Protease (PrA and PrB) and prolyl and arginyl aminopeptidase activities from Debaryomyces hansenii as a function of growth phase and nutrient sources

The effects of nutrient sources and growth phase of Debaryomyces hansenii on the protease (PrA and PrB) and aminopeptidase (prolyl-[PAP] and arginyl-[AAP] aminopeptidases) activities were investigated. These activities were also monitored during growth on a whole sarcoplasmic muscle protein extract (WSPE) and on an equivalent medium but free of compounds under 10 kDa (SPE>10 kDa). The levels of specific protease and aminopeptidase activities were higher when cells were grown in urea and dipeptides than when grown in either ammonium or free amino acids as nitrogen sources. The level of each aminopeptidase (PAP or AAP) activity was preferentially induced by its own substrate (ProLeu or LysAla), suggesting a role in the utilization of exogenous peptides. Higher specific activities for all proteolytic enzymes were detected when using acetate as carbon source. The time course experiments carried out on urea or sarcoplasmic protein-containing media revealed an increase in all activities during transition and advanced stages of stationary phase of growth. In muscle protein extracts, the absence of low molecular mass nutrients (SPE>10 kDa) initially induced the production of PrA, PrB, and AAP activities, possibly involved in the breakdown of muscle oligopeptides.     

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae is glycosylated,sorted and matured in the fission yeast Schizosaccharomyces pombe

Vacuolar carboxypeptidase Y of Saccharomyces cerevisiae (CPY^sc) has been expressed in a Schizosaccharomyces pombe strain devoid of the endogenous equivalent peptidase, employing a 2 μ derived plasmid. Immunoblot analysis revealed that CPY^sc produced in the fission yeast has a higher molecular mass than mature CPY^sc produced by the budding yeast. CPY^sc is glycosylated when expressed in S. pombe and uses four N-linked glycosylation sites as shown by endoglycosidase H digestion. Carbohydrate removal leads to a protein moiety which is indistinguishable in size from deglycosylated CPY^sc produced by S. cerevisiae. CPY^sc isolated from S. pombe soluble extracts is enzymatically active and thus is presumed to undergo correct proteolytic maturation. Subcellular fractionation experiments showed a cofractionation of CPY^sc with the S. pombe endoproteinases PrA and PrB, suggesting that the protein is correctly sorted to the vacuole and that these peptidases might be responsible for zymogen activation.     

Protease B from Debaryomyces hansenii: purification and biochemical properties

The protease B (PrB; EC. 3.4.21.48) of Debaryomyces hansenii CECT 12487 was purified by selective fractionation with protamine sulfate followed by three chromatographic separations. The whole procedure resulted in 324-fold purification with a recovery yield of 1.0%. PrB was active at neutral-basic pH ranging from 6.0 to 12.0 with an optimum at pH 8.0. The molecular mass of the denatured enzyme was 30 kDa. Polyclonal-antibodies raised against PrB from Saccharomyces cerevisiae cross-reacted with the corresponding 30-kDa protein from D. hansenii. The serine protease inhibitor 3,4-DCI and sulphydryl group reagents markedly reduced the enzyme activity. The Km against N-succinyl-Leu-Tyr-7-amido-4-methylcoumarin was 1.79 mM. The presence of endogenous inhibitor for PrB was detected in cell-free extracts of D. hansenii although their inhibitory effect was lost after incubation at 25 degrees C for 20 h. PrB was able to hydrolyze muscle sarcoplasmic proteins by in vitro assays. This is the first endopeptidase purified and characterized from the yeast D. hansenii, whose possible contributions to meat fermentation processes are discussed.     

The alcohol dehydrogenase system in the yeast, Kluyveromyces lactis

We have studied the alcohol dehydrogenase (ADH) system in the yeast Kluyveromyces lactis. Southern hybridization to the Saccharomyces cerevisiae ADH2 gene indicates four probable structural ADH genes in K. lactis. Two of these genes have been isolated from a genomic bank by hybridization to ADH2. The nucleotide sequence of one of these genes shows 80% and 50% sequence identity to the ADH genes of S. cerevisiae and Schizosaccharomyces pombe respectively. One K. lactis ADH gene is preferentially expressed in glucose-grown cells and, in analogy to S. cerevisiae, was named K1ADH1. The other gene, homologous to K1ADH1 in sequence, shows an amino-terminal extension which displays all of the characteristics of a mitochondrial targeting presequence. We named this gene K1ADH3. The two genes have been localized on different chromosomes by Southern hybridization to an orthogonal-field-alternation gel electrophoresis-resolved K. lactis genome. ADH activities resolved by gel electrophoresis revealed several ADH isozymes which are differently expressed in K. lactis cells depending on the carbon source.     

基于乳酸菌生物转化消除酵母抽提物酵母味能力评价

围绕风味物质、呈味物质和氨基酸等指标，开展了基于乳酸乳球菌（Lactococcus lactis）、乳酸链球菌（Streptococcus lactis）和植物乳杆菌（Lactobacillus plantarum）的单菌和混菌发酵体系消除酵母味的研究。结果表明，乳酸菌发酵体系下均有去除酵母味（丙酸和异戊酸）的效果，然而整体的风味具有明显差异。混菌发酵优于单菌发酵，且乳酸乳球菌与乳酸链球菌的混酵效果最佳。与其他6 种发酵体系相比，2 种乳酸菌混合发酵后的酵母抽提物中的酵母味异戊酸降低了86.73%，具有果香和花香的醇类物质从0.77 ng/mL增加至10.73 ng/mL，乳酸质量浓度为10.60 g/L，风味前体氨基酸苯丙氨酸提升了112.91 mg/L，鲜味物质（肌苷酸和鸟苷酸）提升了0.02 g/L，整体风味和呈味协调。本研究将有助于提升酵母抽提物应用的广泛性，为新型酵母抽提物的开发提供理论支撑。     

Vacuolar and extracellular maturation of Saccharomyces cerevisiae proteinase A

The vacuolar aspartyl protease proteinase A (PrA) of Saccharomyces cerevisiae is encoded as a preproenzyme by the PEP4 gene and transported to the vacuole via the secretory route. Upon arrival of the proenzyme proPrA to the vacuole, active mature 42 kDa PrA is generated by specific proteolysis involving the vacuolar endoprotease proteinase B (PrB). Vacuolar activation of proPrA can also take place in mutants lacking PrB activity (prb1). Here an active 43 kDa species termed pseudoPrA is formed, probably by an autocatalytic process. When the PEP4 gene is overexpressed in wild-type cells, mature PrA can be found in the growth medium. We have found that prb1 strains overexpressing PEP4 can form pseudoPrA extracellularly. N-terminal amino acid sequence determination of extracellular, as well as vacuolar pseudoPrA showed that it contains nine amino acids of the propeptide, indicating a cleavage between Phe67 and Ser68 of the preproenzyme. This cleavage site is in accordance with the known substrate preference for PrA, supporting the notion that pseudoPrA is formed by autoactivation. When a multicopy PEP4 transformant of a prb1 mutant was grown in the presence of the aspartyl protease inhibitor pepstatin A, a significant level of proPrA was found in the growth medium. Our analyses show that overexpression of PEP4 leads to the secretion of proPrA to the growth medium where the zymogen is converted to pseudoPrA or mature PrA in a manner similar to the vacuolar processing reactions. Amino acid sequencing of secreted proPrA confirmed the predicted cleavage by signal peptidase between Ala22 and Lys23 of the preproenzyme.     

PROPERTIES OF YEAST PROTEINASES

The properties of yeast proteinases, present in the extract of brewer's yeast, were compared with corresponding properties of plant proteinases, pepsin and trypsin in order to find suitable conditions for their activities to be determined independently. The main differences between yeast and plant proteinases were found in their themostability and activity at pH 3·0. In contrast to plant proteinases, yeast proteinases are thermolabile and active at pH 3·0. The proteolytic activity of yeast proteinases in beer is low and may be detected with sensitive methods using radiolabelled protein substrate. Nevertheless, a very wide range of proteolytic activities (67–1082 nkat/litre) was found in samples of unpasteurized beers.     

Occurrence and characterization of yeasts isolated from artisanal Fiore Sardo cheese

The occurrence of yeast microflora in artisanal Fiore Sardo cheese during ripening was studied. Mean yeast counts ranged from 2.64+/-1 log(10) cfu ml(-1) in milk to 0.65+/-1 log(10) cfu g(-1) in 9 months cheese, with the higher counts observed in 48-h-old cheese. Strains belonging to the prevalent species Debaryomyces hansenii, Kluyveromyces lactis, Geotrichum candidum, Candida zeylanoides and Candida lambica were selected for technological and genotypic characterization. All D. hansenii strains fermented glucose and assimilated lactate, a high percentage assimilated citrate and only a few showed proteolytic and lipolytic activity. All K. lactis strains were able to both assimilate and ferment lactose, to assimilate lactate and to exhibit proteolytic activity on casein. G. candidum assimilated lactate and some strains showed proteolytic and lipolytic activity. C. zeylanoides showed lipolytic activity on tweens and the majority of strains assimilated citrate. C. lambica fermented glucose and assimilated lactate. Considering their diffusion and technological characteristics, an important role for K. lactis and G. candidum in the early stages of the ripening process and for D. hansenii after the first month of ripening can be suggested. RAPD-PCR analysis with M13 primer grouped the isolates in well-separated clusters with their type strains and confirmed the previous phenotypic identification. The high intraspecific homogeneity observed in tested strains could be explained by their isolation from a common substrate and from neighbouring geographical areas. This preliminary study allowed us to isolate autochthon yeast strains showing particular properties which can contribute to the production of typical cheese taste and flavour.     

Production of proteases by psychrotrophic microorganisms.

Six milk-derived psychrotrophic microbial cultures were screened for the ability to grow at refrigerated temperatures and produce proteases in reconstituted skim milk. Of these, two cultures, Pseudomonas fluorescens M3/6 and Pseudomonas fragi K122, produced extracellular protease(s) beginning 7 d postinoculation when the cultures had entered late log or early stationary phases of growth. Further work with these two cultures showed that intracellular proteases were present after only 20-h incubation, before detection of the extracellular proteases. Using H-D-valyl-L-leucyl-L-lysyl-4-nitroanilide (S-2251), a sensitive substrate for plasmin activity, P. fluorescens was shown to have greater intracellular proteolytic activity than extracellular activity at 20 h of incubation. The intracellular enzyme activity remained constant while the extracellular and periplasmic activities increased over the remaining 6-d incubation period. The proteases in crude extracellular extracts from both cultures were characterized and were heat stable with broad temperature (7 to 52 degrees C) and pH (pH 5.5 to 8.5) ranges for activity and were inhibited by the metal chelator, EDTA, indicating that they were metalloproteases.     

Evaluation of Sensory and Microbiological Changes and Identification of Proteolytic Bacteria during the Iced Storage of Farmed Turbot (Psetta maxima)

ABSTRACT Sensory and quantitative microbiological analyses were performed in farmed turbot ( Psetta maxima ) during iced storage. Sensory analyses revealed a shelf life of 19 d for farmed turbot. The production of total volatile base nitrogen (TVB-N) and trimethylamine-nitrogen (TMA-N) was low and reached levels close to 40 mg TVB-N/ 100 g muscle and 3.5 mg /TMA-N/100 g muscle, even after 40 d of refrigerated storage. The pH value of turbot muscle increased from an initial value of 6.3 to close to 7.0 after 29 d of iced storage. Microbial growth was slow in iced turbot: total aerobic counts reached levels below 7 log colony forming units/g units even after 40 d of storage. Lactic acid bacteria—mainly Lactobacillus delbrueckii subsp. delbrueckii and Lactococcus lactis subsp. lactis strains—were predominant among the proteolytic strains isolated from iced turbot. Proteolytic strains of L. lactis subsp. lactis, Brochothrix thermosphacta, Plesiomonas shigelloides, Proteus vulgaris , and Pantoea species were also isolated from temperature-abused turbot, such proteolytic strains being predominant with respect to nonpro-teolytic microorganisms, suggesting a preferential role of such proteolytic bacteria in the spoilage of turbot. The slow bacterial growth, together with the relative predominance of lactic acid bacteria over Gram-negative microorganisms, may be related to the extraordinary maintenance of the quality and extended shelf life of farmed turbot.     

PROTEOLYTIC ACTIVITY OF BACTERIAL STARTER CULTURES FOR MEAT FERMENTATION

Proteolytic activity of fermented meat starter cultures Pediococcus acidiliactic, Lactobacillus sake, Lactobacillus curvatus, Streptomyces griseus, Staphylococcus xylosus and Staphylococcus carnosus was examined after inoculation of sterile beef protein extracts containing 1% dextrose. Analysis for primary amines in extracts generated during fermentation of glucose showed that L. sake had the highest (P<0.05) proteolytic activity. The other cultures ranked from highest to lowest activity as follows : L. curvatus < S. carnosus < S. griseus < P. acidilactici < S. xylosus. All inoculated extracts showed a pH decline by 96 h to pH 3.76-4.31 whereas the control extract remained at 6.0-5.95. All cultures except S. xylosus had CFU increases within 24 h of inoculation. Only L. sake and S. xylosus had counts less than their initial level after 96 h. The results provide evidence that these commercially available starters for meat fermentation do possess significant proteolytic activity when tested using beef protein extracts.     

酸马乳发酵过程中乳酸菌与酵母菌生长的相互影响

研究新疆酸马乳中乳酸乳球菌WLB5、干酪乳杆菌MLS5与马克思克鲁维酵母菌WWMJ1间的相互作用。结果表明：在酸马乳发酵过程中,马克思克鲁维酵母菌WWMJ1可以促进干酪乳杆菌MLS5的生长,干酪乳杆菌MLS5对马克思克鲁维酵母菌WWMJ1的生长有抑制作用,乳酸乳球菌WLB5能促进马克思克鲁维酵母菌WWMJ1的生长。乳酸乳球菌WLB5和干酪乳杆菌MLS5混合发酵有助于提高酸马乳中乳酸菌总活菌数。本研究可为酵母菌在发酵乳制品中的应用及开发新型乳制品提供一定参考。     

Purification and characterisation of proteases A and D from Debaryomyces hansenii

The proteases A (PrA; EC. 3.4.23.25) and D (PrD; EC. 3.4.24.37) of Debaryomyces hansenii CECT 12487 were characterised after their isolation by fractionation with protamine sulfate followed by three chromatographic separations, which included two anion exchange and one gel filtration chromatographic steps. The whole procedures for PrA and PrD resulted in 1349 and 2560 purification-fold with a recovery yield of 1.4 and 1.3%, respectively. PrA was active at acidic-neutral pH with an optimum pH between 5.0 and 6.0. PrD was active at neutral-basic pH with an optimum pH between 7.0 and 8.0. The molecular mass of the native PrA was 55 kDa and (being) 42 kDa in denaturing conditions. Polyclonal-antibodies raised against PrA from Saccharomyces cerevisiae cross-reacted with the corresponding PrA from D. hansenii. PrD showed a native molecular mass of 68 kDa and 65 kDa in denaturing conditions. PrA was an aspartic protease effectively inhibited by pesptatin A while PrD was classified as a metallo protease inhibited by 1,10-phenantroline and affected by some divalent cations such as zinc, cadmium and magnesium. The homology of the PrA to the lisosomal cathepsin D suggests its possible participation in the ripening of fermented meat products.     

The structure and function of Saccharomyces cerevisiae proteinase A

Saccharomyces cerevisiae proteinase A (saccharopepsin; EC 3.4.23.25) is a member of the aspartic proteinase superfamily (InterPro IPR001969), which are proteolytic enzymes distributed among a variety of organisms. Targeted to the vacuole as a zymogen, its activation at acidic pH can occur by two different pathways, a one-step process to release mature proteinase A, involving the intervention of proteinase B, or a step-wise pathway via the autoactivation product known as pseudo-proteinase A. Once active, S. cerevisiae proteinase A is essential to the activities of other yeast vacuolar hydrolases, including proteinase B and carboxypeptidase Y. The mature enzyme is bilobal, with each lobe providing one of the two catalytically essential aspartic acid residues in the active site. The crystal structure of free proteinase A reveals that the flap loop assumes an atypical position, pointing directly into the S(1) pocket of the enzyme. With regard to hydrolysis, proteinase A has a preference for hydrophobic residues with Phe, Leu or Glu at the P1 position and Phe, Ile, Leu or Ala at P1', and is inhibited by IA(3), a natural and highly specific inhibitor produced by S. cerevisiae. This review is the first comprehensive review of S. cerevisiae PrA.     

A FLUOROMETRIC ASSAY FOR PROTEINASE A IN BEER AND ITS APPLICATION FOR THE INVESTIGATION OF ENZYMATIC EFFECTS ON FOAM STABILITY

A previously developed fluorometric assay using synthetic substrate, Succinyl-Arg-Pro-Phe-His-Leu-Leu-Val-Tyr-4-methylcoumaryl-7-amide, for yeast proteinase A (PrA) was modified for the accurate and quick determination for the activity in unpasteurized beer. Employing simple HPLC for the determination of 7-amino-4-methylcoumarine (AMC), a final degradation product on this assay, the activity of PrA in beer was measured without the interference of the fluorogenic and photosensitive substance present in beer. The assay for common unpasteurized beers was completed within 5 hours without any concentration procedure. Its linearity and reproducibility were satisfactory for quantitative purposes. Using a purified PrA from brewer's yeast, the effect of the PrA activity on foam stability during storage was furthermore clarified. The exclusive effect of PrA on foam stability was also demonstrated by proteinase inhibitor test.     

Characterization of bacteriocins from two Lactococcus lactis subsp. lactis isolates

In this study, bacteriocins from two Lactococcus lactis subsp. lactis isolates from raw milk samples in Turkey designated OC1 and OC2, respectively, were characterized and identified. The activity spectra of the bacteriocins were determined by using different indicator bacteria including Listeria, Bacillus and Staphylococcus spp. Bacteriocins were tested for their sensitivity to different enzymes, heat treatments and pH values. Loss of bacteriocin activities after alpha-amylase treatment suggested that they form aggregates with carbohydrates. Molecular masses of the purified bacteriocins were determined by SDS-PAGE. PCR amplification was carried out with specific primers for the detection of their structural genes. As a result of these studies, the two bacteriocins were characterized as nisin and lacticin 481, respectively. Examination of plasmid contents of the isolates and the results of plasmid curing and conjugation experiments showed that in L. lactis subsp. lactis OC1 strain the 39.7-kb plasmid is responsible for nisin production, lactose fermentation and proteolytic activity, whereas the 16.0-kb plasmid is responsible for lacticin 481 production and lactose fermentation in L. lactis subsp. lactis OC2 strain.     

Proteolytic activities of ventral muscle and intestinal content of North Sea herring (Clupea harengus) with full and emptied stomachs

The aim of the work was to examine the effect of allowing herring (Clupea harengus) to empty the stomachs prior to being taken on board the fishing vessels, on the proteolytic activities in extracts of ventral muscle and of intestinal content. The proteolytic activities were examined by gelatin zymography and by incubating the extracts with isolated myosin heavy chain (MHC) protein. Extracts from herring with full stomachs showed strong gelatinolytic and MHC degrading activities particularly in extracts from intestinal contents. Extracts from fish that had been allowed to empty their digestive system for 19 h had reduced activities, which was most noticeable in the ventral muscle extracts. The activities from the ventral muscle did not originate endogenously post-mortem, as shown by the fact that ice storage for 24 h of isolated ventral muscle did not display the activities, while ventral muscle extract from ice-stored whole fish for 24 h did.