Cloning, characterization, controlled overexpression, and inactivation of the major tributyrin esterase gene of Lactococcus lactis.

The gene encoding the major intracellular tributyrin esterase of Lactococcus lactis was cloned using degenerate DNA probes based on 19 known N-terminal amino acid residues of the purified enzyme. The gene, named estA, was sequenced and found to encode a protein of 258 amino acid residues. The transcription start site was mapped 233 nucleotides upstream of the start codon, and a canonical promoter sequence was identified. The deduced amino acid sequence of the estA product contained the typical GXSXG motif found in most lipases and esterases. The protein was overproduced up to 170-fold in L. lactis by use of the nisin-controlled expression system recently developed for lactic acid bacteria. The estA gene was inactivated by chromosomal integration of a temperature-sensitive integration vector. This resulted in the complete loss of esterase activity, which could then be recovered after complementation of the constructed esterase-deficient strain with the wild-type estA gene. This confirms that EstA is the main enzyme responsible for esterase activity in L. lactis. Purified recombinant enzyme showed a preference for short-chain acyl esters, surprisingly also including phospholipids. Medium- and long-acyl-chain lipids were also hydrolyzed, albeit less efficiently. Intermediate characteristics between esterases and lipases make intracellular lactococcal EstA difficult to classify in either of these two groups of esterolytic enzymes. We suggest that, in vivo, EstA could be involved in (phospho)lipid metabolism or cellular detoxification or both, as its sequence showed significant similarity to S-formylglutathione hydrolase (FGH) of Paracoccus denitrificans and human EstD (or FGH), which are part of a universal formaldehyde detoxification pathway.

Specificity of lactococcus lactis subsp. cremoris SK11 proteinase, lactocepin III, in low-water-activity, high-salt-concentration humectant systems and its stability compared with that of lactocepin I

Marked changes in the specificity of hydrolysis of alphas1-, beta-, and kappa-caseins by lactocepin III from Lactococcus lactis subsp. cremoris SK11 were found in humectant systems giving the equivalent water activity (aw) and salt concentration of cheddar cheese. Correlations were noted between certain peptides produced by the activity of lactocepin III in the humectant systems and peptides found in cheddar cheese. The stability of lactocepin III was compared with that of lactocepin I from L. lactis subsp. cremoris HP in the humectant systems at different pHs. Significant differences between the stability of each of the lactocepin types were evident. The relationship between stability and humectant type, aw, pH, and NaCl concentration was complex. Nevertheless, in those systems where aw, pH, and NaCl concentration were equivalent to those in cheddar cheese, lactocepin I was generally more stable than lactocepin III. It was concluded that differences in the specificity and/or stability of various lactocepin types are likely to persist in cheese itself and therefore potentially contribute to differences in the peptide composition of ripened cheese.

A deficiency in aspartate biosynthesis in Lactococcus lactis subsp. lactis C2 causes slow milk coagulation.

A mutant of fast milk-coagulating (Fmc+) Lactococcus lactis subsp. lactis C2, designated L. lactis KB4, was identified. Although possessing the known components essential for utilizing casein as a nitrogen source, which include functional proteinase (PrtP) activity and oligopeptide, di- and tripeptide, and amino acid transport systems, KB4 exhibited a slow milk coagulation (Fmc-) phenotype. When the amino acid requirements of L. lactis C2 were compared with those of KB4 by use of a chemically defined medium, it was found that KB4 was unable to grow in the absence of aspartic acid. This aspartic acid requirement could also be met by aspartate-containing peptides. The addition of aspartic acid to milk restored the Fmc+ phenotype of KB4. KB4 was found to be defective in pyruvate carboxylase and thus was deficient in the ability to form oxaloacetate and hence aspartic acid from pyruvate and carbon dioxide. The results suggest that when lactococci are propagated in milk, aspartate derived from casein is unable to meet fully the nutritional demands of the lactococci, and they become dependent upon aspartate biosynthesis.

Altered specificity of lactococcal proteinase p(i) (lactocepin I) in humectant systems reflecting the water activity and salt content of cheddar cheese.

By using various humectant systems, the specificity of hydrolysis of alpha(s1)-, beta-, and kappa-caseins by the cell envelope-associated proteinase (lactocepin; EC 3.4.21.96) with type P(1) specificity (i.e., lactocepin I) from Lactococcus lactis subsp. lactis BN1 was investigated at water activities (a(w)) and salt concentrations reflecting those in cheddar type cheese. In the presence of polyethylene glycol 20000 (PEG 20000)-NaCl (a(w) = 0.95), hydrolysis of beta-casein resulted in production of the peptides comprising residues 1 to 6 and 47 to 52, which are characteristic of type P(III) enzyme activity (lactocepin III) in buffer. The fragment comprising residues 1 through 166, inclusive (fragment 1-166), which is typical of lactocepin I activity in buffer systems, was not produced. Similarly, peptide 152-160 from kappa-casein, which is usually produced in aqueous buffers exclusively by lactocepin III, was a major product of lactocepin I. Most of the specificity differences obtained in the presence of PEG 20000-NaCl were also obtained in the presence of PEG 20000 alone (a(w) = 0.99). In addition, alpha(s1)-casein, which normally is resistant to lactocepin I activity, was rapidly hydrolyzed in the presence of PEG 20000 alone. Hydrolysis of casein in the presence of PEG 300-NaCl or glycerol-NaCl (both having an a(w) of 0.95) was generally as expected for lactocepin I activity except that beta-casein peptide 47-52 and kappa-casein fragment 1-160 were produced; both of these are normally formed by lactocepin III in buffer. The differences in lactocepin specificity obtained in the humectant systems can be attributed to a combination of a(w) and humectant hydrophobicity, both of which are parameters that are potentially relevant to the cheese-ripening environment.

The effect of cations on the hydrolysis of lactose and the transferase reactions catalysed by beta-galactosidase from six strains of lactic acid bacteria.

beta-Galactosidases from Lactobacillus delbruekii subsp. bulgaricus 20056, Lb. casei 20094, Lactococcus lactis subsp. lactis 7962, Streptococcus thermophilus TS2, Pediococcus pentosaceus PE39 and Bifidobacterium bifidum 1901 were partially purified. The rate of hydrolysis of lactose given by the predominant beta-galactosidase activity from each of the bacteria studied was in all cases enhanced by Mg2+, while the effect of K+ and Na+ differed from strain to strain. The beta-galactosidases from all strains also catalysed trans-galactosylation reactions. The types of oligosaccharides produced appeared to be very similar in each case, but the rates of their production differed. All the beta-galactosidases were also capable of hydrolysing galactosyl-lactose although, unlike the other bacteria studied, Lb. delbruekii subsp. bulgaricus 20056 and Lc. lactis subsp. lactis 7962 were unable to utilise galactosyl-lactose as a carbon source for growth.

Purification of tributyrin esterase from Lactococcus lactis subsp. cremoris E8.

A tributyrin esterase was purified from Lactococcus lactis subsp. cremoris E8 using FPLC chromatography. This was the major esterase activity observed in strain E8 and was associated with a single protein with a subunit molecular mass of 29 kDa and a holoenzyme of molecular mass 109 kDa. The enzyme was active against tributyrin and p-nitrophenyl butyrate. The N-terminal sequence of the enzyme was determined. The enzyme had a pH optimum in the neutral range, was stable on freezing at -20 degrees C, and had a half life of 1 h at 50 degrees C.

Comparative study of methods for the isolation and purification of bovine kappa-casein and its hydrolysis by chymosin.

kappa-Casein was purified from a single batch of whole acid casein (kappa-A variant) using different methods in order to compare their merits in producing a purified material with a carbohydrate and phosphate heterogeneity representative of the whole kappa-casein complement in milk. Ion-exchange methods of purification gave products of higher purity than precipitation techniques involving final purification by ethanol fractionation, but all methods resulted in kappa-caseins of apparently similar heterogeneity and chemical composition. The purified kappa-caseins were hydrolysed with chymosin and the derived macropeptides isolated. These were all virtually identical as determined by reversed-phase chromatography and gel electrophoresis. Some observations on chymosin hydrolysis of kappa-casein were made. In addition to formation of the major para-kappa-casein (Glu1-Phe105) and macropeptide (Met106-Val169), chymosin hydrolysis at pH 6.6 also resulted in two minor para-kappa-caseins with N-termini corresponding to Phe18 and Ser33 of kappa-casein. At pH 5.5 and 4.5 para-kappa-casein was rapidly hydrolysed into at least six fragments, one of which had an N-terminus corresponding to Trp76 of kappa-casein. At pH 6.6, 5.5 and 4.5 the kappa-casein macropeptide was stable to chymosin, but at pH 2.3 it was hydrolysed by chymosin into fragments with N-termini corresponding to Met106, Ile125, Ala138, Val139, Thr145 and Glu147 of kappa-casein.

Involvement of enzyme-substrate charge interactions in the caseinolytic specificity of lactococcal cell envelope-associated proteinases.

Three series of oligopeptides were synthesized to investigate the proposal that a major factor in determining the differences in specificity of the lactococcal cell surface-associated proteinases against caseins is the interactions between charged amino acids in the substrate and in the enzyme. The sequences of the oligopeptides were based on two regions of kappa-casein (residues 98 to 111 and 153 to 169) which show markedly different susceptibilities to PI- and PIII-type lactococcal proteinases. In each series, one oligopeptide had an identical sequence to that of the kappa-casein region, while in the others, one or more charged residues were substituted by an amino acid of opposite charge, i.e., His<-->Glu. Generally, substitution of His by Glu in the oligopeptides corresponding to residues 98 to 111 of kappa-casein resulted in reduced cleavage of susceptible bonds by the PI-type proteinase and increased cleavage of susceptible bonds by the PIII-type proteinase. In the case of the oligopeptide corresponding to residues 153 to 169 of kappa-casein, one major cleavage site was evident, and the bond was hydrolyzed by both types of proteinase (even though this sequence in kappa-casein itself is extremely resistant to the PI-type enzyme). Substitution of Glu by His in this oligopeptide, even in the P7 position, resulted in increased cleavage of the bond by the PI-type proteinase and reduced cleavage by the PIII-type proteinase. C-terminal truncation of this oligopeptide resulted in a 100-fold decrease in the rate of hydrolysis of the susceptible bond and a change in the pattern of cleavage.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris SK11.

An endopeptidase has been purified from Lactococcus lactis subsp. cremoris SK11. The enzyme is a 70 kDa monomer, strongly inhibited by the metalloproteinase inhibitors 1,10-phenanthroline and phosphoramidon but relatively insensitive to EDTA. It is not significantly inhibited by the thiol enzyme inhibitor p-chloromercuribenzoate nor by the serine protease inhibitor phenylmethylsulphonyl fluoride. The action of the endopeptidase in catalysing the hydrolysis of several peptide hormones has been studied and the hydrolysis products identified by sequence analysis. The enzyme catalyses hydrolysis of peptide bonds in which a hydrophobic amino acid (most commonly a Phe or Leu) residue occupies the position immediately C-terminal to the hydrolysed bond. It thus has a specificity very similar to that of thermolysin. Two of the oligopeptides produced during the early stages of beta-casein digestion by the lactococcal cell-wall proteinases were hydrolysed by the endopeptidase, the others were resistant to hydrolysis. Cell fractionation studies have shown that the distribution of endopeptidase activity between the different cell fractions is the same as that of the intracellular marker enzyme fructose bisphosphate aldolase, and thus indicate a cytoplasmic location for the enzyme. These observations argue against a role for this enzyme in the early stages of casein breakdown by the lactococcal proteolytic system.

Specificity of hydrolysis of bovine kappa-casein by cell envelope-associated proteinases from Lactococcus lactis strains.

The cell envelope-associated proteinases from Lactococcus lactis subsp. cremoris H2 (a PI-type proteinase-producing strain) and SK11 (a PIII-type proteinase-producing strain) both actively hydrolyze the kappa-casein component of bovine milk but with significant differences in the specificity of peptide bond hydrolysis. The peptide bonds Ala-23-Lys-24, Leu-32-Ser-33, Ala-71-Gln-72, Leu-79-Ser-80, Met-95-Ala-96, and Met-106-Ala-107 were cleaved by both proteinase types, although the relative rates of hydrolysis at some of these sites were quite different for the two proteinases. Small histidine-rich peptides were formed as early products of the action of the cell envelope-associated proteinases on kappa-casein, implicating this casein as a possible significant source of histidine, which is essential for starter growth. The major difference between the two proteinase types in their action on kappa-casein was in their ability to cleave bonds near the C-terminal end of the molecule. The bond Asn-160-Thr-161 and, to a lesser extent, the bond Glu-151-Val-152 were very rapidly cleaved by the PIII-type proteinase, whereas hydrolysis of these bonds by the PI-type proteinase was barely detectable (even after 24 h of digestion). Differential hydrolysis of kappa-casein at these sites by the two different proteinase types resulted in the formation of distinctive, high-M(r) products detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(ABSTRACT TRUNCATED AT 250 WORDS)

The physiology and biochemistry of the proteolytic system in lactic acid bacteria.

The inability of lactic acid bacteria to synthesize many of the amino acids required for protein synthesis necessitates the active functioning of a proteolytic system in those environments where protein constitutes the main nitrogen source. Biochemical and genetic analysis of the pathway by which exogenous proteins supply essential amino acids for growth has been one of the most actively investigated aspects of the metabolism of lactic acid bacteria especially in those species which are of importance in the dairy industry, such as the lactococci. Much information has now been accumulated on individual components of the proteolytic pathway in lactococci, namely, the cell envelope proteinase(s), a range of peptidases and the amino acid and peptide transport systems of the cell membrane. Possible models of the proteolytic system in lactococci can be proposed but there are still many unresolved questions concerning the operation of the pathway in vivo. This review will examine current knowledge and outstanding problems regarding the proteolytic system in lactococci and also the extent to which the lactococcal system provides a model for understanding proteolysis in other groups of lactic acid bacteria.

The effect of metal ions on the activity and thermostability of the extracellular proteinase from a thermophilic Bacillus, strain EA.1.

The proteinase from the extremely thermophilic Bacillus strain EA.1 exhibits maximum stability at a pH of approx. 6.5. In the presence of calcium ions the half-life at 95 degrees C of the enzyme at this pH was 17 min, and loss of activity followed first-order decay kinetics. The role of metal ions in the activity and stability of the enzyme was studied using the holoenzyme, the metal-depleted apoenzyme, and a zinc-enriched apoenzyme preparation. Zinc and calcium ions were the preferred bivalent cations for the active site and stabilization site(s) respectively. Stabilization by metal ions was not in itself a highly stringent process, but ions other than calcium which stabilized the enzyme generally had a concomitant inhibitory effect on activity. Inhibition and stabilization of the enzyme by cations were concentration-dependent effects and certain ions activated the apoenzyme but not the holoenzyme. Manganese(II) ions conferred some stability and also activated the enzyme, but in the latter case were not as effective as zinc ions. The results are discussed with reference to the ionic radii, co-ordination number and preferred ligand donors of the ions. Mercury(II) ions severely compromised enzyme activity and stability, and the effects of thiol-reactive agents suggest that thiol groups also have a role in enzyme integrity.

Stability and Specificity of the Cell Wall-Associated Proteinase from Lactococcus lactis subsp. cremoris H2 Released by Treatment with Lysozyme in the Presence of Calcium Ions.

The cell wall-associated proteinase from Lactococcus lactis subsp. cremoris H2 (isolate number 4409) was released from the cells by treatment with lysozyme, even in the presence of 50 mM calcium chloride. Cell lysis during lysozyme treatment was minimal. The proteinase activity released by lysozyme treatment fractionated on ion-exchange chromatography as three main forms, the molecular masses of which were determined by gel exclusion chromatography and polyacrylamide gel electrophoresis. Two of the enzyme forms released, 137 and 145 kDa, were the same as those released by incubation of cells in calcium-free phosphate buffer. In the presence of calcium, lysozyme treatment also resulted in the release of a 180-kDa enzyme molecule. The total proteinase activity released by lysozyme treatment (in the presence or absence of calcium) was not only greater than that released by phosphate buffer but was also greater than that initially detectable on the surface of whole cells, suggesting an unmasking of enzyme on the cell surface. The presence of calcium during release treatment resulted in increased stability of the crude enzyme preparations. For the proteinase preparation released by using lysozyme with 50 mM CaCl(2), the half-life of proteinase activity at 37 degrees C was 39 h, compared with 0.22 h for the calcium-free phosphate buffer-released preparation. In all cases, maximum stability was observed at pH 5.5. Comparison of beta-casein hydrolysis by the three forms of the enzyme showed that the products of short-term (5- to 30-min) digestions were very similar, although subtle differences were detected with the 180-kDa form.

Purification and characterization of a thermostable proteinase isolated from Thermus sp. strain Rt41A.

Thermus sp. strain Rt41A produces an extracellular thermostable alkaline proteinase. The enzyme has a high isoelectric point (10.25-10.5) which can be exploited in purification by using cation-exchange chromatography. The proteinase was purified to homogeneity and has a molecular mass of 32.5 kDa by SDS/PAGE. It is a glycoprotein, containing 0.7% carbohydrate as glucose equivalents, and has four half-cystine residues present as two disulphide bonds. Maximum proteolytic activity was observed at pH 8.0 against azocasein and greater than 75% of this activity was retained in the pH range 7.0-10.0. Substrate inhibition was observed with casein and azocasein. The enzyme was stable in the pH range 5.0-10.0 and maximum activity, in a 10-min assay, was observed at 90 degrees C with 5 mM CaCl2 present. No loss of activity was observed after 24 h at 70 degrees C and the half-lives at 80 degrees C and 90 degrees C were 13.5 h and 20 min, respectively. Removal of Ca2+ reduced the temperature for maximum proteolytic activity against azocasein to 60 degrees C and the half-life at 70 degrees C was 2.85 min. The enzyme was stable at low and high ionic strength and in the presence of denaturing reagents and organic solvents. Rt41A proteinase cleaved a number of synthetic amino acid p-nitrophenol esters, the kinetic data indicating that small aliphatic or aromatic amino acids were the preferred residue at the P1 position. The kinetic data for the hydrolysis of a number of peptide p-nitroanilide substrates are also reported. Primary cleavage of the oxidized insulin B chain occurred at sites where the P1' amino acid was aromatic. Minor cleavage sites (24 h incubation) were for amino acids with aliphatic side chains at the P1' position. The esterase and insulin cleavage data indicate the specificity is similar for both the P1 and P1' sites.

The enzymes from extreme thermophiles: bacterial sources, thermostabilities and industrial relevance.

This review on enzymes from extreme thermophiles (optimum growth temperature greater than 65 degrees C) concentrates on their characteristics, especially thermostabilities, and their commercial applicability. The enzymes are considered in general terms first, with comments on denaturation, stabilization and industrial processes. Discussion of the enzymes subsequently proceeds in order of their E.C. classification: oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases. The ramifications of cloned enzymes from extreme thermophiles are also discussed.

The use of proteases from extreme thermophiles for meat tenderisation.

The potential use of the thermophile enzymes E A.1 protease (from Bacillus strain E A.1), 4-1.A protease (from Thermus strain Rt4-1.A) and caldolysin (from Thermus strain T-351) in meat tenderisation has been investigated. Temperature-activity relationships illustrated that E A.1 and 4-1.A proteases were more active on collagen than on meat powder at cooking temperatures (70-90°C), whereas caldoysin was more active on meat powder. The potential of E A.1 and 4-1.A proteases was therefore investigated further using sensory and mechanical evaluation. An Instron Universal Testing Machine was used to quantitatively investigate the effect of cooking temperature and protease concentration on homogenised meat patties. With a cooking time of 30 min, the best protease concentrations (of those used) were found to be 0·75 U/g meat for E A.1 protease and 1·5 U/g meat for 4-1.A protease. The optimum cooking temperature was 80°C in both cases. Sensory analysis confirmed that these concentrations (and also 0·38 U/g meat for E A.1 protease) improved the tenderness significantly. At high concentrations the proteases had a detrimental effect on the mouthfeel of the patties. At lower concentrations this effect was less marked, and good tenderisation was obtained.

Comparison of bovine beta-casein hydrolysis by PI and PIII-type proteinases from Lactococcus lactis subsp. cremoris [corrected].

The action of the cell-wall-associated proteinases from Lactococcus lactis subsp. cremoris strains H2 and SK112 on bovine beta-casein was compared. The proteinase from the H2 strain was characterised as a PI-type proteinase since it did not hydrolyse alpha s1-casein and the initial trifluoroacetic acid-soluble products of beta-casein hydrolysis were identical to those previously identified as hydrolysis products of PI-type lactococcal proteinase action. The time-course of product formation by the proteinase from the H2 strain indicated that the bonds Tyr193-Gln194 and Gln182-Arg183 were the first to be hydrolysed. Cleavage of the bonds Gln175-Lys176, Ser168-Lys169, Ser166-Gln167 and Leu163-Ser164 was also very rapid. Four of the five bonds in beta-casein most susceptible to hydrolysis by the PIII-type proteinase from strain SK112 were different from those cleaved by the PI-type proteinase, initial hydrolysis being at the sites Tyr193-Gln194, Leu192-Tyr193, Asp43-Glu44, Gln46-Asp47 and Phe52-Ala53. Early hydrolysis at the three sites in the N-terminal region of beta-casein, leading to cleavage of the N-terminal phosphopeptide and rapid precipitation of the residual fragment, represents a marked contrast to the action of PI-type proteinases where cleavage at sites in the N-terminal region occurs only very slowly.

Purification and properties of an aryl beta-xylosidase from a cellulolytic extreme thermophile expressed in Escherichia coli.

An aryl beta-xylosidase was purified to homogeneity from an Escherichia coli strain containing a recombinant plasmid carrying a beta-xylosidase (EC 3.2.1.37) gene from the extremely thermophilic anaerobic bacterium isolate Tp8T6.3.3.1 ('Caldocellum saccharolyticum'). It has a pI of 4.3 and shows optimal activity at pH 5.7. The enzyme is highly specific, acting on o- and p-nitrophenyl beta-D-xylopyranosides and minimally on p-nitrophenyl alpha-L-arabinopyranoside. It does not act on xylobiose. The Km for p-nitrophenyl beta-D-xylopyranoside at the optimum pH for activity is 10 mM, and at pH 7.0 is 6.7 mM. Xylose is a competitive inhibitor with Ki 40 mM. Thermal inactivation follows first-order kinetics at 65 and 70 degrees C with t1/2 values of 4.85 h and 40 min respectively. The t1/2 at 70 degrees C is increased 3-fold and 4-fold by the addition of 0.5 mg of BSA/ml and 2 mM-dithiothreitol respectively.

Purification and some properties of a thermostable DNA polymerase from a Thermotoga species.

A stable DNA polymerase (EC 2.7.7.7) has been purified from the extremely thermophilic eubacterium Thermotoga sp. strain FjSS3-B.1 by a five-step purification procedure. First, the crude extract was treated with polyethylenimine to precipitate nucleic acids. The endonuclease activity coprecipitated. DEAE-Sepharose, CM-Sephrarose, and hydroxylapatite column chromatography were used to purify the preparation. As a final step on a small scale, preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis was used. The purified DNA polymerase exhibited a molecular weight of 85,000, as determined by both SDS-polyacrylamide gel electrophoresis and size-exclusion chromatography. Its pH optimum was in the range pH 7.5-8. When assayed over the temperature range 30-80 degrees C, the maximum activity in a 30-min assay was at 80 degrees C. The enzyme was moderately thermostable and exhibited half-lives of 3 min at 95 degrees C and 60 min at 50 degrees C in the absence of substrate. Several additives such as Triton X-100 enhanced thermostability. During storage at 4 degrees C and -70 degrees C, the stability of the enzyme was improved by the addition of gelatin.