADPH, both enzymes showed reductive activities toward at least nine aldehyde substrates, but no NADP(+)-dependent oxidative activities. These two YGL039w proteins thus appear to be aldehyde reductases. In addition, although both enzymes retained more than 70% of their activities after incubation for 30 min at temperatures below 40°C or at pHs between 5.5 and 11.3, YGL039w2 was slightly more thermostable than YGL039w1.