Biosynthesis of PP-V, a monascorubramine homologue, by Penicillium sp. AZ

The biosynthetic pathway of PP-V, a new monascorubramine homologue, was elucidated by ¹³C-labeling studies. The [1-¹³C] of acetate was incorporated into 2-, 3a-, 4a-, 6-, 8-, 9-, 11-, 13-, 15-, 17-, and 19-Cs of PP-V, and the [2-¹³C], into 3-, 4-, 5-, 8a-, 9a-, 10-, 12-, 14-, 16-, 18-, and 20-Cs. These incorporation patterns coincide with those reported in the biosynthesis of a Monascus azaphilone pigment, monascorubrin.     

改造乙酰羟酸合成酶提高L-亮氨酸产量

乙酰羟酸合成酶（acetohydroxy acid synthase,AHAS,编码基因ilvBN）是L-亮氨酸合成途径的第一个限速酶。以谷氨酸棒杆菌XL-3（Corynebacterium glutamicum XL-3）为底盘细胞,通过分析并改造AHAS增加其对底物丙酮酸的偏好性,从而提高L--亮氨酸产量。首先利用AHAS的氨基酸序列进行同源建模,根据蛋白质结构进行丙氨酸扫描,找到突变的潜在位点,通过测定突变体酶活力和重组菌株的L--亮氨酸产量寻找最适突变体。测定结果发现将157位Gln突变成Arg能够有效提高AHAS催化丙酮酸的能力,最终重组菌株的L--亮氨酸产量达到（23.5±1.8）g/L,比出发菌株谷氨酸棒杆菌XL-3增加了51%,同时副产物L--异亮氨酸产量有所下降。因此,通过对AHAS的理性改造促进了L--亮氨酸的合成,该研究结果对后续利用蛋白质工程强化微生物合成L-亮氨酸等支链氨基酸具有重要的参考价值。     

Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae

Nicotinamide adenine dinucleotide (NAD⁺) is synthesized via two major pathways in prokaryotic and eukaryotic systems: the de novo biosynthesis pathway from tryptophan precursors, or the salvage biosynthesis pathway from either extracellular nicotinic acid or various intracellular NAD⁺ decomposition products. NAD⁺ biosynthesis via the salvage pathway has been linked to an increase in yeast replicative lifespan under calorie restriction (CR). However, the relative contribution of each pathway to NAD⁺ biosynthesis under both normal and CR conditions is not known. Here, we have performed lifespan, NAD⁺ and NADH (the reduced form of NAD⁺) analyses on BY4742 wild‐type, NAD⁺ salvage pathway knockout (npt1Δ) and NAD⁺ de novo pathway knockout (qpt1Δ) yeast strains cultured in media containing either 2% glucose (normal growth) or 0.5% glucose (CR). We have utilized ¹⁴C labelled nicotinic acid in the culture media combined with HPLC speciation and both UV and ¹⁴C detection to quantitate the total amounts of NAD⁺ and NADH and the amounts derived from the salvage pathway. We observed that wild‐type and qpt1Δ yeast exclusively utilized extracellular nicotinic acid for NAD⁺ and NADH biosynthesis under both the 2% and 0.5% glucose growth conditions, suggesting that the de novo pathway plays little role if a functional salvage pathway is present. We also observed that NAD⁺ concentrations decreased in all three strains under CR. However, unlike the wild‐type strain, NADH concentrations did not decrease and NAD⁺: NADH ratios did not increase under CR for either knockout strain. Lifespan analyses revealed that CR resulted in a lifespan increase of approximately 25% for the wild‐type and qpt1Δ strains, while no increase in lifespan was observed for the npt1Δ strain. In combination, these data suggest that having a functional salvage pathway is required for lifespan extension under CR. Copyright © 2009 John Wiley & Sons, Ltd.     

Gene cloning and characterization of dihydrolipoamide dehydrogenase from Microbacterium luteolum: A useful enzymatic regeneration system of NAD from NADH

Dihydrolipoamide dehydrogenase (LPD), a useful biocatalyst for regenerating NAD⁺, was purified from Microbacterium luteolum JCM 9174, and the gene encoding LPD was cloned from the genomic DNA. The gene contained an opening reading frame consisting of 1395 nucleotides encoding 465 amino acid residues with a predicted molecular weight of 49912.1 Da, which displayed 36–78% homology to known LPDs. Moreover, the FAD- and NAD⁺-binding sites and the two catalytic residues in the LPDs were conserved. The enzyme was expressed in recombinant Escherichia coli cells and purified to homogeneity by column chromatography. LPD of M. luteolum (MluLPD) accepted not only lipoamide but also some artificial electron acceptors such as dichlorophenolindophenol (DCIP) and nitrotetrazolium blue (NTB), that is, it functions as a diaphorase. NAD⁺ demonstrated a strong activating effect on MluLPD, and the activity was 5.2 times higher than that without NAD⁺. The enzyme was suitable for regenerating NAD⁺ in biocatalytic reactions because of its high affinity for NADH (6.1 μM). An NAD⁺-regenerating system with MluLPD and laccase using 2,5-dimethoxy-1,4-benzoquinone as a hydrogen acceptor was demonstrated.     

Metabolite channeling and compartmentation in the human cell line AGE1.HN determined by 13C labeling experiments and 13C metabolic flux analysis

This study focused on analyzing active pathways and the metabolic flux distribution in human neuronal AGE1.HN cells that is a desirable basis for a rational design and optimization of producing cell lines and production processes for biopharmaceuticals. ¹³C-labeling experiments and ¹³C metabolic flux analysis were conducted using glucose, glutamine, alanine and lactate tracers in parallel experiments. Connections between cytosolic and mitochondrial metabolite pools were verified, e.g., flux from TCA cycle metabolite ¹³C to glycolytic metabolites. It was also found that lactate and alanine are produced from the same pyruvate pool and that consumed alanine is mainly directly metabolized and secreted as lactate. Activity of the pentose phosphate pathway was low being around 2.3% of the glucose uptake flux. This might be compensated in AGE1.HN by high mitochondrial malic enzyme flux producing NADPH. Mitochondrial pyruvate transport was almost zero. Instead pyruvate carbons were channeled via oxaloacetate into the TCA cycle which was mainly fed via α-ketoglutarate and oxaloacetate during the investigated phase. The data indicate that further optimization of this cell line should focus on the improved substrate usage which can be accomplished by an improved connectivity between glycolytic and mitochondrial pyruvate pools or by better control of the substrate uptake.     

Heat-adaptation induced thermotolerance in Staphylococcus aureus: Influence of the alternative factor σ

The role of σ^B in the Staphylococcus aureus heat-shock induced thermotolerance was investigated. Survival curves at 58 °C of S. aureus strain Newman and its isogenic ΔsigB mutant were obtained for native and heat-shocked cells (45 °C for 5–120 min) in exponential and stationary phase of growth. The magnitude of the acquisition of thermotolerance at 58 °C depended on the growth phase and on the duration of the heat shock. Stationary growth phase cells were always more heat tolerant than exponentially growing cells and thermotolerance increased with heat-shock duration up to 120 min. S. aureus cells were able to increase their heat tolerance in the absence of the σ^B factor. In stationary phase, whereas in the parental strain the thermotolerance was increased by a factor of 12 after a heat shock of 120 min at 45 °C (δ values at 58 °C for native and heat-shocked cells were 0.63 and 7.22 min, respectively), in the mutant strain it increased 43 fold (δ values 0.09 and 3.87 min). The addition of chloramphenicol to the adaptation medium resulted in a lower increase in heat tolerance but did not prevent it completely, suggesting that S. aureus can partially increase its thermotolerance without “de novo” protein synthesis. Both the number of non-damaged cells and the proportion of cells able to repair sublethal damage were higher for heat-shocked cells.     

Characterisation and quantification of the reaction(s) catalysed by transglutaminase using the o‐phthaldialdehyde reagent

The potential application of the o‐phthaldialdehyde (OPA) reagent for quantification of the type and extent of the reaction(s) catalysed by transglutaminase (TGase) during incubation with sodium caseinate (NaCN) was investigated. Initial studies were performed to ensure that NH₃, a by‐product of TGase activity, could be determined with the OPA reagent in trichloroacetic acid (TCA) supernatants of NaCN solutions. The detectable concentration of exogenously added NH₃ (at NH₃ concentrations > 10 mM ) was found to decrease during extended incubation at 23, 37 and 50°C and at either pH 7.0 or 8.0 in 4% w/v NaCN solutions, even when taking into account the evaporation of water from the sample. The recovery of NH₃ from 12% w/v TCA supernatants of NaCN solutions spiked with 5 mM NH₃ at 23°C and pH 7.0 was found to be 88%. The release of NH₃ and the decrease in ε‐amino groups on incubating NaCN with TGase was subsequently quantified using the OPA reagent. Incubation of NaCN (4% w/v) with TGase at 23°C resulted in progressive increases and decreases, respectively, in NH₃ and ε‐amino group concentration with increased incubation time. These changes were dependent on TGase : NaCN. It was estimated that approximately 20% of the available Lys residues in NaCN were involved in TGase‐catalysed cross‐links. However, the observed decrease in ε‐amino group concentration was higher than expected. This may be due to concealment of noncross‐linked amino groups in polymerised NaCN, making them unavailable for reaction with the OPA reagent.     

Enzymatic properties of transglutaminase produced by a new strain of Bacillus circulans BL32 and its action over food proteins

The aim of this study was to physicochemically characterize transglutaminase (TGase) from Bacillus circulans BL32, a strain recently isolated from the Amazon basin region, for its application in food systems. The effects of pH and temperature on the enzyme activity were determined by Central Composite Rotatable Design (CCRD), with maximal TGase activities obtained for pH between 5.7 and 8.7 and temperatures of 25-45 °C. This microbial TGase showed to be remarkably stable: over 90% of its activity was retained after 120 min of incubation at 50 °C. The Ca²⁺ and Mg²⁺ cations enhanced enzyme activity and its thermal stability when in concentrations of up to 2 and 1 mol L⁻¹, respectively. Casein, isolated soy protein, and hydrolysed animal protein were treated with this TGase. The decrease in the amount of free amino groups, especially for casein, showed the cross-linking of protein catalysed by this enzyme, while the emulsifying properties of these proteins were improved with treatment. These results suggest that this microbial TGase has a good potential to be used in food and other industrial applications.     

Effect of growth rate reduction and genetic modifications on acetate accumulation and biomass yields in Escherichia coli

Although acetate biosynthesis in Escherichia coli provides an important intermediary for ATP synthesis, its accumulation inhibits both cell growth and protein production. Since pyruvate provides the largest flux to acetate and is central to the problem of acetate production, acetate accumulation could be reduced or abolished if the pyruvate pool for the TCA cycle was reduced. To examine this possibility, various pyruvate kinase (pyk) and phosphotransferase system (pts) mutants were tested for acetate production in batch cultures with glucose as the only carbon source. The pykA pykF mutant exhibited significant reductions in the specific growth rate and acetate production compared with the wild-type strain. Interestingly, in the case of pts and pts pyk mutants in which increased biomass yields were observed in comparison with the wild-type strain, no acetate production was detected. Therefore, these mutants are potentially useful for higher production of recombinant proteins. The results from the continuous cultivation performed using the wild-type strain at various dilution rates, suggest acetate reduction as a consequence of both genetic changes and growth rate diminutions.     

Purification and properties of a heat-stable enzyme of <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> Rm12 from raw milk

Pseudomonas fluorescens Rm12 is a kind of Psychrotrophic bacteria growing in cold raw milk. It produced an extracellular heat resistant protease with an estimated molecular weight of 45 kDa by size exclusion chromatography and SDS-PAGE under both reducing and non-reducing conditions. The enzyme, designated Ht13, was purified to electrophoretic homogeneity from the culture supernatant by sequentially using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography and size exclusion chromatography. The specific activity of the enzyme increased 115.5-folds. The optimum pH value and temperature of Ht13 were 7.5 and 40 °C, respectively. Based on its biochemical characteristics, Ht13 can be included in the group of metalloproteases, which was inhibited by 1, 10-phenanthroline and EDTA but not by pepstatin A, chymostatin, STI, E-64, BBI, PMSF and pAPMSF. Mn²⁺ has positive effect on activity and can increased the heat resistance capability, while Ca²⁺ had a negligible effect. For the hydrolysis of azocasein, the Km was 0.012 mg mL⁻¹. The enzyme showed typical heat-stable behavior. After treatment of 160 °C 20 s, the residual activity was 9%. The half-life of the enzyme at 160 °C in buffer with Mn²⁺ was approximately 12 s. Among several main milk proteins, Ht13 can cleave α_s-casein, β-casein and κ-casein. The sequence of 1st–16th amino acids of N-terminal was MSKVKDKAIVSAAQAS, which was same as those proteases excreted from some other P. fluorescens. However, their molecular weights, the activation ion and amino acid composition were different, suggesting Ht13 from P. fluorescens Rm12 is a novel protease.     

Cloning and characterization of β-agarase AgaYT from Flammeovirga yaeyamensis strain YT

A bacterium with potent agar-degrading capability was isolated from the surface of a red algae, Gracilaria tenuistipitata. Based on phenotypic characteristics, 16S rDNA gene sequence and a phylogenetic analysis, this bacterium was identified and named as Flammeovirga yaeyamensis strain YT. PCR using homology-based degenerate primers was employed to clone any agarase gene belonging to GH16 family encoded in F. yaeyamensis strain YT. The resolved 1512 nucleotides revealed that the cloned gene, namely AgaYT, encodes a protein of 503 amino acids comprising a signal peptide, a glycosyl hydrolase catalytic module and a C-terminal domain with an unknown function. The recombinant protein r-AgaYT is an endo-type β-agarase hydrolyzing agarose to yield neoagarobiose and neoagarotetraose as the main hydrolytic products. The specific activity of r-AgaYT was determined about 178.6 U mg^{− 1} at 40°C and pH 8.0.     

Increased riboflavin production from activated bleaching earth by a mutant strain of Ashbya gossypii

The production of riboflavin from vegetable oil was increased using a mutant strain of Ashbya gossypii. This mutant was generated by treating the wild-type strain with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Riboflavin production was 10-fold higher in the mutant compared to the wild-type strain. The specific intracellular catalase activity after 3 d of culture was 6-fold higher in the mutant than in the wild-type strain. For the mutant, riboflavin production in the presence of 40 mM hydrogen peroxide was 16% less than that in the absence of hydrogen peroxide, whereas it was 56% less for the wild-type strain. The isocitrate lyase (ICL) activity of the mutant was 0.26 mU/mg of protein during the active riboflavin production phase, which was 2.6-fold higher than the wild-type strain. These data indicate that the mutant utilizes the carbon flux from the TCA cycle to the glyoxylate cycle more efficiently than the wild-type strain, resulting in enhanced riboflavin production. This novel mutant has the potential to be of use for industrial-scale riboflavin production from waste-activated bleaching earth (ABE), thereby transforming a useless material into a valuable bioproduct.     

Biological control of Penicillium italicum of Citrus and Botrytis cinerea of Grape by Strain 34–9 of Kloeckera apiculata

The biological control capability of strain 34-9 of Kloeckera apiculata against Penicillium italium (Wehmer), postharvest rot of citrus fruits and Botrytis cinerea, postharvest rot of grape fruits was studied in vitro and in vivo. Strain 34-9 of K. apiculata at 3×10⁸ CFU (colony-forming unit)/ml of washed cells provided complete control of 3×10⁵ spores/ml of P. italium and B. cinerea during storage at 25 °C for 6 d. Antagonist population increased 40, 195 times in citrus fruit wound site and grape fruit wound site at 25 °C for 3 d, respectively, then the population stabilized for the remaining storage period. Cell-free culture filtrate, supernatant fluid and sterilized solution of strain 34-9 of K. apiculata had no antagonist against P. italium of citrus and B. cinerea of grape. These results showed that competition for nutrient, not antibiotic production, played a major role in the biological control capability of strain 34-9 of K. apiculata against P. italium of citrus and B. cinerea of grape.     

Acidic β-mannanase from Penicillium pinophilum C1: Cloning, characterization and assessment of its potential for animal feed application

The β-mannanase gene, man5C1, was cloned from Penicillium pinophilum C1, a strain isolated from the acidic wastewater of a tin mine in Yunnan, China, and expressed in Pichia pastoris. The sequence analysis displayed the gene consists of a 1221-bp open reading frame encoding a protein of 406 amino acids (Man5C1). The deduced amino acid sequence of Man5C1 showed the highest homology of 57.8% (identity) with a characterized β-mannanase from Aspergillus aculeatus belonging to glycoside hydrolase family 5. The purified rMan5C1 had a high specific activity of 1035 U mg^–1 towards locust bean gum (LBG) and showed highest activity at pH 4.0 and 70°C. rMan5C1 was adaptable to a wide range of acidity, retaining > 60% of its maximum activity at pH 3.0–7.0. The enzyme was stable over a broad pH range (3.0 to 10.0) and exhibited good thermostability at 50°C. The K_m and V_max values were 5.6 and 4.8 mg mL^–1, and 2785 and 1608 μmol min^–1 mg^–1, respectively, when LBG and konjac flour were used as substrates. The enzyme had strong resistance to most metal ions and proteases (pepsin and trypsin), and released 8.96 mg g^–1 reducing sugars from LBG in the simulated gastric fluid. All these favorable properties make rMan5C1 a promising candidate for use in animal feed.     

Involvement of cytochrome P450 in hydroxylation of propylbenzene by Fusarium moniliforme strain MS31

Fusarium moniliforme strain MS31 can oxidize propylbenzene to (R)-1-phenylpropanol with what may be a cytochrome P450. Hydroxylation of propylbenzene needed molecular oxygen, and NADPH as a coenzyme gave a higher yield than NADH. The hydroxylation proceeded further when FAD and FMN were added than in their absence, suggesting that the enzyme was a flavo-protein. Carbon monoxide inhibited the hydroxylation, as did other cytochrome P450 inhibitors such as SKF 525A and miconazole. These characteristics matched those of a microsomal cytochrome P450 monooxygenase system that contained NADPH-cytochrome P450 reductase.     

Immunization of the industrial fermentation starter culture strain of Saccharomyces cerevisiae to a contaminating killer toxin-producing Candida tropicalis

K3 killer trait was introduced into the fermentation starter strain of Saccharomyces cerevisiae BSP 1 in order to construct immune industrial strain that produces K3 type killer toxin and was resistant to Candida tropicalis (K⁺) contamination. Protoplasts of respiration-deficient Rho^o strain of S. cerevisiae NCYC 761 (K3) and S. cerevisiae BSP 1 were fused. The resulting respiration-competent hybrid with K3 type killer activity was selected on media containing a non-fermentable carbon source and by a killer zone assay in a plate test, respectively. The fusant was similar to the parent strain in its fermentation and sugar utilization patterns, growth rate, dough-raising properties and osmotolerance. The newly constructed S. cerevisiae BSP 1 (K3) inhibited the growth of C. tropicalis in a pH range from 3.5 to 5.0 and over a temperature range of 20–30°C.     

理性代谢工程改造促进谷氨酸棒杆菌高效合成L-谷氨酰胺

L-谷氨酰胺（L-glutamine,L-Gln）是人体液中含量最丰富的一种半必需氨基酸,具有多种营养和药理功能,被广泛应用于医药、食品添加剂、营养保健品、饲料等领域。目前,微生物发酵法是生产L-Gln的主要方法,发酵产酸效率低、副产物多、菌种性能欠缺等问题严重限制了其工业化应用。为解决这一问题,作者以实验室前期构建的L-Gln生产菌株CGQ03为出发菌株,通过强化谷氨酸脱氢酶表达及辅因子NADPH供应促进前体L-谷氨酸合成、增强辅因子ATP胞内含量、过表达溶氧相关蛋白VHb提高细胞携氧能力、增强关键酶谷氨酰胺合成酶催化活性及发酵工艺优化等策略提高了L-Gln产量。最终的基因工程菌CGQ08/pDXW10-glnASc-gdhCg在5 L发酵罐上补料分批发酵66 h,L-Gln的产量最高达（94.5±1.8） g/L,糖酸转化率为34.8%,生产强度为1.43 g/（L·h）。本研究为L-Gln及其相关化合物的工业化生产提供了借鉴。     

Passage of stable isotope-labeled grass silage fiber and fiber-bound protein through the gastrointestinal tract of dairy cows

Fractional passage rates are required to predict nutrient absorption in ruminants but data on nutrient-specific passage kinetics are largely lacking. With the use of the stable isotope ratio (δ) as an internal marker, we assessed passage kinetics of fiber and fiber-bound nitrogen (N) of intrinsically labeled grass silage from fecal and omasal excretion patterns of δ¹³C and δ¹⁵N. In a 6 × 6 Latin square, lactating dairy cows received grass silages [455 g/kg of total diet dry matter (DM) ] in a 2 × 3 factorial arrangement from ryegrass swards fertilized at low (45 kg of N/ha) or high (90 kg of N/ha) levels of N and harvested at 3 maturity stages. Feed intake (16.7 ± 0.48 kg of DM/d; mean ± standard error of the mean) and milk yield (26.7 ± 0.92 kg/d) increased at the high level of N fertilization and at decreasing maturity. Nutrient digestibility decreased with increasing plant maturity, particularly at the high level of N fertilization, essentially reflecting dietary treatment effects on the nutritional composition of the grass silage. Fractional rumen passage rates (K₁) were highest and total mean retention time in the gastrointestinal tract (TMRT) was lowest when based on the external marker chromium mordanted fiber (Cr-NDF; 0.047/h and 38.0 h, respectively). Fecal δ¹³C in the acid detergent fiber fraction (¹³CADF) provided the lowest K₁ (0.023/h) and the highest TMRT (61.1 h) and highest peak concentration time (PCT; 24.3 h) among markers. In comparison, fecal fiber-bound N (¹⁵NADF) had a considerably higher K₁ (0.032/h) and lower TMRT (46.4 h) than ¹³CADF. Total N (measured with ¹⁵NDM) had a comparable K₁ (0.034/h) to that of ¹⁵NADF but provided the highest fractional passage rates from the proximal colon-cecum (K₂; 0.37/h) and lowest PCT (17.4 h) among markers. A literature review indicated unclear effects of grass silage maturity on K₁ and unknown effects of N fertilization on K₁. Our study indicated no effect of advancing maturity on fecal K₁ and a trend for K₁ to increase with the high level of N fertilization. Parameter K₂ increased, whereas PCT and TMRT generally decreased with the high level of N fertilization. Omasal digesta sampling largely confirmed results based on fecal sampling. Results indicate that the use of δ¹³C and δ¹⁵N can describe fiber-specific passage kinetics of forage.     

Online determination of <Superscript>2</Superscript>H/<Superscript>1</Superscript>H and <Superscript>13</Superscript>C/<Superscript>12</Superscript>C isotope ratios of cinnamaldehyde from different sources using gas chromatography isotope ratio mass spectrometry

The combination of gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) and gas chromatography-pyrolysis-isotope ratio mass spectrometry (GC-P-IRMS) is applied to the authenticity assessment of cinnamaldehyde from various sources. For that reason, cinnamon oils were self-prepared by steam distillation from three different varieties of cinnamon bark on the market, C. ceylanicum (ceylon), C. cassia (cassia) and C. burmanii (cassia vera). Furthermore, the so-called wood cinnamon was investigated, which is produced from the outer bark of older branches of cinnamon of minor quality. Self-prepared oils were analysed from commercial cinnamon powder. In addition several commercial samples of cinnamon oil and cinnamaldehyde, some of them declared to be natural, were investigated. ²_V-SMOW and ¹³C_V-PDB values of cinnamaldehyde were determined and characteristic authenticity ranges were deduced, allowing the differentiation between synthetic and natural samples. By correlation of both the ²_V-SMOW and ¹³C_V-PDB values, characteristic authenticity ranges were defined for ceylon, cassia and wood cinnamon. The ²_V-SMOW and ¹³C_V-PDB values of cassia vera samples are in the range of cassia. By comparing the ²_V-SMOW values of different self-prepared samples (ground bark, distillate) of cinnamon determined by TC/EA-IRMS with the corresponding GC-IRMS values, online GC-IRMS methods are proved to be essential in the authentication of complex natural products.     

KINETIC STUDIES AND MECHANISM OF CATALYSIS OF MALIC DEHYDROGENASE FROM DIOSCOREA ROTUNDATA TUBER

ABSTRACT The kinetic mechanism of catalysis by malic dehydrogenase (EC 1.1.1.37) isolated from yam (Dioscorea rotundata) tuber has been delineated. Initial velocity studies with the enzyme in the presence and absence of products of the reaction revealed an ordered sequential mechanism. The K_m values obtained from secondary plots were 0.05, 0.08, 0.48 and 2.56 mM for NADH, OAA, and NAD⁺ and L-malic acid, respectively. Product inhibition studies in both the forward and backward reactions support an ordered Bi-Bi sequential mechanism. This begins with an obligatory binding of NAD⁺ to form the first binary complex and a final release of NADH.