Alicyclobacillus acidocaldarius thermophilic esterase EST2's activity in milk and cheese models

The aim of this work was to investigate the behavior of thermophilic esterase EST2 from Alicyclobacillus acidocaldarius in milk and cheese models. The pure enzyme was used to compare the EST2 hydrolytic activity to the activity of endogenous esterase EstA from Lactococcus lactis. The results indicate that EST2 exhibits 30-fold-higher esterase activity than EstA. As EstA has thioesterase activity, EST2 was assayed for this activity under the optimal conditions determined for EstA (namely, 30 degrees C and pH 7.5). Although it is a thermophilic enzyme, EST2 exhibited eightfold-higher thioesterase activity than EstA with S-methyl thiobutanoate. The abilities of EST2 and EstA to synthesize short-chain fatty acid esters were compared. Two methods were developed to do this. In the first method a spectrophotometric assay was used to monitor the synthesis of esters by the pure enzymes using p-nitrophenol as the alcohol substrate. The synthetic activities were also evaluated under conditions that mimicked those present in milk and/or cheese. The second method involved evaluation of the synthetic abilities of the enzymes when they were directly added to a model cheese matrix. Substantial ester synthesis by EST2 was observed under both conditions. Finally, esterase and thioesterase activities were evaluated in milk using the purified EST2 enzyme and in the model cheese matrix using a strain of L. lactis NZ9000 harboring the EST2 gene and thus overproducing EST2. Both the esterase and thioesterase activities measured in milk and in the cheese matrix were much greater than the activities of the controls.     

Testing biochemistry revisited: how in vivo metabolism can be understood from in vitro enzyme kinetics

A decade ago, a team of biochemists including two of us, modeled yeast glycolysis and showed that one of the most studied biochemical pathways could not be quite understood in terms of the kinetic properties of the constituent enzymes as measured in cell extract. Moreover, when the same model was later applied to different experimental steady-state conditions, it often exhibited unrestrained metabolite accumulation.Here we resolve this issue by showing that the results of such ab initio modeling are improved substantially by (i) including appropriate allosteric regulation and (ii) measuring the enzyme kinetic parameters under conditions that resemble the intracellular environment. The following modifications proved crucial: (i) implementation of allosteric regulation of hexokinase and pyruvate kinase, (ii) implementation of V(max) values measured under conditions that resembled the yeast cytosol, and (iii) redetermination of the kinetic parameters of glyceraldehyde-3-phosphate dehydrogenase under physiological conditions.Model predictions and experiments were compared under five different conditions of yeast growth and starvation. When either the original model was used (which lacked important allosteric regulation), or the enzyme parameters were measured under conditions that were, as usual, optimal for high enzyme activity, fructose 1,6-bisphosphate and some other glycolytic intermediates tended to accumulate to unrealistically high concentrations. Combining all adjustments yielded an accurate correspondence between model and experiments for all five steady-state and dynamic conditions. This enhances our understanding of in vivo metabolism in terms of in vitro biochemistry.     

Synthesis and posttranslational regulation of pyruvate formate-lyase in Lactococcus lactis

The enzyme pyruvate formate-lyase (PFL) from Lactococcus lactis was produced in Escherichia coli and purified to obtain anti-PFL antibodies that were shown to be specific for L. lactis PFL. It was demonstrated that activated L. lactis PFL was sensitive to oxygen, as in E. coli, resulting in the cleavage of the PFL polypeptide. The PFL protein level and its in vivo activity and regulation were shown by Western blotting, enzyme-linked immunosorbent assay, and metabolite measurement to be dependent on the growth conditions. The PFL level during anaerobic growth on the slowly fermentable sugar galactose was higher than that on glucose. This shows that variation in the PFL protein level may play an important role in the regulation of metabolic shift from homolactic to mixed-acid product formation, observed during growth on glucose and galactose, respectively. During anaerobic growth in defined medium, complete activation of PFL was observed. Strikingly, although no formate was produced during aerobic growth of L. lactis, PFL protein was indeed detected under these conditions, in which the enzyme is dispensable due to the irreversible inactivation of PFL by oxygen. In contrast, no oxygenolytic cleavage was detected during aerobic growth in complex medium. This observation may be the result of either an effective PFL deactivase activity or the lack of PFL activation. In E. coli, the PFL deactivase activity resides in the multifunctional alcohol dehydrogenase ADHE. It was shown that in L. lactis, ADHE does not participate in the protection of PFL against oxygen under the conditions analyzed. Our results provide evidence for major differences in the mechanisms of posttranslational regulation of PFL activity in E. coli and L. lactis.     

Differences in sensitivity to NADH of purified pyruvate dehydrogenase complexes of Enterococcus faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli: Implications for their activity in vivo

Abstract The effect of NADH on the activity of the purified pyruvate dehydrogenase complexes (PDHc) of Enterococcus (Ec.) faecalis, Lactococcus lactis, Azotobacter vinelandii and Escherichia coli was determined in vitro. It was found that the PDHc of E. coli and L. lactis was active only at relatively low NADH/NAD ratios, whereas the PDHc of Ec. faecalis was inhibited only at high NADH/NAD ratios. The PDHc of Azotobacter vinelandii showed an intermediate sensitivity. The organisms were grown in chemostat culture under conditions that led to different intracellular NADH/NAD ratios and the PDHc activities in vivo could be calculated from the specific rates of product formation. Under anaerobic growth conditions, only Ec. faecelis expressed PDHc activity in vivo. The activities in vivo of the complexes of the different organisms were in good agreement with their properties determined in vitro. The physiological consequences of these results are discussed.     

The level of pyruvate-formate lyase controls the shift from homolactic to mixed-acid product formation in Lactococcus lactis

Regulation of pyruvate-formate lyase (PFL) activity in vivo plays a central role in the shift from homolactic to mixed-acid product formation observed during the growth of Lactococcus lactis on glucose and galactose, respectively. Characterisation of L. lactis MG1363 in anaerobic batch cultures showed that the specific in vivo activity (flux) of PFL was 4-fold higher in L. lactis cells grown with galactose, compared with cells grown with glucose. The change in the PFL flux correlated with the observed variation in the PFL enzyme level, i.e. the PFL enzyme level was 3.4-fold higher in L. lactis cells grown on galactose than in those grown on glucose. To investigate whether a variation in the level of PFL was responsible for the shift in pyruvate metabolism, L. lactis strains with altered expression of pfl were constructed. The pfl gene was expressed under the control of different constitutive promoters in L. lactis MG1363 and in the PFL-deficient strain CRM40. Strains with five different PFL levels were obtained. Variation in the PFL level markedly affected the resulting end-product formation in these strains. During growth on galactose, the flux towards mixed-acid products was to a great extent controlled by the PFL level. This demonstrates that a regulated PFL level plays a predominant role in the regulation of the metabolic shift from homolactic to mixed-acid product formation in L. lactis.     

Expression of six peptidases from Lactobacillus helveticus in Lactococcus lactis

For development of novel starter strains with improved proteolytic properties, the ability of Lactococcus lactis to produce Lactobacillus helveticus aminopeptidase N (PepN), aminopeptidase C (PepC), X-prolyl dipeptidyl aminopeptidase (PepX), proline iminopeptidase (PepI), prolinase (PepR), and dipeptidase (PepD) was studied by introducing the genes encoding these enzymes into L. lactis MG1363 and its derivatives. According to Northern analyses and enzyme activity measurements, the L. helveticus aminopeptidase genes pepN, pepC, and pepX are expressed under the control of their own promoters in L. lactis. The highest expression level, using a low-copy-number vector, was obtained with the L. helveticus pepN gene, which resulted in a 25-fold increase in PepN activity compared to that of wild-type L. lactis. The L. helveticus pepI gene, residing as a third gene in an operon in its host, was expressed in L. lactis under the control of the L. helveticus pepX promoter. The genetic background of the L. lactis derivatives tested did not affect the expression level of any of the L. helveticus peptidases studied. However, the growth medium used affected both the recombinant peptidase profiles in transformant strains and the resident peptidase activities. The levels of expression of the L. helveticus pepD and pepR clones under the control of their own promoters were below the detection limit in L. lactis. However, substantial amounts of recombinant pepD and PepR activities were obtained in L. lactis when pepD and pepR were expressed under the control of the inducible lactococcal nisA promoter at an optimized nisin concentration.     

Proteolytic enzyme activities in Cheddar cheese juice made using lactococcal starters of differing autolytic properties

AIMS: To determine proteolytic enzyme activities released in Cheddar cheese juice manufactured using lactococcal starter strains of differing autolytic properties. METHODS AND RESULTS: The activities of residual chymosin, cell envelope proteinase and a range of intracellular proteolytic enzymes were determined during the first 70 days of ripening when starter lactococci predominate the microbial flora. In general, in cell free extracts (CFE) of the strains, the majority of proteolytic activities was highest for Lactococcus lactis HP, intermediate for L. lactis AM2 and lowest for L. lactis 303. However, in cheese juice, as ripening progressed, released proteolytic activities were highest for the highly autolytic strain L. lactis AM2, intermediate for L. lactis 303 and lowest for L. lactis HP. CONCLUSIONS: These results indicate that strain related differences in autolysis influence proteolytic enzyme activities released into Cheddar cheese during ripening. No correlation was found between proteolytic potential of the starter strains measured in CFE prior to cheese manufacture and levels of activities released in cheese juice. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings further support the importance of autolysis of lactococcal starters in determining the levels of proteolytic activities present in cheese during initial stages of ripening.     

Identification, cloning, and characterization of a Lactococcus lactis branched-chain alpha-keto acid decarboxylase involved in flavor formation

The biochemical pathway for formation of branched-chain aldehydes, which are important flavor compounds derived from proteins in fermented dairy products, consists of a protease, peptidases, a transaminase, and a branched-chain alpha-keto acid decarboxylase (KdcA). The activity of the latter enzyme has been found only in a limited number of Lactococcus lactis strains. By using a random mutagenesis approach, the gene encoding KdcA in L. lactis B1157 was identified. The gene for this enzyme is highly homologous to the gene annotated ipd, which encodes a putative indole pyruvate decarboxylase, in L. lactis IL1403. Strain IL1403 does not produce KdcA, which could be explained by a 270-nucleotide deletion at the 3' terminus of the ipd gene encoding a truncated nonfunctional decarboxylase. The kdcA gene was overexpressed in L. lactis for further characterization of the decarboxylase enzyme. Of all of the potential substrates tested, the highest activity was observed with branched-chain alpha-keto acids. Moreover, the enzyme activity was hardly affected by high salinity, and optimal activity was found at pH 6.3, indicating that the enzyme might be active under cheese ripening conditions.     

Regulation of Proteolytic Enzyme Activity in Lactococcus lactis

Two different Lactococcus lactis host strains, L. lactis subsp. lactis MG1363 and L. lactis subsp. cremoris SK1128, both containing plasmid pNZ521, which encodes the extracellular serine proteinase (PrtP) from strain SK110, were used to study the medium and growth-rate-dependent activity of three different enzymes involved in the proteolytic system of lactococci. The activity levels of PrtP and both the intracellular aminopeptidase PepN and the X-prolyl-dipeptidyl aminopeptidase PepXP were studied during batch and continuous cultivation. In both strains, the PrtP activity level was regulated by the peptide content of the medium. The highest activity level was found during growth in milk, and the lowest level was found during growth in the peptide-rich laboratory medium M17. Regulation of the intracellular peptidase activity appeared to be a strain-dependent phenomenon. In cells of strain MG1363, the activity levels of PepN and PepXP were regulated in a similar way to that observed for PrtP. In cells of strain SK1128, the levels of both peptidases were not significantly influenced by the peptide content of the medium. The presence of specific concentrations of the dipeptide prolylleucine could mimic the low activity levels of the regulated proteolytic enzymes, even to the activity level found on M17 medium. The effect of the presence of the dipeptide prolylleucine in the medium on the activity level of the regulated proteolytic enzymes was confirmed at fixed growth rates in chemostat cultures.     

Enzymatic analysis of a thermostabilized mutant of an Escherichia coli hygromycin B phosphotransferase

An Escherichia coli hygromycin B phosphotransferase (HPH) and its thermostabilized mutant protein, HPH5, containing five amino acid substitutions, D20G, A118V, S225P, Q226L, and T246A (Nakamura et al., J. Biosci. Bioeng., 100, 158-163 (2005)), obtained by an in vivo directed evolution procedure in Thermus thermophilus, were produced and purified from E. coli recombinants, and enzymatic comparisons were performed. The optimum temperatures for enzyme activity were 50 and 55 degrees C for HPH and HPH5 respectively, but the thermal stability of the enzyme activity and the temperature for protein denaturation of HPH5 increased, from 36 and 37.2 degrees C of HPH to 53 and 58.8 degrees C respectively. Specific activities and steady-state kinetics measured at 25 degrees C showed only slight differences between the two enzymes. From these results we concluded that HPH5 was thermostabilized at the protein level, and that the mutations introduced did not affect its enzyme activity, at least under the assay conditions.     

Only one of the two annotated Lactococcus lactis fabG genes encodes a functional beta-ketoacyl-acyl carrier protein reductase

The small genome of the Gram-positive bacterium Lactococcus lactis ssp. lactis IL1403 contains two genes that encode proteins annotated as homologues of Escherichia coli beta-hydroxyacyl-acyl carrier protein (ACP) reductase. E. coli fabG encodes beta-ketoacyl-acyl carrier protein (ACP) reductase, the enzyme responsible for the first reductive step of the fatty acid synthetic cycle. Both of the L. lactis genes are adjacent to (and predicted to be cotranscribed with) other genes that encode proteins having homology to known fatty acid synthetic enzymes. Such relationships have often been used to strengthen annotations based on sequence alignments. Annotation in the case of beta-ketoacyl-ACP reductase is particularly problematic because the protein is a member of a vast protein family, the short-chain alcohol dehydrogenase/reductase (SDR) family. The recent isolation of an E. coli fabG mutant strain encoding a conditionally active beta-ketoacyl-ACP reductase allowed physiological and biochemical testing of the putative L. lactishomologues. We report that expression of only one of the two L. lactis proteins (that annotated as FabG1) allows growth of the E. coli fabG strain under nonpermissive conditions and restores in vitro fatty acid synthetic ability to extracts of the mutant strain. Therefore, like E. coli, L. lactis has a single beta-ketoacyl-ACP reductase active with substrates of all fatty acid chain lengths. The second protein (annotated as FabG2), although inactive in fatty acid synthesis both in vivo and in vitro, was highly active in reduction of the model substrate, beta-ketobutyryl-CoA. As expected from work on the E. coli enzyme, the FabG1 beta-ketobutyryl-CoA reductase activity was inhibited by ACP (which blocks access to the active site) whereas the activity of FabG2 was unaffected by the presence of ACP. These results seem to be an example of a gene duplication event followed by divergence of one copy of the gene to encode a protein having a new function.     

Regulation of enzyme activity in the cell: effect of enzyme concentration.

The rapid development in our understanding of the regulation of enzyme activity makes it a high priority to ascertain whether the behavior of purified enzymes reflects their functional characteristics in vivo. Enzyme concentration is usually the most significant difference between routine in vitro assays and in vivo conditions, as it is well known that many intracellular enzymes are present in vivo at much higher concentrations than used in vitro. Various procedures are suitable for kinetic analysis at physiological concentrations of enzyme. Those more frequently used have been cell permeabilization, the utilization of purified enzymes at concentrations close to the in vivo range, and the addition of polyethylene glycol to increase the local protein concentration. In this review we briefly summarize observations on enzymes reported to exhibit concentration-dependent activity. The effect of enzyme concentration has been most thoroughly investigated in the case of phosphofructokinase. These studies may provide insight into the regulation of this important enzyme in the cell. The implications of both homologous and heterologous protein-protein interactions for the effect of enzyme concentration and their roles in the control of enzyme activity in vivo are also discussed.     

Dynamics of pyruvate metabolism in Lactococcus lactis.

The pyruvate metabolism in the lactic acid bacterium Lactococcus lactis was studied in anaerobic cultures under transient conditions. During growth of L. lactis in continuous culture at high dilution rate, homolactic product formation was observed, i.e., lactate was produced as the major end product. At a lower dilution rate, the pyruvate metabolism shifted towards mixed acid-product formation where formate, acetate, and ethanol were produced in addition to lactate. The regulation of the shift in pyruvate metabolism was investigated by monitoring the dynamic behavior of L. lactis in continuous cultures subjected to step changes in dilution rate. Both shift-up and shift-down experiments were carried out, and these experiments showed that the enzyme pyruvate formate-lyase (PFL) plays a key role in the regulation of the shift. Pyruvate formate-lyase in vivo activity was regulated both at the level of gene expression and by allosteric modulation of the enzyme. A simple mathematical model was proposed to estimate the relative significance of the regulatory mechanisms involved.     

Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation.

A gene of Lactococcus lactis subsp. cremoris MG1363 encoding a peptidoglycan hydrolase was identified in a genomic library of the strain in pUC19 by screening Escherichia coli transformants for cell wall lysis activity on a medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells. In cell extracts of L. lactis MG1363 and several halo-producing E. coli transformants, lytic bands of similar sizes were identified by denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gels containing L. lactis or M. lysodeikticus cell walls. Of these clearing bands, corresponding to the presence of lytic enzymes with sizes of 46 and 41 kDa, the 41-kDa band was also present in the supernatant of an L. lactis culture. Deletion analysis of one of the recombinant plasmids showed that the information specifying lytic activity was contained within a 2,428-bp EcoRV-Sau3A fragment. Sequencing of part of this fragment revealed a gene (acmA) that could encode a polypeptide of 437 amino acid residues. The calculated molecular mass of AcmA (46,564 Da) corresponded to that of one of the lytic activities detected. Presumably, the enzyme is synthesized as a precursor protein which is processed by cleavage after the Ala at position 57, thus producing a mature protein with a size of 40,264 Da, which would correspond to the size of the enzyme whose lytic activity was present in culture supernatants of L. lactis. The N-terminal region of the mature protein showed 60% identity with the N-terminal region of the mature muramidase-2 of Enterococcus hirae and the autolysin of Streptococcus faecalis. Like the latter two enzymes, AcmA contains C-terminal repeated regions. In AcmA, these three repeats are separated by nonhomologous intervening sequences highly enriched in serine, threonine, and asparagine. Genes specifying identical activities were detected in various strains of L. lactis subsp. lactis and L. lactis subsp. cremoris by the SDS-polyacrylamide gel electrophoresis detection assay and PCR experiments. By replacement recombination, an acmA deletion mutant which grew as long chains was constructed, indicating that AcmA is required for cell separation.