Characterization of Muscle Sarcoplasmic and Myofibrillar Protein Hydrolysis Caused by Lactobacillus plantarum

1999 ◽  
Vol 65 (8) ◽  
pp. 3540-3546 ◽  
Author(s):  
Silvina Fadda ◽  
Yolanda Sanz ◽  
Graciela Vignolo ◽  
M.-Concepción Aristoy ◽  
Guillermo Oliver ◽  
...  
Keyword(s):  
Lactobacillus Plantarum ◽  
Myofibrillar Protein ◽  
Myofibrillar Proteins ◽  
Muscle Proteins ◽  
Whole Cells ◽  
Sarcoplasmic Proteins ◽  
Cell Extracts ◽  
Hydrophobic Nature ◽  
Hydrolysis Of

ABSTRACT Strains of Lactobacillus plantarum originally isolated from sausages were screened for proteinase and aminopeptidase activities toward synthetic substrates; on the basis of that screening,L. plantarum CRL 681 was selected for further assays on muscle proteins. The activities of whole cells, cell extracts (CE), and a combination of both on sarcoplasmic and myofibrillar protein extracts were determined by protein, peptide, and free-amino-acid analyses. Proteinase from whole cells initiated the hydrolysis of sarcoplasmic proteins. The addition of CE intensified the proteolysis. Whole cells generated hydrophilic peptides from both sarcoplasmic and myofibrillar proteins. Other peptides of a hydrophobic nature resulted from the combination of whole cells and CE. The action of both enzymatic sources on myofibrillar proteins caused maximal increases in lysine, arginine, and leucine, while the action of those on sarcoplasmic proteins mainly released alanine. In general, pronounced hydrolysis of muscle proteins required enzyme activities from whole cells in addition to those supplied by CE.

scholarly journals Characterization of a Feruloyl Esterase from Lactobacillus plantarum

2013 ◽  
Vol 79 (17) ◽  
pp. 5130-5136 ◽  
Author(s):  
María Esteban-Torres ◽  
Inés Reverón ◽  
José Miguel Mancheño ◽  
Blanca de las Rivas ◽  
Rosario Muñoz
Keyword(s):  
Lactobacillus Plantarum ◽  
Bacterial Species ◽  
Hydrolytic Activity ◽  
Feruloyl Esterase ◽  
Gene Encoding ◽  
Content Type ◽  
Cell Extracts ◽  
Methyl Ferulate ◽  
Methyl Caffeate

ABSTRACTLactobacillus plantarumis frequently found in the fermentation of plant-derived food products, where hydroxycinnamoyl esters are abundant.L. plantarumWCFS1 cultures were unable to hydrolyze hydroxycinnamoyl esters; however, cell extracts from the strain partially hydrolyze methyl ferulate and methylp-coumarate. In order to discover whether the protein Lp_0796 is the enzyme responsible for this hydrolytic activity, it was recombinantly overproduced and enzymatically characterized. Lp_0796 is an esterase that, among other substrates, is able to efficiently hydrolyze the four model substrates for feruloyl esterases (methyl ferulate, methyl caffeate, methylp-coumarate, and methyl sinapinate). A screening test for the detection of the gene encoding feruloyl esterase Lp_0796 revealed that it is generally present amongL. plantarumstrains. The present study constitutes the description of feruloyl esterase activity inL. plantarumand provides new insights into the metabolism of hydroxycinnamic compounds in this bacterial species.

Characterization of lactococci and lactobacilli isolated from semihard goats' cheese

1991 ◽  
Vol 58 (1) ◽  
pp. 137-145 ◽  
Author(s):  
Teresa Requena ◽  
Carmen Peláez ◽  
Michel J. Desmazeaud
Keyword(s):  
Leucine Aminopeptidase ◽  
Sodium Dodecyl ◽  
Skim Milk ◽  
Titratable Acidity ◽  
Aminopeptidase Activity ◽  
Whole Cells ◽  
Alanine Aminopeptidase ◽  
Triton X ◽  
Hydrolysis Of

SummarySeveral strains ofLactococcus lactissubsp.lactis, Lactobacillus caseiandLactobacillus plantarumisolated from traditional goats' cheese have been studied for titratable acidity, proteolysis in milk and enzymic activities. Aminopeptidasc activities were measured with whole cells and cells permeabilized with Triton X-100. Caseinolytic activity was investigated using electrophoresis in polyacrylamide gel with sodium dodecyl sulphate.Lc. lactissubsp.lactishad a level of proteolytic activity in skim milk greater than that ofLb. casei, while this activity inLb. plantarumwas very low. Alanine aminopeptidase activity was almost non-existent for all strains tested, while lysine aminopeptidase activity appeared to be of fundamentally intracellular origin. Leucine aminopeptidase activity was also greater in cells that had been permeabilized than in whole cells forLb. caseiandLb. plantarum. Lc. lactissubsp.lactisleucine aminopeptidase activity was greater in whole cells. No significant hydrolysis of casein was found withLb. caseiI FPL 725 andLb. plantarumIFPL 722 permeabilized with Triton X-100 after 24 h incubation with whole bovine casein.

scholarly journals Characterization of S-Triazine Herbicide Metabolism by a Nocardioides sp. Isolated from Agricultural Soils

2000 ◽  
Vol 66 (8) ◽  
pp. 3134-3141 ◽  
Author(s):  
Edward Topp ◽  
Walter M. Mulbry ◽  
Hong Zhu ◽  
Sarah M. Nour ◽  
Diane Cuppels
Keyword(s):  
Agricultural Soils ◽  
Sole Source ◽  
Bacterial Strains ◽  
Corn Production ◽  
Whole Cells ◽  
Carbon And Nitrogen ◽  
Cell Extracts ◽  
Hydrolytic Reaction ◽  
Central Canada

ABSTRACT Atrazine, a herbicide widely used in corn production, is a frequently detected groundwater contaminant. Nine gram-positive bacterial strains able to use this herbicide as a sole source of nitrogen were isolated from four farms in central Canada. The strains were divided into two groups based on repetitive extragenic palindromic (rep)-PCR genomic fingerprinting with ERIC and BOXA1R primers. Based on 16S ribosomal DNA sequence analysis, both groups were identified as Nocardioides sp. strains. None of the isolates mineralized [ring-U-14C]atrazine. There was no hybridization to genomic DNA from these strains usingatzABC cloned from Pseudomonas sp. strain ADP or trzA cloned from Rhodococcus corallinus. S-Triazine degradation was studied in detail inNocardioides sp. strain C190. Oxygen was not required for atrazine degradation by whole cells or cell extracts. Based on high-pressure liquid chromatography and mass spectrometric analyses of products formed from atrazine in incubations of whole cells with H2 18O, sequential hydrolytic reactions converted atrazine to hydroxyatrazine and then to the end productN-ethylammelide. Isopropylamine, the putative product of the second hydrolytic reaction, supported growth as the sole carbon and nitrogen source. The triazine hydrolase from strain C190 was isolated and purified and found to have a Km for atrazine of 25 μM and a V max of 31 μmol/min/mg of protein. The subunit molecular mass of the protein was 52 kDa. Atrazine hydrolysis was not inhibited by 500 μM EDTA but was inhibited by 100 μM Mg, Cu, Co, or Zn. Whole cells and purified triazine hydrolase converted a range of chlorine or methylthio-substituted herbicides to the corresponding hydroxy derivatives. In summary, an atrazine-metabolizingNocardioides sp. widely distributed in agricultural soils degrades a range of s-triazine herbicides by means of a novel s-triazine hydrolase.

Regulation of myofibrillar protein turnover during maturation in normal and undernourished rat pups

2000 ◽  
Vol 278 (4) ◽  
pp. R845-R854 ◽  
Author(s):  
Marta L. Fiorotto ◽  
Teresa A. Davis ◽  
Peter J. Reeds
Keyword(s):  
Protein Synthesis ◽  
Protein Turnover ◽  
Myofibrillar Protein ◽  
Synthesis Rate ◽  
Protein Synthesis Rate ◽  
Myofibrillar Proteins ◽  
Sarcoplasmic Proteins ◽  
Rat Pups ◽  
Sarcoplasmic Protein ◽  
Degradation Rates

The study tested the hypothesis that a higher rate of myofibrillar than sarcoplasmic protein synthesis is responsible for the rapid postdifferentiation accumulation of myofibrils and that an inadequate nutrient intake will compromise primarily myofibrillar protein synthesis. Myofibrillar (total and individual) and sarcoplasmic protein synthesis, accretion, and degradation rates were measured in vivo in well-nourished (C) rat pups at 6, 15, and 28 days of age and compared at 6 and 15 days of age with pups undernourished (UN) from birth. In 6-day-old C pups, a higher myofibrillar than sarcoplasmic protein synthesis rate accounted for the greater deposition of myofibrillar than sarcoplasmic proteins. The fractional synthesis rates of both protein compartments decreased with age, but to a greater degree for myofibrillar proteins (−54 vs. −42%). These decreases in synthesis rates were partially offset by reductions in degradation rates, and from 15 days, myofibrillar and sarcoplasmic proteins were deposited in constant proportion to one another. Undernutrition reduced both myofibrillar and sarcoplasmic protein synthesis rates, and the effect was greater at 6 (−25%) than 15 days (−15%). Decreases in their respective degradation rates minimized the effect of undernutrition on sarcoplasmic protein accretion from 4 to 8 days and on myofibrillar proteins from 13 to 17 days. Although these adaptations in protein turnover reduced overall growth of muscle mass, they mitigated the effects of undernutrition on the normal maturational changes in myofibrillar protein concentration.

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.

scholarly journals Myofibrillar protein turnover. Synthesis rates of myofibrillar and sarcoplasmic protein fractions in different muscles and the changes observed during postnatal development and in response to feeding and starvation

1983 ◽  
Vol 214 (2) ◽  
pp. 587-592 ◽  
Author(s):  
P C Bates ◽  
D J Millward
Keyword(s):  
Protein Synthesis ◽  
Steady State ◽  
Postnatal Development ◽  
Turnover Rate ◽  
Myofibrillar Protein ◽  
Protein Fractions ◽  
Myofibrillar Proteins ◽  
Muscle Proteins ◽  
Sarcoplasmic Protein ◽  
Myofibrillar Protein Synthesis

Measurement of rates of synthesis of skeletal-muscle proteins in adult rats shows that the faster overall rate of turnover in diaphragm and soleus muscles compared with several other, more glycolytic, muscles is also exhibited by the myofibrillar proteins, since the ratio of sarcoplasmic to myofibrillar protein synthesis is similar for all muscles. Further, throughout postnatal development, when the overall turnover rate falls with age, parallel changes occur for the myofibrillar proteins, as indicated by a constant ratio of sarcoplasmic to myofibrillar protein synthesis (2.06) in the steady state after overnight starvation. Only in the youngest (4 weeks old) rats is a slightly lower ratio observed (1.72). These results indicate that, when changes in the overall turnover rate of muscle proteins occur, the relative turnover of the two major protein fractions stays constant. However, measurements in the non-steady state during growth and after starvation for 4 days show that the relative synthesis rates of the two fractions change as a result of a disproportionate increase in myofibrillar protein synthesis during growth and decrease during starvation. Thus the synthesis rate of the slower-turning-over myofibrillar protein fraction is more sensitive to nutritional state than is that of the sarcoplasmic protein. It is suggested that such responses may help to maintain constant tissue composition during non-steady-state conditions of growth and atrophy.

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 Characterization of a Novel Zinc-Containing, Lysine-Specific Aminopeptidase from the Hyperthermophilic Archaeon Pyrococcus furiosus

2005 ◽  
Vol 187 (6) ◽  
pp. 2077-2083 ◽  
Author(s):  
Sherry V. Story ◽  
Claudia Shah ◽  
Francis E. Jenney ◽  
Michael W. W. Adams
Keyword(s):  
Metal Ion ◽  
Specific Activity ◽  
Pyrococcus Furiosus ◽  
High Specific Activity ◽  
Native Form ◽  
Genome Database ◽  
Hyperthermophilic Archaeon ◽  
Cell Extracts ◽  
Hydrolysis Of

ABSTRACT Cell extracts of the proteolytic, hyperthermophilic archaeon Pyrococcus furiosus contain high specific activity (11 U/mg) of lysine aminopeptidase (KAP), as measured by the hydrolysis of l-lysyl-p-nitroanilide (Lys-pNA). The enzyme was purified by multistep chromatography. KAP is a homotetramer (38.2 kDa per subunit) and, as purified, contains 2.0 ± 0.48 zinc atoms per subunit. Surprisingly, its activity was stimulated fourfold by the addition of Co2+ ions (0.2 mM). Optimal KAP activity with Lys-pNA as the substrate occurred at pH 8.0 and a temperature of 100°C. The enzyme had a narrow substrate specificity with di-, tri-, and tetrapeptides, and it hydrolyzed only basic N-terminal residues at high rates. Mass spectroscopy analysis of the purified enzyme was used to identify, in the P. furiosus genome database, a gene (PF1861) that encodes a product corresponding to 346 amino acids. The recombinant protein containing a polyhistidine tag at the N terminus was produced in Escherichia coli and purified using affinity chromatography. Its properties, including molecular mass, metal ion dependence, and pH and temperature optima for catalysis, were indistinguishable from those of the native form, although the thermostability of the recombinant form was dramatically lower than that of the native enzyme (half-life of approximately 6 h at 100°C). Based on its amino acid sequence, KAP is part of the M18 family of peptidases and represents the first prokaryotic member of this family. KAP is also the first lysine-specific aminopeptidase to be purified from an archaeon.

scholarly journals Hydrolysis of Pork Muscle Sarcoplasmic Proteins byLactobacillus curvatus and Lactobacillus sake

1999 ◽  
Vol 65 (2) ◽  
pp. 578-584 ◽  
Author(s):  
Silvina Fadda ◽  
Yolanda Sanz ◽  
Graciela Vignolo ◽  
M.-Concepción Aristoy ◽  
Guillermo Oliver ◽  
...  
Keyword(s):  
Amino Acids ◽  
Free Amino Acids ◽  
Free Amino ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Lactobacillus Curvatus ◽  
Whole Cells ◽  
Sarcoplasmic Proteins ◽  
Lactobacillus Sake ◽  
Cell Extracts

ABSTRACT Lactobacillus curvatus CECT 904 and Lactobacillus sake CECT 4808 were selected on the basis of their proteolytic activities against synthetic substrates. Further, the effects of whole cells, cell extracts, and a combination of both enzymatic sources on muscle sarcoplasmic proteins were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase high-performance liquid chromatography analyses. Strains of both species displayed proteinase activities on five sarcoplasmic proteins. The inoculation of whole cells caused a degradation of peptides, whereas the addition of cell extracts resulted in the generation of both hydrophilic and hydrophobic peptides. This phenomenon was remarkably more pronounced when L. curvatus was involved. Whole cells also consumed a great amount of free amino acids, while the addition of intracellular enzymes contributed to their generation.L. sake accounted for a greater release of free amino acids. In general, cell viability and also proteolytic events were promoted when cell suspensions were provided with cell extracts as an extra source of enzymes.

scholarly journals Purification and Characterization of an Inverting Stereo- and Enantioselective sec-Alkylsulfatase from the Gram-Positive Bacterium Rhodococcus ruber DSM 44541

2003 ◽  
Vol 69 (5) ◽  
pp. 2810-2815 ◽  
Author(s):  
Mateja Pogorevc ◽  
Kurt Faber
Keyword(s):  
Enzymatic Hydrolysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rhodococcus Ruber ◽  
Whole Cells ◽  
Enantiomeric Ratio ◽  
Alkyl Sulfates ◽  
Substrate Tolerance ◽  
Hydrolysis Of

ABSTRACT Whole cells of Rhodococcus ruber DSM 44541 were found to hydrolyze (±)-2-octyl sulfate in a stereo- and enantiospecific fashion. When growing on a complex medium, the cells produced two sec-alkylsulfatases and (at least) one prim-alkylsulfatase in the absence of an inducer, such as a sec-alkyl sulfate or a sec-alcohol. From the crude cell-free lysate, two proteins responsible for sulfate ester hydrolysis (designated RS1 and RS2) were separated from each other based on their different hydrophobicities and were subjected to further chromatographic purification. In contrast to sulfatase RS1, enzyme RS2 proved to be reasonably stable and thus could be purified to homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single band at a molecular mass of 43 kDa. Maximal enzyme activity was observed at 30°C and at pH 7.5. Sulfatase RS2 showed a clear preference for the hydrolysis of linear secondary alkyl sulfates, such as 2-, 3-, or 4-octyl sulfate, with remarkable enantioselectivity (an enantiomeric ratio of up to 21 [23]). Enzymatic hydrolysis of (R)-2-octyl sulfate furnished (S)-2-octanol without racemization, which revealed that the enzymatic hydrolysis proceeded through inversion of the configuration at the stereogenic carbon atom. Screening of a broad palette of potential substrates showed that the enzyme exhibited limited substrate tolerance; while simple linear sec-alkyl sulfates (C7 to C10) were freely accepted, no activity was found with branched and mixed aryl-alkyl sec-sulfates. Due to the fact that prim-sulfates were not accepted, the enzyme was classified as sec-alkylsulfatase (EC 3.1.6.X).

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