The growth-promoting effects of protein hydrolysates and their derived peptides on probiotics: structure-activity relationships, mechanisms and future perspectives.

Lactic acid bacteria (LAB) are the main probiotics currently available in the markets and are essential for maintaining gut health. To guarantee probiotic function, it is imperative to boost the culture yield of probiotic organisms, ensure the sufficient viable cells in commercial products, or develop effective prebiotics. Recent studies have shown that protein hydrolysates and their derived peptides promote the proliferation of probiotic in vitro and the abundance of gut flora. This article comprehensively reviews different sources of protein hydrolysates and their derived peptides as growth-promoting factors for probiotics including Lactobacillus, Bifidobacterium, and Saccharomyces. We also provide a preliminary analysis of the characteristics of LAB proteolytic systems focusing on the correlation between their elements and growth-promoting activities. The structure-activity relationship and underlying mechanisms of growth-promoting peptides and their research perspectives are thoroughly discussed. Overall, this review provides valuable insights into growth-promoting protein hydrolysates and their derived peptides for proliferating probiotics in vivo or in vitro, which may inspire researchers to explore new options for industrial probiotics proliferation, dairy products fermentation, and novel prebiotics development in the future.

Lactic acid production using cheese whey based medium in a stirred tank reactor by a ccpA mutant of Lacticaseibacillus casei.

In lactobacilli, CcpA is known to modulate the expression of genes involved in sugar metabolism, stress response and aerobic adaptation. This study aimed to evaluate a ccpA mutant of Lacticaseibacillus casei BL23 to increase lactic acid production using cheese whey. The ccpA derivative (BL71) showed better growth than the L. casei wild-type in the whey medium. In a stirred tank reactor, at 48 h, lactate production by BL71 was eightfold higher than that by BL23. In batch fermentations, the final values reached were 44.23 g L-1 for BL71 and 27.58 g L-1 for BL23. Due to a decrease in the delay of lactate production in the mutant, lactate productivity increased from 0.17 g (L.h)-1 with BL23 to 0.80 g (L.h)-1 with BL71. We found that CcpA would play additional roles in nitrogen metabolism by the regulation of the proteolytic system. BL71 displayed higher activity of the PepX, PepQ and PrtP enzymes than BL23. Analysis of prtP expression confirmed this deregulation in BL71. Promoter analysis of the prtP gene revealed CcpA binding sites with high identity to the cre consensus sequence and the interaction of CcpA with this promoter was confirmed in vitro. We postulate that deregulation of the proteolytic system in BL71 allows a better exploitation of nitrogen resources in cheese whey, resulting in enhanced fermentation capacity. Therefore, the ccpA gene could be a good target for future technological developments aimed at effective and inexpensive lactate production from dairy industrial wastes.

Editing of the Proteolytic System of Lactococcus lactis Increases Its Bioactive Potential.

Large-scale mass spectrometry-based peptidomics for bioactive-peptide discovery is relatively unexplored because of challenges in intracellular peptide extraction and small-peptide identification. Here, we present an analytical pipeline for large-scale intracellular peptidomics of Lactococcus lactis It entails an optimized sample preparation protocol for L. lactis, used as an "enzyme complex" to digest ß-casein, an extraction method for its intracellular peptidome, and a peptidomics data analysis and visualization procedure. In addition, we proofread the publicly available bioactive-peptide databases and obtained an optimized database of bioactive peptides derivable from bovine ß-casein. We used the pipeline to examine cultures of L. lactis MG1363 and a set of 6 isogenic multiple peptidase mutants incubated with ß-casein. We observed a clearly strain-dependent accumulation of peptides with several bioactivities, such as angiotensin-converting enzyme (ACE)-inhibitory, dipeptidyl peptidase 4 (DPP-IV)-inhibitory, and immunoregulatory functions. The results suggest that both the number of different bioactive peptides and the bioactivity diversity can be increased by editing the proteolytic system of L. lactis This comprehensive pipeline offers a model for discovery of bioactive peptides in combination with other proteins and might be applicable to other bacteria.IMPORTANCE Lactic acid bacteria (LAB) are very important for the production of safe and healthy human and animal fermented foods and feed and, increasingly more, in the functional food industry. The intracellular peptidomes of LAB are promising reservoirs of bioactive peptides. We show here that targeted genetic engineering of the peptide degradation pathway allows steering the composition of the peptide pool of the LAB Lactococcus lactis and production of peptides with interesting bioactivities. Our work could be used as a guideline for modifying proteolytic systems in other LAB to further explore their potential as cell peptide factories.

Insight into the bovine milk peptide LPcin-YK3 selection in the proteolytic system of Lactobacillus species.

Antimicrobial peptides are class of small, positively charged peptides known for their broad-spectrum antimicrobial activity. Antimicrobial activities for most antimicrobial peptides have largely remained elusive, particularly in the lactic acid bacteria. However, recently our investigation using LPcin-YK3, an antimicrobial peptide from bovine milk, suggests that in vitro antimicrobial activity was reduced over 100-fold compared with pathogenic bacteria. Additionally, for the structural study of how antimicrobial peptide undergoes its reaction at the proteolytic pathway of lactic acid bacteria based on degradation assay and propidium iodide staining, we performed molecular docking for interaction between oligopeptide-binding protein A and LPcin-YK3 peptide. Given that degradation related to the LPcin-YK3 peptide in lactic acid bacteria proteolytic system, the inhibitory inactivity of LPcin-YK3 against beneficial lactic acid bacteria strains may be one of the primary pharmacological properties of recombinant peptide discovered in bovine milk. These results provide structural and functional insights into the proteolytic mechanism and possibility as a putative substrate of oligopeptide-binding protein A in respect of LPcin-YK3 peptide.

Effects of lead, cadmium and zinc on protein changes in Silene vulgaris shoots cultured in vitro.

In the present research, Silene vulgaris as a representative species growing on both unpolluted and heavy metal (HM) polluted terrains were used to identify ecotype-specific responses to metallic stress. Growth, cell ultrastructure and element accumulations were compared between non-metallicolous (NM), calamine (CAL) and serpentine (SER) specimens untreated with HMs and treated with Pb, Cd and Zn ions under in vitro conditions. Moreover, proteins' modifications related to their level, carbonylation and degradations via vacuolar proteases were verified and linked with potential mechanisms to cope with ions toxicity. Our experiment revealed diversified strategy of HM uptake in NM and both metallicolous ecotypes, in which antagonistic relationship of Zn and Pb/Cd ions provided survival benefits for the whole organism. Despite this similarity, growth rate and metabolic pathways induced in CAL and SER shoots varied significantly. Exposition to HMs in CAL culture led to drop in protein level by approximately 16% compared to the control. This parameter nearly correlated with the enhanced activity of proteases at pH 5.2 as well as possible glutamate changes to proline and reduced glutathione, resulting in intensified growth and first signs of cell senescence. In turn, SER shoots were characterized by growth retardation (to 53% of the control), although protein level and carbonylation were not modified, while a deeper insight into protein network showed its remodeling towards production of polyamines and 2-oxoglutarate delivered to the Krebs cycle. Contrary, an uncontrolled HM influx in NM shoots contributed to morpho-structural disorders accompanied by an increase activity of proteases involved in the degradation of oxidized proteins, what pointed to metal-induced autophagy. Taken together, S. vulgaris ecotypes respond to stress by triggering various mechanisms engaged their survival and/or death under HM treatment.

Mdm2 and MdmX RING Domains Play Distinct Roles in the Regulation of p53 Responses: A Comparative Study of Mdm2 and MdmX RING Domains in U2OS Cells.

Dysfunction of the tumor suppressor p53 occurs in most human cancers. Mdm2 and MdmX are homologous proteins from the Mdm (Murine Double Minute) protein family, which play a critical role in p53 inactivation and degradation. The two proteins interact with one another via the intrinsic RING (Really Interesting New Gene) domains to achieve the negative regulation of p53. The downregulation of p53 is accomplished by Mdm2-mediated p53 ubiquitination and proteasomal degradation through the ubiquitin proteolytic system and by Mdm2 and MdmX mediated inhibition of p53 transactivation. To investigate the role of the RING domain of Mdm2 and MdmX, an analysis of the distinct functionalities of individual RING domains of the Mdm proteins on p53 regulation was conducted in human osteosarcoma (U2OS) cell line. Mdm2 RING domain was observed mainly localized in the cell nucleus, contrasting the localization of MdmX RING domain in the cytoplasm. Mdm2 RING was found to possess an endogenous E3 ligase activity, whereas MdmX RING did not. Both Mdm2 and MdmX RING domains were able to dimerize with endogenous full-length Mdm2 and MdmX protein and affect their cellular function. The results showed that overexpression of the Mdm2 or MdmX RING domains interfered with the endogenous full-length Mdm2 and MdmX activity and resulted in p53 stabilization and p53 target gene activation. However, both Mdm RING domains showed oncogenic activity in a colony formation assay, suggesting that the Mdm RING domains possess p53-independent oncogenic properties. This study highlights the distinct structural and functional traits of the RING domain of Mdm2 and MdmX and characterized their role in cellular responses through interfering with p53 dependent signaling pathway.

Transcription activity of lactic acid bacterial proteolysis-related genes during cheese maturation.

The catabolism of milk protein in cheese is one way how the microorganisms influence the sensorial characteristics of the final product. In this investigation, we paid attention to four genes [prtP (cell-envelope proteinase gene), pepX (X-prolyl dipeptidyl aminopeptidase gene), pepN (aminopeptidase gene) and bcaT (branched chain aminotransferase gene)] responsible for the production of volatile aroma-active compounds from milk proteins by lactic acid bacteria (LAB). We studied the dynamics of these genes and their corresponding LAB host, during the maturation of a raw ewes' milk-based cheese, using metagenomics and metatranscriptomics approaches. The transcriptome-oriented experiments included the analysis of total RNA (at three stages of cheese maturation) and also the construction of specific cDNA sub-libraries of the abovementioned genes. The proteolytic transcriptome analysis was supported by following the transcription activity of 16S rRNA gene and by metagenomic investigation. The combination of the described methods permitted to screen the dynamics of targeted genes throughout the cheese production. Lactococci were the major players in the LAB group, but the analysis provided also information on the role and properties of members of the genus Lactobacillus, such as Lb. rhamnosus, Lb. helveticus, Lb. pentosus, Lb. curvatus, Lb. parabuchneri, Lb. plantarum, Lb. brevis, Lb. delbrueckii, Lb. paracasei, Lb. fermentum and Lb. heilongjiangensis, proteolysis-related genes of which were active during cheese ripening.

Protective Effect of Pyropia yezoensis Peptide on Dexamethasone-Induced Myotube Atrophy in C2C12 Myotubes.

Dexamethasone (DEX), a synthetic glucocorticoid, causes skeletal muscle atrophy. This study examined the protective effects of Pyropia yezoensis peptide (PYP15) against DEX-induced myotube atrophy and its association with insulin-like growth factor-I (IGF-I) and the Akt/mammalian target of rapamycin (mTOR)-forkhead box O (FoxO) signaling pathway. To elucidate the molecular mechanisms underlying the effects of PYP15 on DEX-induced myotube atrophy, C2C12 myotubes were treated for 24 h with 100 µM DEX in the presence or absence of 500 ng/mL PYP15. Cell viability assays revealed no PYP15 toxicity in C2C12 myotubes. PYP15 activated the insulin-like growth factor-I receptor (IGF-IR) and Akt-mTORC1 signaling pathway in DEX-induced myotube atrophy. In addition, PYP15 markedly downregulated the nuclear translocation of transcription factors FoxO1 and FoxO3a, and inhibited 20S proteasome activity. Furthermore, PYP15 inhibited the autophagy-lysosomal pathway in DEX-stimulated myotube atrophy. Our findings suggest that PYP15 treatment protected against myotube atrophy by regulating IGF-I and the Akt-mTORC1-FoxO signaling pathway in skeletal muscle. Therefore, PYP15 treatment appears to exert protective effects against skeletal muscle atrophy.

Genomic and functional characterisation of two Enterococcus strains isolated from Cotija cheese and their potential role in ripening.

Enterococcus spp. are present in the native microbiota of many traditional fermented foods. Their ability to produce antibacterial compounds, mainly against Listeria monocytogenes, has raised interest recently. However, there is scarce information about their proteolytic and lipolytic potential, and their biotechnological application is currently limited because enterococcal strains have been related to nosocomial infections. In this work, next-generation sequencing and optimised bioinformatic pipelines were used to annotate the genomes of two Enterococcus strains-one E. faecium and one E. faecalis-isolated from the Mexican artisanal ripened Cotija cheese. A battery of genes involved in their proteolytic system was annotated. Genes coding for lipases, esterases and other enzymes whose final products contribute to cheese aroma and flavour were identified as well. As for the production of antibacterial compounds, several peptidoglycan hydrolase- and bacteriocin-coding genes were identified in both genomes experimentally and by bioinformatic analyses. E. faecalis showed resistance to aminoglycosides and E. faecium to aminoglycosides and macrolides, as predicted by the genome functional annotation. No pathogenicity islands were found in any of the strains, although traits such as the ability of biofilm formation and cell aggregation were observed. Finally, a comparative genomic analysis was able to discriminate between the food strains isolated and nosocomial strains. In summary, pathogenic strains are resistant to a wide range of antibiotics and contain virulence factors that cause host damage; in contrast, food strains display less antibiotic resistance, include genes that encode class II bacteriocins and express virulence factors associated with host colonisation rather than invasion.

Pyropia yezoensis Protein Prevents Dexamethasone-Induced Myotube Atrophy in C2C12 Myotubes.

Glucocorticoids (GCs), which are endocrine hormones released under stress conditions, can cause skeletal muscle atrophy. This study investigated whether Pyropia yezoensis crude protein (PYCP) inhibits synthetic GCs dexamethasone (DEX)-induced myotube atrophy associated with proteolytic systems. Mouse skeletal muscle C2C12 myotubes were treated with DEX in the presence or absence of PYCP. DEX exposure (100 µM) for 24 h significantly decreased myotube diameter and myogenin expression, which were all increased by treatment with 20 and 40 µg/mL PYCP. Additionally, PYCP significantly reduced the nuclear expression of the forkhead box transcription factors, FoxO1 and FoxO3a, and ubiquitin-proteasome pathway activation. Further mechanistic research revealed that PYCP inhibited the autophagy-lysosome pathway in DEX-induced C2C12 myotubes. These findings indicate that PYCP prevents DEX-induced myotube atrophy through the regulation of FoxO transcription factors, followed by the inhibition of the ubiquitin-proteasome and autophagy-lysosome pathways. Therefore, we suggest that inhibiting these two proteolytic processes with FoxO transcription factors is a promising strategy for preventing DEX-related myotube atrophy.

Fast and Cost-Effective Biochemical Spectrophotometric Analysis of Solution of Insect "Blood" and Body Surface Elution.

Using insect hemolymph ("blood") and insect body surface elutions, researchers can perform rapid and cheap biochemical analyses to determine the insect's immunology status. The authors of this publication describe a detailed methodology for a quick marking of the concentration of total proteins and evaluation of the proteolytic system activity (acid, neutral, and alkaline proteases and protease inhibitors), as well as a methodology for quick "liver" tests in insects: alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and urea and glucose concentration analyses. The meaning and examples of an interpretation of the results of the presented methodology for biochemical parameter determination are described for the example of honey bees.

Disassembly of the lens fiber cell nucleus to create a clear lens: The p27 descent.

The eye lens is unique among tissues: it is transparent, does not form tumors, and the majority of its cells degrade their organelles, including their cell nuclei. A mystery for over a century, there has been considerable recent progress in elucidating mechanisms of lens fiber cell denucleation (LFCD). In contrast to the disassembly and reassembly of the cell nucleus during mitosis, LFCD is a unidirectional process that culminates in destruction of the fiber cell nucleus. Whereas p27Kip1, the cyclin-dependent kinase inhibitor, is upregulated during formation of LFC in the outermost cortex, in the inner cortex, in the nascent organelle free zone, p27Kip1 is degraded, markedly activating cyclin-dependent kinase 1 (Cdk1). This process results in phosphorylation of nuclear Lamins, dissociation of the nuclear membrane, and entry of lysosomes that liberate DNaseIIß (DLAD) to cleave chromatin. Multiple cellular pathways, including the ubiquitin proteasome system and the unfolded protein response, converge on post-translational regulation of p27Kip1. Mutations that impair these pathways are associated with congenital cataracts and loss of LFCD. These findings highlight new regulatory nodes in the lens and suggest that we are close to understanding this fascinating terminal differentiation process. Such knowledge may offer a new means to confront proliferative diseases including cancer.

Coenzyme Q10 treatments influence the lifespan and key biochemical resistance systems in the honeybee, Apis mellifera.

Natural bioactive preparations that will boost apian resistance, aid body detoxification, or fight crucial bee diseases are in demand. Therefore, we examined the influence of coenzyme Q10 (CoQ10, 2,3-dimethoxy, 5-methyl, 6-decaprenyl benzoquinone) treatment on honeybee lifespan, Nosema resistance, the activity/concentration of antioxidants, proteases and protease inhibitors, and biomarkers. CoQ10 slows age-related metabolic processes. Workers that consumed CoQ10 lived longer than untreated controls and were less infested with Nosema spp. Relative to controls, the CoQ10-treated workers had higher protein concentrations that increased with age but then they decreased in older bees. CoQ10 treatments increased the activities of antioxidant enzymes (superoxide dismutase, GPx, catalase, glutathione S-transferase), protease inhibitors, biomarkers (aspartate aminotransferase, alkaline phosphatase, alanine aminotransferase), the total antioxidant potential level, and concentrations of uric acid and creatinine. The activities of acidic, neutral, and alkaline proteases, and concentrations of albumin and urea were lower in the bees that were administered CoQ10. CoQ10 could be taken into consideration as a natural diet supplement in early spring before pollen sources become available in the temperate Central European climate. A response to CoQ10 administration that is similar to mammals supports our view that Apis mellifera is a model organism for biochemical gerontology.

Proteome analysis of Lactobacillus helveticus H9 during growth in skim milk.

Lactobacillus helveticus H9 was isolated from traditionally fermented yak milk in Tibet (China) with the ability to produce the antihypertensive peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP) during milk fermentation. To understand the changes in the protein expression of L. helveticus H9, proteome analysis was performed at 3 different growth stages, lag phase (pH 6.1), log phase (pH 5.1), and stationary phase (pH 4.5) using 2-dimensional electrophoresis (2-DE). Further analysis showed that 257 differential protein spots were found and 214 protein spots were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS). The cellular functions of the differentially expressed proteins were complex. Interestingly, the proteolytic system-related proteins aminopeptidase N (PepN), aminopeptidase E (PepE), endopeptidase O2 (PepO2), and oligopeptide transport system permease protein (OppC) were observed only on the maps of pH 5.1 and pH 4.5, which was consistent with the presence of angiotensin I-converting enzyme (ACE)-inhibitory peptides VPP and IPP during these 2 growth stages (log phase and stationary phase). These results, combined with a previous study of gene expression of the proteolytic system, led us to conclude that the Opp transport system, pepE, and pepO2 are likely related to the production of ACE-inhibitory peptides.

Glycative stress as a cause of macular degeneration.

People are living longer and rates of age-related diseases such as age-related macular degeneration (AMD) are accelerating, placing enormous burdens on patients and health care systems. The quality of carbohydrate foods consumed by an individual impacts health. The glycemic index (GI) is a kinetic measure of the rate at which glucose arrives in the blood stream after consuming various carbohydrates. Consuming diets that favor slowly digested carbohydrates releases sugar into the bloodstream gradually after consuming a meal (low glycemic index). This is associated with reduced risk for major age-related diseases including AMD, cardiovascular disease, and diabetes. In comparison, consuming the same amounts of different carbohydrates in higher GI diets, releases glucose into the blood rapidly, causing glycative stress as well as accumulation of advanced glycation end products (AGEs). Such AGEs are cytotoxic by virtue of their forming abnormal proteins and protein aggregates, as well as inhibiting proteolytic and other protective pathways that might otherwise selectively recognize and remove toxic species. Using in vitro and animal models of glycative stress, we observed that consuming higher GI diets perturbs metabolism and the microbiome, resulting in a shift to more lipid-rich metabolomic profiles. Interactions between aging, diet, eye phenotypes and physiology were observed. A large body of laboratory animal and human clinical epidemiologic data indicates that consuming lower GI diets, or lower glycemia diets, is protective against features of early AMD (AMDf) in mice and AMD prevalence or AMD progression in humans. Drugs may be optimized to diminish the ravages of higher glycemic diets. Human trials are indicated to determine if AMD progression can be retarded using lower GI diets. Here we summarized the current knowledge regarding the pathological role of glycative stress in retinal dysfunction and how dietary strategies might diminish retinal disease.

Functions and distribution of calpain-calpastatin system components in brain during mammal ontogeny.

Calpain and calpastatin are the key components of the calcium-dependent proteolytic system. Calpains are regulatory, calcium-dependent, cytoplasmic proteinases, and calpastatin is the endogenous inhibitor of calpains. Due to the correlation between changes in the activity of the calpain-calpastatin system in the brain and central nervous system (CNS) pathology states, this proteolytic system is a prime focus of research on CNS pathological processes, generally characterized by calpain activity upregulation. The present review aims to generalize existing data on cerebral calpain distribution and function through mammalian ontogenesis. Special attention is given to the most recent studies on the topic as more information on calpain-calpastatin system involvement in normal CNS development and functioning has become available. We also discuss data on calpain and calpastatin activity and production in different brain regions during ontogenesis as comparative analysis of these results in association with ontogeny processes can reveal brain regions and developmental stages with pronounced function of the calpain system.

Characterization of an Aminopeptidase A from Tetragenococcus halophilus CY54 Isolated from Myeolchi-Jeotgal.

In this study, a pepA gene encoding glutamyl (aspartyl)-specific aminopeptidase (PepA; E.C. 3.4.11.7) was cloned from Tetragenococcus halophilus CY54. The translated PepA from T. halophilus CY54 showed very low similarities with PepAs from Lactobacillus and Lactococcus genera. The pepA from T. halophilus CY54 was overexpressed in E. coli BL21(DE3) using pET26b(+). The recombinant PepA was purified by using an Ni- NTA column. The size of the recombinant PepA was 39.13 kDa as determined by SDS-PAGE, while its optimum pH and temperature were pH 5.0 and 60°C, respectively. In addition, the PepA was completely inactivated by 1 mM EDTA, indicating its metallopeptidase nature. The Km and Vmax of the PepA were 0.98 ± 0.006 mM and 0.1 ± 0.002 mM/min, respectively, when Glu-pNA was used as the substrate. This is the first report on PepA from Tetragenococcus species.

Bioactive peptides produced by engineered probiotics and other food-grade bacteria: A review.

Synthetic biology is employed for the study and design of engineered microbes with new and improved therapeutic functions. The main advantage of synthetic biology is the selective genetic manipulation of living organisms with desirable beneficial effects such as probiotics. Engineering technologies have contributed to the edition of metabolic processes involved in the mechanisms of action of probiotics, such as the generation of bioactive peptides. Hence, current information related to bioactive peptides, produced by different engineering probiotics, with antimicrobial, antiviral, antidiabetic, and antihypertensive activities, as well as their potential use as functional ingredients, is discussed here. Besides, the effectiveness and safety aspects of these bioactive peptides were also described.

Effect of Comb Cell Width on the Activity of the Proteolytic System in the Hemolymph of Apis mellifera Workers.

This study is a continuation of the innovative research of the impact of rearing of bee colonies simultaneously on standard- and small-cell combs on the traits of worker bees and bee colonies. Its aim was to compare the activities of proteases and their inhibitors in the hemolymph of workers reared in a small-cell comb (SMC) and a standard-cell comb (STC) in colonies kept simultaneously on standard- and small-cell combs. The width of comb cells in which workers are reared has a significant effect on the protein concentration and the proteolytic system in the hemolymph, which is reflected in the activities of proteases and their inhibitors. The protein concentrations in the 1-day-old workers were always higher (p ≤ 0.05) in the SMC than STC workers. The opposite was found in the older bee workers (aged 7, 14 and 21 d). The activities of proteases and their inhibitors in the 1-day-old workers were always higher (usually significantly at p ≤ 0.05) in STC than SMC workers, and opposite results were observed in the groups of the older workers (aged 7, 14 and 21 d). The differences between the workers from small-cell combs and those reared in standard-cell combs may be related to their different tasks. Workers reared in small-cell combs probably work as foragers outside the nest, whereas bees reared in standard-cell combs work in the nest. This hypothesis requires confirmation. To reduce the impact of accidental determinants on the results of single-season research on honeybees, it is advisable that such investigations should be conducted for several consecutive years.

Modeled Structure of the Cell Envelope Proteinase of Lactococcus lactis.

The cell envelope proteinase (CEP) of Lactococcus lactis is a large extracellular protease covalently linked to the peptidoglycan of the cell wall. Strains of L. lactis are typically auxotrophic for several amino acids and in order to grow to high cell densities in milk they need an extracellular protease. The structure of the entire CEP enzyme is difficult to determine experimentally due to the large size and due to the attachment to the cell surface. We here describe the use of a combination of structure prediction tools to create a structural model for the entire CEP enzyme of Lactococcus lactis. The model has implications for how the bacterium interacts with casein micelles during growth in milk, and it has implications regarding the energetics of the proteolytic system. Our model for the CEP indicates that the catalytic triad is activated through a structural change caused by interaction with the substrate. The CEP of L. lactis might become a useful model for the mode of action for enzymes belonging to the large class of S8 proteinases with a PA (protease associated) domain and a downstream fibronectin like domain.