Basic characterization and partial purification of ß-glucosidase from cell-free extracts of Oenococcus oeni ST81.

AIMS: This study was designed to characterize a ß-glucosidase of Oenococcus oeni ST81, a strain isolated from a Spanish wine of the origin appellation Ribeira Sacra. METHODS AND RESULTS: The ß-glucosidase of O. oeni ST81 seems to have a periplasmic localization into the cells. This activity was strongly inhibited by gluconic acid, partially inhibited by glucose and not inhibited by fructose, lactate, malate, mannitol or sorbitol. Ethanol increased the activity of this enzyme up to 147%. Among the several metal ions assayed, only Fe²âº (10 mmol l⁻¹) and Cu²âº (5 mmol l⁻¹) exhibited a partial inhibitory effect (40%). This enzyme was partially purified using a combination of ammonium sulfate precipitation and chromatographic methods. The single peak because of ß-glucosidase in all chromatographic columns indicates the presence of a single enzyme with an estimated molecular mass of 140 kDa. The calculated K(m) and V(max) values for 4-nitrophenyl-ß-D-glucopyranoside were 0·38 mmol l⁻¹ and 5·21 nmol min⁻¹, respectively. The enzyme was stable at pH 5·0 with a value of t(1/2) = 50 days for the crude extract. CONCLUSIONS: The ß-glucosidase of O. oeni ST81 is substantially different from those characterized from other wine-related lactic acid bacteria (LAB), such as Lactobacillus plantarum and Lactobacillus brevis; however, it appears to be closely related to a ß-glucosidase from O. oeni ATCC BAA-1163 cloned into Escherichia coli. The periplasmic localization of the enzyme together with its high tolerance to ethanol and fructose, the low inhibitory effect of some wine-related compounds on the enzymatic activity and long-term stability of the enzyme could be of interest for winemaking. SIGNIFICANCE AND IMPACT OF THE STUDY: Information regarding a ß-glucosidase from O. oeni ST81 is presented. Although the release of aroma compounds by LAB has been demonstrated, little information exists concerning the responsible enzymes. To our knowledge, this study contains the first characterization of a native ß-glucosidase purified from crude extracts of O. oeni ST81.

Low-salt restructured fish products from Atlantic mackerel (Scomber scombrus) with texture resembling turkey breast.

Atlantic mackerel (Scomber scombrus) is a pelagic and migratory species that is usually caught with other fish as bycatch. The aim of this work was to obtain low-salt restructured fish products from Atlantic mackerel resembling turkey breast using transglutaminase (0.2 U/g) as binder. NaCl concentration (0-20 g/kg), temperature (25-40 °C) and time of incubation (30-90 min) were assayed. The texture parameters (Warner-Bratzler force and Warner-Bratzler work) and expressible water were compared to those of turkey breast. Mathematical models were obtained to determine the effect of these variables on the texture of Atlantic mackerel restructured products. Optimal conditions to obtain a similar texture than turkey breast were found. The overall optimization point out that the treatment at 31.8 °C for 63.35 min using a NaCl concentration of 8.45 g/kg allowed to obtain restructured products from Atlantic mackerel with texture and expressible water similar to those of turkey breast. Color parameters (L*, a* and b*) of the product were also similar to those of turkey breast. The results showed the feasibility of producing low-salt restructured products from Atlantic mackerel resembling turkey breast using transglutaminase.

Characterization of lactic acid bacteria from musts and wines of three consecutive vintages of Ribeira Sacra.

AIMS: This study was designed to isolate and characterize the lactic acid microbiota of the musts and wines of a young denomination of origin area, Ribeira Sacra in north-west Spain. METHODS AND RESULTS: Over three consecutive years (2007, 2008 and 2009), we examined musts and wines from four cellars in different zones of the region. Through biochemical and genetic tests, 459 isolates of lactic acid bacteria (LAB) were identified as the following species: Lactobacillus alvei (0·7%), Lactobacillus brevis (1·7%), Lactobacillus frumenti (0·9%), Lactobacillus kunkeei (12%), Lactobacillus plantarum (6·5%), Lactobacillus pentosus (0·9%), Lactococcus lactis ssp. lactis (3%), Leuconostoc citreum (0·7%), Leuconostoc fructosum (synon. Lactobacillus fructosum) (3·7%), Leuconostoc mesenteroides ssp. mesenteroides (2·8%), Leuconostoc pseudomesenteroides (0·2%), Oenococcus oeni (59%), Pediococcus parvulus (7%) and Weisella paramesenteroides (synon. Leuconostoc paramesenteroides) (0·9%). Of these species, O. oeni was the main one responsible for malolactic fermentation (MLF) in all cellars and years with the exception of Lact. plantarum, predominant in 2007, in one cellar, and Lact. brevis, Lact. frumenti and Ped. parvulus coexisting with O. oeni in one cellar in 2009. Different strains (84) of LAB species (14) were identified by biochemical techniques (API strips, the presence of plasmids, enzyme activities and MLF performance) and molecular techniques (PCR). All assays were carried out with every one of the 459 isolates. To select candidates for use as culture starters, we assessed malolactic, ß-glucosidase and tannase activities, the presence of genes involved in biogenic amine production and plasmid content. CONCLUSIONS: A high diversity of LAB is present in the grape musts of Ribeira Sacra but few species are responsible for MLF; however, different strains of such species are involved in the process. As far as we are aware, this is the first report of Lact. frumenti thriving in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on LAB populations in must and wine is presented. A large collection of well-characterized strains of LAB are available as starter cultures to winemakers.

Nucleotide sequence analysis of pRS2 and pRS3, two small cryptic plasmids from Oenococcus oeni.

Nucleotide sequence analysis of two cryptic plasmids, pRS2 (2544 bp) and pRS3 (3948 bp), from Oenococcus oeni revealed the presence in both of three major open reading frames with significant similarity to other small cryptic plasmids from O. oeni. The results suggest that those plasmids could be separated into two subfamilies, one represented by pLo13 and pRS3, the other represented by pOg32, pRS1, and pRS2.

Nucleotide sequence analysis of pRS1, a cryptic plasmid from Oenococcus oeni.

A new cryptic plasmid, pRS1, from an Oenococcus oeni strain isolated from Spanish wines is reported. Nucleotide sequence analysis (2523 bp) revealed the presence of three major open reading frames (ORFs) whose nucleotide sequence and encoded proteins exhibit high homology with those of pOg32, a previously described plasmid of O. oeni. Common features in other plasmids from O. oeni (i.e., pLo13 and pOg32) have been found in pRS1. They have three major ORFs in the same strand; the putative encoded proteins by two of these ORFs exhibit homology with the replication (Rep) and the recombination (Pre) proteins, respectively, of the pT181 plasmid family and related gram-positive bacteria plasmids; these plasmids contain the DNA sequences required for plasmid replication by the rolling circle mechanism and for recombination (i.e., double-strand origin, DSO; single-strand origin, SSO; recombination-specific sites, RSA and RSB); and finally, all these plasmids have a third ORF of unknown function. These features suggest that pRS1 could constitute together with pLo13 and pOg32 a family of small cryptic plasmids of O. oeni.

Cloning of Frankia species putative tRNA(Pro) genes and their efficacy for pSAM2 site-specific integration in Streptomyces lividans.

pSAM2 is a conjugative Streptomyces ambofaciens mobile genetic element that can transfer and integrate site specifically in the genome. The chromosomal attachment site (attB) for pSAM2 site-specific recombination for two Frankia species was analyzed. It overlaps putative proline tRNA genes having a 3'-terminal CCA sequence, an uncommon feature among actinomycetes. pSAM2 is able to integrate into a cloned Frankia attB site harbored in Streptomyces lividans. The integration event removes the 3'-terminal CCA sequence and introduces a single nucleotide difference in the T psi C loop of the putative Frankia tRNA(Pro) gene. Major differences between the attP sequence from pSAM2 and the Frankia attB sequence restrict the identity segment to a 43-bp-long region. Only one mismatch is found between these well-conserved att segments. This nucleotide substitution makes a BstBI recognition site in Frankia attB and was used to localize the recombination site in a 25-bp region going from the anticodon to the T psi C loop of the tRNA(Pro) sequence. Integration of pSAM2 into the Frankia attB site is the first step toward introduction of pSAM2 derivatives into Frankia spp.

Induction of DNA amplification in the Bacillus subtilis chromosome.

A system allowing the induction of DNA amplification in Bacillus subtilis was developed, based on a thermosensitive plasmid, pE194, stably integrated in the bacterial chromosome. An amplification unit, comprising an antibiotic resistance marker flanked by directly repeated sequences, was placed next to the integrated plasmid. Activation of pE194 replication led to DNA amplification. Two different amplification processes appeared to take place: one increased the copy number of all sequences in the vicinity of the integrated plasmid and was possibly of the onion skin type, while the other increased the copy number of the amplification unit only and generated long arrays of amplification units. These arrays were purified and shown to consist mainly of directly repeated amplification units but to also contain non-linear regions, such as replication forks and recombination intermediates. They were attached to the chromosome at one end only, and were, in general, not stably inherited, which suggests that they are early amplification intermediates. Longer arrays were detected before the shorter ones during amplification. When the parental amplification unit contained repeats which differed by a restriction site the arrays which derived thereof contained in a majority of cases only a single type of repeat. We propose that the amplified DNA is generated by rolling circle replication, and that such a process might underlie a number of amplification events.

Hypersecretion of a cellulase from Clostridium thermocellum in Bacillus subtilis by induction of chromosomal DNA amplification.

We have inserted a DNA fragment composed of (i) the promoter and the export signal of the Bacillus subtilis levansucrase gene; (ii) the sequence encoding the mature part of the Clostridium thermocellum endoglucanase A gene in a specific site of the B. subtilis chromosome. The insert was flanked by directly repeated pBR322 sequences of 3.9 kb. Plasmid pE194, which has a thermosensitive replication, was integrated adjacent to one of the repeats. When the integrated plasmid was allowed to replicate, the insert and one of the repeats was amplified up to a level of about 250 copies per chromosome. Endoglucanase A was efficiently synthesized in, and secreted from, cells containing the amplified structure, since the heterologous fusion protein was the major extracellular protein in a B. subtilis sacUh strain. The NH2-terminal sequence of the secreted protein revealed three different cleavage sites in the vicinity of the signal peptidase recognition sequence.

Characterization and partial purification of L-asparaginase from Corynebacterium glutamicum.

A high L-asparaginase (L-asparagine amidohydrolase: EC 3.5.1.1) activity was found under conditions of lysine overproduction in cultures of Corynebacterium glutamicum. L-Asparaginase was purified 98-fold by protamine sulphate precipitation. DEAE-Sephacel anion exchange, ammonium sulphate precipitation and Sephacryl S-200 gel filtration. The asparaginase protein was subjected to PAGE under non-denaturing conditions, identified by an in situ reaction and eluted from the gel in an active form. The estimated Mr from gel filtration and SDS-PAGE was 80,000. The L-asparaginase activity was inhibited by the L-asparagine analogue 5-diazo-4-oxo-L-norvaline. Neither D-asparagine nor L-glutamine was a substrate for the enzyme. L-Asparaginase was produced constitutively: its role may be that of an overflow enzyme, converting excess asparagine into aspartic acid, the direct precursor of lysine and threonine.