D-lactic acid production by metabolically engineered Saccharomyces cerevisiae

Poly D-lactic acid is an important polymer because it improves the thermostability of poly L-lactic acid by the stereo complex formation. We constructed a metabolically engineered Saccharomyces cerevisiae that produces D-lactic acid efficiently. In this recombinant, the coding region of pyruvate decarboxylase 1 (PDC1) was completely deleted, and two copies of the D-lactate dehydrogenase (D-LDH) gene from Leuconostoc mesenteroides subsp. mesenteroides strain NBRC3426 were introduced into the genome. The D-lactate production reached 61.5 g/l, the amount of glucose being transformed into D-lactic acid being 61.2% under neutralizing conditions. Additionally, the yield of free D-lactic acid was also shown to be 53.0% under non-neutralizing conditions. It was confirmed that D-lactic acid of extremely high optical purity of 99.9% or higher. Our finding obtained the possibility of a new approach for pure d-lactic acid production without a neutralizing process compared with other techniques involving lactic acid bacteria and transgenic Escherichia coli.     

Effect of six organic acids on staphylococcal growth and enterotoxin production.

Four Staphylococcus aureus strains were incubated at 37 degrees C for 24 h in broth progressively acidified with lactic, citric, ascorbic, acetic, pyruvic and propionic acids, and their survival rate and enterotoxin producing ability was studied. Acids were chosen based on their frequent use by the food industry. Periodically, samples were withdrawn to determine counts, pH and the presence of enterotoxins A, B, C, and D. For a given acid, the effect on growth and enterotoxin synthesis was different. The most inhibitory acid for the growth of strains FRI-100 and FRI-472 was pyruvic acid, for strain FRI-137 was lactic acid, all six acids were equally effective on strain S6. Lactic acid was very inhibitory to enterotoxin synthesis, but the effect on this parameter of acetic and citric acids was almost nil. Enterotoxins were seen to be inactivated at acid pH values; enterotoxin B was the most resistant to inactivation.     

Chiral Liquid Chromatography for Resolving Malic Acid Enantiomers in Adulterated Apple Juice

Reversed-phase HPLC with an aqueous mobile phase containing the chiral ligand-exchanger Cu¹¹/ (N,N-dimethyl-L-valine)₂ resolved the enantiomeric a-hydroxy acids, D- and L-malate. Post-column detection with acidic Fe¹¹¹ resulted in specific detection of α-hydroxy acids, so filtered apple juice gave a simple profile. D-Malic acid in apple juices suspected of being adulterated with synthetic DL-malic acid is presently determined from the difference between DL-malic acid (HPLC assay) and L-malic acid (L-malate dehydrogenase assay). The potential of the chiral HPLC method relative to the indirect method was evaluated and additional possibilities for direct and more sensitive determination of D-malate were suggested.     

Classification and identification of strains of Lactobacillus brevis based on electrophoretic characterization of D-lactate dehydrogenase: relationship between D-lactate dehydrogenase and beer-spoilage ability

The cell-free extracts of 60 strains which were identified phenotypically as being those of Lactobacillus brevis, including 48 isolates from the environment and 12 reference strains, were applied to polyacrylamide gel electrophoresis for extracting their NAD-dependent D- and L-lactate dehydrogenases (LDH). These strains were divided into 5 groups, i.e., Groups A, B, C, D, and E, on the basis of the electrophoretic mobilities of their D-LDH. The strains showed variations in their carbohydrate fermentation patterns. No relationship between the profile of D-LDH and the carbohydrate fermentation pattern was recognized. However, there appeared to be a relationship between the D-LDH profile and the beer-spoilage ability, because 40 out of 44 beer-spoilage strains identified as L. brevis were classified to Group B. We purified D-LDHs from the so-called complete beer-spoilage strain SBC 8002 of LDH Group B and from the non beer-spoilage strains JCM 1059T of LDH Group A and AHU 1508 of LDH Group C. Although the purified D-LDHs had the same molecular weight (84 kDa), each possessed a different optimum pH, optimum temperature, and isoelectric point. The aforementioned parameter values for the enzyme from the so-called complete beer-spoilage strain SBC 8002 of LDH Group B were 10.0, 50 degrees C, and 4.1, respectively; this strain was discriminated from the D-LDHs of the other two non beer-spoilage strains especially by its optimum temperature (50 degrees C).     

Transport of lactic acid in Kluyveromyces marxianus: evidence for a monocarboxylate uniport.

Lactic acid-grown cells of a strain of Kluyveromyces marxianus transported D- and L-lactic acid by a saturable mechanism that was partially inducible and subject to glucose repression, with the following kinetic parameters at pH 5.4: Vmax = 1.00 (+/- 0.13) mmol h-1 per g dry weight and Ks = 0.42 (+/- 0.08) mM. Lactic acid transport was competitively inhibited by pyruvic, glycolic, acetic and bromoacetic acids. The latter, a non-metabolizable analogue, was transiently accumulated, the extent depending on the extracellular pH. The pH dependence of the Ks values for undissociated lactic acid and for the lactate anion indicated that the latter was the transported species. Lactate uptake was not accompanied by the simultaneous uptake of protons, potassium ions or sodium ions excluding symport mechanisms. Initial lactic acid uptake led to transient membrane hyperpolarization as measured with a fluorescent dye excluding also an electroneutral anion antiport mechanism. It was concluded that lactate anions use a monocarboxylate uniport and that the counter anion, possibly bicarbonate, uses a separate channel, the coupling being electrical and loose.     

Purification and characterization of a dehydrogenase catalyzing conversion of N alpha-benzyloxycarbonyl-L-aminoadipic-delta-semialdehyde to N alpha-benzyloxycarbonyl-L-aminoadipic acid from rhodococcus sp. AIU Z-35-1

The enzyme catalyzing conversion of N alpha-benzyloxycarbonyl-L-aminoadipic-delta-semialdehyde (N alpha-Z-L-AASA) to N alpha-benzyloxycarbonyl-L-aminoadipic acid (N alpha-Z-L-AAA) in Rhodococcus sp. AIU Z-35-1 was identified, and its characteristics were revealed. This reaction was catalyzed by a dehydrogenase with a molecular mass of 59 kDa. The dehydrogenase exhibited enzyme activity on not only N alpha-Z-L-AASA but also N alpha-Z-D-AASA and short chain aliphatic aldehydes, but not on aromatic aldehydes and alcohols. The apparent Km values for N alpha-Z-L-AASA, N alpha-Z-D-AASA and NAD+ were estimated to be 3.8 mM, 14.1 mM and 0.16 mM, respectively. The NH2 terminal amino acid sequence of this enzyme exhibited a similarity to those of a piperidein-6-carboxylate dehydrogenase from Streptomyces clavuligerus and a putative dehydrogenase from Rhodococcus sp. RHA 1, but not to those of other microbial aldehyde dehydrogenases.     

Effect of added α-ketoglutaric acid,pyruvic acid or pyridoxal phosphate on proteolyis and quality of Cheddar cheese

Cheddar cheese curds were supplemented with 1, 5 or 20 g of α-ketoglutarate or pyruvic acid or 1.2 g pyridoxal-5¹-phosphate/kg cheese curd. The higher levels of keto-acids (5 or 20 g/kg curd) caused undesirable changes in the physico-chemical properties of resultant cheese. All levels of α-ketoglutarate reduced the pH of the cheese and promoted syneresis during pressing, while pyruvic acid increased the pH of the cheese. The numbers of starter and non-starter lactic acid bacteria were not affected by the addition of keto-acids or pyridoxal-5¹-phosphate. α-Ketoglutarate or pyruvic acid, at 1 g/kg, or pyridoxal-5¹-phosphatase, at 1.2 g/kg cheese curd, did not influence primary proteolysis in the cheese. The highest and lowest concentrations of total and individual free amino acids were found in the cheeses treated with pyruvic acid or α-ketoglutarate, respectively. The concentrations of most amino acids were lower in the cheeses treated with pyridoxal-5¹-phosphate than in the control. The results of this study suggest that α-ketoglutarate and pyridoxal-5¹-phosphate enhanced the degradation of most amino acids in Cheddar cheese while pyruvic acid promoted the formation of amino acids. The cheeses treated with α-ketoglutarate were more mature than the control cheese of the same age while pyruvic acid-treated cheese had a better flavour than the control.     

Influence of yeast strain on binding of sulphur dioxide in wines,and on its formation during fermentation

The amounts of sulphur dioxide bound by acetaldehyde, pyruvic acid and α-ketoglutaric acid during fermentation of three grape juices by eight wine yeasts (Saccharomyces sp.) are reported. These constituents accounted for 49–83 % (mean 69) of the measured bound SO₂, depending on the yeast strain and juice. the maximum range of concentrations of the binding components for individual wines were 10–48 ppm for acetaldehyde, 9–77 ppm for pyruvic acid and 5–63 ppm for α-ketoglutaric acid, depending on yeast strain and grape juice. the validity of the calculations was verified by an experiment with SO₂ and the three binding compounds in a multicomponent model system. The acetaldehyde content was related to the total SO₂ present, which itself was determined by the strain of yeast. SOz bound in the wines after a further SO₂ addition was correlated significantly with pyruvic and α-ketoglutaric acids, but not with acetaldehyde. Certain yeasts produced SO₂ during fermentation in grape juice and in synthetic media with defined sulphur sources. More SO₂ was produced at pH 3.6 than 3.0 in the absence of added sulphate in grape juice. Sulphate was the best sulphur source for SO₂ production in synthetic media, although some yeasts were able to produce smaller amounts of SO₂ from l-cysteine and reduced glutathione.     

Lipolysis, proteolysis and formation of volatile components during ripening of a fermented sausage with Pediococcus pentosaceus and Staphylococcus xylosus as starter cultures

Bacterial growth, formation of acids, lipolysis, proteolysis, fat oxidation, formation of volatile compounds and flavour characteristics were followed during ripening and storage of a fermented sausage. The starter culture used was composed of Pediococcus pentosaceus and Staphylococcus xylosus. The number of Pediococcus sp. increased by 1.5 log cfu/g during the first day of processing and remained constant at this level for 3 weeks. The corresponding initial increase in the numbers of Staphylococcus sp., 0.4 log cfu/g, was followed by a rapid decrease in the viable numbers. Lactic acid, mainly d-lactic acid, and acetic acid were formed during ripening. The triglycerides were hydrolysed to 1,2-diglycerides and free fatty acids at the beginning of ripening, followed by the formation of 1,3-diglycerides and monoglycerides, indicating lipolytic activity. Moreover, the nonprotein nitrogen increased during ripening as a result of the proteolytic activity. Most of the changes with respect to pH, formation of d-lactic acid, acetic acid, peroxides and flavour development occurred during the initial 3 days of ripening, when growth of Pediococcus sp. and Staphylococcus sp. occurred. Lipolysis as well as proteolysis continued after this initial period. The volatile compounds identified belonged to several chemical families, viz. aliphatic and aromatic hydrocarbons, aldehydes, ketones, alcohols, phenols, carboxylic acids, esters, nitrogen compounds, sulphur compounds, chloride compounds, terpenes and furans. Many of the volatile compounds probably originated from smoke and seasoning (onion/garlic and pepper), while others were a result of the activities of muscle enzymes and bacteria.     

Purification and characterization of an l-amino acid oxidase from Pseudomonas sp. AIU 813

An l-amino acid oxidase was found from a newly isolated strain, Pseudomonas sp. AIU 813. This enzyme was remarkably induced by incubation with l-lysine as a nitrogen source, and efficiently purified using an affinity chromatography with l-lysine as ligand. The enzyme oxidized l-lysine, l-ornithine and l-arginine, but not other l-amino acids and d-amino acids. The oxidase activity for l-lysine was detected in a wide pH range, and its optimal was pH 7.0. In contrast, the oxidase activity for l-ornithine and l-arginine was not shown in acidic region from pH 6.5, and optimal pH for both substrates was 9.0. The enzyme was a flavoprotein and composed of two identical subunits with molecular mass of 54.5?kDa. The N-terminal amino acid sequence was similar to that of putative flavin-containing amine oxidase and putative tryptophan 2-monooxygenase, but not to that of l-amino acid oxidases.     

Taste compounds in chorizo and their changes during ripening

Taste compounds in chorizo—a dry fermented sausage—prepared by both traditional and industrial methods, i.e. D- and L-lactic acid, acetic acid, free amino acids and ATP derivatives, were analysed. Industrial chorizo contained larger amounts of D-lactic, L-lactic and acetic acids, several amino acids (e.g. γ-aminobutyric acid, phenylalanine) and hypoxanthine (Hx) than the traditional one (p < 0.05). The variations in taste compounds during the ripening of chorizo were tracked. D-Lactic and acetic acid increased, as well as most of the amino acids. ATP derivatives showed characteristic changes, IMP decreased on the first day and inosine (Ino) changed gradually to Hx during ripening. The taste compounds contents of garlic and paprika were studied. There was an important contribution of asparagine from spices to the initial mixture of sausage.     

缬氨酸转氨酶拆分DL-缬氨酸的催化条件

利用具有缬氨酸转氨酶活性的工程菌对DL-缬氨酸进行拆分,考察了反应温度、pH值、底物摩尔比、底物浓度和金属离子对酶活性和底物转化率的影响。结果显示,该催化反应的最适反应条件为:反应温度是45℃,pH=9,L-缬氨酸与丙酮酸的摩尔比1∶8,DL-缬氨酸初始浓度为0.6 mol/L、丙酮酸初始浓度为2.4 mol/L,0.5 mmol/L的Mg2+和Na+对酶活性有明显的促进作用。     

MALIC DEHYDROGENASES FROM DIOSCOREA ROTUNDATA TUBERS: PURIFICATION AND PROPERTIES OF TWO ISOZYMES

Two molecular forms of the enzyme malate dehydrogenase (MDH₁ and MDH₂) have been purified from Dioscorea rotundata tubers. The enzymes were resolved on a DEAE-cellulose column and found to be homogeneous with differential mobilities on polyacrylamide gels. Both enzymes appeared to be dimeric proteins with subunit molecular weights of 30, 100 ± 50 and 31,600 ± 250 for MDH₁ and MDH₂, respectively. The enzymes exhibited maximal catalytic activity within the pH range 6.57.5 at 37C and were inhibited by oxaloacetic acid at concentrations greater than 0.25 mM as well as other naturally occurring metabolites (citric acid, isocitric acid, a-hetoglutaric acid, succinic acid, pyruvic acid, aspartic acid, glutamic acid, ATP and ADP). The thermal stability and storability of the enzymes at different conditions were significantly different.     

Sulphite-binding power of wines and ciders. I. Equilibrium constants for the dissociation of carbonyl bisulphite compounds

The apparent Equilibrium Constants were determined at pH3 and 4 for the dissociation of the bisulphite compounds of acetaldehyde, pyruvic acid, 2-ketoglutaric acid, L -xylosone, D -threo-2,5-hexodiulose, 2,5-diketogluconic acid, 2-ketogluconic acid and galacturonic acid. Determinations were also made over a wider pH range for acetaldehyde (pH 1 to 13) and pyruvic acid (pH 1.5 to 8).     

一株产大豆蛋白酶的芽孢杆菌发酵条件的优化

对一株产大豆蛋白酶芽孢杆菌(Bacillussp.D-16-9)的发酵条件进行了优化,研究各种碳源、氮源及无机盐对产酶的影响,应用正交试验优化发酵培养基组成。结果发现菌株D-16-9可以利用无机氮源生长,但用来产酶则很差;有机氮源利于生长和产酶,适当添加诱导物更利于产酶;酶合成模式属于滞后合成型,大量收获在菌体生长的稳定期。通过试验确定发酵培养基的最佳组成:5.0%玉米粉、3.0%大豆豆粕、0.05%氯化钾0、.05%硫酸镁;确定最适发酵条件为:起始pH10,培养温度30℃、种龄24 h、接种量10%,250 mL三角瓶中装入100 mL发酵培养基,摇瓶转速170 r/min,发酵时间120 h。综合最佳的培养基组成和培养条件,最终大豆蛋白酶活达6 500 U/mL。优化后蛋白酶活性由4423.20U/mL提高到6505.60U/mL。     

Analysis of selective production of Nalpha-benzyloxycarbonyl-L-aminoadipate-delta-semialdehyde and Nalpha-benzyloxycarbonyl-L-aminoadipic acid by Rhodococcus sp. AIU Z-35-1

The factors for selective production of N(alpha)-benzyloxycarbonyl-L-aminoadipate-delta-semialdehyde (N(alpha)-Z-L-AASA) and N(alpha)-benzyloxycarbonyl-L-aminoadipic acid (N(alpha)-Z-L-AAA) from N(alpha)-benzyloxycarbonyl-L-lysine (N(alpha)-Z-L-lysine) by Rhodococcus sp. AIU Z-35-1 were analyzed. The cultivation time was important for selective production of N(alpha)-Z-L-AASA, since N(alpha)-Z-L-lysine oxidizing enzyme reached maximum at the early stage of cell growth and then decreased. The differences of enzyme activities of N(alpha)-Z-L-lysine oxidizing enzyme and N(alpha)-Z-L-AASA dehydrogenase in pH and temperature also affected the selective production of N(alpha)-Z-L-AASA. For efficient production of N(alpha)-Z-L-AAA, it was important for cultivation time that N(alpha)-Z-L-AASA dehydrogenase activity be higher than N(alpha)-Z-L-lysine oxidizing enzyme activity, since a high concentration of N(alpha)-Z-L-AASA inhibited N(alpha)-Z-L-AASA dehydrogenase activity. The optimum pH of N(alpha)-Z-L-AAA production was affected by the instability of N(alpha)-Z-L-AASA dehydrogenase in the alkaline pH region.     

An enzymic assay for β-hydroxybutyric acid in liquid whole egg

A rapid biochemical method for the determination of D-β-hydroxybutyric acid using a specific enzyme (D-β-hydroxybutyric dehydrogenase) is described. The assay is based on the measurement of reduced adenine dinucleotide formed during the enzymic conversion of D-β-hydroxybutyric acid to acetoacetic acid. Using the biochemical assay, samples of liquid whole egg obtained from 18-day-old incubator reject eggs contained 1.81–2.29 mmol litre-¹ D-β-hydroxybutyric acid; the limit of reliable detection being estimated as 20 μmol litre-¹ for this method.     

耐热菌D-乳酸脱氢酶突变体的可视化建模和大肠杆菌中突变体对产苯乳酸的影响

以生物信息学为基础,分析D-乳酸脱氢酶（D-LDH）的保守氨基酸残基、活性中心氨基酸残基、蛋白质三维结构和同源建模,可视化比较建模突变体空间构象,优选最佳突变体模型。结果显示,在D-LDH的20个保守氨基酸中,4个与酶活性中心有关。比较突变体模型发现,49和297位的phe或try的苯环形成空间位阻,F49A或Y279A及F49A和Y279A双突变体可解除位阻。对已构建的三个突变体初步发酵显示,IPTG和乳糖都能诱导突变体酶在大肠杆菌中产生苯乳酸,静置培养比摇振培养产量高,用乳糖诱导时,突变体F49A（A.a D-LDH-F49A株）苯乳酸的量比野生型（A.a.D-LDH株）的高。优选可视化突变体可作为高效构建工程菌的方法之一。     

反相高效液相色谱法(RP-HPLC)测定啤酒中有机酸

建立了一种利用反相高效液相色谱法同时测定啤酒中 9种有机酸的方法。应用反相C18色谱柱Nucleosil 10 0C18　 2 5 0× 4 0mm ,以 0 1mol/LKH2 PO4(用H3 PO4调 pH3 0 )为流动相 ,紫外检测 2 15nm ,将啤酒中草酸、丙酮酸、苹果酸、α 酮戊二酸、乳酸、乙酸、柠檬酸、富马酸、琥珀酸一次性分离。除柠檬酸外各种有机酸的回收率在 85 %以上 ,变异系数 <5 %。该方法简便、快速、准确。此外 ,对影响有机酸分离的因素进行了研究 ,并测定了国内市售的十几种啤酒中的有机酸含量     

Influence of different pH values and inoculation time on the growth and malolactic activity of a strain of Oenococcus oeni

The present study was undertaken to evaluate the influence of medium pH and inoculation time on the growth and malolactic activity of an Oenococcus oeni culture. Samples of a commercial white grape juice adjusted to pH 3.2, 3.4 or 3.6, and inoculated with Saccharomyces cerevisiae strain CY3079, were inoculated with a malolactic culture ( Oenococcus oeni strain 31) at the beginning, middle, and end of alcoholic fermentation. The results obtained from this single case study show that it is possible to inoculate the bacterial culture at the three different times during alcoholic fermentation without slowing down or stopping alcoholic fermentation or causing failure of MLF. However, pH and timing of bacterial inoculation were critical to how rapidly MLF starts. At pH 3.2 a lowering of bacterial viability was observed, but a more important reduction was recorded at all tested pH levels when the bacteria were inoculated halfway through alcoholic fermentation. When inoculation was carried out at the end of alcoholic fermentation, the presence of yeast seemed to favour bacterial viability and activity and bacteria performed MLF even in difficult conditions such as pH values around 3. In all wines malolactic fermentation was accompanied by total degradation of malic and citric acids and production of L-lactic acid, D-lactic and acetic acids.