Purification and Characterization of a Substrate-Size-Recognizing Metalloendopeptidase from Streptococcus cremoris H61

During the ripening of Gouda-type cheese, two kinds of endopeptidases were found to participate in the degradation of αs1-CN(f1-23), a specific product from αs1-casein hydrolyzed by chymosin. One of the endopeptidases, lactic acid bacteria endopeptidase (LEP-II), which can recognize the size of its substrates, has already been purified and characterized (T. R. Yan, N. Azuma, S. Kaminogawa, and K. Yamauchi, Eur. J. Biochem. 163:259-265, 1987). The other endopeptidase, LEP-I, was purified to homogeneity by conventional chromatographic techniques from Streptococcus cremoris H61. The enzyme appeared to be monomeric, with an apparent molecular weight of 98,000, and its isoelectric point was 5.1. For the hydrolysis of αs1-CN(f1-23), the enzyme had an optimum pH and temperature of 7.0 to 7.5 and 40°C, respectively. Its activity was inhibited by such chelating agents as EDTA and 1,10-phenanthrolin, and it could be fully reactivated by Mn²⁺. Inhibitors specific for serine and thiol proteases had no effect on the protease activity. The enzyme showed a high affinity toward the Glu-Asn peptide bond of αs1-CN(f1-23) and αs1-CN(f91-100) but showed no hydrolysis activity toward αs1-CN(f1-52), αs1-CN(61-122), αs1-CN(136-196), αs1-casein, β-casein, κ-casein, α-lactalbumin, and β-lactoglobulin. The K_m and V_max of LEP-I for αs1-CN(f1-23) were 14.2 pM and 139 U, respectively.     

Isolation and Characterization of Plasmid DNA in Streptococcus cremoris

Nine industrially important strains of Streptococcus cremoris (HP, AM₂, ML₁, WC, C₃, R₁, E₈, KH, and Wg₂) were shown to possess a diversity of plasmid molecules. Molecular weights of plasmids were determined from their relative mobilities after agarose gel electrophoresis and via electron microscopy. To illustrate the varied plasmid sizes, strain HP contained plasmids of 26, 18, 8.5, 3.3, and 2 megadaltons (Mdal); strain ML₁ contained plasmids of 29, 18, 9, 4, 2.2, and 1.8 Mdal; and strain AM₂ had plasmids of 42, 27, 16, and 8.4 Mdal. The numbers of plasmids observed in the other strains were 6, 5, 5, 7, 5, and 4 for C₃, E₈, KH, R₁, WC, and Wg₂, respectively. A spontaneous proteinase-negative (Prt⁻) mutant of HP was missing the 8.5-Mdal plasmid, which suggests that in this strain proteinase activity could be linked to this particular plasmid. A lactose-negative (Lac⁻) Prt⁻ mutant of ML₁ lacked the 2.2-Mdal plasmid. Under the conditions employed, antibiotic sensitivity and heavy-metal susceptibility did not correlate with the missing plasmid in Prt⁻ HP or in the Lac⁻ Prt⁻ ML₁. Curing experiments with AM₂, using acridine dyes and elevated temperatures, did not yield Lac⁻ variants. AM₂ was also cultured at high dilution rates in a chemostat for 168 h by using a buffered milk or lactic broth at 18 or 32°C with no selection of Lac⁻ derivatives. The inability to obtain Lac⁻ variants under conditions known to facilitate plasmid elimination suggests that lactose metabolism is not plasmid-mediated in AM₂.     

Purification and Characterization of a Dipeptidase from Streptococcus cremoris Wg2

A dipeptidase was purified to homogeneity from a crude cell extract of Streptococcus cremoris Wg2 by DEAE-Sephacel column chromatography followed by preparative disc gel electrophoresis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band with a molecular weight of 49,000. The dipeptidase is capable of hydrolyzing a range of dipeptides, but not peptides with longer chains. The enzyme was shown to be a metallo-Mn²⁺ enzyme with a pH optimum of 8 and a temperature optimum of 50°C. The enzyme is strongly inhibited by thiol-reducing reagents but not by sulfhydryl reagents. Kinetic studies indicated that the enzyme has a relatively low affinity for leucyl-leucine and alanyl-alanine (K_m, 1.6 and 7.9 mM, respectively) but can hydrolyze these substrates at very high rates (V_max, 3,700 and 13,000 μmol/min per mg of protein, respectively).     

Purification and characterization of a novel metalloendopeptidase from Streptococcus cremoris H61. A metalloendopeptidase that recognizes the size of its substrate

An endopeptidase (LEP-II), which has a unique substrate specificity, was purified to homogeneity by conventional chromatographic techniques from Streptococcus cremoris H61. The enzyme was a metalloendopeptidase since it was inhibited by EDTA and 1,10-phenanthroline; the metal-depleted enzyme could be fully reactivated by micromolar levels of Zn2+ and was not inhibited by specific inhibitors for serine or thiol protease. The molecular mass of the enzyme was estimated to be 80 kDa by Sephacryl S-300 gel filtration and high-performance liquid chromatography with a TSK-G3000SW column. The enzyme consisted of two identical subunits and the N-terminal sequence of LEP-II was determined up to the 19th residue. Although the enzyme had a broad substrate specificity it specifically hydrolyzed the peptide bonds involving the amino groups of hydrophobic amino acid residues. Various small polypeptides, such as alpha s1-CN(f1-23), alpha s1-CN(f91-100), oxidized insulin B chain, glucagon and some biologically active peptides were hydrolyzed. However, a variety of larger polypeptides or proteins, such as alpha s1-CN(f1-54), alpha s1-CN(f61-123), alpha s1-CN(f136-196), alpha s1-casein, beta-casein, and kappa-casein were not hydrolyzed. LEP-II recognized the size of its substrates, which were limited below a molecular mass of about 3.5 kDa.     

Isolation and characterization of the plasma membrane from slime-forming,encapsulated Streptococcus cremoris

• 1.1. The plasma membrane of slime-forming, encapsulated Streptococcus cremoris from “viili” was isolated in hypotonie conditions in the presence of lysozyme (EC 3.2.1.17) using density gradient centrifugation as the last purification step.
• 2.2. The membrane yield was 15.8% of wet weight cells and the preparation contained 64.4% protein. 19.1% carbohydrate, 5.8% aminosugars, 5.1% RNA and 0.07% DNA.
• 3.3. Buffered 1% (w/v) Triton X-100 solubilized 33.6% of membrane proteins. The number of polypeptides detected by SDS-polyacrylamide gel electrophoresis was 59 when the membrane was isolated without a protease inhibitor and 44 in the presence of a protease inhibitor.
• 4.4. The molecular weights of the polypeptides varied from 13,500 to 100,000.
• 5.5. Ultrathin-layer electrofocusing analysis revealed the range of protein pi values to be between 3.50 and 5.85 concerning 77.3% of proteins and between pI 5.85 and 8.15 concerning 18.2% of proteins.
• 6.6. The isoelectric point of the only basic protein component was 9.3.

     

Kinetic Properties of a Dipeptidase from Streptococcus cremoris

Some kinetic properties of a dipeptidase purified from a cell-free extract of Streptococcus cremoris H 61 were investigated. The Km values of this enzyme for various dipeptides were divided into 3 groups. Group 1 comprised mainly of neutral dipeptides, such as Leu-Gly, Leu-Leu and Leu-Ala, which had relatively low Km values (in the range 4.0-6.6 mm). Group 2 consisted of dipeptides with aromatic large amino acids either at the N- or C-terminal positions, like Leu-Phe, Phe-Ala and Leu-Tyr, which had very low Km values (in the range 1.0-2.4 mm). Group 3 was made up by dipeptides with acidic or basic amino acids at the N-terminals; His-Ala and Glu-Val were typical of this group. These had very high Km values (in the range 10–20 mm). Substantial substrate competition was found to exist in the presence of His-Ala. Bestatin inhibited the enzyme competitively with Leu-Gly and was found to have an apparent Ki value of 3.0 × 10^?8 m for the enzyme. Further, the enzyme was completely inhibited by EDTA at a concentration of 2.0 × 10^?5 m. On the other hand, once the activity was inhibited by EDTA, it could be restored by Co²⁺ and Zn²⁺ in the acidic pH side, and by Ca²⁺ and Mn²⁺ in the alkaline pH side.     

ScrFI: a new sequence-specific endonuclease from Streptococcus cremoris

A novel sequence-specific endonuclease has been isolated from Streptococcus cremoris F. ScrFI recognises the sequence: (formula; see text) and cleaves as indicated by the arrow ( ). It is the first enzyme to recognise this sequence and the first endonuclease reported from the lactic streptococci used in dairy fermentations.     

Purification and Properties of Intracellular Proteinase from Streptococcus cremoris

Proteolytic activity in the extract from the cells of Streptococcus cremoris increased in the presence of casein, lactose, glucose, and CaCl₂ in the media but was negligibly detectable in the extract of the cells harvested from the culture containing succinate or citrate. The intracellular proteinase from S. cremoris harvested from tomato medium was purified 150-fold in this experiment. The enzyme had a molecular weight of 140,000, optimum pH at 6.5 to 7.0, and maximum activity at 30 C. The proteinase was activated by Ca²⁺ and inhibited by Zn²⁺, Cu²⁺, Hg²⁺, Fe²⁺, ethylenediaminetetraacetate, and sodium lauryl sulfate. The K_m value of the enzyme towards each casein fraction was almost the same, and the V_max of the enzyme towards α_s-casein was smaller than those towards the other casein fractions.     

Prophage-Cured Derivatives of Streptococcus lactis and Streptococcus cremoris

Prophage curing was achieved in Streptococcus lactis and Streptococcus cremoris, and the cured derivatives were shown to be indicators for their temperate bacteriophages. Relysogenization of these cured derivatives completed the first formal demonstration of the lysogenic state in lactic streptococci.     

Purification and Some Properties of a Membrane-Bound Aminopeptidase A from Streptococcus cremoris

A membrane-bound l-alpha-glutamyl (aspartyl)-peptide hydrolase (aminopeptidase A) (EC 3.4.11.7) from Streptococcus cremoris HP has been purified to homogeneity. The free gamma-carboxyl group rather than the amino group of the N-terminal l-alpha-glutamyl (aspartyl) residue appeared to be essential for catalysis. No endopeptidase activity could be established with this enzyme. The native enzyme is a polymeric, most probably trimeric, metalloenzyme (relative molecular weight, approximately 130,000) which shows on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels apparent high relative molecular weight values due to (lipid?) material dissociable with butanol. The subunit (relative molecular weight, approximately 43,000) is catalytically inactive. The enzyme is inactivated completely by dithiothreitol, chelating agents, and the bivalent metal ions Cu and Hg. Of the sulfhydryl-blocking reagents tested, only p-hydroxymercuribenzoate appeared to inhibit the enzyme. Activity lost by treatment with a chelating agent could be restored by Co and Zn. The importance of the occurrence of an aminopeptidase A in S. cremoris with respect to growth in milk is discussed.     

Isolation and Characterization of Plasmid Deoxyribonucleic Acid from Streptococcus mutans

A small plasmid with a molecular weight of approximately 3.0 x 10(6) and present to the extent of about 16 copies per chromosomal genome equivalent was isolated from Streptococcus mutans strain LM-7.     

Purification and Some Properties of Diplococcin from Streptococcus cremoris 346

Eleven of 150 Streptococcus cremoris strains examined produced the bacteriocin diplococcin. The diplococcin activity spectrum was restricted to S. cremoris and Streptococcus lactis strains, and none of a wide range of other gram-positive or gram-negative strains were inhibited. The diplococcin produced by S. cremoris 346 was purified by ammonium sulfate precipitation and column chromatography. Purified diplococcin was very unstable at room temperature and lost 75% of its activity after heating at 100°C for 1 min. The proteolytic enzymes trypsin, pronase, and α-chymotrypsin completely inactivated diplococcin. The amino acid composition showed a high content of acidic and neutral acids and a correspondingly low content of basic amino acids, including one residue of ornithine per mole. From the amino acid analysis a molecular weight of 5,300 was estimated. Diplococcin was readily distinguished from the S. lactis bacteriocin nisin by its restricted activity spectrum, its biological properties, and by cross-reaction experiments.     

Bitter Peptide Isolated from Milk Cultures of Streptococcus cremoris

Certain cultures of Streptococcus cremoris produced a bitter taste that occurred in the whey portion of milk cultures. Whey from a culture which produced bitterness was fractionated on Sephadex. The fraction in which the bitter taste was concentrated was chromatographed successively on paper with butanol-acetic acid-water (5:1:4), and then butanol-2-butanone-water (2:2:1). In each instance, the bitter component was in the most rapidly moving band that gave a positive ninhydrin test. The bitterness was observed to be caused by a peptide containing the following numbers of each amino acid: arginine, 1; glutamic acid, 2; glycine, 2; isoleucine, 2; leucine, 2; phenylalanine, 1; proline, 5; and valine, 4. N-terminal amino acids could be detected by coupling with 2,4-dinitrofluorobenzene or phenylisothiocyanate, or by hydrolysis with leucine aminopeptidase. When treated with carboxypeptidase, only leucine and valine appeared at the C-terminal end, and these were detected simultaneously.     

Cloning and expression of a Streptococcus cremoris proteinase in Bacillus subtilis and Streptococcus lactis

Previously, curing experiments suggested that plasmid pWV05 (17.5 megadaltons [Md]) of Streptococcus cremoris Wg2 specifies proteolytic activity. A restriction enzyme map of pWV05 was constructed, the entire plasmid was subcloned in Escherichia coli with plasmids pBR329 and pACYC184. A 4.3-Md HindIII fragment could not be cloned in an uninterrupted way in E. coli but could be cloned in two parts. Both fragments showed homology with the 9-Md proteinase plasmid of S. cremoris HP. The 4.3-Md HindIII fragment was successfully cloned in Bacillus subtilis on plasmid pGKV2 (3.1 Md). Crossed immunoelectrophoresis of extracts of B. subtilis carrying the recombinant plasmid (pGKV500; 7.4 Md) showed that the fragment specifies two proteins of the proteolytic system of S. cremoris Wg2. PGKV500 was introduced in a proteinase-deficient Streptococcus lactis strain via protoplast transformation. Both proteins were also present in cell-free extracts of S. lactis(pGKV500). In S. lactis, pGKV500 enables the cells to grow normally in milk with rapid acid production, indicating that the 4.3-Md HindIII fragment of plasmid pWV05 specifies the proteolytic activity of S. cremoris Wg2.