Biochemical and phylogenetic analyses of a cold-active beta-galactosidase from the lactic acid bacterium Carnobacterium piscicola BA

We are investigating glycosyl hydrolases from new psychrophilic isolates to examine the adaptations of enzymes to low temperatures. A beta-galactosidase from isolate BA, which we have classified as a strain of the lactic acid bacterium Carnobacterium piscicola, was capable of hydrolyzing the chromogen 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-Gal) at 4 degrees C and possessed higher activity in crude cell lysates at 25 than at 37 degrees C. Sequence analysis of a cloned DNA fragment encoding this activity revealed a gene cluster containing three glycosyl hydrolases with homology to an alpha-galactosidase and two beta-galactosidases. The larger of the two beta-galactosidase genes, bgaB, encoded the 76.8-kDa cold-active enzyme. This gene was homologous to family 42 glycosyl hydrolases, a group which contains several thermophilic enzymes but none from lactic acid bacteria. The bgaB gene from isolate BA was subcloned in Escherichia coli, and its enzyme, BgaB, was purified. The purified enzyme was highly unstable and required 10% glycerol to maintain activity. Its optimal temperature for activity was 30 degrees C, and it was inactivated at 40 degrees C in 10 min. The K(m) of freshly purified enzyme at 30 degrees C was 1.7 mM, and the V(max) was 450 micromol. min(-1). mg(-1) with o-nitrophenyl beta-D-galactopyranoside. This cold-active enzyme is interesting because it is homologous to a thermophilic enzyme from Bacillus stearothermophilus, and comparisons could provide information about structural features important for activity at low temperatures.     

Photobacterium sp. JT-ISH-224 produces two sialyltransferases, alpha-/beta-galactoside alpha2,3-sialyltransferase and beta-galactoside alpha2,6-sialyltransferase

A novel bacterium, Photobacterium sp. JT-ISH-224, that produces alpha-/beta-galactoside alpha2,3-sialyltransferase and beta-galactoside alpha2,6-sialyltransferase, was isolated from the gut of a Japanese barracuda. The genes that encode the enzymes were cloned from the genomic library of the bacterium using the genes encoding alpha-/beta-galactoside alpha2,3-sialyltransferase from P. phosphoreum and beta-galactoside alpha2,6-sialyltransferase from P. damselae as probes. The nucleotide sequences were determined, and open reading frames of 1,230 and 1,545 bp for encoding an alpha2,3-sialyltransferase and an alpha2,6-sialyltransferase of 409- and 514-amino acid residues, respectively, were identified. The alpha2,3-sialyltransferase had 92% amino acid sequence identity with the P. phosphoreum alpha2,3-sialyltransferase, whereas the alpha2,6-sialyltransferase had 54% amino acid sequence identity with the P. damselae alpha2,6-sialyltransferase. For both enzymes, the DNA fragments that encoded the full-length protein and its truncated form lacking the putative signal peptide sequence were amplified by a polymerase chain reaction and cloned into an expression vector. Each gene was expressed in Escherichia coli, and the lysate from each strain had enzymatic activity. The alpha2,3-sialyltransferase catalysed the transfer of N-acetylneuraminic acid (NeuAc) from CMP-NeuAc to lactose, alpha-methyl-galactopyranoside and beta-methyl-galactopyranoside with low apparent K(m) and the alpha2,6-sialyltransferase catalysed the transfer of NeuAc from CMP-NeuAc to lactose with low apparent K(m).     

Rat small-intestinal galactosidases. Separation by ion-exchange chromatography and gel filtration

1. The chromatography of rat small-intestinal beta-galactosidase activities on gel-filtration and ion-exchange columns has been studied. Five different substrates were used to measure beta-galactosidase activity (lactose, phenyl beta-galactoside, o-nitrophenyl beta-galactoside, p-nitrophenyl beta-galactoside and 6-bromo-2-naphthyl beta-galactoside) and the activity was measured at one acid and one more neutral pH value. 2. By gel filtration one acid beta-galactosidase, hydrolysing lactose and the hetero-beta-galactosides at about the same rate, and one more neutral beta-galactosidase, hydrolysing lactose much more rapidly than the hetero-beta-galactosides, were separated. 3. By ion-exchange chromatography the acid enzyme was fractionated into two components. These may be individual enzymes or different forms of the same enzyme.     

Cloning of alpha- and beta-galactosidase genes from an extreme thermophile, Thermus strain T2, and their expression in Thermus thermophilus HB27.

The genes encoding thermostable alpha- and beta-galactosidases from an extremely thermophilic bacterium, Thermus strain T2, were cloned in Escherichia coli. The alpha-galactosidase gene was located just downstream from the beta-galactosidase gene. The genes were introduced into Thermus thermophilus HB27 with the aid of Thermus cryptic plasmid pTT8, and beta-galactosidases were expressed constitutively.     

Characterization of beta-galactosidase from a special strain of Aspergillus oryzae.

beta-Galactosidase [EC 3.2.1.23] was isolated from a partially purified preparation obtained from cultured cells of a special strain of Aspergillus oryzae, RT 102 (FERM-P1680). The enzyme preparation gave a single protein band on polyacrylamide gel electrophoresis and was free from alpha-galactosidase, alpha- and beta-mannosidase, alpha- and beta-N-acetylhexosaminidase, and protease activities. The beta-galactosidase was capable of acting on aryl beta-galactosides, lactose, and lactosides. It also hydrolyzed beta-galactosyl linkages in urinary glycoasparagines and asialo alpha1-acid glycoprotein. The enzyme was rather stable in aqueous solution, retaining full activity at 4 degrees for at least several months. At pH 4.5, the optimum pH for the enzyme activity, and 37 degrees, full activity was maintained for several days.     

Rat small-intestinal β-galactosidases. Kinetic studies with three separated fractions

1. Three fractions of beta-galactosidase activity from the rat small-intestinal mucosa were separated chromatographically. Two of these fractions had an acid pH optimum at 3-4, and the third one had a more neutral pH optimum at 5.7. 2. The two ;acid' beta-galactosidase fractions had considerably lower K(m) values for hetero beta-galactosides than for lactose. The V(max.) values were similar for all the substrates used (lactose, phenyl beta-galactoside, o-nitrophenyl beta-galactoside, p-nitrophenyl beta-galactoside and 6-bromo-2-naphthyl beta-galactoside). No difference could be detected between the two ;acid' fractions with respect to their enzymic properties (pH optimum, K(m) for the different substrates, K(i) for lactose as an inhibitor of the hydrolysis of hetero beta-galactosides, K(i) for phenyl beta-galactoside as an inhibitor of the hydrolysis of lactose, and relative V(max.) for the hydrolysis of different substrates). These two fractions probably represent different forms of the same enzyme. 3. The ;neutral' fraction had similar K(m) values for all the substrates hydrolysed, but with lactose as substrate the V(max.) was much higher than with the hetero beta-galactosides. This fraction did not split phenyl beta-galactoside or 6-bromo-2-naphthyl beta-galactoside at a measurable rate. 4. Lactose was a competitive inhibitor of the hetero beta-galactosidase activities of all the three fractions, and K(i) for lactose as an inhibitor in each case was the same as K(m) for the lactase activity. Phenyl beta-galactoside was a competitive inhibitor of the lactase activity of all the three fractions. These facts strongly indicate that in all the three fractions lactose is hydrolysed by the same active sites as the hetero beta-galactosides. 5. Human serum albumin stabilized the separated enzymes against inactivation by freezing and thawing.     

Cloning chromosomal lac genes of Klebsiella pneumoniae

The chromosomal gene for beta-galactosidase from Klebsiella pneumoniae strain T17R1 and associated regulatory genes have been cloned as a 5-kb HindIII fragment in the pBR322 plasmid vector. The beta-galactoside permease gene is not present in a functional form in the 5-kb fragment. The K. pneumoniae genes are expressed in an Escherichia coli host. The synthesis of beta-galactosidase is inducible by isopropyl-beta-D-galactosidase (IPTG) and is sensitive to catabolite repression. There appears to be greater homology between the K. pneumoniae and E. coli structural genes for beta-galactosidase than there is between the respective repressor genes.     

Characterization of a psychrotrophic Arthrobacter gene and its cold-active beta-galactosidase.

Enzymes with high specific activities at low temperatures have potential uses for chemical conversions when low temperatures are required, as in the food industry. Psychrotrophic microorganisms which grow at low temperatures may be a valuable source of cold-active enzymes that have higher activities at low temperatures than enzymes found for mesophilic microorganisms. To find cold-active beta-galactosidases, we isolated and characterized several psychrotrophic microorganisms. One isolate, B7, is an Arthrobacter strain which produces beta-galactosidase when grown in lactose minimal media. Extracts have a specific activity at 30 degrees C of 2 U/mg with o-nitrophenyl-beta-D-galactopyranoside as a substrate. Two isozymes were detected when extracts were subjected to electrophoresis in a nondenaturing polyacrylamide gel and stained for activity with 5-bromo-4-chloro-indolyl-beta-D-galactopyranoside (X-Gal). When chromosomal DNA was prepared and transformed into Escherichia coli, three different genes encoding beta-galactosidase activity were obtained. We have subcloned and sequenced one of these beta-galactosidase genes from the Arthrobacter isolate B7. On the basis of amino acid sequence alignment, the gene was found to have probable catalytic sites homologous to those from the E. coli lacZ gene. The gene encoded a protein of 1,016 amino acids with a predicted molecular mass of 111 kDa. The enzyme was purified and characterized. The beta-galactosidase from isolate B7 has kinetic properties similar to those of the E. coli lacZ beta-galactosidase but has a temperature optimum 20 degrees C lower than that of the E. coli enzyme.     

Heterologous expression of the lactacin F peptides by Carnobacterium piscicola LV17.

The lactacin F complex, composed of LafA and LafX peptides, is produced by Lactobacillus johnsonii VPI 11088 and is active against five other Lactobacillus species and Enterococcus faecalis. The genetic determinants encoding the lactacin F complex are organized in a 1-kb polycistronic operon which comprises three genes, lafA, lafX, and ORFZ (encoding the putative immunity protein). The lafA and lafX genes encode the bacteriocin precursors with N-terminal extensions characterized by a Gly-Gly-1*Xaa+1 cleavage site (*). The Gly-Gly motif is conserved in several other bacteriocins, including carnobacteriocins A, BM1, and B2. Carnobacterium piscicola LV17 produces carnobacteriocins which are active against Listeria monocytogenes and other lactic acid bacteria. In this study, the lactacin F operon was introduced into C. piscicola LV17. The transformants produced lactacin F concurrently with the carnobacteriocins. When the lafA and lafX genes were separated and cloned individually into LV17, production of either LafA or LafX by C. piscicola LV17 was detected by complementation with L. johnsonii clones producing LafX or LafA, respectively. Transformants of C. piscicola LV17 which produced lactacin F, LafA, or LafX, in combination with the carnobacteriocins, were assayed for an increased and expanded inhibitory spectrum. The recombinant organisms were only active against lactacin F- and carnobacteriocin-sensitive strains. A plasmidless derivative of LV17 which does not produce the carnobacteriocins failed to produce lactacin F, LafA, or LafX when transformed with the appropriate recombinant plasmids. The ability of C. piscicola LV17 to produce lactacin F demonstrates that the machinery for the carnobacteriocins is capable of processing and exporting bacteriocins from both systems.     

Molecular cloning of lactose genes in dairy lactic streptococci: the phospho-beta-galactosidase and beta-galactosidase genes and their expression products

The mesophilic (S. lactis and S. cremoris) and thermophilic (S. thermophilus) dairy lactic streptococci, which are used in industrial dairy fermentations, contain two different lactose hydrolysing enzymes, a phospho-beta-galactosidase and a beta-galactosidase. The central role of these enzymes in the pathways used for lactose transport and degradation is discussed along with their properties and distributions in lactic streptococci. In addition, recent results on the cloning, expression and sequence organization of the genes for the mesophilic phospho-beta-galactosidase and thermophilic beta-galactosidase are reviewed. Original data are presented concerning heterologous gene expression in the study of lactose hydrolysis in lactic streptococci. These include 1) the purification of the S. lactis phospho-beta-galactosidase from an overproducing Escherichia coli, and 2) the expression of the E. coli beta-galactosidase (lacZ) gene in S. lactis employing a lactic streptococcal expression vector.     

Metabolic Flux and Fitness

Studies of Escherichia coli under competition for lactose in chemostat cultures have been used to determine the selective effects of variation in the level of the beta-galactoside permease and the beta-galactosidase enzyme. The results determine the adaptive topography of these gene products relative to growth in limiting lactose and enable predictions concerning the selective effects of genetic variants found in natural populations. In the terms of metabolic control theory, the beta-galactosidase enzyme at wild-type-induced levels has a small control coefficient with respect to fitness (C = 0.018), and hence genetic variants resulting in minor changes in enzyme activity have disproportionately small effects on fitness. However, the apparent control coefficient of the beta-galactoside permease at wild-type-induced levels is large (C = 0.551), and hence even minor changes in activity affect fitness. Therefore, we predict that genetic polymorphisms in the lacZ gene are subject to less effective selection in natural populations than are those in the lacY gene. The beta-galactoside permease is also less efficient than might be expected, and possible forces resulting in selection for an intermediate optimum level of permease activity are considered. The selective forces that maintain the lactose operon in a regulated state in natural populations are also discussed.     

Three forms of thermostable lactose-hydrolase from Thermus sp. IB-21: cloning, expression, and enzyme characterization

Three thermostable lactose-hydrolases, namely, two beta-glycosidases (bglA and bglB) and one beta-galactosidase (bgaA) genes were cloned from the genomic library of Thermus sp. IB-21. The bglA, bglB, and bgaA consisted of 1311 bp (436 amino acid residues), 1296 bp (431 aa), and 1938 bp (645 aa) of nucleotides with predicted molecular masses of 49,066, 48,679, and 72,714 Da, respectively. These enzymes were overexpressed in Escherichia coli BL21(DE3) using pET21b(+) vector system. The recombinant enzymes were purified to homogeneity by a heat precipitation (70 degrees C, 40 min) and a Ni2+-affinity chromatography. The molecular masses of the purified enzymes estimated by SDS-PAGE agreed with their predicted values. All the purified enzymes showed their optimal pH at around 5.0-6.0. In contrast, the temperature profiles for activity and thermostability patterns were different for each enzyme. BglB beta-glycosidase displayed the best lactose hydrolysis activity of the three enzymes without substrate inhibition up to 200 mM lactose at 70 degrees C and pH 7.0. The specific activities (U/mg) of BglA, BglB, and BgaA on 138 mM lactose at 70 degrees C and pH 7.0 were 36.8, 160.3, and 8.5, respectively.     

Salt tolerance of lactose-grown Vibrio parahaemolyticus carrying Escherichia coli lac genes

Lac- strains of Vibrio parahaemolyticus were converted to Lac+ on receiving a hybrid plasmid containing the lactose utilization genes of Escherichia coli K-12. A V. parahaemolyticus strain containing this hybrid plasmid exhibited optimal growth rates on glucose and other carbon sources in the presence of 0.2 to 0.4 M NaCl. Growth of the same strain on lactose was inhibited at similar concentrations of NaCl. The altered growth rate responses in lactose medium appeared to be attributable to effects of NaCl on the activity of lactose permease, and possibly on that of beta-galactosidase, rather than on the levels of these enzymes in V. parahaemolyticus cells.     

Salt tolerance of lactose-grown Vibrio parahaemolyticus carrying Escherichia coli lac genes.

Lac- strains of Vibrio parahaemolyticus were converted to Lac+ on receiving a hybrid plasmid containing the lactose utilization genes of Escherichia coli K-12. A V. parahaemolyticus strain containing this hybrid plasmid exhibited optimal growth rates on glucose and other carbon sources in the presence of 0.2 to 0.4 M NaCl. Growth of the same strain on lactose was inhibited at similar concentrations of NaCl. The altered growth rate responses in lactose medium appeared to be attributable to effects of NaCl on the activity of lactose permease, and possibly on that of beta-galactosidase, rather than on the levels of these enzymes in V. parahaemolyticus cells.     

Cold-active acid beta-galactosidase activity of isolated psychrophilic-basidiomycetous yeast Guehomyces pullulans

In the present study, psychrophilic yeasts, which grow on lactose as a sole carbon source at low temperature and under acidic conditions, were isolated from soil from Hokkaido, Japan. The phenotypes and sequences of 28S rDNA of the isolated strains indicated a taxonomic affiliation to Guehomyces pullulans. The isolated strains were able to grow on lactose at below 5 degrees C, and showed cold-active acid beta-galactosidase activity even at 0 degrees C and pH 4.0 in the extracellular fractions. Moreover, K(m) of beta-galactosidase activity for lactose in the extracellular fraction from strain R1 was found to be 50.5 mM at 10 degrees C, and the activity could hydrolyze lactose in milk at 10 degrees C. The findings in this study indicate the possibility that the isolated strains produce novel acid beta-galactosidases that are able to hydrolyze lactose at low temperature.     

Genetic diversity in the lactose operons of Lactobacillus helveticus strains and its relationship to the role of these strains as commercial starter cultures

Two novel insertion sequence elements, ISLhe1 and ISLhe15, were located upstream of the genes encoding the beta-galactosidase enzyme in Lactobacillus helveticus commercial starter strains. Strains with the IS982 family element, ISLhe1, demonstrated reduced beta-galactosidase activity compared to the L. helveticus type strain, whereas strains with the ISLhe15 element expressed beta-galactosidase in the absence of lactose.     

Isozymes of alpha-galactosidase from Bacillus stearothermophilus

Two molecular forms of alpha-galactosidase (EC 3.2.1.22) synthesized constitutively by Bacillus stearothermophilus, strain AT-7, have been purified. alpha-Galactosidase I (with the substrate p-nitrophenyl alpha-D-galactopyranoside (PNPG)) has a pH optimum of 6 and half-life at 65 degrees C of > 2 h at low protein concentration. alpha-Galactosidase II has a pH optimum of 7 with PNPG and a half-life at 65 degrees C of about 3 min. The isozymes also differ with respect to their Km with PNPG and melibiose. Both enzymes are inhibited competitively by D-galactose, melibiose, and Tris. With the beta-glycosides cellobiose and lactose either noncompetitive or mixed-type inhibition is observed, with the pattern dependent on both the pH and the isozyme. The two isozymes have similar Arrhenius activation energies (about 20 kcal/mol, 1 kcal = 4.184 kJ). Their molecular weights, estimated by disc gel electrophoresis, are alpha-galactosidase I, 280 000 +/- 30 000 and alpha-galactosidase II, 325 000 +/- 15 000. Dodecyl sulfate gel electrophoresis gave a single band for each enzyme. The respective molecular weights, 81 000 +/- 500 for alpha-galactosidase I and 84 000 +/- 500 for alpha-galactosidase II, suggest that both enzymes consist of four subunits.     

Endocytotic internalization of alpha-2-macroglobulin: alpha-galactosidase conjugate by cultured fibroblasts derived from Fabry hemizygote

Endocytotic internalization of alpha-galactosidase by cultured fibroblasts derived from a patient with Fabry's disease was achieved via receptor-mediated endocytosis of alpha-2-macroglobulin (alpha-2-M). alpha-galactosidase of coffee beans was conjugated to alpha-2-M when the latter was treated with trypsin. Internalization of the conjugate resulted in an increase of alpha-galactosidase activity in the crude cell extracts. The observed internalization was blocked by the presence of bacitracin, an inhibitor of binding between alpha-2-M and its receptor on the cell surface. When the cells were incubated at 4 degrees C with the conjugate, internalization was also inhibited. The alpha-galactosidase activity in the cells was saturated when the concentration of the conjugate in the medium was 40 micrograms/ml. Since non-conjugated alpha-galactosidase was not effectively internalized, the observed internalization of the conjugate was mediated by recognition of alpha-2-M by its receptor. The effective internalization of alpha-galactosidase described in this paper has a potential use in the enzyme replacement therapy of Fabry's disease.     

Mobilization and expression of bacteriocin plasmids from Carnobacterium piscicola isolated from meat

Mobilization and expression of bacteriocin plasmids from Carnobacterium piscicola isolated from meat. The nonconjugative plasmids pCP40 and pCP49 associated with bacteriocin production in Carnobacterium piscicola LV17, a lactic acid bacterium isolated from meat, were mobilized by the wide host range conjugative plasmid pAMβ1 by two stage conjugation. At the first stage, pAMβ1 was conjugally transferred into C. piscicola LV17 containing the two plasmids associated with bacteriocin production and a cryptic plasmid. Mobilization of the two bacteriocin plasmids by pAMβ1 was done by the second stage conjugation between the pAMβ1-containing C. piscicola LV17 and chloramphenicol (Cm)-resistant Bac^- mutant of C. piscicola LV17. The transconjugants had either partial bacteriocin activity associated with acquisition of pCP40 or pCP49, or complete bacteriocin activity associated with acquisition of all three of the resident plasmids from C. piscicola LV17 or an 89 MDa cointegrated plasmid derived from pCP40 and pCP49. Further manipulation of the transconjugants and a mutant strain of C. piscicola LV17 resulted in separate strains with only pCP40 or pCP49 which produce different bacteriocins. The bacteriocin gene from pCP49 was cloned into pCaT, a chloramphenicol resistance-encoding vector, and electrotransformed into another bacteriocin-producing strain of C. piscicola , enhancing the antagonistic spectrum of the recipient strain.     

Heterologous expression of lactose- and galactose-utilizing pathways from lactic acid bacteria in Corynebacterium glutamicum for production of lysine in whey

The genetic determinants for lactose utilization from Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842 and galactose utilization from Lactococcus lactis subsp. cremoris MG 1363 were heterologously expressed in the lysine-overproducing strain Corynebacterium glutamicum ATCC 21253. The C. glutamicum strains expressing the lactose permease and beta-galactosidase genes of L. delbrueckii subsp. bulgaricus exhibited beta-galactosidase activity in excess of 1000 Miller units/ml of cells and were able to grow in medium in which lactose was the sole carbon source. Similarly, C. glutamicum strains containing the lactococcal aldose-1-epimerase, galactokinase, UDP-glucose-1-P-uridylyltransferase, and UDP-galactose-4-epimerase genes in association with the lactose permease and beta-galactosidase genes exhibited beta-galactosidase levels in excess of 730 Miller units/ml of cells and were able to grow in medium in which galactose was the sole carbon source. When grown in whey-based medium, the engineered C. glutamicum strain produced lysine at concentrations of up to 2 mg/ml, which represented a 10-fold increase over the results obtained with the lactose- and galactose-negative control, C. glutamicum 21253. Despite their increased catabolic flexibility, however, the modified corynebacteria exhibited slower growth rates and plasmid instability.