Malolactic enzyme

Some enzymes contain bound NAD+ which oxidizes a substrate alcohol to facilitate a reaction step and is then regenerated. For example, the malolactic enzyme found in some lactic acid bacteria and also in Ascaris decarboxylates L-malate to lactate (Eq. 15-12). This reaction is similar to those of isocitrate dehydrogenase,110-112 6-phosphogluconate dehydrogenase,113 and the malic enzyme (Eq. 13-45)114 which utilize free NAD+ to first dehydrogenate the substrate to a bound oxoacid whose (3 carbonyl group facilitates decarboxylation. Likewise, the bound NAD+ of the malolactic... 

This is the main reaction of MLR Chemically it consists of a simple decarboxylation of the L-malic acid in wine into L-lactic acid. Biochemically, it is the result of activity of the malolactic enzyme, characteristic of lactic acid bacteria. This transformation has a dual effect. On the one hand, it deacidifies the wine, in other words, it raises the pH, an effect that is greater at higher initial quantities of malic acid. It also gives the wine a smoother taste, replacing the acidic and astringent flavour of the malic acid, by the smoother flavour of the lactic acid. 

Battermann, G, Radler, F. (1991). A comparative study of malolactic enzyme and malic enzyme of different lactic acid bacteria. Can. J. Microbiol., 37, 211-217. 

Labarre, C., Guzzo, J., Cervin, J. R, Divies, C. (1996). Cloning and characterization of the genes encoding the malolactic enzyme and the malate permease of Leuconostoc oenos. Appl. Environ. Microbiol, 62, 1274-1282. 

Lonvaud-Eunel, A., Strasser de Saad, A. M. (1982). Purification and properties of a malolactic enzyme from a strain of Leuconostoc mesenteroides isolated from grapes. Appl Environ. Microbiol, 43, 357-361. 

Naouri, P., Chagnaud, P., Amaud, A., Galzy, P. (1990). Purification and properties of a malolactic enzyme from Leuconostoc oenos ATCC 23278. J. Basic Microbiol, 30, 577-585. 

Cox, D J. and Henick-Kling, T. 1990. A comparison of lactic acid bacteria for energy-yielding (ATP) malolactic enzyme systems. Am. J. Enol. Vitic. 41, 215-218. 

Zapparoli, G., Torriani, S., Pesente, P., and Dellaglio, F. 1998. Design and evaluation of malolactic enzyme gene targeted primers for rapid identification and detection of Oenococcus oeni in wine. Lett. Appl. Microbiol. Tl, 243-246. 

Most bacteria convert malic acid to lactic acid with an intermediate formation of pyruvic acid, while Oenococcus oeni (previously classified as Leuconostoc oenos) expresses the malolactic enzyme to directly convert malic acid in one-step reaction. O. oeni has been extensively studied for controlled MLF of wine due to its higher tolerance to ethanol, low pH, and Yeasts like Schizosaccharomyces pombe and Saccharomyces strains can also convert malic acid through a maloethanolic-type fermentation. Lactic acid is less acidic than malic acid, and as a conseqnence, MLF leads to improvement of the sensory properties and biological stability of the wines. Additionally, the production of various other by-products of the MLF reaction may affect wine flavor positively. 

Vaillant, H. and Formisyn, P. (1996) Purification of the malolactic enzyme from a Leuconostoc oenos strain and use in a membrane reactor for achieving the malolactic fermentation of wine. Biotechnol Appl. Biochem., 24, 217-223. 

Madin-Darby canine kidney (MDCK) 658 malolactic enzyme 419 mammalian cells... 

Figure 9.19 There are two options to genetically engineer extraneous malate utilisation in order to deacidify wine. One approach utilises the Schizosaccharomyces pombe malate transporter gene (mael) and the O. oeni malolactic enzyme gene (mleA), enabling yeast to perform malolactic fermentation in parallel with alcoholic fermentation. Alternatively, Saccharomyces cerevisiae can be modified by the introduction of mael and the S. pombe malic enzyme gene (mae2), thereby enabling the conversion of malate into ethanol.

The reaction is catalyzed by malate carboxylyase, the so-called malolactic enzyme, and requires the coenzyme NAD as well as Mn. Unlike formation of lactic acid from growth on glucose, only the L isomer is produced during MLF. 

Therefore, the hypothesis of the existence of an enzyme catalyzing the direct decarboxylation of L-malic acid into L-lactic acid was made. The enzyme, called the malolactic enzyme, was isolated for the first time in Lactobacillus plantarum (Lonvaud, 1975 Schiitz and Radler, 1974). From acellular bacterial extracts and thanks to successive purification stages, the authors obtained purified fractions responding to the fnnctional criteria of the malolactic enzyme. L-Malic acid is transformed stoichiometrically into L-lactic acid. These fractions do not have an LDH activity. 

At least in L. mesenteroides and L. oenos (0. oeni), the malolactic enzyme is inducible. Cultivated without mafic acid during numerous generations, the cells conserve a very small residual activity. They regain their maximum activity as... 

The malolactic enzyme purified from Lactococ-cus lactis, a lactic bacterium of milk origin, has... 

The nucleotide sequence encoding the malolactic enzyme is therefore known and shows a strong resemblance to the malic enzyme. The binding sites of the coenzyme on the protein have also been located (Figure 5.5). Finally, after having been inserted into a vector, this gene was transferred into E. coli and also into laboratory strains of S. cerevisiae yeast the gene was expressed in these conditions (Ansanay et al., 1993 Denayrolles et al, 1995). 

Fig. 5.5. Nucleotidic sequence of the DNA fragment carrying the malolactic enzyme (mLeS) gene and proteic sequences coded by this fragment. Certain proteic zones particularly well conserved between the malolactic enzyme and malic enzymes have been underlined. A potential function has been specified for some...

Minerals such as Mg " ", Mn +, K+, and Na+ are necessary. The first two are often used as key enzyme cofactors of the metabolism (kinases, malolactic enzyme). The following trace elements are involved in the nutrition of lactococci Cu +, Fe " ", Mo and Se. Yet the role of these metal... 

Having witnessed the difficulty of obtaining lactic acid bacteria growth in wine, Lafon-Lafourcade (1970) studied the possibility of obtaining malic acid degradation by using a biomass sufficiently abundant and rich in malolactic enzyme so that the reaction can occur without cellular multiplication. 

When the evolution of an inoculated bacteria population in wine is studied, an abrupt drop in the number of viable cells is observed in the first hours. Afterwards, the decline is slower. After several days, bacterial growth may occur, but this growth is too uncertain to be used as a technique for initiating malolactic fermentation. Yet, during the decline of the population, the malolactic enzyme supplied by the bacteria induces the partial degradation of malic acid. In this case, the bacteria do not act as a fermentation starter but rather as a potential enzymatic support. 

Many authors have used this idea of reactivation. Although many different procedures have been proposed, that of Lafon-Lafourcade et al. (1983) is the most used. Non-sulfited grape juice is diluted to half its original concentration (80 g/1 of sugar per liter) a commercial yeast autolysate is added (5 g/1) and the pH is adjusted to 4.5 with CaCOs. After several hours, commercial biomasses inoculated at 10 cells/ml at 25°C produce fermentation starters rich in malolactic enzymes. These starters are also more resistant in wine than non-reactivated starters. Populations increase to 10, 10 and 10 cells/ml after 2 hours, 24 hours and 6 days of reactions, respectively. 

L-malic acid is decarboxylated to L-lactic acid by LAB, a reaction known as MLF. Wine pH increases by several tenths of units depending on the initial malic acid concentration, which may range from 1 to 8 g/1. Although the MLF reaction has been known for a long time (Seifert 1901) only since the 1970s has the malolactic enzyme been purified and characterized, first from Lact. plcmtarum... 

Carboxylic acids Malic Malolactic enzyme L-Lactic acid Almost all LAB strains Increase of softness... 

Lyases are enzymes that remove certain groups from substrates by mechanisms other than hydrolysis. Decarboxylases and ammonia lyases can be immobilized onto pCOa or pNHa electrodes, respectively, for amino acid determination. Malate is determined by fixing the malolactic enzyme from Leuconostoc cenos to a pC02 electrode. The enzyme catalyses the following reaction ... 

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