scholarly journals Identification of Endopeptidase Genes from the Genomic Sequence of Lactobacillus helveticus CNRZ32 and the Role of These Genes in Hydrolysis of Model Bitter Peptides

2005 ◽  
Vol 71 (6) ◽  
pp. 3025-3032 ◽  
Author(s):  
Vidya R. Sridhar ◽  
Joanne E. Hughes ◽  
Dennis L. Welker ◽  
Jeffery R. Broadbent ◽  
James L. Steele
Keyword(s):  
Genomic Sequence ◽  
Cheddar Cheese ◽  
Lactobacillus Helveticus ◽  
Model Systems ◽  
Cheese Ripening ◽  
Bitter Peptides ◽  
Cell Extracts ◽  
Genes Encoding ◽  
Hydrolysis Of

ABSTRACT Genes encoding three putative endopeptidases were identified from a draft-quality genome sequence of Lactobacillus helveticus CNRZ32 and designated pepO3, pepF, and pepE2. The ability of cell extracts from Escherichia coli DH5α derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, β-casein (β-CN) (f193-209) and αS1-casein (αS1-CN) (f1-9), under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10°C) was examined. CNRZ32 PepO3 was determined to be a functional paralog of PepO2 and hydrolyzed both peptides, while PepE and PepF had unique specificities towards αS1-CN (f1-9) and β-CN (f193-209), respectively. CNRZ32 PepE2 and PepO did not hydrolyze either peptide under these conditions. To demonstrate the utility of these peptidases in cheese, PepE, PepO2, and PepO3 were expressed in Lactococcus lactis, a common cheese starter, using a high-copy vector pTRKH2 and under the control of the pepO3 promoter. Cell extracts of L. lactis derivatives expressing these peptidases were used to hydrolyze β-CN (f193-209) and αS1-CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides αS1-CN (f1-9), αS1-CN (f1-13), and αS1-CN (f1-16) were identified from Cheddar cheese serum and included in the defined peptide mix. Our results demonstrate that in all systems examined, PepO2 and PepO3 had the highest activity with β-CN (f193-209) and αS1-CN (f1-9). Cheese-derived peptides were observed to affect the activity of some of the enzymes examined, underscoring the importance of incorporating such peptides in model systems. These data indicate that L. helveticus CNRZ32 endopeptidases PepO2 and PepO3 are likely to play a key role in this strain's ability to reduce bitterness in cheese.

scholarly journals Genetic Characterization of a Cell Envelope-Associated Proteinase from Lactobacillus helveticus CNRZ32

1999 ◽  
Vol 181 (15) ◽  
pp. 4592-4597 ◽  
Author(s):  
Jeffrey A. Pederson ◽  
Gerald J. Mileski ◽  
Bart C. Weimer ◽  
James L. Steele
Keyword(s):  
Cell Surface ◽  
Deletion Mutant ◽  
Cell Envelope ◽  
Physiological Role ◽  
Lactobacillus Helveticus ◽  
Whole Cells ◽  
A Cell ◽  
Hydrolysis Of

ABSTRACT A cell envelope-associated proteinase gene (prtH) was identified in Lactobacillus helveticus CNRZ32. TheprtH gene encodes a protein of 1,849 amino acids and with a predicted molecular mass of 204 kDa. The deduced amino acid sequence of the prtH product has significant identity (45%) to that of the lactococcal PrtP proteinases. Southern blot analysis indicates thatprtH is not broadly distributed within L. helveticus. A prtH deletion mutant of CNRZ32 was constructed to evaluate the physiological role of PrtH. PrtH is not required for rapid growth or fast acid production in milk by CNRZ32. Cell surface proteinase activity and specificity were determined by hydrolysis of αs1-casein fragment 1-23 by whole cells. A comparison of CNRZ32 and its prtH deletion mutant indicates that CNRZ32 has at least two cell surface proteinases that differ in substrate specificity.

Accelerated cheese ripening with heat treated cells of Lactobacillus helveticus and a commercial proteolytic enzyme

1988 ◽  
Vol 55 (2) ◽  
pp. 239-245 ◽  
Author(s):  
Ylva Ardö ◽  
Hans-Erik Pettersson
Keyword(s):  
Proteolytic Enzyme ◽  
Bitter Taste ◽  
Proteolytic Enzymes ◽  
Lactobacillus Helveticus ◽  
Cheese Ripening ◽  
Heat Treated ◽  
Hydrolysed Casein ◽  
Hydrolysis Of ◽  
Microbial Sources ◽  
Hard Cheese

SummarySynergic effects of proteolytic enzymes from two different microbial sources on the ripening of Swedish hard cheese were studied. When extracellular proteolytic enzymes from Bacillus subtilis (Neutrase) and/or heat treated cells of Lactobacillus helveticus (now L. delbrueckii subsp. helveticus) were added to the cheese milk, cheese ripening was accelerated; Neutrase effectively hydrolysed casein to give a softer body. Addition of heat treated lactobacilli did not accelerate hydrolysis of casein, but accelerated the breakdown of peptides which increased the amount of amino acid N in the cheese and also enhanced the intensity of cheese flavour. A bitter taste which developed in cheeses with added Neutrase could be eliminated by the simultaneous addition of heat treated lactobacilli.

Role of Penicillium roqueforti proteinases during blue cheese ripening

1981 ◽  
Vol 48 (3) ◽  
pp. 479-487 ◽  
Author(s):  
Dominique Le Bars ◽  
Jean-Claude Gripon
Keyword(s):  
Electrophoretic Mobility ◽  
Casein Hydrolysate ◽  
Penicillium Roqueforti ◽  
Protein Nitrogen ◽  
Blue Cheese ◽  
Cheese Ripening ◽  
Electrophoretic Patterns ◽  
Casein Hydrolysates ◽  
Hydrolysis Of

SummaryThe hydrolysis of isolated αs1- and β-caseins by Penicillium roqueforti aspartyl proteinase produced comparable quantities of pH 4·6 soluble N. The amount of non-protein nitrogen obtained with β-casein was clearly lower than that obtained with αs1-casein, showing that few low molecular weight peptides were released when this casein was hydrolysed. Electrophoresis of αs1-casein hydrolysates produced by aspartyl proteinase showed 5 bands of mobility close to or higher than that of αs1-casein. β-Casein hydrolysates gave 4 bands, 2 of which (βPrapl and βPrap2) showed low electrophoretic mobility. The products corresponding to βPrapl and βPrap2 were purified from a β-casein hydrolysate and identified as fragments Val98-Val209 and Glu100-Val209 of β-casein respectively. The occurrence of the βPrapl and βPrap2 bands in electrophoretic patterns obtained from sterile curd with aspartyl proteinase and controlled-flora curd, where P. roqueforti was the only micro-organism developing, showed the presence of aspartyl proteinase synthesis and activity in cheese. A band of very low electrophoretic mobility (βPrmpl) was present in electrophoregrams of controlled-flora curd inoculated with P. roqueforti. This band, resulting from the action of the metalloproteinase on β-casein, revealed that this enzyme was both synthesized in and active in cheese.

scholarly journals Characterization of β-galactosidase and α-galactosidase activities from the halophilic bacterium Gracilibacillus dipsosauri

2021 ◽  
Vol 71 (1) ◽  
Author(s):  
Charles E. Deutch ◽  
Amy M. Farden ◽  
Emily S. DiCesare
Keyword(s):  
Gel Filtration ◽  
Halophilic Bacterium ◽  
Peptide Sequencing ◽  
Exchange Chromatography ◽  
Galactosidase Activity ◽  
Ph Optimum ◽  
Whole Cells ◽  
Cell Extracts ◽  
Genes Encoding ◽  
Hydrolysis Of

Abstract Purpose Gracilibacillus dipsosauri strain DD1 is a salt-tolerant Gram-positive bacterium that can hydrolyze the synthetic substrates o-nitrophenyl-β-d-galactopyranoside (β-ONP-galactose) and p-nitrophenyl-α-d-galactopyranoside (α-PNP-galactose). The goals of this project were to characterize the enzymes responsible for these activities and to identify the genes encoding them. Methods G. dipsosauri strain DD1 was grown in tryptic soy broth containing various carbohydrates at 37 °C with aeration. Enzyme activities in cell extracts and whole cells were measured colorimetrically by hydrolysis of synthetic substrates containing nitrophenyl moieties. Two enzymes with β-galactosidase activity and one with α-galactosidase activity were partially purified by ammonium sulfate fractionation, ion-exchange chromatography, and gel-filtration chromatography from G. dipsosauri. Coomassie Blue-stained bands corresponding to each activity were excised from nondenaturing polyacrylamide gels and subjected to peptide sequencing after trypsin digestion and HPLC/MS analysis. Result Formation of β-galactosidase and α-galactosidase activities was repressed by d-glucose and not induced by lactose or d-melibiose. β-Galactosidase I had hydrolytic and transgalactosylation activity with lactose as the substrate but β-galactosidase II showed no activity towards lactose. The α-galactosidase had hydrolytic and transgalactosylation activity with d-melibiose but not with d-raffinose. β-Galactosidase I had a lower Km with β-ONP-galactose as the substrate (0.693 mmol l−1) than β-galactosidase II (1.662 mmol l−1), was active at more alkaline pH, and was inhibited by the product d-galactose. β-Galactosidase II was active at more acidic pH, was partially inhibited by ammonium salts, and showed higher activity with α-PNP-arabinose as a substrate. The α-galactosidase had a low Km with α-PNP-galactose as the substrate (0.338 mmol l−1), a pH optimum of about 7, and was inhibited by chloride-containing salts. β-Galactosidase I activity was found to be due to the protein A0A317L6F0 (encoded by gene DLJ74_04930), β-galactosidase II activity to the protein A0A317KZG3 (encoded by gene DLJ74_12640), and the α-galactosidase activity to the protein A0A317KU47 (encoded by gene DLJ74_17745). Conclusions G. dipsosauri forms three intracellular enzymes with different physiological properties which are responsible for the hydrolysis of β-ONP-galactose and α-PNP-galactose. BLAST analysis indicated that similar β-galactosidases may be formed by G. ureilyticus, G. orientalis, and G. kekensis and similar α-galactosidases by these bacteria and G. halophilus.

scholarly journals Hydrolysis of Casein-Derived Peptides αS1-Casein(f1-9) and β-Casein(f193-209) by Lactobacillus helveticus Peptidase Deletion Mutants Indicates the Presence of a Previously Undetected Endopeptidase

2003 ◽  
Vol 69 (2) ◽  
pp. 1283-1286 ◽  
Author(s):  
Jeffrey E. Christensen ◽  
Jeffery R. Broadbent ◽  
James L. Steele
Keyword(s):  
Mass Spectrometry ◽  
High Performance Liquid Chromatography ◽  
High Performance ◽  
Lactobacillus Helveticus ◽  
Deletion Mutants ◽  
Reverse Phase ◽  
Peptide Bonds ◽  
Cell Extracts ◽  
Hydrolysis Products ◽  
Hydrolysis Of

ABSTRACT Peptides derived from hydrolysis of αS1-casein(f1-9) [αS1-CN(f1-9)] and β-CN(f193-209) with cell extracts of Lactobacillus helveticus CNRZ32 and single-peptidase mutants (ΔpepC, ΔpepE, ΔpepN, ΔpepO, and ΔpepX) were isolated by using reverse-phase high-performance liquid chromatography and were characterized by mass spectrometry. The peptides identified suggest that there was activity of an endopeptidase, distinct from previously identified endopeptidases (PepE and PepO), with specificity for peptide bonds C terminal to Pro residues. Identification of hydrolysis products derived from a carboxyl-blocked form of β-CN(f193-209) confirmed that the peptides were derived from the activity of an endopeptidase.

THE CHARACTERIZATION OF LACTOBACILLI FROM CHEDDAR CHEESE: I. AN EVALUATION OF PHYSIOLOGICAL AND BIOCHEMICAL TESTS

1962 ◽  
Vol 8 (5) ◽  
pp. 727-735 ◽  
Author(s):  
R. E. Smith ◽  
J. D. Cunningham
Keyword(s):  
Preliminary Investigation ◽  
Skim Milk ◽  
Cheddar Cheese ◽  
Titratable Acidity ◽  
Lactobacillus Brevis ◽  
Lactobacillus Helveticus ◽  
Biochemical Tests ◽  
Morphological Studies ◽  
Hydrolysis Of

Characterization studies were conducted on 230 cultures of lactobacilli isolated from Canadian Cheddar cheese, and on an additional 15 named cultures from various sources. Preliminary investigation included reactions with 19 carbohydrates, yeast glucose litmus milk, and arginine, hippurate, and aesculin broths. This resulted in the appearance of six major groups, tentatively designated as Lactobacillus plantarum, Lactobacillus casei, Lactobacillus helveticus, Lactobacillus brevis, Lactobacillus fermenti, and an unclassifiable group. Subgroups of the divisions were noted. Sixty-eight cultures were chosen for detailed study. Tests performed included the production of catalase, nitrite, hydrogen sulphide, indole, and polysaccharide; the hydrolysis of starch, gelatin, Tweens 40 and 60, polypectate, and casein; and tolerance of growth temperatures, sodium chloride, and phenol. Titratable acidity in skim milk was determined, and morphological studies were carried out. Accumulated data indicated that the group previously designated as L. helveticus, and the unclassified group, consisted of variants of L. plantarum, L. casei, or intermediates.

Making Cheddar cheese on a small scale under controlled bacteriological conditions

1959 ◽  
Vol 26 (2) ◽  
pp. 105-112 ◽  
Author(s):  
L. A. Mabbitt ◽  
Helen R. Chapman ◽  
M. Elisabeth Sharpe
Keyword(s):  
Cheddar Cheese ◽  
Small Scale ◽  
Ripening Period ◽  
Cheese Ripening ◽  
Micro Organisms

A cover for a 40 gal. cheese vat has been specially designed to allow cheese to be made under conditions which prevent the entry of micro-organisms into the vat from the dairy environment. The technique employed is described. Using milk free from lactobacilli it has been possible to make Cheddar cheese in which no lactobacilli grew during the ripening period. An assessment of the role of these and other micro-organisms in cheese ripening should now be possible.The technique should also be of help in other cheese investigations where control of the microflora is desired.

scholarly journals Genetic Diversity in the Lactose Operons of Lactobacillus helveticus Strains and Its Relationship to the Role of These Strains as Commercial Starter Cultures

2005 ◽  
Vol 71 (3) ◽  
pp. 1655-1658 ◽  
Author(s):  
M. J. Callanan ◽  
T. P. Beresford ◽  
R. P. Ross
Keyword(s):  
Genetic Diversity ◽  
Type Strain ◽  
Insertion Sequence ◽  
Starter Cultures ◽  
Lactobacillus Helveticus ◽  
Galactosidase Activity ◽  
Genes Encoding ◽  
Sequence Elements ◽  
Insertion Sequence Elements

ABSTRACT Two novel insertion sequence elements, ISLhe1 and ISLhe15, were located upstream of the genes encoding the β-galactosidase enzyme in Lactobacillus helveticus commercial starter strains. Strains with the IS982 family element, ISLhe1, demonstrated reduced β-galactosidase activity compared to the L. helveticus type strain, whereas strains with the ISLhe15 element expressed β-galactosidase in the absence of lactose.

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