Expression of a β-galactosidase gene from Lactobacillus sake in Escherichia coli

Summary A -galactosidase gene from Lactobacillus sake coding for lactose hydrolysis was cloned and expressed in Escherichia coli. Chromosomal DNA from L. sake was partially digested with the restriction enzyme Sau3AI, and the 3–6 Kb fragment was ligated to the cloning vector pSP72 digested with BamHI. One E. coli transformant expressing -galactosidase was isolated on X-gal plates. It contained a plasmid with an insertion of approx. 4 Kb. The restriction map of the recombinant plasmid was constructed. The characteristics of the recombinant -galactosidase were compared with those of the wild type. The optima pH and temperature for both enzymes was 6.5 and 50°C, respectively. Stability of the enzymes at different temperatures and activity on lactose were determined.     

Synthesis of a ‘direct-linked’ C-disaccharide from a pyranulose glycoside

Syntheses of five ‘direct linked’ C-disaccharides 8a–e were reported. The (Et₃SiH/BF₃·Et₂O) reduction of pyranulose glycoside 1 yielded (6S)- and (6R)-6-(2,3,5-tri-O-benzoyl-β-d-ribofuranosyl)pyran-3(2H,6H)-one (2a and 2b) in a ratio of ca. 2:1 and in 88% combined yield. The absolute stereochemistry of each was determined from its CD spectrum. The reduction of 2a with NaBH₄ in methanol afforded two allylic alcohols 6a and 6b in 14 and 73% yield, respectively. The reduction of 2b with NaBH₄ afforded 6c and 6d in 30 and 56% yield, respectively. Cis hydroxylation of the double bond in compounds 6a–d with osmium tetroxide gave 7a–e. The stereoisomers 7a–e were separated and their configuration was established by ¹H NMR spectroscopy. Debenzoylation of compounds 7a–e with aqueous sodium carbonate produced deprotected C-disaccharides 8a–e.     

Expression in Escherichia coli of a cloned β-glucanase gene from Bacillus amyloliquefaciens

Summary The recombinant phage G1 has been identified by screening 700 plaques of a Charon 4A library, containing DNA of Bacillus amyloliquefaciens, for phage clones directing the hydrolysis of lichenan in Escherichia coli, as indicated by haloes surrounding plaques on lichenan agar. The gene coding for an endo--1.3–1.4-glucanase was recloned within a 3.6 kb EcoRI fragment into the EcoRI site of plasmid pBR322, in both orientations.The location and extent of the bgl gene on the 3.6 kb Bacillus DNA insert was estimated by insertion mutagenesis with transposon Tn5 and restriction mapping of Tn5 insertions within or near to the bgl gene.The -glucanase synthesized by E. coli harbouring plasmids pEG1 or pEG2 was shown to accumulate mainly in the periplasmic space but -glucanase activities were also detected extracellulary and in the cytoplasm. The molecular weight of the enzyme synthesized in E. coli harbouring pEG1 was estimated by SDS-polyacrylamide gel electrophoresis to be about 24000. It was shown that the level of bgl gene expression in E. coli varies about 10-fold, depending on the orientation of the 3.6 kb DNA-fragment cloned within the EcoRI site of pBR322. After insertion of HindIII-DNA fragments from phage into the HindIII site of the -glucanase-high-expression plasmid pEG1, we obtained clones also showing an approximately 10-fold reduction in -glucanase activites. It was thus concluded that on plasmid pEG1 the leftward acting Ap^r (PI) promotor of plasmid pBR322 strongly increases the expression in E. coli of the cloned B. amyloliquefaciens bgl gene.Abbreviations Ap ampicillin, Km, kanamycin - kd kilodalton - kb kilobase pairs - moi multiplicity of infection - pfu plaque forming units - SDS sodium dodecylsulphate - Tc tetracycline     

Synthesis of α3 phage DNA in replication mutants of Escherichia coli

Host functions for DNA replication of bacteriophage α3, a representative of group A microvirid phages, were studied using dna and rep mutants of Escherichia coli. In dna⁺ cells, conversion of phage α3 single-stranded DNA (SS) into the double-stranded replicative form (RF) was insensitive to 30–150 μg/ml of chloramphenicol, 200 μg/ml of rifampicin, 50 μg/ml of nalidixic acid, or 200 μg/ml of novobiocin. At 43°C, synthesis of the parental RF was inhibited in dnaG and dnaZ mutants, but not in dnaE and rep strains. Replication of phage α3 progeny RF was prevented by 50 μg/ml of mitomycin C (in hcr⁺ bacteria), 50 μg/ml of nalidixic acid or 200 μg/ml of novoviocin, but neither by 30 μg/ml of chloramphenicol nor by 200 μg/ml of rifampicin. Besides dnaG and dnaZ gene products, dnaE and rep functions were essential for the progeny RF synthesis. Host factor dependence of α3 was relatively simple and, in contrast with phages øX174 and G4, α3 did not require dnaB and dnaC(D) activities.     

High-level expression of extracellular secretion of a β-xylosidase gene from Paecilomyces thermophila in Escherichia coli

A novel β-xylosidase gene (designated as PtXyl43) from thermophilic fungus Paecilomycesthermophila was cloned and extracellularly expressed in Escherichia coli. PtXyl43 belonging to glycoside hydrolase (GH) family 43 has an open reading frame of 1017 bp, encoding 338 amino acids without a predicted signal peptide. No introns were found by comparison of the PtXyl43 genomic DNA and cDNA sequences. The recombinant β-xylosidase (PtXyl43) was secreted into the culture medium in E. coli with a yield of 98.0 U mL(-1) in shake-flask cultures. PtXyl43 was purified 1.2-fold to homogeneity with a recovery yield of 61.5% from the cell-free culture supernatant. It appeared as a single protein band on SDS-PAGE with a molecular mass of approx 52.3 kDa. The enzyme exhibited an optimal activity at 55 °C and pH 7.0, respectively. This is the first report on the cloning and expression of a GH family 43 β-xylosidase gene from thermophilic fungi.     

Behavior of λ Bacteriophage in a Recombination Deficient Strain of Escherichia coli

The behavior of lambda phage in the Rec(-) strain JC-1569 is compared with that in the Rec(+) strain JC-1557. No difference deemed significant was noted in the adsorption rate, latent period, burst size, frequency of lysogenization, and frequency of vegetative phage recombination. The location of the prophage and its mode of insertion in the Rec(-) lysogen of wild-type lambda (lambda(+)) were inferred to be normal from the results of conjugational crosses. Spontaneous and ultraviolet (UV) irradiation induction of lambda(+) were markedly reduced in the Rec(-) lysogen. On the other hand, thermal induction of a mutant lambda (lambdacI857) lysogen of the Rec(-) strain was not reduced and was only slightly affected by UV irradiation. Phage subject to inhibition by lambda immunity failed to multiply in UV-irradiated cells of the Rec(-) lambda(+) lysogen, whereas those not inhibited by this immunity did multiply. It was concluded that the failure of UV to induce lambda(+) in the Rec(-) lysogen was not due to damage to the prophage, but rather to the inability of the irradiated cells to respond by lifting immunity. Preliminary evidence indicates that a single mutation confers recombination deficiency and the inability to lift immunity after UV irradiation. Possible relationships between recombination and the lifting of immunity are enumerated.     

Over-production of β-carotene from metabolically engineered Escherichia coli

To produce recombinant β-carotene in vitro, synthetic operons encoding genes governing its biosynthesis were engineered into Escherichia coli. Constructs harboring these operons were introduced into either a high-copy or low-copy cloning vector. β-Carotene production from these recombinant E. coli cells was either constitutive or inducible depending upon plasmid copy number. The most efficient β-carotene production was with the low-copy based vector. The process was increased incrementally from a 5 l to a 50 l fermentor and finally into a 300 l fermentor. The maximal β-carotene yields achieved using the 50 l and 300 l fermentor were 390 mg l⁻¹ and 240 mg l⁻¹, respectively, with overall productivities of 7.8 mg l⁻¹ h⁻¹ and 4.8 mg l⁻¹ h⁻¹.     

Purification and properties of a β-1,4-xylanase from a cellulolytic extreme thermophile expressed in Escherichia coli

• 1.1. An endoxylanase (EC 3.2.1.8) was purified from an Escherichia coli strain carrying a xylanase gene from the extreme thermophile “Caldocellum saccharolyticum strain Tp8T6.3.3.1. It was found to have an M_r of 42,000 and an isoelectric point of approx. 5.0.
• 2.2. The enzyme showed optimum activity at pH 5.0–7.7 and had an activation energy of 44 kJ mol⁻¹. It was stable at room temperature at pH 4.5–11.5 in the presence of 0.5 mg ml⁻¹ bovine serum albumin. The half-life of the enzyme at 75°C was 20 min at pH 6.0 in the presence of 0.5 mg ml⁻¹ bovine serum albumin.
• 3.3. The xylanase had highest activity on oat spelts xylan, releasing xylobiose and some xylotriose. The K_m for oat spelts xylan was 0.021% (w/v) at pH6.0.
• 4.4. The enzyme had high activity on sugar cane bagasse hemicelluloses A and B, lower activity on larchwood xylan and also hydrolysed carboxymethylcellulose, 4-methylumbelliferyl β-D-cellobioside and p-nitrophenyl β-D-cellobioside, but could not hydrolyse xylobiose.
• 5.5. It showed transferase activity on p-nitrophenyl β-D-xylopyranoside. Xylose did not inhibit the enzyme.

     

Growth peculiarities of commensal Escherichia coli isolates from the gut microflora in Crohn’s disease patients

Verhulst’s logistic differential equation, popular in mathematical ecology, is used in modeling of population growth, neural networks, statistics, reaction models, Fermi distribution, modeling of tumor growth, etc. We used this function to characterize growth of commensal Escherichia coli isolates from gut microflora in Crohn’s disease patients. The results of our investigations show differences in growth parameters of commensal E. coli, isolated from the gut microflora in Crohn’s disease patients and healthy volunteers; it is most likely explained by the influence of chronic inflammatory processes on growth and reproduction of these bacteria. It has been established that the used mathematical model well characterizes growth of patients’ gut E. coli isolates, and it can be important for the expedient probiotics’ application during the disease.     

Cloning of the thermostable α-amylase gene from Pyrococcus woesei in Escherichia coli

Pyrococcus woesei (DSM 3773) α-amylase gene was cloned into pET21d(+) and pYTB2 plasmids, and the pET21d(+)α-amyl and pYTB2α-amyl vectors obtained were used for expression of thermostable α-amylase or fusion of α-amylase and intein in Escherichia coli BL21(DE3) or BL21(DE3)pLysS cells, respectively. As compared with other expression systems, the synthesis of α-amylase in fusion with intein in E. coli BL21(DE3)pLysS strain led to a lower level of inclusion bodies formation—they exhibit only 35% of total cell activity—and high productivity of the soluble enzyme form (195,000 U/L of the growth medium). The thermostable α-amylase can be purified free of most of the bacterial protein and released from fusion with intein by heat treatment at about 75°C in the presence of thiol compounds. The recombinant enzyme has maximal activity at pH 5.6 and 95°C. The half-life of this preparation in 0.05 M acetate buffer (pH 5.6) at 90°C and 110°C was 11 h and 3.5 h, respectively, and retained 24% of residual activity following incubation for 2 h at 120°C. Maltose was the main end product of starch hydrolysis catalyzed by this α-amylase. However, small amounts of glucose and some residual unconverted oligosaccharides were also detected. Furthermore, this enzyme shows remarkable activity toward glycogen (49.9% of the value determined for starch hydrolysis) but not toward pullulan.     

Growth of a Capsid Mutant of Bacteriophage φX174 in a Temperature-Sensitive Strain of Escherichia coli

A capsid mutant of X174 is capable of forming replicative form and synthesizing single strands at the restrictive temperature in a dnaB mutant of Escherichia coli. Under similar conditions, the wild-type bacteriophage is incapable of either step in viral synthesis.     

Translation of Virus mRNA: Synthesis of Bacteriophage Qβ Proteins in a Cell-Free Extract from Wheat Embryo

The RNA from bacteriophage Qbeta can be translated by cell-free extracts from wheat embryos. This translation, by 80S ribosomes, occurs at a low magnesium ion concentration. Three products are synthesized which coelectrophorese with Qbeta proteins synthesized in Escherichia coli extracts. The smallest of these has been identified as coat protein. Although the polycistronic bacteriophage message is translated with fidelity, the efficiency is much less than when the monocistronic brome mosaic virus coat protein message is translated.     

Expression Optimization and Biochemical Characterization of a Recombinant γ-Glutamyltranspeptidase from Escherichia coli Novablue

A truncated Escherichia coli Novablue γ-glutamyltranspeptidase (EcGGT) gene lacking the first 48-bp coding sequence for part of the signal sequence was amplified by polymerase chain reaction and cloned into expression vector pQE-30 to generate pQE-EcGGT. The maximum production of His₆-tagged enzyme by E. coli M15 (pQE-EcGGT) was achieved with 0.1 mM IPTG induction for 12 h at 20 °C. The overexpressed enzyme was purified to homogeneity by nickel-chelate chromatography to a specific transpeptidase activity of 4.25 U/mg protein and a final yield of 83%. The molecular masses of the subunits of the purified enzyme were estimated to be 41 and 21 kDa respectively by SDS-PAGE, indicating EcGGT still undergoes the post-translational cleavage even in the truncation of signal sequence. The optimum temperature and pH for the recombinant enzyme were 40 °C and 9, respectively. The apparent K _m and V _max values for γ-glutamyl-p-nitroanilide as γ-glutamyl donor in the transpeptidation reaction were 37.9 μM and 53.7 × 10⁻³ mM min⁻¹, respectively. The synthesis of L-theanine was performed in a reaction mixture containing 10 mM L-Gln, 40 mM ethylamine, and 1.04 U His₆-tagged EcGGT/ml, pH 10, and a conversion rate of 45% was obtained.     

Structure of the β-Galactosidase Gene from Thermus sp. Strain T2: Expression in Escherichia coli and Purification in a Single Step of an Active Fusion Protein

The nucleotide sequence of both the bgaA gene, coding for a thermostable β-galactosidase of Thermus sp. strain T2, and its flanking regions was determined. The deduced amino acid sequence of the enzyme predicts a polypeptide of 645 amino acids (M_r, 73,595). Comparative analysis of the open reading frames located in the flanking regions of the bgaA gene revealed that they might encode proteins involved in the transport and hydrolysis of sugars. The observed homology between the deduced amino acid sequences of BgaA and the β-galactosidase of Bacillus stearothermophilus allows us to classify the new enzyme within family 42 of glycosyl hydrolases. BgaA was overexpressed in its active form in Escherichia coli, but more interestingly, an active chimeric β-galactosidase was constructed by fusing the BgaA protein to the choline-binding domain of the major pneumococcal autolysin. This chimera illustrates a novel approach for producing an active and thermostable hybrid enzyme that can be purified in a single step by affinity chromatography on DEAE-cellulose, retaining the catalytic properties of the native enzyme. The chimeric enzyme showed a specific activity of 191,000 U/mg at 70°C and a K_m value of 1.6 mM with o-nitrophenyl-β-d-galactopyranoside as a substrate, and it retained 50% of its initial activity after 1 h of incubation at 70°C.β-d-Galactosidase (EC 3.2.1.23) catalyzes the hydrolysis of β-1,4-d-galactosidic linkages. This enzyme is distributed in numerous microorganisms, plants, and animal tissues. The application of β-galactosidase to the hydrolysis of lactose in dairy products, such as milk and cheese whey, has received much attention (7, 21), and in this regard, thermostable β-galactosidases have attracted increasing interest because of their potential usefulness in the industrial processing of lactose-containing products (21). Thermostable enzymes have a number of generally recognized advantages in industrial applications, such as associated chemical resistance and reduced chances of microbial growth at high temperatures (15, 19). Nevertheless, relatively few studies have been conducted on β-galactosidases from thermotolerant or thermophilic bacteria, and as far as we know, only four genes encoding these enzymes have been cloned (5, 10, 11, 13, 16, 18).An important property that has received little attention in the literature is the level of purity of commercial preparations of β-galactosidases, especially with regard to the presence of other enzymes, such as proteases. These contaminants could have a severe impact on the stability of the enzyme, leading to undesirable changes in dairy products during storage (21). To prevent these, a new method was developed to purify the β-galactosidase (LacZ) of Escherichia coli by fusing to its N terminus the choline-binding domain (ChBD) of the pneumococcal autolytic amidase LytA (23). This system allowed the purification of E. coli β-galactosidase in a single step by affinity chromatography on DEAE-cellulose (23). Thus, it appeared interesting to test whether this procedure could also be used in the purification of a thermostable enzyme in order to circumvent contamination problems.This paper reports the molecular characterization of the bgaA gene, encoding the β-galactosidase (BgaA) of Thermus sp. strain T2, and describes the construction of a ChBD-BgaA chimera which retains the biochemical properties of the native enzyme and can be purified in a single chromatographic step.     

Cloning-Independent Expression and Analysis of ω-Transaminases by Use of a Cell-Free Protein Synthesis System

Herewith we report the expression and screening of microbial enzymes without involving cloning procedures. Computationally predicted putative ω-transaminase (ω-TA) genes were PCR amplified from the bacterial colonies and expressed in a cell-free protein synthesis system for subsequent analysis of their enzymatic activity and substrate specificity. Through the cell-free expression analysis of the putative ω-TA genes, a number of enzyme-substrate pairs were identified in a matter of hours. We expect that the proposed strategy will provide a universal platform for bridging the information gap between nucleotide sequence and protein function to accelerate the discovery of novel enzymes.Recent advances in genome sequencing technology have accumulated enormous amounts of sequence information (12). Although protein function encoded in nucleotide sequences can be annotated using computational alignment tools, in many cases, significant similarity to proteins with known function is hard to establish (5, 18). To understand the biological function of these unknown proteins, as well as to validate the computer-annotated results, efficient methods that enable rapid translation of genetic information into protein function are in high demand. The availability of high-throughput method for protein generation is also essential for accelerating the discovery and evolution of biocatalysts (3, 4, 6, 14, 22, 23) used in industry. While gene cloning and cultivation of transformed cells have long been used as standard methods for production of recombinant proteins, the vast amount of sequence information from various genome sequencing projects is now demanding a throughput of protein expression that exceeds that of the present in vivo expression techniques.Compared to cell-based gene expression, cell-free protein synthesis offers substantial advantages in speed and flexibility for the simultaneous expression of multiple proteins (7, 9, 13, 16, 19, 21). As a part of our efforts to extend the application of cell-free protein synthesis into the field of enzyme technology, we report in this paper an integrated methodology for fast expression screening of enzymes using ω-transaminases (ω-TAs) as a model target. Transaminases are pyridoxal-5′-phosphate (PLP)-dependent enzymes that catalyze reversible transfer of amine groups to keto acids, producing diverse proteogenic or nonproteogenic amino acids (1).In this work, ω-TA genes from microbial colonies were amplified by PCR and directly expressed in a cell-free protein synthesis system. Expressed enzymes were then screened for their activity toward different amine donors by colorimetric measurement of the changes in the concentration of pyruvate, which was used as a common amine acceptor. As a result, analysis of the substrate specificities of the enzymes encoded by 11 ω-TA genes toward 16 amine-donating compounds were completed within a matter of hours, identifying a number of enzyme-substrate matches.We started by examining whether sufficient amount of proteins could be generated for enzymatic analysis of ω-TAs when the PCR products amplified from the bacterial colonies were used as the template for cell-free synthesis reactions. The efficiency of protein synthesis was compared for reactions programmed with a plasmid-cloned ω-TA gene from Vibrio fluvialis (Vfω-TA) (pIVEX2.3d ω-TA Vf) and reactions programmed with the same gene prepared by two-step PCR from a bacterial colony (Vibrio fluvialis JS17 [20]), as depicted in Fig. ​Fig.1.1. The ω-TA genes examined in this study are listed in Table S1 in the supplemental material along with their bacterial sources.Open in a separate windowFIG. 1.Experimental scheme for cloning-independent cell-free expression screening of ω-transaminases. The expression templates for cell-free synthesis were prepared through two-step amplification of the target open reading frame (ORF) from bacterial genomes. PCR products were translated into corresponding enzymes in microtiter plates as described in the text. Upon completion of the synthesis reaction, the reaction mixture was sequentially supplied with the assay mixture and chromogenic compound to determine the residual pyruvate concentration after the amine transfer reaction. Abbreviations: For and Rev Primer, forward and reverse primers, respectively; 5′-UTR, 5′ UTR; T7P, T7 promoter; RBS, ribosome binding site; T7T, T7 terminator; ω-TAp, putative ω-transaminase; ω-TA Rs, ω-transaminase from Rhodobacter sphaeroides; ω-TA At, ω-transaminase from Agrobacterium tumefaciens.The templates for cell-free synthesis of ω-TA were prepared by colony PCR and subsequent second-round PCR using the MEGA primers flanking the T7 promoter, ribosome binding site, polyhistidine tag, and the T7 terminator. All of the PCRs were carried out using LA Taq DNA polymerase (Takara Bio Inc., Otsu, Japan). PCR products were directly used as the template for protein synthesis without purification. The standard cell-free reaction mixture consisted of the following components in a final volume of 50 μl: 57 mM HEPES-KOH (pH 7.5); 1.2 mM ATP; 0.85 mM (each) CTP, GTP, and UTP; 1.7 mM dithiothreitol; 0.17 mg/ml Escherichia coli total tRNA mixture (from strain MRE600); 90 mM potassium glutamate; 80 mM ammonium acetate; 12 mM magnesium acetate; 34 μg/ml l-5-formyl-5,6,7,8-tetrahydrofolic acid (folinic acid); 1.5 mM (each) 20 amino acids; 2% polyethylene glycol 8000 (PEG 8000); 67 mM creatine phosphate; 3.2 μg/ml creatine kinase; 10 μM l-[U-¹⁴C]leucine (11.3 GBq/mmol); 0.5 μg/ml PCR-amplified DNA; and 14 μl of the S12 extract (11). Cell-free synthesized proteins were quantified by measuring trichloroacetic acid (TCA)-insoluble radioactivity (10), and the size and relative solubility of the synthesized protein were determined by Western blot analysis on a 12% Tricine-SDS-polyacrylamide gel (17). Mouse anti-histidine-tagged IgG antibody (Merck KGaA, Darmstadt, Germany) and rabbit anti-mouse IgG conjugated to horseradish peroxidase (HRP) (Sigma, St. Louis, MO) were used as the primary and secondary antibodies, respectively. The PCR products served as translation substrates appropriate for producing as much protein as the corresponding plasmid-cloned gene when expressed in the reaction mixture (see Fig. S1 in the supplemental material).We next proceeded to amplify 11 ω-TA genes, including 9 putative ones from the colonies of bacterial origins (see Table S2 in the supplemental material for the list of primers used in this study), and express them in the reaction mixture prepared in two microtiter plates. Each of the 11 target genes was added to the plates by column on the plate (columns 2 through 12). Column 1 was used for negative-control reactions without any template DNAs (Fig. ​(Fig.1).1). After the PCR-amplified template DNAs were added to the plates, the plates were sealed with a plastic film to prevent evaporation and incubated at 37°C. From the measurements of TCA-insoluble radioactivity, it was estimated that 301 (±13) to 501 (±9) μg/ml of the encoded enzymes were produced after 90 min of incubation (see Fig. S2A in the supplemental material). Although most of the cell-free synthesized ω-TAs was highly insoluble in the initial experiments conducted under standard reaction conditions, the relative amounts of the soluble enzymes were markedly improved by using the GroEL/ES-enriched S12 extract (8) (Fig. S2B).Instead of the conventional high-performance liquid chromatography (HPLC) methods which have limited throughput for handling many reaction samples from different enzyme-substrate combinations, in this study, we used a simple colorimetric method for combinatorial analysis of the cell-free synthesized ω-TAs with different amine-donating substrates. Using Vfω-TA as a model ω-TA, it was first examined whether the progress of amine transfer reaction can be assayed quantitatively by colorimetric measurement of the residual pyruvate concentration. On the basis of our previous finding that Vfω-TA takes amine donors containing aryl groups as effective substrates (20), cell-free synthesized Vfω-TA was incubated with α-methylbenzylamine or benzylamine in the presence of pyruvate, and the residual pyruvate concentration in the assay mixture was determined. In brief, the amine transfer reaction was initiated by adding 50 μl of assay mixture (50 mM Tris-HCl buffer [pH 7.2], 10 mM sodium pyruvate, 10 mM [each] amine donors, and 20 μM pyridoxal-5′-phosphate) to the completed cell-free synthesis reaction mixtures (50 μl) in a 96-well plate. After 3 h of incubation at 37°C, the assay mixture was diluted with an equal volume of distilled water. When 100 μl of the diluted solution was transferred to a fresh plate containing 50 μl of 0.5 mM 2,4-dinitrophenylhydrazine (DNP), a yellow precipitate of pyruvate-dinitrophenylhydrazone (PA-DNPH) derivative was formed instantly. The absorbance at 450 nm was measured in a microplate reader (Bio-Rad, Hercules, CA) and compared with a standard curve to determine the amount of residual pyruvate in each well. Although the sensitivity of the residual pyruvate assay was as low as 0.01 mM, considering the error range, it was determined that a sensitivity of 0.1 mM should be used instead. When the optical density at 450 nm (OD₄₅₀) was measured after the addition of DNP and referred to a standard curve, the conversion yield based on the amount of residual pyruvate concentration showed good correlation with the results from standard HPLC assay (Table ​(Table11 and Fig. ​Fig.2A,2A, insets) where acetophenone or benzaldehyde generated from the corresponding substrates was separated in the Discovery HS F5 (5-μm particle size; 150- by 4.6-mm inner diameter [i.d.]; Supelco, Bellefonte, PA) column and measured at 254 nm. In addition, the relative amount of pyruvate was also able to be compared visually by adding 100 μl of 4 N NaOH solution, which turned the color of PA-DNPH to dark red (2, 15).Open in a separate windowFIG. 2.(A) Reactivity of 16 amine donors toward Vibrio fluvialis ω-TA. Reduced amounts of pyruvate concentration after the amine transfer reactions are plotted. Substrate number abbreviations: S01, α-methylbenzylamine; S02, α-ethylbenzylamine; S03, benzylamine; S04, 3-phenyl-1-propylamine; S05, phenylbutylamine; S06, 1-aminoindan; S07, ethylamine; S08, propylamine; S09, butylamine; S10, amylamine; S11, isopropylamine; S12, sec-butylamine; S13, β-alanine; S14, 3-amino-n-butyric acid; S15, phenylalanine; S16, 3-amino-3-phenylpropionic acid. The insets show HPLC traces of acetophenone and benzaldehyde after termination of transamination reaction by use of Vibrio fluvialis ω-TA against α-methylbenzylamine and benzylamine. mAU, milliabsorbance units. (B) Photo image of the assay plate after the addition of DNP and NaOH. ω-TA Vf, ω-TA from Vibrio fluvialis.

TABLE 1.

Activity comparison of Vibrio fluvialis ω-TA by HPLC and colorimetric method