Production of beta-galactosidase from Streptococcus thermophilus grown in whey.

beta-D-Galactosidase (EC 3.2.1.23) was extracted from Streptococcus thermophilus grown in deproteinized cheese whey. Cultural conditions optimum for maximum enzyme production were pH 7.0, 40 degrees C, and 24 h. Proteose peptone (2.0%, wt/vol) and corn steep liquor (2.8%, wt/vol) were highly stimulatory, increasing the enzyme units available in their absence from 660 U/liter of medium to 18,200 and 10,000 U/liter of medium, respectively, in their presence. There was an insignificant increase in the production of enzyme in the presence of added inorganic nitrogen and phosphorus sources. Enzymatic hydrolysis for recuction of lactose content in aqueous solution and in skim milk was studied.     

Production of polyhydroxyalkanoates from whey by Thermus thermophilus HB8

The thermophilic bacterium Thermus thermophilus HB8 is able to utilize lactose from whey-based media for the biosynthesis of polyhydroxyalkanoates (PHAs) under nitrogen limitation. T. thermophilus can utilize both, glucose and galactose, the products of lactose hydrolysis. When T. thermophilus HB8 was grown in culture media containing 24% (v/v) whey, PHA was accumulated up to 35% (w/w) of its biomass after 24 h of cultivation. The effect of initial phosphate concentration on the PHA production was also investigated. Using an initial phosphate concentration of 50 mM the PHA accumulation was enhanced. Analysis of the produced PHA from T. thermophilous HB8 grown in whey-based media revealed a novel heteropolymer consisting of the short chain length 3-hydroxyvalerate (3HV; 38 mol%) and the medium chain length, 3-hydroxyheptanoate (3HHp; 9.89 mol%), 3-hydroxynanoate (3HN; 16.59 mol%) and 3-hydroxyundecanoate (3HU; 35.42 mol%). Despite the low molecular weight of the produced PHA by T. thermophilus, whey could be an excellent substrate for the production of heteropolymers with unique properties.     

Small-scale analysis of exopolysaccharides from Streptococcus thermophilus grown in a semi-defined medium

Background  

Exopolysaccharides (EPSs) produced by lactic acid bacteria are important for the texture of fermented foods and have received a great deal of interest recently. However, the low production levels of EPSs in combination with the complex media used for growth of the bacteria have caused problems in the accurate analysis of the EPS. The purpose of this study was to find a growth medium for physiological studies of the lactic acid bacterium Streptococcus thermophilus, and to develop a simple method for qualitative and quantitative analysis of EPSs produced in this medium.     

Hydrolysis of whey proteins by Lactobacillus acidophilus, Streptococcus thermophilus and Lactobacillus delbrueckii ssp. bulgaricus grown in a chemically defined medium

AIMS: To evaluate the ability of themophilic lactic acid bacteria (LAB) to hydrolyse the whey proteins beta-lactoglobulin (BLG) and alpha-lactalbumin (ALA) in a chemically defined medium (CDM). METHODS AND RESULTS: The ability of three LAB strains to hydrolyse BLG and ALA was studied in a CDM supplemented with these proteins or whey protein concentrate (WPC). Protein hydrolysis was determined by Tricine/SDS-PAGE and RP-HPLC. Maximum BLG (21%) and ALA (26%) degradation by LAB was observed using WPC. Under starving conditions, BLG degradation was greater for Lactobacillus delbrueckii ssp. bulgaricus CRL 454 than for Lactobacillus acidophilus CRL 636 and Streptococcus thermophilus CRL 804. All three strains showed different peptide profiles and were not able to hydrolyse ALA under starvation. CONCLUSIONS: The assayed LAB strains were able to degrade BLG during growth in a CDM and under starving conditions. The different peptide profiles obtained indicate distinct protease specificities. SIGNIFICANCE AND IMPACT OF THE STUDY: These strains could be used as adjunct cultures to increase BLG digestibility in whey-based or whey-containing foods. To our knowledge, this is the first report on the ability of a Lact. acidophilus strain to degrade BLG.     

Production by and isolation of exopolysaccharides from Streptococcus thermophilus grown in a milk medium and evidence for their growth-associated biosynthesis

Four Streptococcus thermophilus strains ( Strep. thermophilus BTC, Strep. thermophilus LY03, Strep. thermophilus 480 and Strep. thermophilus Sfi20) have been examined for their exopolysaccharide production capacity. All strains produced a polymer composed of the neutral sugars glucose and galactose, but in different ratios. It was clearly shown that the biosynthesis of exopolysaccharides from Strep. thermophilus LY03 is growth-associated and hence displays primary metabolite kinetics. The monomer ratio of the exopolysaccharide synthesized did not vary throughout the fermentation cycle. The production kinetics and exopolysaccharide yields were strongly dependent on the fermentation conditions. Physical factors such as temperature, pH and oxygen tension as well as chemical factors (medium composition, initial lactose concentration, carbon/nitrogen levels) were of utmost importance.     

Galactokinase activity in Streptococcus thermophilus.

ATP-dependent phosphorylation of [14C]galactose by 11 strains of Streptococcus thermophilus indicated that these organisms possessed the Leloir enzyme, galactokinase (galK). Activities were 10 times higher in fully induced, galactose-fermenting (Gal+) strains than in galactose-nonfermenting (Gal-) strains. Lactose-grown, Gal- cells released free galactose into the medium and were unable to utilize residual galactose or to induce galK above basal levels. Gal+ S. thermophilus 19258 also released galactose into the medium, but when lactose was depleted growth on galactose commenced, and galK increased from 0.025 to 0.22 micromol of galactose phosphorylated per min per mg of protein. When lactose was added to galactose-grown cells of S. thermophilus 19258, galK activity rapidly decreased. These results suggest that galK in Gal+ S. thermophilus is subject to an induction-repression mechanism, but that galK cannot be induced in Gal- strains.     

Galactose transport in Streptococcus thermophilus.

Although Streptococcus thermophilus accumulated [14C]lactose in the absence of an endogenous energy source, galactose-fermenting (Gal+) cells were unable to accumulate [14C]galactose unless an additional energy source was added to the test system. Both Gal+ and galactose-nonfermenting (Gal-) strains transported galactose when preincubated with sucrose. Accumulation was inhibited 50 or 95% when 10 mM sodium fluoride or 1.0 mM iodoacetic acid, respectively, was added to sucrose-treated cells, indicating that ATP was required for galactose transport activity. Proton-conducting ionophores also inhibited galactose uptake, although N,N'-dicyclohexyl carbodiimide had no effect. The results suggest that galactose transport in S. thermophilus occurs via an ATP-dependent galactose permease and that a proton motive force is involved. The galactose permease in S. thermophilus TS2b (Gal+) had a Km for galactose of 0.25 mM and a Vmax of 195 micromol of galactose accumulated per min per g (dry weight) of cells. Several structurally similar sugars inhibited galactose uptake, indicating that the galactose permease had high affinities for these sugars.     

Enhanced Exopolysaccharide Production by Metabolic Engineering of Streptococcus thermophilus

It is possible that the low levels of production of exopolysaccharides (EPSs) by lactic acid bacteria could be improved by altering the levels of enzymes in the central metabolism that influence the production of precursor nucleotide sugars. To test this hypothesis, we identified and cloned the galU gene, which codes for UDP glucose pyrophosphorylase (GalU) in Streptococcus thermophilus LY03. Homologous overexpression of the gene led to a 10-fold increase in GalU activity but did not have any effect on the EPS yield when lactose was the carbon source. However, when galU was overexpressed in combination with pgmA, which encodes phosphoglucomutase (PGM), the EPS yield increased from 0.17 to 0.31 g/mol of carbon from lactose. A galactose-fermenting LY03 mutant (Gal⁺) with increased activities of the Leloir enzymes was also found to have a higher EPS yield (0.24 g/mol of carbon) than the parent strain. The EPS yield was further improved to 0.27 g/mol of carbon by overexpressing galU in this strain. However, the highest EPS yield, 0.36 g/mol of carbon, was obtained when pgmA was knocked out in the Gal⁺ strain. Measurements of the levels of intracellular metabolites in the cultures revealed that the Gal⁺ strains had considerably higher glucose 1-phosphate levels than the other strains, and the strain lacking PGM activity had threefold-higher levels of glucose 1-phosphate than the other Gal⁺ strains. These results show that it is possible to increase EPS production by altering the levels of enzymes in the central carbohydrate metabolism.     

Cloning the gene of beta-galactosidase from the industrial strain of Streptococcus lactis 111 in E. coli cells and conjugated transfer of this gene to Streptococcus thermophilus cells

The ability of the industrial strains of Streptococcus lactis to synthesize the enzyme beta-galactosidase was studied. Five strains among sixteen were found to produce high levels of the enzyme. The beta-galactosidase gene in the most active strain Streptococcus lactis 111 was shown to be located on the 50 kb conjugative plasmid. The plasmid was transferred by conjugation into Streptococcus thermophilus cells and subsequently the gene for beta-galactosidase was studied in transconjugants. The beta-galactosidase gene from Streptococcus lactis 111 was subcloned in Escherichia coli cells on the plasmid pBR322. The gene was localized on the 4.8 kb BgIII fragment of DNA. Following the restriction of DNA by the Sau3A the gene was subcloned on the birepliconed plasmid vector pCB20 capable of replication in the Gram-negative as well as Gram-positive microorganisms. The recombinant derivatives of pCB20 were isolated that carry the beta-galactosidase gene on the DNA fragments of different size.     

The activity of the lactose transporter from Streptococcus thermophilus is increased by phosphorylated IIA and the action of beta-galactosidase

The metabolism of lactose by Streptococcus thermophilus is highly regulated, allowing the bacterium to prefer lactose over glucose as main source of carbon and energy. In vitro analysis of the enzymes involved in transport and hydrolysis of lactose showed that the transport reaction benefits from the hydrolysis of lactose at the trans side of the membrane. Furthermore, the activity of LacS is modulated by PEP-dependent phosphorylation of the IIA domain via the general energy coupling proteins of the PTS, Enzyme I and HPr. To determine whether unphosphorylated LacS-IIA inhibited, or the phosphorylated form stimulated lactose counterflow, a LacS-IIA truncation mutant of LacS was constructed. Detailed analyses of transport in whole cells and in proteoliposomes indicated that unphosphorylated LacS-IIA does not functionally interact with the carrier domain. Instead, interaction of the phosphorylated form of LacS-IIA with the carrier stimulates lactose counterflow transport. The proposed mode of regulation thus proceeds via a mechanism opposite to the inducer exclusion type of regulation in gram-negative bacteria, where transporters are inhibited by binding of the unphosphorylated form of IIA(Glc).     

Production of β-galactosidase fromkluyveromyces fragilis grown on whey

Optimum conditions for β-galactosidase production byK. fragilis were studied. Enzyme production has a maximum after 8 –12 h of incubation. Composition of whey (from different sources) did not affect enzyme production. Different heat treatments also had no effect. Whey reconstituted to 8 –12 % total solids and adjusted to pH 4.0 afforded maximum enzyme production. Whereas inorganic nitrogen sources (specially ammonium salts) only slightly stimulated enzyme production, organic nitrogen sources (specially partially digested proteins) provided a nearly four-fold increase in enzyme production. Yeast extract and beef extract and industrial by-products like corn-steep liquor significantly stimulated enzyme production. Manganese and magnesium salts had a very little stimulation effect.     

Cloning of the Streptococcus thermophilus beta-galactosidase gene and its expression in Escherichia coli and Bacillus subtilis cells

The beta-galactosidase gene from the chromosome of Streptococcus thermophilus, strain 6 kb, has been cloned on a vector plasmid pBR322. The corresponding gene has been found to be located on the Pst1 DNA fragment. The restriction map of this 6 kb fragment has been constructed. The shortening of the DNA fragment carrying the beta-galactosidase gene has been achieved by digestion of the recombinant derivative of pBR322 by the restriction endonuclease Sau3A under the conditions of incomplete hydrolysis. The obtained fragments have been cloned into the BamHI site in the berepliconed shuttle vector pCB20 for grampositive and gramnegative bacteria. The obtained recombinant plasmids contained the beta-galactosidase gene in the inserted fragments of different length. Expression of the cloned beta-galactosidase gene in Escherichia coli and Bacillus subtilis cells has been studied.     

Characterization of superoxide dismutase in Streptococcus thermophilus.

Streptococcus thermophilus AO54 possesses a single manganese-containing superoxide dismutase (MnSOD). The enzyme was found to be insensitive to cyanide or to a modified H2O2 treatment. The enzyme is expressed in a growth-phase-dependent fashion, increasing three- to fourfold upon entry into stationary phase. The specific activity for MnSOD was the same under anaerobic or aerobic conditions and was not induced by the presence of paraquat under aerobic conditions.     

Isolation and partial characterization of plasmid DNA from Streptococcus thermophilus.

Of 23 strains of Streptococcus thermophilus examined, 5 were found to contain a single small cryptic plasmid, designated pHM1 through pHM5. Through analysis by restriction endonuclease mapping and DNA-DNA hybridization, the five plasmids were found to be closely related. They were present in 4 to 18 copies per cell and ranged in size from 1.4 to 2.2 megadaltons. Plasmids pHM1 and pHM5, as well as pHM2 and pHM4, were found to be identical. Single restriction endonuclease sites were observed on each plasmid for PvuII and MboI. Plasmids pHM2 and pHM3 each had an additional single site for HhaI, and pHM1 had an additional single site for HindIII. The characteristics of these plasmids may make them useful for the development of cloning vectors for use in S. thermophilus.     

Phosphofructokinases from Lactobacteriaceae. II. Purification and properties of phosphofructokinase from Streptococcus thermophilus

Phosphofructokinase (ATP : D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from Streptococcus thermophilus has been purified. It is a tetramer composed of identical subunits of molecular weight 36 000 and exhibits Michaelis-Menten kinetics. Compared to the phosphofructokinases from taxonomically related bacteria, the enzyme from S. thermophilus is more stable at high temperatures. In addition, it has been demonstrated that the phosphofructokinases from lactobacteria and also from Bacillus stearothermophilus show immunologic cross-reaction. In spite of the significantly different kinetic properties and the different thermostability of these enzymes, this finding indicates great structural resemblance.     

Characterization of a theta-replicating plasmid from Streptococcus thermophilus

Plasmids of Streptococcus thermophilus were previously classified, based on DNA homology, into at least four groups (A-D). Here, we report the characterization of plasmids of group B and D. The sequence analysis of pSMQ173b (group D) indicates that this plasmid contains 4449 bp, five open reading frames (ORFs) and replicates via the rolling-circle mechanism of the pGI3 family. The plasmid pSMQ308 (group B) contains 8144 bp and six ORFs. Two ORFs likely encode a primase/helicase and an integrase. Northern blot experiments demonstrate that these two orfs are transcribed within the three strains containing plasmids of group B. Two-dimensional agarose gel electrophoresis shows that pSMQ308 replicates via a theta mechanism. To our knowledge, this is the first report of a plasmid replicating via a theta mode in S. thermophilus. Finally, a classification of 20 sequenced S. thermophilus plasmids into six groups based on their mode of replication is proposed.     

Analysis of the lacZ sequences from two Streptococcus thermophilus strains: comparison with the Escherichia coli and Lactobacillus bulgaricus beta-galactosidase sequences

The lacZ gene from Streptococcus thermophilus A054, a commercial yogurt strain, was cloned on a 7.2 kb PstI fragment in Escherichia coli and compared with the previously cloned lacZ gene from S. thermophilus ATCC 19258. Using the dideoxy chain termination method, the DNA sequences of both lacZ structural genes were determined and found to be 3071 bp in length. When the two sequences were more closely analysed, 21 nucleotide differences were detected, of which only nine resulted in amino acid changes in the proteins, the remainder occurring in wobble positions of the respective codons. Only three bases separated the termination codon for the lacS gene from the initiation codon for lacZ, suggesting that the lactose utilization genes are organized as an operon. The amino acid sequence of the beta-galactosidase, derived from the DNA sequence, corresponds to a protein with a molecular mass of 116860 Da. Comparison of the S. thermophilus amino acid sequences with those from Lactobacillus bulgaricus, E. coli and Klebsiella pneumoniae showed 48, 35 and 32.5% identity respectively. Although little sequence homology was observed at the DNA level, many regions conserved in the amino acid sequence were identified when the beta-galactosidase proteins from S. thermophilus, E. coli and L. bulgaricus were compared.