Expression and characterization of Kluyveromyces lactis β-galactosidase in Escherichia coli

Kluyveromyces lactis -galactosidase gene, LAC4, was expressed in Escherichia coli as a soluble His-tagged recombinant enzyme under the optimized culture conditions. The expressed protein was multimeric with a subunit molecular mass of 118 kDa. The dimeric form of the -galactosidase was the major fraction but had a lower activity than those of the multimeric forms. The purified enzyme required Mn²⁺ for activity and was inactivated irreversibly by imidazole above 50 mM. The activity was optimal at 37 and 40 °C for o-nitrophenyl--d-galactopyranoside (oNPG) and lactose, respectively. The optimum pH value is 7. The K _m and V _max values of the purified enzyme for oNPG were 1.5 mM and 560 mol min^–1 mg^–1, and for lactose 20 mM and 570 mol min^–1 mg^–1, respectively.     

Isolation and characterization of lactose non-utilizing mutants in Aspergillus nidulans

Summary A number ofAspergillus nidulans mutants unable to grow on lactose or growing very poorly on this sugar have been isolated. They may be divided into two major groups: to the first belong mutants in which -galactosidase can be induced by galactose but not by lactose. Mutants of the second group are induced neither by lactose nor by galactose. Mutants of the first group showed an impaired lactose-permease system, while those of the second group most likely concern -galactosidase structural or regulatory genes as they show a normal rate of lactose uptake. Genetic analysis revealed that mutants from the first group fall into three different loci and those from the second into four loci. No mutant has been found so far with the lactose-permease system and -galactosidase simultaneously impaired, or with a constitutive level of either activity.The wild-type strain ofAspergillus nidulans grows on lactose as the sole carbon source. The two enzymes necessary for the utilization of lactose, that is lactose permease (which is likely to be a complex system) and -galactosidase show an inductive response to lactose and galactose (Paszewskiet al., 1970). Mycelia grown on glucose show a low level of permease activity which rises 7–10-fold upon induction by lactose, and no activity of -galactosidase. Induction of both enzymes is not time-coordinated — the induction of permease preceeds the induction of -galactosidase. In contrast toNeurospora crassa (Bates and Woodward, 1964; Bateset al., 1967; Lester and Byers, 1965) only one type of -galactosidase with pH optimum 7.5–7.6 was found inAspergillus nidulans.A number of mutants unable to grow on lactose or growing very poorly on this sugar have been isolated. Their genetic and enzymatic characterization is given in this paper.     

Studies on Tn951 (lac+) expression in Agrobacterium

Summary None of the Agrobacterium tumefaciens and A. rubi strains tested produces detectable amounts of -galactosidase although they are capable of utilizing lactose as sole source of carbon. This opportunity was taken to investigate the expression of lac transposon Tn951 (Cornelis et al. 1978) in Agrobacterium with the ultimate goal of using this system to investigate alien gene expression. When the transposon was introduced with the help of a broad-host range plasmid, RP1, the transconjugants produced significant quantities of -galactosidase which was inducible by isopropyl--D-thiogalactopyranoside. Tn951 was capable of restoring the Lac⁺ phenotype to an A. tumefaciens mutant not capable of using lactose. Cellobiose, a known inducer of aldohexopyranoside: cytochrome c oxidoreductase which regulates the characteristic 3-ketolactose production in Agrobacterium: van Beeumen and De Ley (1968), had no effect on -galactosidase activity.Abbreviations NCPPB National Collection of Plant Pathogenic Bacteria, Harpenden - km kanamycin resistance - str streptomycin resistance - rif ^r rifampicin resistance     

Induction of β galactosidase in the yeast Kluyveromyces lactis

Summary The inductive effect of lactose, -methyl-thio-D-galactopyranoside, (TMG) and glucose on galactosidase synthesis in Kluyveromyces lactis has been studied. Whereas TMG gave a five fold stimulation of the rate of -galactosidase synthesis, lactose only gave a small stimulation. Glucose caused represssion at levels above 10^-3M but stimulated -galactosidase synthesis when added at lower concentrations.     

Mutations conferring quantitative and qualitative increases in beta-galactosidase activity in Escherichia coli

Summary Sodium lactobionate is not utilized as a carbon source byEscherichia coli because it is only poorly bound and hydrolyzed by -galactosidase and it does not induce the formation of the enzyme. However, treatment with N-methyl-N-nitro-N-nitrosoguanidine produced 32 independent mutants able to grow on lactobionate. Most of the mutants formed -galactosidase constitutively, 29 of them having mutations in the regulatory gene and one possibly in the operator. In addition, the mutants possessed quantitatively—or qualitatively—altered -galactosidase. In 28 mutants the -galactosidase activity was 1.5 to 4.5 times that of the wild-type. The enzymes of these mutants were unaltered in thermostability and substrate binding. One enzyme that was titrated immunologically possessed a molecular activity indentical with the wild-type enzyme. These mutants appear to contain extra copies of the gene for -galactosidase. The spontaneous mutation rate to constitutivity was 6.3x10^-3 and to the formation of apparently extra genes, 9.2x10^-3.The -galactosidases of three mutants were qualitatively changed as judged from their increased thermosensitivity, altered substrate-binding constants and greatly increased ability to hydrolyze lactose and lactobionate. Affinity for 0-nitrophenyl--galactoside and galactose was increased by the mutations while that for lactose was decreased; maximum velocities for the hydrolysis of 0-nitrophenyl--galactoside were also decreased. Relative to their rates of hydrolysis of 0-nitrophenyl--galactoside, these altered enzymes hydrolyzed lactose at 6 to 8 times, and lactobionate up to 23 times, the rate given by the normal enzyme. The mutations appear to increase the hydrophobic nature of the enzyme near the aglycon binding site and facilitate the hydrolysis of more hydrophilic galactosides. The lactobionic acid positive character could be transferred to other bacteria by sexual conjugation when the enzyme changes were qualitative, but not when they were quantitative.     

Mutants which make more malT product, the activator of the maltose regulon in Escherichia coli

Summary Some of the previously described malT-lacZ fusion strains (Débarbouillé and Schwartz, 1979) produce very low amounts of -galactosidase activity and hence grow poorly on lactose. Spontaneous mutants growing faster on lactose have been isolated. Some of the mutations map in, or close to, the promoter of the hybrid gene. They lead to an increased production of the hybrid proteins, which then become detectable on polyacrylamide gels. This effect is cis dominant. When the mutations, called malT ^q+, are transduced into a malT ⁺⁺ background the resulting transductants express the three maltose operons in a partially constitutive way. The malT ^q+ mutations therefore represent a new type of constitutive mutation. Their existence provides further evidence for the previously proposed model of positive regulation in the maltose regulon. In addition they should facilitate the purification of the malT product, and the indentification of the malT promoter on the DNA.     

Expression of the Alcaligenes eutrophus poly(β-hydroxybutyric acid)-synthetic pathway in Pseudomonas sp.

The 12.5-kb EcoRI restriction fragment PP1 of Alcaligenes eutrophus strain H16, which encodes for -ketothiolase, NADP-dependent acetoacetyl-CoA reductase and poly(-hydroxybutyric acid)-synthase was mobilized to six different species of the genus Pseudomonas belonging to the rRNA homology group I. Pseudomonas aeruginosa, P. fluorescens, P. putida, P. oleovorans, P. stutzeri and P. syringae, which are unable to synthesize and accumulate poly(-hydroxybutyric acid), PHB, were employed as recipients. Whereas the A. eutrophus PHB-synthetic enzymes were only marginally expressed in P. stutzeri, they were readily expressed in the other species. For example, the specific activity of PHB-synthase was 1.8 U/g protein in transconjugants of P. stutzeri but was between 21 and 77 U/mg protein in transconjugants of the other species. All recombinant strains harboring plasmid pVK101::PP1 except those of P. stutzeri accumulated PHB; the PHB content of the cells grown on gluconate under nitrogen limitation varied between 8 and 24.3% of the cellular dry mass.Abbreviations PHB poly(-hydroxybutyric acid) - PHA poly(hydroxyalkanoic acid)     

Polymorphism of the MHC class II Eb gene determines the protection against collagen-induced arthritis

Collagen-induced arthritis (CIA) is an animal model of auto immune polyarthritis, sharing similarities with rheumatoid arthritis (RA). Paradoxally, susceptibility to mouse CIA is controlled by the H2A loci (DQ homologous) while RA is linked to HLA.DR genes (H2E homologous). We recently showed that the E^d molecule prevents CIA development in susceptible H2 ^q mice. We addressed the question of whether H2Eb polymorphism will influence CIA incidence as HLA.DRB1 polymorphism does in RA. In F₁ mice, only H2Eb^d and H2Eb^s molecules showed protection. Using recombinant B10.RDD (Eb ^d/b) mice, we found that CIA protection was mediated by the first domain of the E^d molecule. Using peptides covering the third hypervariable region of the E chain, we found a perfect correlation between presentation of E peptides by the H2A^q molecule and protection on CIA. Therefore, the mechanism by which H2Eb protects against CIA seems to rely on the affinity of E peptides for the H2A_q molecule.     

Is the lactose-utilization ability ofRhizobium ofSesbania procumbens a vestigeal function?

Rhizobium SBS-R100, isolated from the stem nodules ofSesbania procumbens, synthesized -galactosidase constitutively. Transposon mutagenesis by Tn9 induced mutants defective in lactose utilization; the mutations did not interfere with growth, nodulation or N₂ fixation. Mouse monoclonal antibody raised against -galactosidase ofEscherichia coli reacted with soluble proteins of wild typeRhizobium SBS-R100. Anin vivo constructed recombinant plasmid pSBS-4 complemented aRhizobium mutant defective in lactose utilization.     

Immobilization of β-galactosidase (K. lactis) on solid phase Ni(II)-chelate

Summary Ni(II)-iminodiacetate-agarose achieved a near quantitative adsorption of -galactosidase (Kluyveromices lactis) with a yield of 96% of activity applied. A high percentage (90%) of -galactosidase activity was preserved after immobilization on the solid phase. The insoluble derivative could be used for lactose hydrolysis either in solution or in whey permeate.     

The chromosomal localization of human β-galactosidase revisited: a locus for β-galactosidase on human chromosome 3 and for its protective protein on human chromosome 22

Summary A series of man-Chinese hamster and man-mouse somatic cell hybrids was investigated to study the localization of the genes coding for the human lysosomal enzyme -galactosidase (EC 3.2.1.23) and for its protective protein. Using a monoclonal antibody, raised against human placental -galactosidase, it was observed that the structural locus for the -galactosidase polypeptide is located on chromosome 3. The nature of the involvement of chromosome 22 in the expression of human -galactosidase was elucidated by metabolic labelling of the hybrids with radioactive amino acids, immunoprecipitation with monoclonal and polyclonal antibodies against -galactosidase, followed by analysis via gel electrophoresis and fluorography.The data show that the presence of chromosome 22 coincides with the presence of a 32 kd protein. This polypeptide, the protective protein was previously shown to be intimately associated with human -galactosidase. In addition, the protective protein was found to be essential for the in vivo stability of -galactosidase by aggregating -galactosidase monomers into high molecular weight multimes. Both chromosome 3 and 22 are therefore necessary to obtain normal levels og -galactosidase activity in human cells.     

Significance of β-galactosidase repression in glucose inhibition of lactose utilization inEscherichia coli

The role of -galactosidase repression in glucose inhibition of lactose utilization was studied inEscherichia coli. Escherichia coli 3300 constitutively produces -galactosidase even in the presence of glucose. When this strain was grown in a mixture of glucose and lactose, lactose utilization did not occur until glucose was depleted. The addition of glucose to a 3300 culture grown in lactose immediately caused a permanent inhibition of lactose utilization and only a mild transient repression of -galactosidase. Exogenous cyclic adenosine monophosphate (AMP) did not overcome the glucose inhibition of lactose utilization but did relieve the transient repression. Thus glucose inhibition of lactose utilization is not related to -galactosidase repression and is independent of cyclic AMP.     

Purification and characterization of a thermostable β-galactosidase with high transgalactosylation activity from Saccharopolyspora rectivirgula

We purified an extracellular thermostable -galactosidase of Saccharopolyspora rectivirgula strain V2-2, a thermophilic actinomycete, to homogeneity and characterized it to be a monomeric enzyme with a relative molecular mass of 145 000 and s°_20,w of 7.1 s. In addition to the hydrolytic activity of 1-O-substituted -d-galactopyranosides such as lactose [a Michaelis constant K _m=0.75 mm and molecular activity (k _cat)= 63.1 s^–1 at pH 7.2 and 55° C] and p-nitrophenyl -d-galactopyranoside (K _m=0.04 mm k _cat= 55.8 s^–1), the enzyme had a high transgalactosylation activity. The enzyme reacted with 1.75 m lactose at 70°C and pH 7.0 for 22 h to yield oligosaccharides in a maximum yield (other than lactose) of 41% (w/w). A general structure for the major transgalactosylic products could be expressed as (Gal)_c-Glc, where n is 1, 2, 3, and 4 with a glucose at a reducing terminal. These oligosaccharides could selectively promote the growth of the genus Bifidobacterium found in human intestines. S. rectivirgula -galactosidase was stable at pH 7.2 up to 60°C (for 4 h in the presence of 10 m MnCl₂) or 70°C (for 22 h in the presence of 1.75 m lactose and 10 m MnCl₂). Thus the enzyme is applicable to an immobilized enzyme system at high temperatures (60°C <) for efficient production of the oligosaccharides from lactose. Correspondence to: T. Nakayama     

β-Globin gene polymorphism in the Saudi Arab population

Summary DNA mapping with the restriction endonucleases, Hpa I and Mst II, has been used to investigate -globin gene polymorphism in the Saudi Arab population. Using Hpa I digestion, 13.0kb and 7.6kb fragments were found in association with the ^A and ^S genes. The frequency of the polymorphic forms in two regions investigated vary significantly. In Al-Hafouf and the surrounding villages, situated in the Eastern Province of Saudi Arabia, the frequencies of association of the ^S gene with the Hpa I 7.6kb, 7.0kb, and 13.0kb fragments were 0.866, 0.043, and 0.071, respectively. The frequency of association of ^A with the 7.6kb and 13.0kb fragments resulting from the Hpa I digestion were 0.875 and 0.125. In Khaiber, Tehamat-Aseer, and surrounding villages, in the Western Province, the frequency of association of ^S with 7.6kb, 7.0kb, and 13.0kb fragments were 0.836, 0.027, and 0.0136, respectively, while that of ^S was 0.250 and 0.750 with 7.0kb and 13.0kb Hpa I fragments, respectively. Using Mst II digestion, ^A was found to be linked to a 1.15kb fragment, while ^s was linked to a 1.35kb fragment. The normal (Hb AA), heterozygotes (Hb AS), and homozygotes (Hb SS) gave 1.15, 1.15/1.35, and 1.35kb fragments, respectively. The results of this study show extensive polymorphism at the Hpa I restriction site of the ^A and ^S globin genes with the different polymorphic forms existing at a variable frequency in different regions of Saudi Arabia.     

Classification and intragenic position of mutations in the beta-galactosidase gene of Escherichia coli

Summary Following treatment with N-methyl-N-nitro-N-nitrosoguanidine, 1,257 mutants of Escherichia coli K12 were isolated on lactose-tetrazolium medium. Of these mutants, 345 were lactose-negative and lacked appreciable -galactosidase activity. About half of these enzyme-deficient mutants had lost the whole lactose operon; the remainder (174) were point mutations within the -galactosidase gene. With the exception of 42 which could not be classified, the mutations were identified either as chain-terminating (UAG 57, UAA 6, UGA 60) or missense (9). There were no mutations of the reading frame and no short deletions. The unclassifiable mutants do not form crossreacting protein and are probably a type of chain-terminating mutant.Each of the point mutations was assigned by sexduction to one of 36 deletion regions. The map distribution shows a deficiency of chain-terminating mutants in the operator-distal part of the gene and a localization of missense mutations in regions corresponding to the position of the apparent substrate-binding sites of the enzyme.     

Microwave-assisted synthesis of galacto-oligosaccharides from lactose with immobilized beta-galactosidase from Kluyveromyces lactis

Galacto-oligosaccharides (GOS) were synthesized from lactose by immobilized and free -galactosidase from Kluyveromyces lactis (Lactozym 3000 L HP-G) using either focused microwave irradiation or conventional heating. Immobilization of the -galactosidase on to Duolite A-568 increased the synthesis of GOS. GOS selectivity (GOS synthesis/lactose hydrolysis ratio) increased when the water activity of the media was reduced, notably with a high initial lactose concentration but also by using co-solvents in the media. The advantage of microwave heating on GOS formation was also examined. Addition of solvent and carrying out the reaction under microwave irradiation resulted an increase in the production of GOS. The selectivity for GOS synthesis can be increased by 217-fold under microwave irradiation, using immobilized -glucosidase and with added co-solvents such as hexanol.     

O-Glycosylhydrolases of Marine Filamentous Fungi: β-1,3-Glucanases of Trichoderma aureviride

The ability to produce extracellular O-glycosylhydrolases was studied in 14 strains of marine filamentous fungi sampled from the bottom sediments of the South China Sea. The following activities were detected in the culture liquids of the fungi: N-acetyl--D-glucosaminidase, -D-glucosidase, -D-galactosidase, -1,3-glucanase, amylase, and pustulanase. -1,3-Glucanases were isolated by ultrafiltration, hydrophobic interaction chromatography, and ion exchange chromatography, and their properties were studied. Data on products of enzymatic digestion of laminaran, absence of transglycosylation activity, and the pattern of action of natural inhibitors confirmed that -1,3-glucanase belonged to the exo type. Inhibitor analysis demonstrated the role of a thiol group and tryptophan and tyrosine residues in the catalytic activity.     

Metabolic engineering for direct lactose utilization by Saccharomyces cerevisiae

A recombinant strain of Saccharomyces cerevisiae, secreting -galactosidase from Kluyveromyces lactis, grew efficiently with more than 60 g lactose l^–1. The growth rate (0.23 h^–1) in a cheese-whey medium was close to the highest reported hitherto for other recombinant S. cerevisiae strains that express intracellular -galactosidase and lactose-permease genes. The conditions for growth and -galactosidase secretion in this medium were optimized in a series of factorial experiments. Best results were obtained at 23 °C for 72 h. Since the recombinant strain produced less than 3% ethanol from the lactose, it was also assayed for the production of fructose 1,6-bisphosphate from cheese whey, and 0.06 g l^–1 h^–1 were obtained.     

Conditional lysis ofEscherichia coli by the fusion of extracellular (STA) to periplasmic (LTB) enterotoxins: Apparent phenotypic suppression of lactose permease

The expression of a methanol-soluble, heat-stable enterotoxin (ST_A) fused to the B subunit of the heat-labile enterotoxin (LT_B) at 35°C or higher temperatures caused strains ofEscherichia coli deficient in lactose permease to behave on indicator media as Lac⁺; however, at 33°C or lower temperatures the original Lac^– phenotype of the host strains was maintained. The apparent phenotypic suppression oflacY was shown to be due to lysis of a fraction of the bacteria and the consequent release of active-galactosidase to the culture supernatant. After incubation at 37°C for 1 h, the cultures were committed to lyse. Plasmid and chromosomal mutants that do not show this phenotype were isolated by selecting Lac^– colonies at the unpermissive temperature. The mutations on the plasmids were localized in both the heat-stable and the heat-labile enterotoxin genes. Chromosomal mutants that show normal levels of-galactosidase and fused toxins have also been isolated.     

Cloning and nucleotide sequence of the β-galactosidase gene from Lactococcus lactis ssp. Lactis ATCC7962

The gene encoding -galactosidase of Lactococcus lactis ssp. lactis ATCC7962 was cloned and its nucleotide sequence was determined. The -galactosidase of L. lactis was expressed in Escherichia coli and transformants containing this gene fragment appeared as blue colonies on LB plates containing X-gal. The -galactosidase activity of E. coli transformant was thirty times higher than that of L. lactis. The gene for the 115 kDa -galactosidase has a 2991-bp open reading frame preceded by a putative ribosome binding site. The deduced amino acid sequence show a high degree of homology to the -galactosidase of E. coli, and the putative active site residues are conserved (Glu-429 and Tyr-475)