Identification of a Class of Protein ADP-Ribosylating Sirtuins in Microbial Pathogens.

Sirtuins are an ancient family of NAD(+)-dependent deacylases connected with the regulation of fundamental cellular processes including metabolic homeostasis and genome integrity. We show the existence of a hitherto unrecognized class of sirtuins, found predominantly in microbial pathogens. In contrast to earlier described classes, these sirtuins exhibit robust protein ADP-ribosylation activity. In our model organisms, Staphylococcus aureus and Streptococcus pyogenes, the activity is dependent on prior lipoylation of the target protein and can be reversed by a sirtuin-associated macrodomain protein. Together, our data describe a sirtuin-dependent reversible protein ADP-ribosylation system and establish a crosstalk between lipoylation and mono-ADP-ribosylation. We propose that these posttranslational modifications modulate microbial virulence by regulating the response to host-derived reactive oxygen species.

Characteristics of the Proteolytic Enzymes Produced by Lactic Acid Bacteria.

Over the past several decades, we have observed a very rapid development in the biotechnological use of lactic acid bacteria (LAB) in various branches of the food industry. All such areas of activity of these bacteria are very important and promise enormous economic and industrial successes. LAB are a numerous group of microorganisms that have the ability to ferment sugars into lactic acid and to produce proteolytic enzymes. LAB proteolytic enzymes play an important role in supplying cells with the nitrogen compounds necessary for their growth. Their nutritional requirements in this regard are very high. Lactic acid bacteria require many free amino acids to grow. The available amount of such compounds in the natural environment is usually small, hence the main function of these enzymes is the hydrolysis of proteins to components absorbed by bacterial cells. Enzymes are synthesized inside bacterial cells and are mostly secreted outside the cell. This type of proteinase remains linked to the cell wall structure by covalent bonds. Thanks to advances in enzymology, it is possible to obtain and design new enzymes and their preparations that can be widely used in various biotechnological processes. This article characterizes the proteolytic activity, describes LAB nitrogen metabolism and details the characteristics of the peptide transport system. Potential applications of proteolytic enzymes in many industries are also presented, including the food industry.

A Plasmid-Encoded Putative Glycosyltransferase Is Involved in Hop Tolerance and Beer Spoilage in Lactobacillus brevis.

Lactobacillus brevis beer-spoiling strains harbor plasmids that contain genes such as horA, horC, and hitA which are known to confer hop tolerance. The L. brevis beer-spoiling strain UCCLBBS124, which possesses four plasmids, was treated with novobiocin, resulting in the isolation of UCCLBBS124 derivatives exhibiting hop sensitivity and an inability to grow in beer. One selected derivative was shown to have lost a single plasmid, here designated UCCLBBS124_D, which harbors the UCCLBBS124_pD0015 gene, predicted to encode a glycosyltransferase. Hop tolerance and growth in beer were restored when UCCLBBS124_pD0015 was introduced in one of these hop-sensitive derivatives on a plasmid. We hypothesize that this gene modifies the surface composition of the polysaccharide cell wall, conferring protection against hop compounds. Furthermore, the introduction of this gene in trans in L. brevis UCCLB521, a strain that cannot grow in and spoil beer, was shown to furnish the resulting strain with the ability to grow in beer, while its expression also conferred phage resistance. This study underscores how the acquisition of certain mobile genetic elements plays a role in hop tolerance and beer spoilage for strains of this bacterial species.IMPORTANCELactobacillus brevis is a member of the lactic acid bacteria and is often reported as the causative agent of food or beverage spoilage, in particular, that of beer. Bacterial spoilage of beer may result in product withdrawal or recall, with concomitant economic losses for the brewing industry. A very limited number of genes involved in beer spoilage have been identified and primarily include those involved in hop resistance, such as horA, hitA, and horC However, since none of these genes are universal, it is clear that there are likely (many) other molecular players involved in beer spoilage. Here, we report on the importance of a plasmid-encoded glycosyltransferase associated with beer spoilage by L. brevis that is involved in hop tolerance. The study highlights the complexity of the genetic requirements to facilitate beer spoilage and the role of multiple key players in this process.

Technically relevant enzymes and proteins produced by LAB suitable for industrial and biological activity.

Lactic acid bacteria (LAB) are a unique subset of microorganisms that have co-evolved with humans since the beginning of agricultural practices and animal domestication and throughout our never-ending quest for food preservation, digestibility, and flavor enhancement. LAB have historically played a preponderant role in our foods. In this review, we focus on the enzymatic activities and current or potential applications of LAB in our lives. A description of each of the enzymatic systems in LAB is included. Glycosidases, which hydrolyze the most abundant food molecules and as sources of carbon, sustain the lives of organisms on Earth as well as ensure microbial innocuity by the production of lactic acid from the uniquely mammalian carbohydrate, lactose. Lipases and proteases or proteinases are of fundamental importance in food fermentations and in dairy foods for flavor development. Bacteriocins and peptidoglycan hydrolases are part of the enzymatic system of LAB that has evolved to make these bacteria fierce competitors in various microbiomes, which are highly important for the human gut. In this review, we also present an explanation on how the versatility of the genetics of LAB can adapt to the matrix where they are placed with the advantage of not having any toxicity to humans. The systematic study of LAB enzymes has allowed for some unique applications in foods and biopharmaceutical industries. Here, we summarize how different enzyme systems in LAB are classified, and thus, facilitate much-needed further studies to understand the fundamentals and translate them into applications to improve our lives.

Short communication: An inducible CRISPR/dCas9 gene repression system in Lactococcus lactis.

Lactococcus lactis, one of the most important probiotic lactic acid bacteria (LAB), is widely used in the dairy industry as a cell factory for recombinant protein production. Currently, a nisin-controlled inducible expression system is used for this purpose and represents the only commercial expression system in LAB. However, the available genetic modification methods are rather limited for modulating gene expression in L. lactis. Here, we developed a 2-plasmid system for gene transcription repression in L. lactis NZ9000 that uses inducible clustered regularly interspaced short palindromic repeats (CRISPR)-dCas9. An inducible promoter Pnisin was used to drive the expression of dCas9 from Streptococcus pyogenes, whereas a strong constitutive promoter P44 drove single guide RNA expression for single or multiple target genes. dCas9 enabled CRISPR interference-mediated silencing of single or multiple target genes with significant reduction of gene expression, up to 99%. In addition, LLNZ_07335, a putative penicillin acylase, was identified as bile salt hydrolase for bile salt resistance in NZ9000 using this system. To our knowledge, this report is the first for a functional gene for bile salt tolerance in L. lactis. Overall, our work introduces a new gene repression tool for various applications in L. lactis or other LAB.

Partial characterization and purification of phytase from Lactobacillus plantarum CRL1964 isolated from pseudocereals.

Cereals and pseudocereals are a rich source of nutrients and trace elements, but their dietary bioavailability is low due to the presence of phytate (IP6), an antinutritional compound with the ability to chelate cations and proteins. Phytase is an enzyme that catalyzes the hydrolysis of IP6 and it is used as an additive improving the nutritional quality of grain-based foods. The aim of this study was to select lactic acid bacteria (LAB) isolated from pseudocereals with phytase activity, characterize their production and activity, and purify the enzyme. LAB strains isolated from grains and spontaneous sourdough of quinoa and amaranth were grown in the Man Rogosa and Sharpe medium where the inorganic phosphate (Pi) was replaced by 1% of IP6. Phytase activity was determined by measuring the Pi released from IP6. Phytase of Lactobacillus (L.) plantarum CRL1964 (PhyLP) showed the highest specific activity from 73 LAB evaluated. IP6 induces PhyLP production, which is at its maximum at the end of the exponential phase. PhyLP was thermostable and maintained its activity under acidic conditions. The enzymatic activity is stimulated by ethylenediaminetetraacetic acid, Co2+, and ascorbic acid. PhyLP was partially purified and showed a molecular mass of 55 kDa. L. plantarum CRL1964 and/or PhyLP have the potential to be included in the processing of cereal/pseudocereals based products for animal feed and/or the food industry improving its nutritional value.

Bile salt hydrolase activity is present in nonintestinal lactic acid bacteria at an intermediate level.

It is generally considered that bile salt hydrolase (BSH) activity is hardly detected in nonintestinal lactic acid bacteria (LAB). The aim of this study was to investigate the distribution and intensity of BSH activity in LAB isolated from naturally fermented vegetables and milk. A total of 624 lactic acid bacterial strains classified into 6 genera and 50 species were isolated from 144 naturally fermented vegetable samples and 103 naturally fermented milk samples, and their BSH activity was screened by gas chromatography with electron capture detection. The BSH-positive strains were further analyzed quantitatively for their deconjugation ability against six human-conjugated bile salts by HPLC based on the disappearance of the conjugated bile salts from the reaction mixture. The results showed that 39% of the strains possessed BSH activity distributed in 24 lactic acid bacterial species. The strains of the fermented vegetable origin showed a 0.5-fold higher incidence of BSH-positive strains than those of the fermented milk origin, and the lactic acid bacilli exhibited 2.5-fold higher incidence of BSH-positive strains than the lactic acid cocci in general. The strains of the fermented vegetable origin generally had greater bile salt deconjugation ability than those of the fermented milk origin. More than 97% and 93% of the BSH-positive strains exhibited a greater substrate preference for glycoconjugated bile salts than tauroconjugated bile salts and for dihydroxy bile salts than trihydroxy bile salts, respectively. This study demonstrated that BSH activity was also present in nonintestinal LAB.

Lactic acid bacteria isolated from dairy products as potential producers of lipolytic, proteolytic and antibacterial proteins.

Regular consumption of fermented dairy products helps maintain a healthy microbiota and prevent gut dysbiosis-linked diseases. The lactic acid bacteria (LAB) present in food enhance the digestibility of proteins, moderate the release of fatty acids, and support human health through inhabiting the gastrointestinal tract. These desirable properties of LAB are attributed, in part, to their metabolic processes involving enzymes such as lipases, proteases, and antibacterial proteins. The LAB strains presenting higher enzymatic activities may offer improved functionality for applications in foods. The first aim of this work was to isolate and identify LAB from diverse dairy products and select those with enhanced enzymatic activities. Secondly, this work aimed to investigate the subcellular organization and identity of these enzymes after semi-purification. Out of the total 137 LAB strains isolated and screened, 50.3% and 61.3% of the strains exhibited lipolytic and proteolytic activities, respectively. Seven strains displaying high enzymatic activities were selected and further characterized for the cellular organization of their lipases, proteases, and antibacterial proteins. The lipolytic and proteolytic activities were exhibited predominantly in the extracellular fraction; whereas, the antibacterial activities were found in various cellular fractions and were capable of inhibiting common undesirable microorganisms in foods. In total, two lipases, seven proteases, and three antibacterial proteins were identified by LC-MS/MS. Characterization of LAB strains with high enzymatic activity has potential biotechnological significance in fermentative processes and in human health as they may improve the physicochemical characteristics of foods and displace strains with weaker enzymatic activities in the human gut microbiota.

First Insight into the Technological Features of Lactic Acid Bacteria Isolated from Algerian Fermented Wheat Lemzeiet.

Fermented cereals are part of the main traditional diets of many people in Africa, usually obtained from artisanal production. The intensification of their manufacturing, responding to the consumers demand, requires a better control to ensure their sanitary, nutritional, and taste qualities, hence, the need of selecting accurate and safe starter cultures. In the present study, 48 lactic acid bacteria (LAB) strains, previously isolated from Algerian fermented wheat lemzeiet, were analyzed for different technological properties. 14 LAB strains, belonging to Pediococcus pentosaceus, Enterococcus faecium, Lactobacillus curvatus, Lactobacillus brevis, and Leuconostoc mesenteroides species, decreased rapidly the pH of the flour extract broth close to 4 or below. 91% of strains showed extracellular protease activity, but only 12% were amylolytics. 18 LAB strains inhibited or postponed the growth of three fungal targets Rhodotorula mucilaginosa UBOCC-A-216004, Penicillium verrucosum UBOCC-A-109221, and Aspergillus flavus UBOCC-A-106028. The strains belonging to Lactobacillus spp., Leuconostoc fallax, L. mesenteroides, and Weissella paramesenteroides were the most antifungal ones. Multiplex PCR for biogenic amines' production did not reveal any of the genes involved in the production of putrescine, histamine, and tyramine for 17 of the 48 strains. The obtained results provided several candidates for use as starter culture in the future production of lemzeiet.

d-Alanyl-d-Alanine Ligase as a Broad-Host-Range Counterselection Marker in Vancomycin-Resistant Lactic Acid Bacteria.

The peptidoglycan composition in lactic acid bacteria dictates vancomycin resistance. Vancomycin binds relatively poorly to peptidoglycan ending in d-alanyl-d-lactate and binds with high affinity to peptidoglycan ending in d-alanyl-d-alanine (d-Ala-d-Ala), which results in vancomycin resistance and sensitivity, respectively. The enzyme responsible for generating these peptidoglycan precursors is dipeptide ligase (Ddl). A single amino acid in the Ddl active site, phenylalanine or tyrosine, determines depsipeptide or dipeptide activity, respectively. Here, we established that heterologous expression of dipeptide ligase in vancomycin-resistant lactobacilli increases their sensitivity to vancomycin in a dose-dependent manner and overcomes the effects of the presence of a native d-Ala-d-Ala dipeptidase. We incorporated the dipeptide ligase gene on a suicide vector and demonstrated that it functions as a counterselection marker (CSM) in lactobacilli; vancomycin selection allows only those cells to grow in which the suicide vector has been lost. Subsequently, we developed a liquid-based approach to identify recombinants in only 5 days, which is approximately half the time required by conventional approaches. Phylogenetic analysis revealed that Ddl serves as a marker to predict vancomycin resistance and consequently indicated the broad applicability of the use of Ddl as a counterselection marker in the genus Lactobacillus Finally, our system represents the first "plug and play" counterselection system in lactic acid bacteria that does not require prior genome editing and/or synthetic medium.IMPORTANCE The genus Lactobacillus contains more than 200 species, many of which are exploited in the food and biotechnology industries and in medicine. Prediction of intrinsic vancomycin resistance has thus far been limited to selected Lactobacillus species. Here, we show that heterologous expression of the enzyme Ddl (dipeptide ligase)-an essential enzyme involved in peptidoglycan synthesis-increases sensitivity to vancomycin in a dose-dependent manner. We exploited this to develop a counterselection marker for use in vancomycin-resistant lactobacilli, thereby expanding the poorly developed genome editing toolbox that is currently available for most strains. Also, we showed that Ddl is a phylogenetic marker that can be used to predict vancomycin resistance in Lactobacillus; 81% of Lactobacillus species are intrinsically resistant to vancomycin, which makes our tool broadly applicable.

LPMOs in cellulase mixtures affect fermentation strategies for lactic acid production from lignocellulosic biomass.

Enzymatic catalysis plays a key role in the conversion of lignocellulosic biomass to fuels and chemicals such as lactic acid. In the last decade, the efficiency of commercial cellulase cocktails has increased significantly, in part due to the inclusion of lytic polysaccharide monooxygenases (LPMOs). However, the LPMOs' need for molecular oxygen to break down cellulose demands reinvestigations of process conditions. In this study, we evaluate the efficiency of lactic acid production from steam-exploded birch using an LPMO-containing cellulase cocktail in combination with lactic acid bacteria, investigating both separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF). While the SSF set up generally has been considered to be more efficient because it avoids sugar accumulation which may inhibit the cellulases, the SHF set up in our study yielded 26-32% more lactic acid than the SSF. This was mainly due to competition for oxygen between LPMOs and the fermenting organisms in the SSF process, which resulted in reduced LPMO activity and thus less efficient saccharification of the lignocellulosic substrate. By means of aeration it was possible to activate the LPMOs in the SSF, but less lactic acid was produced due to a shift in metabolic pathways toward production of acetic acid. Overall, this study shows that lactic acid can be produced efficiently from lignocellulosic biomass, but that the use of LPMO-containing cellulase cocktails in fermentation processes demands re-thinking of traditional process set ups due to the requirement of oxygen in the saccharification step. Biotechnol. Bioeng. 2017;114: 552-559. © 2016 Wiley Periodicals, Inc.

KOMODO: a web tool for detecting and visualizing biased distribution of groups of homologous genes in monophyletic taxa.

The enrichment analysis is a standard procedure to interpret 'omics' experiments that generate large gene lists as outputs, such as transcriptomics and protemics. However, despite the huge success of enrichment analysis in these classes of experiments, there is a surprising lack of application of this methodology to survey other categories of large-scale biological data available. Here, we report Kegg Orthology enrichMent-Online DetectiOn (KOMODO), a web tool to systematically investigate groups of monophyletic genomes in order to detect significantly enriched groups of homologous genes in one taxon when compared with another. The results are displayed in their proper biochemical roles in a visual, explorative way, allowing users to easily formulate and investigate biological hypotheses regarding the taxonomical distribution of genomic elements. We validated KOMODO by analyzing portions of central carbon metabolism in two taxa extensively studied regarding their carbon metabolism profile (Enterobacteriaceae family and Lactobacillales order). Most enzymatic activities significantly biased were related to known key metabolic traits in these taxa, such as the distinct fates of pyruvate (the known tendency of lactate production in Lactobacillales and its complete oxidation in Enterobacteriaceae), demonstrating that KOMODO could detect biologically meaningful differences in the frequencies of shared genomic elements among taxa. KOMODO is freely available at http://komodotool.org.

Phylloquinone (vitamin K(1) ) biosynthesis in plants: two peroxisomal thioesterases of Lactobacillales origin hydrolyze 1,4-dihydroxy-2-naphthoyl-CoA.

It is not known how plants cleave the thioester bond of 1,4-dihydroxy-2-naphthoyl-CoA (DHNA-CoA), a necessary step to form the naphthoquinone ring of phylloquinone (vitamin K(1) ). In fact, only recently has the hydrolysis of DHNA-CoA been demonstrated to be enzyme driven in vivo, and the cognate thioesterase characterized in the cyanobacterium Synechocystis. With a few exceptions in certain prokaryotic (Sorangium and Opitutus) and eukaryotic (Cyanidium, Cyanidioschyzon and Paulinella) organisms, orthologs of DHNA-CoA thioesterase are missing outside of the cyanobacterial lineage. In this study, genomic approaches and functional complementation experiments identified two Arabidopsis genes encoding functional DHNA-CoA thioesterases. The deduced plant proteins display low percentages of identity with cyanobacterial DHNA-CoA thioesterases, and do not even share the same catalytic motif. GFP-fusion experiments demonstrated that the Arabidopsis proteins are targeted to peroxisomes, and subcellular fractionations of Arabidopsis leaves confirmed that DHNA-CoA thioesterase activity occurs in this organelle. In vitro assays with various aromatic and aliphatic acyl-CoA thioester substrates showed that the recombinant Arabidopsis enzymes preferentially hydrolyze DHNA-CoA. Cognate T-DNA knock-down lines display reduced DHNA-CoA thioesterase activity and phylloquinone content, establishing in vivo evidence that the Arabidopsis enzymes are involved in phylloquinone biosynthesis. Extraordinarily, structure-based phylogenies coupled to comparative genomics demonstrate that plant DHNA-CoA thioesterases originate from a horizontal gene transfer with a bacterial species of the Lactobacillales order.

Methionine metabolism: major pathways and enzymes involved and strategies for control and diversification of volatile sulfur compounds in cheese.

For economical reasons and to accommodate current market trends, cheese manufacturers and product developers are increasingly interested in controlling cheese flavor formation and developing new flavors. Due to their low detection threshold and diversity, volatile sulfur compounds (VSCs) are of prime importance in the overall flavor of cheese and make a significant contribution to their typical flavors. Thus, the control of VSCs formation offers considerable potential for industrial applications. This paper gives an overview of the main VSCs found in cheese, along with the major pathways and key enzymes leading to the formation of methanethiol from methionine, which is subsequently converted into other sulfur-bearing compounds. As these compounds arise primarily from methionine, the metabolism of this amino acid and its regulation is presented. Attention is focused in the enzymatic potential of lactic acid bacteria (LAB) that are widely used as starter and adjunct cultures in cheese-making. In view of industrial applications, different strategies such as the enhancement of the abilities of LAB to produce high amounts and diversity of VSCs are highlighted as the principal future research trend.

Computational analysis of cysteine and methionine metabolism and its regulation in dairy starter and related bacteria.

Sulfuric volatile compounds derived from cysteine and methionine provide many dairy products with a characteristic odor and taste. To better understand and control the environmental dependencies of sulfuric volatile compound formation by the dairy starter bacteria, we have used the available genome sequence and experimental information to systematically evaluate the presence of the key enzymes and to reconstruct the general modes of transcription regulation for the corresponding genes. The genomic organization of the key genes is suggestive of a subdivision of the reaction network into five modules, where we observed distinct differences in the modular composition between the families Lactobacillaceae, Enterococcaceae, and Leuconostocaceae, on the one hand, and the family Streptococcaceae, on the other. These differences are mirrored by the way in which transcription regulation of the genes is structured in these families. In the Lactobacillaceae, Enterococcaceae, and Leuconostocaceae, the main shared mode of transcription regulation is methionine (Met) T-box-mediated regulation. In addition, the gene metK, encoding S-adenosylmethionine (SAM) synthetase, is controlled via the S(MK) box (SAM). The S(MK) box is also found upstream of metK in species of the family Streptococcaceae. However, the transcription control of the other modules is mediated via three different LysR-family regulators, MetR/MtaR (methionine), CmbR (O-acetyl[homo]serine), and HomR (O-acetylhomoserine). Redefinition of the associated DNA-binding motifs helped to identify/disentangle the related regulons, which appeared to perfectly match the proposed subdivision of the reaction network.

Evidence of two functionally distinct ornithine decarboxylation systems in lactic acid bacteria.

Biogenic amines are low-molecular-weight organic bases whose presence in food can result in health problems. The biosynthesis of biogenic amines in fermented foods mostly proceeds through amino acid decarboxylation carried out by lactic acid bacteria (LAB), but not all systems leading to biogenic amine production by LAB have been thoroughly characterized. Here, putative ornithine decarboxylation pathways consisting of a putative ornithine decarboxylase and an amino acid transporter were identified in LAB by strain collection screening and database searches. The decarboxylases were produced in heterologous hosts and purified and characterized in vitro, whereas transporters were heterologously expressed in Lactococcus lactis and functionally characterized in vivo. Amino acid decarboxylation by whole cells of the original hosts was determined as well. We concluded that two distinct types of ornithine decarboxylation systems exist in LAB. One is composed of an ornithine decarboxylase coupled to an ornithine/putrescine transmembrane exchanger. Their combined activities results in the extracellular release of putrescine. This typical amino acid decarboxylation system is present in only a few LAB strains and may contribute to metabolic energy production and/or pH homeostasis. The second system is widespread among LAB. It is composed of a decarboxylase active on ornithine and l-2,4-diaminobutyric acid (DABA) and a transporter that mediates unidirectional transport of ornithine into the cytoplasm. Diamines that result from this second system are retained within the cytosol.

Relationship between bile salt hydrolase activity, changes in the internal pH and tolerance to bile acids in lactic acid bacteria.

The effect of the conjugated bile acid (BA) on the microbial internal pH (pHin) values in lactic acid bacteria with and without ability to hydrolyze bile salts (BSH[+] and BSH[-] strains, respectively) was evaluated. BSH(+) strains showed a gradual increase in the pHin following the addition of conjugated BA; this behavior was more pronounced with GDCA than with TDCA may be due to the higher affinity of BSH for the glyco-conjugates acids. Conversely, the BSH(-) strains showed a decrease in internal pH probably as a consequence of weak acid accumulation. As expected, a decrease in the cytoplasmatic pH affected the cell survival in this last group of strains, while the BSH(+) strains were more resistant to the toxic effect of BA. PURPOSE OF WORK: To evaluate bile salt hydrolase activities, changes in the internal pH and cell survival to bile acids in lactic acid bacteria to establish the relationship between these parameters.

Screening for glycosidase activities of lactic acid bacteria as a biotechnological tool in oenology.

The aim of this study was to evaluate the ability from a number of lactic acid bacteria isolated from different sources to produce glycosidase enzymes. Representative isolates (225) from clusters obtained after genotyping, using randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) analysis, of 1,464 isolates, were screened for ß-D-glucosidase activity. Thirty-five of them were selected for subsequent analysis. These strains were able to hydrolyze α-D-glucopyranoside, ß-D-xylopyranoside and α-L-arabinofuranoside although ß-D-glucosidase activity was the predominant activity for 22 of the selected strains. Only some of them did so with α-L-rhamnopyranoside. All of these were from wine samples and were identified as belonging to the Oenococcus oeni species using Amplification and Restriction Analysis of 16S-rRNA gene (16S-ARDRA). When the influence of pH, temperature and ethanol or sugars content on ß-D-glucosidase activity was assayed, a strain-dependent response was observed. The ß-D-glucosidase activity occurred in both whole and sonicated cells but not in the supernatants from cultures or obtained after cell sonication. Strains 10, 17, 21, and 23 retained the most ß-D-glucosidase activity when they were assayed at the conditions of temperature, pH, ethanol and sugar content used in winemaking. These results suggest that these strains could be used as a source of glycosidase enzymes for use in winemaking.

An overview of functional genomics and relevance of glycosyltransferases in exopolysaccharide production by lactic acid bacteria.

There are many reports on exopolysaccharides of lactic acid bacteria (LAB EPS) such as isolation, production and applications. The LAB EPS have been proved to exhibit significantly improved texture and rheological properties in order to prevent syneresis of fermented foods. Furthermore, they are known to have many biological properties such as mouthwatering flavors, antioxidant activity, cholesterol lowering and antimicrobial activities. Considering their GRAS status, LAB EPS need to be explored for better titre and improved biological properties, where strain improvement by genetic engineering has a major role for making tailor-made EPS. The genetic overview of the EPS production by LAB is an auxiliary area of interest as the process and the biosynthetic pathway involves numerous genes and their proteins. Among them Glycosyltransferases (gtfs) are the key enzymes involved in EPS biosynthesis. Current knowledge of gtfs of LAB and its manipulation is limited. The present review spotlights the importance of glycosyltransferases and their specific role on the biosynthesis of LAB EPS and addresses the functionality and applicability of these enzymes and their products. It enfold the available literature including some patents in recent past to underline the fact that glycosyltransferases are un-reluctantly the key proteins involved in the EPS biosynthesis.

Integrated approach for obtaining bioactive peptides from whey proteins hydrolysed using a new proteolytic lactic acid bacteria.

In this study, we investigated the ability of Enterococcus faecalis 2/28, isolated from artisan cheese, to release biopeptides from whey proteins. We used an in silico approach for predicting the bioactivities of peptides generated by E. faecalis. The results of the in vitro study showed that the whey protein hydrolysates (WPHs) obtained had angiotensin-I-converting enzyme (ACE) and dipeptidyl peptidase IV (DPP-IV) inhibitory activities, with inhibition of ACE being stronger than that of DPP-IV. To identify peptides that may be potential inhibitors of ACE, WPH with the highest ACE inhibitory activity was analysed using Sephadex G-75 gel filtration chromatography, Superdex peptide 10/300 GL size exclusion chromatography, and liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS). Among the identified peptides were ACE-inhibitory peptides (LDAQSAPLR, LKGYGGVSLPEW, and LKALPMH), antimicrobial peptides (AASDISLLDAQSAPLR, IIAEKTKIPAVF, IDALNENK, and VLVLDTDYK), DPP-IV-inhibitory peptides (LKALPMH, LKPTPEGDLEIL, LKGYGGVSLPE, LKPTPEGDLE, ILDKVGINY, and VLVLDTDYK), proliferation stimulating peptide (IDALNENK), and cytotoxic peptide (LIVTQTMK).