Internal Amino Acid Sequences Research Articles

Purification, characterization, and molecular gene cloning of an antifungal protein from Ginkgo biloba seeds

A novel basic protein with antifungal activity was isolated from the seeds of Ginkgo biloba and purified to homogeneity. The protein inhibited the growth of some fungi (Fusarium oxysporum, Trichoderma reesei, and Candida albicans) but did not exhibit antibacterial action against Escherichia coli. Furthermore, this protein showed weak inhibitory activity against the aspartic protease pepsin. To design primers for gene amplification, the NH(2)-terminal and partial internal amino acid sequences were determined using peptides obtained from a tryptic digest of the oxidized protein. The full-length cDNA of the antifungal protein was cloned and sequenced by RT-PCR and rapid amplification of cDNA ends (RACE). The cDNA contained a 402-bp open reading frame encoding a 134-aa protein with a potential signal peptide (26 residues), suggesting that this protein is synthesized as a preprotein and secreted outside the cells. The antifungal protein shows approximately 85% identity with embryo-abundant proteins from Picea abies and Picea glauca at the amino acid level; however, there is no homology between this protein and other plant antifungal proteins, such as defensin, and cyclophilin-, miraculin- and thaumatin-like proteins.

Cloning, Expression and Purification of Arylsulfate Sulfotransferase from Eubacterium A-44

A gene (astA) encoding arylsulfate sulfotransferase (ASST), which transfers a sulfate group from phenolic sulfate esters to phenolic acceptors, was cloned from a Eubacterium A-44 genomic library. The probe (1.5 kb fragment) for the astA gene was prepared from the PCR product of the primers produced using two internal amino acid sequences of ASST, which had been purified from Eubacterium A-44. The astA gene was cloned into the pKF3 vector. Its sequence revealed a 1863 bp open reading frame (ORF) encoding a protein containing 620 amino acids with a secretary signal peptide, and showed 91% homology (identity) to Eubacterium rectale IIIH previously reported. The cloned astA gene was expressed under the T7 promoter of the expression vectors, pET-39b(+) and pET-26b(+), in Escherichia coli BL21 (DE3), and the expressed ASSTs were purified using His Bind column chromatography. The specific activities of the purified ASSTs were 25.6 micromol/min/mg and 37.1 micromol/min/mg, respectively.

Characterization of a Novel d -Lyxose Isomerase from Cohnella laevoribosii RI-39 sp. nov

A newly isolated bacterium, Cohnella laevoribosii RI-39, could grow in a defined medium with L-ribose as the sole carbon source. A 21-kDa protein isomerizing L-ribose to L-ribulose, as well as D-lyxose to D-xylulose, was purified to homogeneity from this bacterium. Based on the N-terminal and internal amino acid sequences of the purified enzyme obtained by N-terminal sequencing and quantitative time of flight mass spectrometry-mass spectrometry analyses, a 549-bp gene (lyxA) encoding D-lyxose (L-ribose) isomerase was cloned and expressed in Escherichia coli. The purified endogenous enzyme and the recombinant enzyme formed homodimers that were activated by Mn(2+). C. laevoribosii D-lyxose (L-ribose) isomerase (CLLI) exhibits maximal activity at pH 6.5 and 70 degrees C in the presence of Mn(2+) for D-lyxose and L-ribose, and its isoelectric point (pI) is 4.2 (calculated pI, 4.9). The enzyme is specific for D-lyxose, L-ribose, and D-mannose, with apparent K(m) values of 22.4 +/- 1.5 mM, 121.7 +/- 10.8 mM, and 34.0 +/- 1.1 mM, respectively. The catalytic efficiencies (k(cat)/K(m)) of CLLI were 84.9 +/- 5.8 mM(-1) s(-1) for D-lyxose (V(max), 5,434.8 U mg(-1)), 0.2 mM(-1) s(-1) for L-ribose (V(max), 75.5 +/- 6.0 U mg(-1)), and 1.4 +/- 0.1 mM(-1) s(-1) for D-mannose (V(max), 131.8 +/- 7.4 U mg(-1)). The ability of lyxA to permit E. coli cells to grow on D-lyxose and L-ribose and homology searches of other sugar-related enzymes, as well as previously described sugar isomerases, suggest that CLLI is a novel type of rare sugar isomerase.

Inositol-1 (or 4)-monophosphatase from Glycine max embryo axes is a phosphatase with broad substrate specificity that includes phytate dephosphorylation

A phosphate-hydrolyzing activity from Glycine max embryo axes was purified by a series of chromatographic steps and electroelution from activity gels, and demonstrated to be an inositol-1 (or 4)-monophosphatase by partial internal amino acid sequence. This enzyme hydrolyzed ATP, sodium pyrophosphate (NaPPi), inositol hexakisphosphate, and inositol 1-monophosphate, but not p-nitrophenyl phosphate, ADP, AMP or glucose 6-P. Using NaPPi as substrate, the highly purified protein hydrolyzed up to 0.4 mmol phosphate min − 1 mg − 1 protein and had a Km avg of 235 μM for NaPPi. Since NaPPi is relatively inexpensive and readily available, we used this as substrate for the subsequent characterization. We observed the following: (a) specific inhibition by Li and NaF but not by butanedione monoxime, or orthovanadate; (b) activation by Cu 2+ and Mg 2+; (c) optimum activity at pH 7.4; and (d) temperature stability after 1-h incubations at 37–80 °C, with maximum activity at 37 °C. The partially purified protein was detected by in-gel activity assays and the band was electroeluted to yield a highly purified protein. Analysis by SDS-PAGE and native IEF-PAGE yielded a single major polypeptide of 29 kDa and pI ∼ 5.9, respectively. In addition, in-gel activity from embryo axes and whole hypocotyls at early germination times revealed one high and one intermediate molecular weight isoform, but only the intermediate one corresponded to IMPase. Throughout the post-imbibition period, the activity of the high molecular weight isoform disappeared and IMPase increased, indicating an increasing expression of the enzyme as germination and growth proceeded. These data indicate that the inositol-1 (or 4)-monophosphatase present in the embryo axis of G. max has a wide phosphate substrate specificity, and may play an important role in phosphate metabolism during the germination process.

A Rice Family 9 Glycoside Hydrolase Isozyme with Broad Substrate Specificity for Hemicelluloses in Type II Cell Walls

An auxin analog, 2,4-D, stimulates the activity of endo-1,4-beta-glucanase (EGase) in rice (Oryza sativa L.). The auxin-induced activity from three protein fractions was purified to homogeneity from primary root tissues (based on SDS-PAGE and isoelectric focusing after Coomassie brilliant blue staining). Amino acid sequencing indicated that the 20 N-terminal amino acid sequence of the three proteins was identical, suggesting that these proteins may be cognates of one EGase gene. An internal amino acid sequence of the the rice EGase (LVGGYYDAGDNVK) revealed that this enzyme belongs to glycosyl hydrolase family 9 (GHF9). The major isoform of this rice GHF9 [molecular weight based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS): 51,216, isoelectric point (pI): 5.5] specifically hydrolyzed 1,4-beta-glycosyl linkages of carboxymethyl (CM)-cellulose, phosphoric acid-swollen cellulose, 1,3-1,4-beta-glucan, arabinoxylan, xylan, glucomannan, cellooligosaccharides [with a degree of polymerization (DP) >3] and 1,4-beta-xylohexaose, indicating a broader substrate range compared with those of other characterized GHF9 enzymes or EGases from higher plants. Hydrolytic products of two major hemicellulosic polysaccharides in type II cell walls treated with the purified enzyme were profiled using high-performance anion exchange chromatography (HPAEC). The results suggested that endolytic attack by rice EGase is not restricted to either the cellulose-like domain of 1,3-1,4-beta-glucan or the unsubstituted 1,4-beta-xylosyl backbone of arabinoxylan, but results in the release of smaller oligosaccharides (DP <6) from graminaceous hemicelluloses. The comparatively broader substrate range of this EGase with respect to beta-1,4-glycan backbones (glucose and xylose) may partly reflect different roles of gramineous and non-gramineous GHF9 enzymes.

Storage protein profiles in Spanish and runner market type peanuts and potential markers

BackgroundProteomic analysis has proven to be the most powerful method for describing plant species and lines, and for identification of proteins in complex mixtures. The strength of this method resides in high resolving power of two-dimensional electrophoresis (2-DE), coupled with highly sensitive mass spectrometry (MS), and sequence homology search. By using this method, we might find polymorphic markers to differentiate peanut subspecies.ResultsTotal proteins extracted from seeds of 12 different genotypes of cultivated peanut (Arachis hypogaea L.), comprised of runner market (A. hypogaea ssp. hypogaea) and Spanish-bunch market type (A. hypogaea ssp. fastigiata), were separated by electrophoresis on both one- and two-dimensional SDS-PAGE gels. The protein profiles were similar on one-dimensional gels for all tested peanut genotypes. However, peanut genotype A13 lacked one major band with a molecular weight of about 35 kDa. There was one minor band with a molecular weight of 27 kDa that was present in all runner peanut genotypes and the Spanish-derivatives (GT-YY7, GT-YY20, and GT-YY79). The Spanish-derivatives have a runner-type peanut in their pedigrees. The 35 kDa protein in A13 and the 27 kDa protein in runner-type peanut genotypes were confirmed on the 2-D SDS-PAGE gels. Among more than 150 main protein spots on the 2-D gels, four protein spots that were individually marked as spots 1–4 showed polymorphic patterns between runner-type and Spanish-bunch peanuts. Spot 1 (ca. 22.5 kDa, pI 3.9) and spot 2 (ca. 23.5 kDa, pI 5.7) were observed in all Spanish-bunch genotypes, but were not found in runner types. In contrast, spot 3 (ca. 23 kDa, pI 6.6) and spot 4 (ca. 22 kDa, pI 6.8) were present in all runner peanut genotypes but not in Spanish-bunch genotypes. These four protein spots were sequenced. Based on the internal and N-terminal amino acid sequences, these proteins are isoforms (iso-Ara h3) of each other, are iso-allergens and may be modified by post-translational cleavage.ConclusionThese results suggest that there may be an association between these polymorphic storage protein isoforms and peanut subspecies fastigiata (Spanish type) and hypogaea (runner type). The polymorphic protein peptides distinguished by 2-D PAGE could be used as markers for identification of runner and Spanish peanuts.

Pea extracellular Cu/Zn-superoxide dismutase responsive to signal molecules from a fungal pathogen

We previously reported that the release of O2 − from isolated pea cell walls was enhanced by a 70-kDa glycoprotein elicitor but was suppressed by mucin-type glycopeptide suppressors (supprescins A and B) prepared from pycnospore germination fluid of Mycosphaerella pinodes, causal agent of Mycosphaerella blight of pea. Here, we show that superoxide dismutase (SOD) in the apoplast fluid/cell wall of pea seedlings responds to the fungal elicitor and suppressor molecules. In a pharmacological study and with internal amino acid sequencing, the apoplastic SOD in a pea cultivar Midoriusui was found to be a Cu/Zn type SOD. We cloned a full-length cDNA of the Cu/Zn-SOD and designated it as PsCu/Zn-SOD1. An increase in PsCu/Zn-SOD1 mRNA and the PsCu/Zn-SOD1 protein was induced by treatment with the elicitor more intensively than by wounding. Such induction by the elicitor or wounding, however, was inhibited by the concomitant presence of supprescins. The SOD activity of recombinant PsCu/Zn-SOD1 was regulated directly by these signal molecules in a manner similar to their effect on the SOD activity in the apoplastic fluid and in the cell wall-bound proteins. Based on these findings, we discuss a role for PsCu/Zn-SOD1 in the pea defense response.

Homology between ABH-carrier α2-seminoglycoprotein and Mac-2 binding protein

α2-seminoglycoprotein (α2-SGP), purified from human seminal plasma, is a carrier of glycoprotein for the ABO blood grouping. The α2-SGP exists in the secretions of the seminal vesicle and various glands. However, the function of α2-SGP is, as yet, unknown. In this study, we determined that two internal amino acid sequences of 8 and 12 residues of α2-SGP were Ala-Val-Asp-Thr-Trp-Ser-Trp-Gly and Thr-Leu-Gln-Ala-Leu-Glu-Phe-His-Thr-Val-Pro-Phe. These sequences were completely coincident with the domain 3 of human Mac-2 binding protein (M2BP), which was identified as a tumor-associated antigen. In addition, we also confirmed an α2-SGP binding activity to galectin-3 that was one of a ligand for M2BP, and the immunological cross-reactivity between α2-SGP and M2BP. These findings strongly suggested that α2-SGP was identical with M2BP.

Purification of a novel aminopeptidase from the pollen of Parietaria judaica that alters epithelial integrity and degrades neuropeptides

Parietaria judaica pollen is a common cause of pollinosis in the Mediterranean area. This study sought to purify and characterize the peptidase responsible for the majority of proteolytic activity present in the pollen extract of P judaica, and to investigate its contribution to the allergic response. A serial of chromatographic steps was applied to isolate the peptidase from P judaica's pollen, and its biochemical properties were determined. Bioactive peptides present in the airways were incubated with the peptidase, and their degradation was visualized by direct protein sequencing. In addition, we measured the cellular detachment, by methylene blue binding assay, of an airway-derived epithelial cell line (A549) in the presence of the peptidase, and visualized, by Western blot, the degradation of proteins from intercellular junctions. We purified a 98-kDa peptidase from the pollen of P judaica that was classified as an aminopeptidase on the basis of its biochemical properties and internal amino acid sequence. The aminopeptidase was able to degrade bioactive peptides. Moreover, the aminopeptidase caused cellular detachment of A549 cell line and degradation of occludin and E-cadherin. Our results suggest that the P judaica aminopeptidase can alter the integrity of the epithelium barrier by degrading occludin as well as E-cadherin. In addition, P judaica aminopeptidase can degrade bioactive peptides, which can exacerbate the overall bronchoconstrictive effect detected in asthmatic lungs. The novel aminopeptidase described here could constitute a relevant therapeutic target in the treatment of allergic disorders induced by the pollen of P judaica.

NADP-dependent Mannitol Dehydrogenase, a Major Allergen of Cladosporium herbarum

Cladosporium herbarum is an important allergenic fungal species that has been reported to cause allergic diseases in nearly all climatic zones. 5-30% of the allergic population displays IgE antibodies against molds. Sensitization to Cladosporium has often been associated with severe asthma and less frequently with chronic urticaria and atopic eczema. However, no dominant major allergen of this species has been found so far. We present cloning, production, and characterization of NADP-dependent mannitol dehydrogenase of C. herbarum (Cla h 8) and show that this protein is a major allergen that is recognized by IgE antibodies of approximately 57% of all Cladosporium allergic patients. This is the highest percentage of patients reacting with any Cladosporium allergen characterized so far. Cla h 8 was purified to homogeneity by standard chromatographic methods, and both N-terminal and internal amino acid sequences of protein fragments were determined. Enzymatic analysis of the purified natural protein revealed that this allergen represents a NADP-dependent mannitol dehydrogenase that interconverts mannitol and d-fructose. It is a soluble, non-glycosylated cytoplasmic protein. Two-dimensional protein analysis indicated that mannitol dehydrogenase is present as a single isoform. The cDNA encoding Cla h 8 was cloned from a lambda-ZAP library constructed from hyphae and spores. The recombinant non-fusion protein was expressed in Escherichia coli and purified to homogeneity. Its immunological and biochemical identity with the natural protein was shown by enzyme activity tests, CD spectroscopy, IgE immunoblots with sera of patients, and by skin prick testing of Cladosporium allergic patients. This protein therefore is a new major allergen of C. herbarum.

Purification and characterization of anticoagulant protein from the Tabanus,Tabanus bivittatus

Tabanus anticoagulant protein (TAP) was isolated from the whole body of the tabanus, Tabanus bivittatus, using three purification steps (ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, and ion exchange chromatography on DEAE Sephadex gel). The purified TAP, with a molecular weight of 65 kDa, was assessed to be homogeneous by SDS-polyacrylamide gel electrophoresis, and an isoelectric point of 7.9 was determined by isoelectric focusing. The internal amino acid sequence of the purified protein was composed of Ser-Leu-Asn-Asn-Gln-Phe-Ala-Ser-Phe-Ile-Asp-Lys-Val-Arg. The protein was activated by Cu2+ and Zn2+, and the optimal conditions were found to be at pH 3-6 and 40-70 degrees C. Standard coagulation screen assays were used to determine thrombin time and activated partial thromboplastin time. Chromogenic substrate assays were performed for thrombin and factor Xa activity. TAP considerably prolonged human plasma clotting time, especially activated partial thromboplastin time in a dose-dependent manner; it showed potent and specific antithrombin activity in the chromogenic substrate assay. Specific anti-factor Xa activity in TAP was not detected. Overall, this result suggested that TAP has significant anticoagulant activity on blood coagulation system.

Mass spectrometric sequencing of endotoxin proteins of Bacillus thuringiensis ssp. konkukian extracted from polyacrylamide gels

The amino acid sequences of the crystal proteins of Bacillus thuringiensis ssp. konkukian strain HL-47 are unknown. We used 1-D denaturing polyacrylamide electrophoresis, nano-ESI-Q-TOF-MS, and protein database searching to analyze these proteins. On SDS-PAGE gels, a preparation of purified crystal proteins exhibited 110, 102, 76, 55, 37, and 30 kDa protein bands. Immunoblotting of the gel with antiserum raised to this preparation revealed that four crystal proteins, of 110, 102, 55, and 37 kDa, reacted with the specific antiserum. The 102-kDa major protein reacted strongly. The other crystal proteins showed weak immunoreactivity. The 102 and 55 kDa proteins were analyzed by ESI-MS. The internal amino acid sequence of the 102-kDa major protein has similarity to the sequences of the surface layer protein of B. thuringiensis ssp. finitimus and B. anthracis. However, the internal amino acid sequences of the 55 kDa protein did not show any homology to proteins in the databases. Proteomic analysis of these proteins leads to the conclusion that the sequence data provided the protein databases of the crystal proteins of the konkukian ssp.

Disulfide Proteome Analysis of Buckwheat Seeds to Screen Putative Allergens

ABSTRACTAccumulating evidence suggests a correlation between disulfide bonding and the allergenicity of proteins. Also, a significant characteristic of most food allergens is that they are stable to proteases. We sought to identify putative allergens of buckwheat seed comprehensively by combining a disulfide proteome technique with an in vitro digestibility test. First, a dilute acetic acid fraction of buckwheat seed was found to be rich in disulfide proteins. Internal amino acid sequence analyses of these proteins showed that most of them were known allergens or putative allergens sharing high amino acid sequence similarities to known allergens. Next, the fraction was subjected to in vitro protease digestion, which revealed relatively large fragments that were resistant to prolonged enzymatic digestion. These protease‐resistant fragments contained disulfide bonds that should have protected the potential enzyme cleavage sites by forming compact structures. These results confirm and extend our knowledge of the correlations among the disulfide bonding of proteins, their protease stability, and their allergenicity. Also, these observations suggest a new strategy to identify putative allergens by proteomic approaches as well as to mitigate them.

Liver argininosuccinate synthase binds to bacterial lipopolysaccharides and lipid A and inactivates their biological activities

The liver is known to clear and detoxify circulating lipopolysaccharide (LPS). To characterize the molecules involved in this process in the liver, we attempted to purify mouse liver protein(s) that can interact with lipid A, a biologically active portion of LPS. By partially purifying the inactivating activity against a synthetic lipid A analog, we observed the enrichment of a 45-kDa protein in the active fractions. The internal amino acid sequences of the protein were identical with those of argininosuccinate synthase (EC 6.3.4.5). To examine whether argininosuccinate synthase can interact with lipid A, we purified the enzyme from mouse liver and found the co-elevation of the specific enzyme activity and specific lipid A-inactivating activity, indicating that argininosuccinate synthase is the major lipid A-interacting protein in liver. Argininosuccinate synthase also inhibited the biological activities (macrophage activation and Limulus test) of natural lipid A and rough-type LPS but not smooth-type LPS. The enzyme activity was inhibited by lipid A and rough-type LPS and also by smooth-type LPS. Native gel electrophoresis of a mixture of argininosuccinate synthase and LPS and immunoprecipitation of a mixture of argininosuccinate synthase and [(3)H]-LPS with anti-argininosuccinate synthase antiserum showed that argininosuccinate synthase stably bound lipid A and LPS. These findings, together, indicate that argininosuccinate synthase can effectively bind LPS in the liver.

Liver argininosuccinate synthase binds to bacterial lipopolysaccharides and lipid A and inactivates their biological activities

The liver is known to clear and detoxify circulating lipopolysaccharide (LPS). To characterize the molecules involved in this process in the liver, we attempted to purify mouse liver protein(s) that can interact with lipid A, a biologically active portion of LPS. By partially purifying the inactivating activity against a synthetic lipid A analog, we observed the enrichment of a 45-kDa protein in the active fractions. The internal amino acid sequences of the protein were identical with those of argininosuccinate synthase (EC 6.3.4.5). To examine whether argininosuccinate synthase can interact with lipid A, we purified the enzyme from mouse liver and found the co-elevation of the specific enzyme activity and specific lipid A-inactivating activity, indicating that argininosuccinate synthase is the major lipid A-interacting protein in liver. Argininosuccinate synthase also inhibited the biological activities (macrophage activation and Limulus test) of natural lipid A and rough-type LPS but not smooth-type LPS. The enzyme activity was inhibited by lipid A and rough-type LPS and also by smooth-type LPS. Native gel electrophoresis of a mixture of argininosuccinate synthase and LPS and immunoprecipitation of a mixture of argininosuccinate synthase and [3H]-LPS with anti-argininosuccinate synthase antiserum showed that argininosuccinate synthase stably bound lipid A and LPS. These findings, together, indicate that argininosuccinate synthase can effectively bind LPS in the liver.

Ovalbumin-Related Gene Y Protein Bears Carbohydrate Chains of the Ovomucoid Type

We have recently established the monoclonal antibodies (mAbs) specific to the major food allergen, ovomucoid, as mAb 7D, recognizing the carbohydrate moiety of ovomucoid, and mAb 6H, the peptide moiety (Biosci. Biothechnol. Biochem., 68, 2490-2497, (2004)). Using these mAbs, we found commercially available ovalbumin preparations contaminated with a considerable amount of ovomucoid together with other glycoproteins. To examine the contaminants, egg white was subjected to cation-exchange chromatography. An unidentified protein was found in egg white that reacted with mAb 7D but not with mAb 6H, having a molecular size of about 52 kDa and a blocked N-terminus. Two internal amino acid sequences of the fragments obtained after a lysyl endopeptidase and a hydroxylamine treatment revealed the protein to be ovalbumin Y (ovalbumin-related gene Y protein). We conclude that ovalbumin Y is a unique chimeric glycoprotein having an amino acid sequence similar to that of ovalbumin, but having a carbohydrate moiety similar to that of ovomucoid.

Acetate generation in rat liver mitochondria; acetyl-CoA hydrolase activity is demonstrated by 3-ketoacyl-CoA thiolase

Acetate has been found as an endogenous metabolite of β-oxidation of fatty acids in liver. In order to investigate the regulation of acetate generation in liver mitochondria, we attempted to purify a mitochondrial acetyl-CoA hydrolase in rat liver. This acetyl-CoA-hydrolyzing activity in isolated mitochondria was induced by the treatment of rats with di(2-ehtylhexyl)phthalate (DEHP), a peroxisome proliferator which induces expression of several peroxisomal and mitochondrial enzymes involved in β-oxidation of fatty acids. The purified enzyme was 43-kDa in molecular mass by SDS/PAGE. Internal amino acid sequencing of this enzyme revealed that it was identical with mitochondrial 3-ketoacyl-CoA thiolase, suggesting that this enzyme has two kinds of activities, 3-ketoacyl-CoA thiolase and acetyl-CoA hydrolase activities. Kinetic studies clearly indicated that this enzyme had the both activities and each activity was inhibited by the substrates of the other activity, that is, 3-ketoacyl-CoA thiolase activity was inhibited by acetyl-CoA, on the other hand, acetyl-CoA hydrolase activity was inhibited by acetoacetyl-CoA in a competitive manner. These findings suggested that acetate generation in liver mitochondria is a side reaction of this known enzyme, 3-ketoacyl-CoA thiolase, and this enzyme may regulate its activities depending on each substrate level.

Triphenylmethane Reductase from Citrobacter sp. Strain KCTC 18061P: Purification, Characterization, Gene Cloning, and Overexpression of a Functional Protein in Escherichia coli

We purified to homogeneity an enzyme from Citrobacter sp. strain KCTC 18061P capable of decolorizing triphenylmethane dyes. The native form of the enzyme was identified as a homodimer with a subunit molecular mass of about 31 kDa. It catalyzes the NADH-dependent reduction of triphenylmethane dyes, with remarkable substrate specificity related to dye structure. Maximal enzyme activity occurred at pH 9.0 and 60 degrees C. The enzymatic reaction product of the triphenylmethane dye crystal violet was identified as its leuco form by UV-visible spectral changes and thin-layer chromatography. A gene encoding this enzyme was isolated based on its N-terminal and internal amino acid sequences. The nucleotide sequence of the gene has a single open reading frame encoding 287 amino acids with a predicted molecular mass of 30,954 Da. Although the deduced amino acid sequence displays 99% identity to the hypothetical protein from Listeria monocytogenes strain 4b H7858, it shows no overall functional similarity to any known protein in the public databases. At the N terminus, the amino acid sequence has high homology to sequences of NAD(P)H-dependent enzymes containing the dinucleotide-binding motif GXXGXXG. The enzyme was heterologously expressed in Escherichia coli, and the purified recombinant enzyme showed characteristics similar to those of the native enzyme. This is the first report of a triphenylmethane reductase characterized from any organism.

Gene cloning of an NADPH-dependent menadione reductase from Candida macedoniensis, and its application to chiral alcohol production

The gene encoding an NADPH-dependent menadione reductase of Candida macedoniensis AKU4588 was cloned and sequenced. A 1035 bp nucleotide fragment ( mer) was confirmed to be the gene encoding the enzyme based on the agreement of N-terminal and internal amino acid sequences. The mer encodes 345 amino acid residues, and the deduced amino acid sequence shows high similarity with those of hypothetical proteins from Debaryomyces, Candida and Saccharomyces, and ketoreductase from Zygosaccharomyces. It includes NADPH-binding motif GXXGXXA in its N-terminal region. These findings suggest that the enzyme belongs to the dihydroflavonol-4-reductase superfamily. An expression vector, pETMER, which contains the full length of the mer, was constructed. Escherichia coli cells harboring pETMER exhibits a 127-fold increase in specific menadione-reducing activity under the control of T7 promoter as compared with that of C. macedoniensis. The asymmetric reduction of 4-chloro-3-oxobutanoate ethyl ester to ( S)-4-chloro-3-hydroxybutanoate ethyl ester (CHBE) with E. coli cells, in which both the mer and the glucose dehydrogenase gene were co-expressed, as a catalyst was investigated. The ( S)-CHBE formed amounted to 1680 mM (281 mg/ml), the molar yield being 92.2%. The optical purity of the product was 91.6% enantiomeric excess for the ( S)-isomer. The calculated turnover number of NADP + added to CHBE formed was 12,900 mol/mol.

The Surface-Associated Protein of Staphylococcus saprophyticus Is a Lipase

Staphylococcus saprophyticus surface-associated protein (Ssp) was the first surface protein described for this organism. Ssp-positive strains display a fuzzy layer of surface-associated material in electron micrographs, whereas Ssp-negative strains appear to be smooth. The physiologic function of Ssp, however, has remained elusive. To clone the associated gene, we determined the N-terminal sequence, as well as an internal amino acid sequence, of the purified protein. We derived two degenerate primers from these peptide sequences, which we used to identify the ssp gene from genomic DNA of S. saprophyticus 7108. The gene was cloned by PCR techniques and was found to be homologous to genes encoding staphylococcal lipases. In keeping with this finding, strains 7108 and 9325, which are Ssp positive, showed lipase activity on tributyrylglycerol agar plates, whereas the Ssp-negative strain CCM883 did not. Association of enzyme activity with the cloned DNA was proven by introducing the gene into Staphylococcus carnosus TM300. When wild-type strain 7108 and an isogenic mutant were analyzed by transmission electron microscopy, strain 7108 exhibited the fuzzy surface layer, whereas the mutant appeared to be smooth. Lipase activity and the surface appendages could be restored by reintroduction of the cloned gene into the mutant. Experiments using immobilized collagen type I did not provide evidence for the involvement of Ssp in adherence to this matrix protein. Our experiments thus provided evidence that Ssp is a surface-associated lipase of S. saprophyticus.