Separation and characterization of four enzyme forms of beta-galactosidase from Saccharomyces lactis

beta-Galactosidase from Saccharomyces lactis has been purified to serve as a model for the kinetic behavior of human lactase in adult lactase deficiency. Enzymes from both species are neutral and follow Michaelis-Menten kinetics. beta-Galactosidase of S. lactis is more readily available than the human lactase. An enzyme preparation from S. lactis (Maxilact 40,000), which is used commercially to hydrolyze lactose in milk, has been found to contain four isozymes of beta-galactosidase. Methods have been developed for the separation and purification of each of the four enzymes. The enzymes were found to differ in molecular mass, kinetic behavior, isoelectric point, response to pH, specific volume and sensitivity to metal ions. The four enzymes had apparent molecular masses of 630 kDa, 550 kDa, 41 kDa and 19 kDa. Their specificity constants (kcat/Km) were found to be 42.0, 355.2, 0.38 and 0.48 mM-1 s-1, respectively. The techniques of reiterated ultrafiltration used for the isolation of these isozymes may be applicable to other purification processes.     

Anomerization and hydrolysis of lactose by beta-galactosidase from Saccharomyces lactis

Beta-Galactosidase from Saccharomyces lactis was found to be able to catalyze both the anomerization of alpha-lactose and the hydrolysis of beta-lactose; the rate of hydrolysis appeared to be four times higher with a 1:1 mixture of alpha and beta lactose than with a freshly prepared solution of alpha-lactose. The enzyme was also found to be unable to hydrolyze alpha-lactose. Thus, it appears that beta-galactosidase from S. lactis has its hydrolytic activity on lactose adapted only to the naturally more abundant beta-lactose.     

Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts

Bacterial isolates from bean-sprouts were screened for anti- Listeria monocytogenes bacteriocins using a well diffusion method. Thirty-four of 72 isolates inhibited the growth of L.monocytogenes Scott A. One, HPB 1688, which had the biggest inhibition zone against L.monocytogenes Scott A, was selected for subsequent analysis. Both ribotyping and DNAsequencing of 16S ribosomal RNA gene demonstrated that the isolate was Lactococcus lactis subsp. lactis . Polymerase chain reaction and nucleotide sequencing revealed that thegenomic DNA of the bean-sprout isolates contained a nisin Z structural gene. In MRS broth,bean-sprout isolate HPB 1688 survived at 3–4·5°C for at least 20 d, grew at 4°Cand produced anti-listerial compoundsat 5°C. When co-cultured with L. monocytogenes in MRS broth, the isolate inhibited thegrowth of L. monocytogenes at 4°C after 14d and at 10°C after 2 d. When co-inoculatedwith 10²cells g⁻¹ of L.monocytogenes on fresh-cut ready-to-eat Caesar salad, L. lactis subsp. lactis (10⁸cells g⁻¹) was able to reduce the number of L. monocytogenes by 1–1·4 logs after storage for 10 d at 7° and 10°C. A bacteriocin-producing Enterococcusfaecium was also able to reduce the numbers of L. monocytogenes onCaesar salad, butdid not act synergistically when co-inoculated with L. lactis subsp. lactis .     

Expression of a foreign eukaryotic gene in Saccharomyces cerevisiae: beta-galactosidase from Kluyveromyces lactis

Three recombinant DNA vectors carrying the β-galactosidase structural gene, LAC4, from the yeast Kluyveromyces lactis were constructed and transformed into Saccharomyces cerevisiae. All transformants expressed the β-galactosidase activity of LAC4. However, the level of enzyme activity varied, being highest in cells transformed with vectors which are maintained as multicopy plasmids and lowest in cells transformed with a vector which integrates into chromosomes. Enzyme levels probably reflect gene dosage. LAC4 is very stable when integrated into a chromosome, but unstable when carried on a plasmid. Therefore, stability is a property of the recombinant vector rather than of LAC4, LAC4-coded β-galactosidase synthesized in either S. cerevisiae or in K. lactis is the same as judged by two-dimensional polyacrylamide gel electrophoresis. However, S. cerevisiae transformed with     

Electroinduced extraction of beta-galactosidase from Kluyveromyces lactis

A new methodology for the extraction of beta-galactosidase from the yeast Kluyveromyces lactis was obtained by electropulsation. The application of a series of electric pulses (2 ms duration, 1 Hz frequency, and 4-4.5 kV/cm field strength) to fresh cells suspended in deionized water, followed by incubation in PBS, led to a spontaneous slow release of enzyme at a yield of 75-80% without any further treatment. Most of the enzyme was extracted within 8 h after electropulsation. This release was dependent on the growth phase. The specific activity of beta-galactosidase in the supernatant of pulsed cells was higher by a factor of 1.5-1.7 in comparison with crude extract.     

Isolation and characterization of psychrophiles producing cold-active beta-galactosidase

AIMS: The present study was conducted to screen for psychrophilic micro-organisms that are able to hydrolyse lactose at low temperature, and to examine the cold-active beta-galactosidase produced by the isolated psychrophilic micro-organisms. METHODS AND RESULTS: Psychrophilic bacteria, which grow on lactose as a sole carbon source, were isolated from soil from Hokkaido, Japan. The phenotype and sequence of 16S rDNA of the isolated strains indicated a taxonomic affiliation to Arthrobacter psychrolactophilus. The isolated A. psychrolactophilus strains were able to grow on lactose at below 5 degrees C, and showed cold-active beta-galactosidase activity, which was highly specific at even 0 degrees C. CONCLUSIONS: Facts in this study may indicate the possibility that the isolated strains produce novel beta-galactosidases that are able to hydrolyse lactose at low temperature, although some strains have isozymes. SIGNIFICANCE AND IMPACT OF THE STUDY: It may be possible that the cold active beta-galactosidases from the isolated strains can be applied to the food industry, e.g. processing of milk and whey below 5 degrees C.     

Isolation and characterization of jack bean beta-galactosidase.

A simple procedure has been devised to isolate beta-galactosidase from jack bean meal. The final preparation gives one major protein banc in disc gel electrophoresis. The substrate specificity of this enzyme toward some natural oligosaccharides, glycoproteins, and sphingoglycolipids has been examined in detail. Among three isomers of N-acetyllactosamine, Galbeta1leads to4GlcNAc; while Galbeta1leads to3GlcNAc was hydrolyzed very slowly. This property can be used to distinguish the galactose linkage in asialo-GM1 (Galbeta1leads to3GalNAcbeta1leads to4Galbeta1leads to4Glcleads toCer) and that in lacto-N-neotetraosylceramide (Galbeta1leads to4GlcNAcbeta1leads to 3Galbeta1leads to4Glcleads toCer). For hydrolyzing glycolipids, the effect of sodium taurodeoxycholate and sodium taurochenodeoxycholate on the rate of hydrolysis was carefully examined. This enzyme hydrolyzes lactosylceramide and asialo-GM1 faster than GM1. These results suggest that in addition to the type and linkage of the penultimate sugar unit, the sugar unit at the distal position of the saccharide chain also affects the hydrolysis rate. It also readily liberates 80% D-galactosyl units from asialo alpha1-acid glycoprotein. Escherichia coli beta-galactosidase on the other hand cannot hydrolyze asialo-alpha1-acid glycoprotein, lactosylceramide, GM1, asialo-GM1, and lacto-N-neotetraosylceramide. The molecular weight of this enzyme is about 75,000 and the isoelectric point is pH 8.0. With p-nitrophenyl beta-D-galactopyranoside as substrate, optimal activity occurs at pH 2.8 with glycine-HCl buffer and at pH 3.5 with citrate-phosphate buffer. With lactose as substrate, the pH optimum in these two buffers are 2.8 and 4.0, respectively. Km values for p-nitrophenyl beta-D-galactopyranoside, o-nitrophenyl beta-D-galactopyranoside and lactose are 0.51 mM, 0.63 mM, and 12.23 mM, respectively. Many inhibitors for this enzyme including inorganic ions, monosaccharides, and glycosides are investigated. In contrast to E. coli beta-galactosidase, jack bean beta-galactosidase is not inhibited by p-aminophenyl thio-beta-D-galactopyranoside.     

Isolation and characterization of the genes encoding delta(8)-sphingolipid desaturase from Saccharomyces kluyveri and Kluyveromyces lactis

Saccharomyces kluyveri IFO 1685 and Kluyveromyces lactis IFO 1090 synthesize cerebroside containing 9-methyl- trans-4, trans-8-sphingadienine as a sphingoid base. From the genome of the two strains, the regions encompassing Delta(8)-sphingolipid desaturase were amplified and sequenced. The nucleotide sequences of these regions revealed single open reading frames of 1707 bp for S. kluyveri and 1722 bp for K. lactis, encoding polypeptides of 568 and 573 amino acids with molecular weights of 66.5 and 67.1 kDa, respectively. Conversion of 4-hydroxysphinganine to 4-hydroxy- trans-8-sphingenine in the cells of Saccharomyces cerevisiae was observed by the expressed gene from K. lactis and not by that from S. kluyveri. These findings may be explained by the difference in substrate specificity for the sphingoid base moiety between Delta(8)-sphingolipid desaturases of S. kluyveri and K. lactis.     

Isolation and characterization of cold-adapted strains producing beta-galactosidase

beta-Galactosidase is extensively employed in the manufacture of dairy products, including lactose-reduced milk. Here, we have isolated two gram-negative and rod-shaped coldadapted bacteria, BS 1 and HS 39. These strains were able to break down lactose at low temperatures. Although two isolates were found to grow well at 10 degrees , the BS 1 strain was unable to grow at 37 degrees . Another strain, HS-39, evidenced retarded growth at 37 degrees . The biochemical characteristics and the results of 16S rDNA sequencing identified the BS 1 isolate as Rahnella aquatilis, and showed that the HS 39 strain belonged to genus Buttiauxella. Whereas the R. aquatilis BS 1 strain generated maximal quantities of beta-galactosidase when incubated for 60 h at 10 degrees , Buttiauxella sp. HS-39 generated beta-galactosidase earlier, and at slightly lower levels, than R. aquatilis BS 1. The optimum temperature for beta-galactosidase was 30 degrees for R. aquatilis BS-1, and was 45 degrees for Buttiauxella sp. HS-39, thereby indicating that R. aquatilis BS-1 was able to generate a cold-adaptive enzyme. These two cold-adapted strains, and most notably the beta-galactosidase from each isolate, might prove useful in some biotechnological applications.     

Isolation and characterization of Lactococcus lactis subsp. lactis promoters.

DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp. lactis. For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L. lactis. Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements. From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B. subtilis sigma 43 promoters were identified. Another set of promoters, together with a signal sequence, were also isolated from the same organism. These fragments promoted secretion of TEM beta-lactamase from L. lactis. When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA). By changing the promoter part of the promoter-signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained.     

Isolation and characterization of Lactococcus lactis subsp. lactis promoters.

DNA fragments with promoter activity were isolated from the chromosome of Lactococcus lactis subsp. lactis. For the isolation, a promoter probe vector based on the cat gene was constructed, which allowed direct selection with chloramphenicol in Bacillus subtilis and L. lactis. Four of the putative promoters (P1, P2, P10, and P21) were analyzed further by sequencing, mapping of the 5' end of the mRNA, Northern (RNA blot) hybridization, and chloramphenicol acetyltransferase activity measurements. From these fragments, -10 and -35 regions resembling the consensus Escherichia coli sigma 70 and B. subtilis sigma 43 promoters were identified. Another set of promoters, together with a signal sequence, were also isolated from the same organism. These fragments promoted secretion of TEM beta-lactamase from L. lactis. When the two sets of promoters were compared, it was found that the ones isolated with the cat vector were more efficient (produced more mRNA). By changing the promoter part of the promoter-signal sequence fragment giving the best TEM beta-lactamase secretion into a more efficient one (P2), a 10-fold increase in enzyme production was obtained.     

Isolation and characterization of nuclear envelopes from the yeast Saccharomyces

We have developed a large scale enrichment procedure to prepare yeast nuclear envelopes (NEs). These NEs can be stripped of peripheral proteins to produce a heparin-extracted NE (H-NE) fraction highly enriched in integral membrane proteins. Extraction of H-NEs with detergents revealed previously uncharacterized ring structures associated with the NE that apparently stabilize the grommets of the nuclear pore complexes (NPCs). The high yields obtained throughout the fractionation procedure allowed balance-sheet tabulation of the subcellular distribution of various NE and non-NE proteins. Thus we found that 20% of endoplasmic reticulum (ER) marker proteins are localized at the NE. Using a novel monospecific mAb made against proteins in the H-NE fraction and found to be directed against the pore membrane protein POM152, we showed that while the majority of POM152 is localized in the NE at the NPC, a proportion of this protein is also present in the ER. This ER pool of POM152 is likely to be involved in the duplication of nuclear pores and NPCs during S-phase. Both the NEs and H-NEs were found to be competent for the in vitro posttranslational translocation of prepro-alpha-factor. They may also be suitable to investigate other ER- and NE-associated functions in cell-free systems.     

Biochemical and genetic characterization of -glucosidase mutants in Saccharomyces lactis

Mutants with reduced activity for beta-glucosidase (beta-d-glucoside glucohydrolase EC 3.2.1.21) were isolated from the haploid yeast Saccharomyces lactis. Tetrad analysis indicated that in each mutant a single genetic factor, closely linked or allelic to the structural gene for beta-glucosidase (B locus), is responsible for the decreased activity. beta-Glucosidases produced by wild-type and mutant strains are similar in molecular size and charge but differ in catalytic properties, thermal stability, and serological specificity, indicating that mutants are in the structural gene. All mutants retained their capacity to be induced by either methyl-beta-d-glucoside or glucose. In all cases, the mutant phenotype was dominant in heterozygous diploids.     

Purification and properties of Streptococcus lactis beta-galactosidase

beta-Galactosidase of Streptococcus lactis 7962 was partially purified, and its properties were studied. Enzyme from only this strain of numerous lactic streptococci tested was stable in cell exudates prepared by various means. Cell-free extracts of the 7962 strain were prepared by sonic treatment of washed cells previously grown in presence of lactose to fully induce enzyme synthesis. Protamine sulfate precipitation of the nucleic acids and ammonium sulfate precipitation of protein were used for partial purification of the enzyme. The resulting enzyme, when resuspended in cold (5 C) phosphate buffer, was extremely labile. However, ammonium sulfate in high concentrations (0.85 m) stabilized and stimulated beta-galactosidase activity. Sephadex G-200 gel filtration was used to achieve further purification and to monitor homogeneity of the enzyme. Separation of the beta-galactosidase in buffer at 5 C yielded an enzyme elution pattern showing two peaks of activity. However, addition of the enzyme solution in 0.85 m ammonium sulfate to the column equilibrated with the same salt concentration yielded only one peak of enzyme activity. The data suggested that the native enzyme was dissociating into active subunits which were stabilized in the presence of the ammonium sulfate.     

Expression of a beta-galactosidase gene from Clostridium acetobutylicum in Lactococcus lactis subsp. lactis

A beta-galactosidase gene from Clostridium acetobutylicum NCIB 2951 was expressed after cloning into pSA3 and electroporation into derivatives of Lactococcus lactis subsp. lactis strains H1 and 7962. When the clostridial gene was introduced into a plasmid-free derivative of the starter-type Lact. lactis subsp. lactis strain H1, the resulting construct had high beta-galactosidase activity but utilized lactose only slightly faster than the recipient. beta-galactosidase activity in the construct decreased by over 50% if the 63 kb Lac plasmid pDI21 was also present with the beta-galactosidase gene. Growth rates of Lac+ H1 and 7962 derivatives were not affected after introduction of the clostridial beta-galactosidase, even though beta-galactosidase activity in a 7962 construct was more than double that of the wild-type strain. When pDI21 was electroporated into a plasmid-free variant of strain 7962, the recombinant had high phospho-beta-galactosidase activity and a growth rate equal to that of the H1 wild-type strain. The H1 plasmid-free strain grew slowly in T5 complex medium, utilized lactose and contained low phospho-beta-galactosidase activity. We suggest that beta-galactosidase expression can be regulated by the lactose phosphotransferase system-tagatose pathway and that Lact. lactis subsp. lactis strain H1 has an inefficient permease for lactose and contains chromosomally-encoded phospho-beta-galactosidase genes.     

Isolation and characterization of valine transfer RNA from Saccharomyces cerevisiae.

Two procedures for isolating valine tRNA from commercial bakers' yeast were investigated. The first involved: (a) counter double current distribution; (b) chromatography on benzoyl-DEAE-cellulose; (c) reverse phase chromatography on Chromosorb G saturated with trioctylpropylammonium bromide (Oakridge System 3). The material isolated lacked the 3'-terminal adenylic acid residue. The second procedure involved the first two steps above followed by: (a) enzymatic aminoacylation with a partially purified yeast extract; (b) derivatization with N-phenoxyacetoxysuccinimide; (c) chromatography on benzoyl-DEAE-cellulose; (d) reverse phase chromatography, System 3. The product was intact tRNA. It was a mixture of isoacceptors (59:41) differing by a modification (uracil leads to dihydrouracil) at position 48. It was free of denatured material; specific activity 1,825 pmol of valine/A260 unit of tRNA. Sequence analysis confirmed the recently corrected structure (Bonnet, J., Ebel, J. P., Dirheimer, G., Shershneva, L. P., Krutilina, A. I., Venkstern, T. V., and Bayev, A. A. (1974) Biochimie 56, 1211-1213). A preliminary study of the alkaline hydrolysis of the 7-methylguanosine residue that occurs at position 47 showed that at least two products are formed instead of only one as usually quoted in the literature. A rapid, ultramicro, chromatographic system for separating these products and measuring them quantitatively is described.     

Isolation and characterization of a dinucleoside triphosphatase from Saccharomyces cerevisiae.

An enzyme able to cleave dinucleoside triphosphates has been purified 3,750-fold from Saccharomyces cerevisiae. Contrary to the enzymes previously shown to catabolize Ap4A in yeast, this enzyme is a hydrolase rather than a phosphorylase. The dinucleoside triphosphatase molecular ratio estimated by gel filtration is 55,000. Dinucleoside triphosphatase activity is strongly stimulated by the presence of divalent cations. Mn2+ displays the strongest stimulating effect, followed by Mg2+, Co2+, Cd2+, and Ca2+. The Km value for Ap3A is 5.4 microM (50 mM Tris-HCl [pH 7.8], 5 mM MgCl2, and 0.1 mM EDTA; 37 degrees C). Dinucleoside polyphosphates are substrates of this enzyme, provided that they contain more than two phosphates and that at least one of the two bases is a purine (Ap3A, Ap3G, Ap3C, Gp3G, Gp3C, m7Gp3A, m7Gp3G, Ap4A, Ap4G, Ap4C, Ap4U, Gp4G, and Ap5A are substrates; AMP, ADP, ATP, Ap2A, and Cp4U are not). Among the products, a nucleoside monophosphate is always formed. The specificity of cleavage of methylated dinucleoside triphosphates and the molecular weight of dinucleoside triphosphatase indicate that this enzyme is different from the mRNA decapping enzyme previously characterized (A. Stevens, Mol. Cell. Biol. 8:2005-2010, 1988).