Induction of the Synthesis of Endo-1,4-β-xylanase and β-Galactosidase in Original and Recombinant Strains of the Fungus Penicillium canescens

The induction of synthesis of the secreted enzymes endo-1,4--xylanase (EC 3.2.1.8) and -galactosidase (EC 3.2.1.23) in original and recombinant Penicillium canescens strains has been studied. In all producer strains, the synthesis of these enzymes was induced by arabinose and its metabolite arabitol. The two enzymes differed in the concentration of arabinose required for induction: the synthesis of -galactosidase was most pronounced at 1 mM, whereas maximum synthesis of endo-1,4--xylanase was observed at 5–10 mM. An increase in the number of endo-1,4--xylanase copies in the high-copy-number strain of the fungus suppressed the synthesis of -galactosidase; the synthesis of endo-1,4--xylanase in the high-copy-number recombinant producing -galactosidase was affected to a lesser extent. The amount of enzymes synthesized did not depend on the saccharide used as the sole source of carbon for growing the mycelium prior to its transfer to the inducer-containing medium.     

Substrate specificity and distribution of acid β-galactosidase activities in seizure-susceptible and non-susceptible strains of mice

The properties and distribution of -galactosidase were studied in the mouse brain using the artificial substrate methylumbelliferyl--galactoside. Enzyme activities were compared between an audiogenic seizure-susceptible mouse strain (DBA/2) and three non-susceptible strains of mice (BALB/c, C3H/He and Swiss A2G). At all ages, DBA/2 mice have significantly lower -galactosidase activity compared with the three other mouse strains: this is attributed to the different alleles present at the Bgs locus. The low activity of -galactosidase is also evident when the natural substrate GMI-ganglioside is hydrolyzed. In contrast to this low GMI-ganglioside--galactosidase activity, there is no difference in the activity of the second form of acid -galactosidase, galactosylceramidase, in DBA/2 mice at 7 and 14 days. However, at 21 and 28 days the activity is significantly lower in DBA/2 mice compared with the other strains of mice. These results on -galactosidase activity in the brain of seizure-susceptible and non-susceptible mice are discussed in relation to published levels of GMI-ganglioside and galactosylceramide present in the developing mouse brain.Dedicated to Henry McIlwain.     

DNA content and enzymic activities in the auditory regions of seizuresusceptible and non-susceptible strains of mice

The developmental profiles of four glycosidase enzymes (-D-glucosidase, -D-glucuronidase, -D-N-acetylglucosaminidase and -D-galactosidase) in the cochleas, cochlear nuclei and inferior colliculi of four strains of mice were investigated. The strains used were an audiogenically seizure-susceptible strain (DBA/2) and three non-susceptible strains (BALB/c, C3H/He and Swiss/A₂G). The enzymic activities fell to varying degrees from 7 to 28 days of age. Two significant observations were made—-D-glucuronidase was low in the regions of the C₃H/He strain, and -D-galactosidase was particularly low in the regions of the DBA/2 strain. The very low activity of -D-galactosidase in the DBA/2 mice is discussed in relation to the ganglioside patterns known to be present in these seizure-susceptible mice. Studies on the DNA contents of these auditory regions in the four strains showed no correlation with seizure sensitivity.This paper is dedicated to Dr. Derek Richter on his seventy-fifth birthday.     

α-and β-Glycosidases in maize roots

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for -glucosidase, 4.4 for -galactosidase, 6.4 for -glucosidase and 6.0 for -galactosidase. The -glucosidase showed 4-fold higher activity than the -galactosidase. The distribution of the -glucosidase activity was signifcantly different from that of the -galactosidase, -glucosidase and -galactosidase.Abbreviations -Glu -glucosidase - -Gal -galactosidase - -Glu -glucosidase - -Gal -galactosidase     

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.     

Histochemical Discrimination of Endogenous Mammalian β-galactosidase Activity from that Resulting from lac-Z Gene Expression

Minces of several organs from the transgenic mouse ROSA-gal 26 (ROSA-26), which robustly expresses bacterial lac-Z in most tissues, were exposed to 4-bromo, 5-chloro, 3-indoyl, -D-galactopyrosanide (X-gal) at pH ranging from 7.5 to 9.5 to determine the optimal pH for in situ demonstration of bacterial -galactosidase activity (neutral pH optimum) while minimizing detection of potentially confounding endogenous mammalian -galactosidase (acidic pH optimum). Similar studies were performed with organ minces from C57BL/6 mice, Sprague-Dawley rats, New Zealand white rabbits, and macaques to confirm the effect of pH on minimizing detection of endogenous mammalian -galactosidase. In all organs evaluated; heart, liver, spleen, kidney, brain, and skeletal muscle, endogenous -galactosidase activity was rarely detected following incubation at pH greater than 7.5. In contrast, bacterial -galactosidase activity in the ROSA-26 mice was strongly detected in organ minces following incubation at pH 8.0–9.0. These findings are similar to previous observations we have made in lung minces and confirm that a simple alteration of a commonly used histochemical technique for detecting in situ -galactosidase activity, raising the reaction buffer pH to weakly alkaline range, can reliably distinguish between endogenous activity and that resulting from exogenous bacterial gene expression.     

Synthetic multifunctional proteins

Summary Several E. coli mutants were isolated which produce triple chimeras between one of the trp enzymes lac, repressor and -galactosidase. The mutants were isolated as TonB^- Lac⁺ derivatives of a phenotypically Lac^- TrpR^- strain carrying a lac I ⁺-Z⁺ fusion on a 80dlac phage. The phage is integrated into the chromosome in such a way that the lac and the trp genes are transcribed in the same direction. Of a total of 58 candidates 2 TrpA^- and 3 Trp^- strains produce triple chimeras. The chimeras from the two TrpA^- strains were further examined. They consist of tryptophan synthetase -subunit, lac repressor and -galactosidase. In crude extracts of these strains the tryptophan synthetase -subunit part can be identified by its ability to aggregate with the -subunit since some of the -subunit activity can be precipitated with antiserum against -galactosidase. Furthermore -galactosidase precipitates with antiserum against tryptophan synthetase -subunit. The lac repressor part is able to bind IPTG, but not lac operator DNA in vitro. The -galactosidase part is as unaffected as in the original lac repressor--galactosidase chimera. The molecular weigths of both chimeras are 175,000 when determined by SDS gel electrophoresis. The chimeras are partially degraded giving rise to fragments of distinct molecular weights.     

Studies on the cell cycle of Myxobacter AL-1

The properties of seven enzymes were studied in extracts from Myxobacter AL-1. The enzymes were isocitrate dehydrogenase (E.C. 1.1.1.42), succinate dehydrogenase (E.C. 1.3.99.1), alkaline phosphatase (E.C. 3.1.3.1), -glucosidase (E.C. 3.2.1.20), -glucosidase (E.C. 3.2.1.21), -galactosidase (E.C. 3.2.1.23), and N-acetyl-glucosaminidase (E.C. 3.2.1.30). Four of these enzymes: isocitrate dehydrogenase, -glucosidase, -glucosidase, and -galactosidase are cytosolic enzymes. Succinate dehydrogenase was found to be located on the cytoplasmic membrane system, whereas alkaline phosphatase and N-acetyl-glucosaminidase were considered as enzymes which bind the outer membranes resp. the cell wall. During the cell cycle, all enzymes have a pattern of discontinuous activity increase. Succinate dehydrogenase and isocitrate dehydrogenase exhibit a stepwise increase of activity, whereas the other enzymes follow the pattern of a peak enzyme.     

Differential induction of β-galactosidase and phospho-β-galactosidase activities in the fermentation of whey permeate by Clostridium acetobutylicum

Summary Strains of Clostridium acetobutylicum were tested for the presence of -galactosidase and phospho--galactosidase activities when grown on lactose. All strains, except C. acetobutylicum ATCC 824, showed both enzyme activities. Only phospho--galactosidase activity was detected with C. acetobutylicum ATCC 824. C. acetobutylicum strains P262 and ATCC 824 showed no detectable -galactosidase or phospho--galactosidase activities when grown on glucose. In the fermentation of whey permeate C. acetobutylicum P262 showed an early induction of phospho--galactosidase associated with the acidogenic phase. The -galactosidase activity peaked at a later stage of the fermentation (22 h) coinciding with the solvent production phase. Similar induction of phospho--galactosidase at the early stages (13 h) of fermentation of whey permeate by C. acetobutylicum ATCC 824 was also shown. No -galactosidase activity was detected during the entire course of fermentation by strain ATCC 824.     

Enzymatic syntheses of GlcNAcβ1-2Man and Galβ1-4GlcNAcβ1-2Man as components of complex type sugar chains

GlcNAc1-2Man and GlcNAc1-6Man were synthesized using the reverse hydrolysis activity of -N-acetylglucosaminidase from both jack beans and Bacillus circulans. In turn, Gal1-4GlcNAc1-2Man and Gal1-4GlcNAc1-6Man were synthesized regioselectively using the transglycosylation activity of -galactosidase from Diplococcus pneumoniae and B. circulans, respectively. These di- and trisaccharides are important components of complex type sugar chains and will be used as intermediates in our synthetic studies. Abbreviations: pNp--GlcNAc, p-nitrophenyl 2-acetamido-2-deoxy--D-glucopyranoside; pNp--Gal, p-nitrophenyl -D-galacto-pyranoside     

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)     

Improved in situ β-galactosidase staining for histological analysis of transgenic mice

The present study describes a novel method for the histochemical demonstration of -galactosidase activity on tissue sections. We have replaced 5-bromo-4-chloro-3-indolyl--D-galactoside (X-Gal) with 5-bromoindolyl--o-galactopyranoside (Bluo-Gal) as a chromogenic substrate for the bacterial -galactosidase (lacZ). After -galactosidic cleavage, Bluo-Gal precipitates in form of fine birefringent crystals, whereas X-gal gives rise to an amorphous precipitate. Upon microscopic examination under polarized light, the crystals emit a strong signal consisting of yellow reflected light. This property of Bluo-Gal results in greatly enhanced sensitivity of the staining method for -galactosidase and allows for optimal morphological resolution. To exemplify the applications of this technique, the expression is demonstrated in transgenic mice of -galactosidase driven by a fragment of the human tissue-type plasminogen activator promoter.     

Enzymic activities and galactomannan mobilisation in germinating seeds of fenugreek (Trigonella fœnum-graecum L. Leguminosae)

Summary The activities of -galactosidase, -mannosidase and -mannosidase were determined in extracts from the endosperm and from the embryo of fenugreek seeds at different stages of germination. Endosperm homogenates contained little or no activity of the above enzymes in the early stages of germination, before the reserve galactomannan began to be mobilised. The onset of galactomannan breakdown coincided with the appearance of -galactosidase and -mannosidase activities, which increased throughout the period of galactomannan degradation and then remained constant. A similar rise in -galactosidase and -mannosidase activities occurred during galactomannan breakdown in dry-isolated endosperms incubated under germination conditions. The increase could be suppressed by metabolic inhibitors which also inhibit galactomannan breakdown. Embryo homogenates contained high -galactosidase, high -mannosidase and some -mannosidase activity at all stages of germination.No oligomannosyl -1,4 phosphorylase activity could be detected either in the endosperm or in the embryo.It is concluded that the galactomannan of fenugreek is broken down by a series of hydrolases secreted by the aleurone layer of the endosperm. They include -galactosidase, -mannosidase and probably also endo--mannanase.This is part four in a series of papers dealing with galactomannan metabolism. Part three: Planta (Berl.) 106, 44–60 (1972).     

Histochemical demonstration of the intestinal hetero-β-galactosidase (glucosidase)

Summary The histochemical demonstration of hetero--galactosidase (glucosidase) has been attempted in sections and zymograms of rabbit, monkey and human intestine and of rat kidney.The leakage of this enzyme from unfixed sections was prevented by the use of cold microtome sections adherent to semipermeable membranes. Methods with -D-glucosides and galactosides of 6-Br-2-naphthol (postincubation azocoupling with Fast Blue B as well as simultaneous azocoupling with hexazonium-p-rosaniline), of -naphthol (simultaneous azocoupling with hexazonium-p-rosaniline) and of 4-Cl-5-Br-3-indolyl (with ferricyanide, phenazonium methosulfate or nitro BT and without any oxidation agent) were used an evaluated concerning the specificity, localization ability and inhibition of enzyme activity. Pretreatment of sections with distilled water or saline and inhibition by p-Cl-mercuribenzoate, glucono- and galactono-lactones were used for the characterization of the demonstrated enzyme activity.6-Br-2-naphthyl--D-glucoside is the most specific substrate for hetero--galactosidase. It is not split by lactase and acid -galactosidase. Only lysosomal -glucosidase can interfere. Because the latter enzyme is membrane-bound the difference in color intensity between untreated and prewashed sections are due to hetero--galactosidase. Only localization on the cellular (not intracellular) level can be achieved, however.The simultaneous azocoupling method with -naphthyl--D-glucoside and hexazonium-p-rosaniline enables a very good localization of hetero--galactosidase in the rabbit intestine. Due to a great inhibition exerted by hexazonium-p-rosaniline on the enzyme activity the method is unsuitable for the detection of hetero--galactosidase in zymograms and in the human intestine. Interference of lactase (or lactase-phlorizine hydrolase complex) is to be considered. The lysosomal -glucosidase does not seem to interfere.Indigogenic methods are not sensitive either. With ferricyanide as an oxidation agent it was not possible to detect the activity of hetero--galactosidase in zymograms and in sections. This is possibly due to overoxidation of indigo. The same holds true for phenazonium methosulfate used for the processing of zymograms. However, it was possible to reveal the activity of hetero--galactosidase in sections of the rabbit and monkey intestine with phenazonium methosulfate as oxidation agent. Nitro BT enhanced the coloration both in zymograms and in sections. In the latter case diffusion artifacts cannot be prevented, however. The interference of lactase, lysosomal -galactosidase and possibly of lysosomal -glucosidase (depending on the glycoside used) is always to be considered.Hetero--galactosidase was localized in the cytoplasm (particularly in the supranuclear region) of differentiated enterocytes covering the villi of the rabbit (the highest activity), monkey and human (the lowest activity) intestine. In crypt enterocytes and in cells of Brunner's glands the activity was lower. The occurrence of a low activity of hetero--galactosidase in the brush border of enterocytes of the rabbit intestine was also demonstrated.A proximodistal gradient was observed in the rabbit and monkey intestine, the upper jejunum displaying the highest activity.In jejunal biopsies of patients with celiac sprue (in the acute stage of the disease) the activity of hetero--galactosidase was lowered. No changes of activity were observed in jejunal biopsies of patients with isolated deficiencies of lactase or sucrase.In the rat kidney the enzyme was demonstrated particularly in the cytoplasm of cells of proximal convoluted tubules.     

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.     

Effects of glycine supplement on protein production and release in recombinant Escherichia coli

Summary The effect of glycine supplement to growth media on protein expression and release in a recombinant strain RR1 of E. coli was investigated. Addition of glycine to the growth media in moderate amount (up to 1%) was observed to enhance significantly the release of periplasmic proteins from the cell to the broth. The extracellular activities of the model enzymes -amylase and -lactamase were increased by a factor of 16.3 and 3.8 respectively in the presence of glycine. These activities corresponded to about 50% of the total production for each protein. Furthermore, with glycine supplement the total enzyme activity of both -amylase, -lactamase as well as -galactosidase were increased by a factor of about 2.5. Cell growth characteristics and low extracellular activity of the cytoplasmic protein -galactosidase are indicative that glycine does not cause significant cell-lysis for a concentration below 0.7%.     

Synthesis of Galβ1-3GlcNAc and Galβ1-3GlcNAcβ-SEt by an enzymatic method comprising the sequential use of β-galactosidases from bovine testes andEscherichia coli

Gal1-3GlcNAc (1) and Gal1-3GlcNAc-SEt (2) were synthesized on a 100 mg scale by the transgalactosylation reaction of bovine testes -galactosidase with lactose as donor andN-acetylglucosamine and GlcNAc-SEt as acceptors. In both cases the product mixtures contained unwanted isomers and were treated with -galactosidase fromEscherichia coli which has a different specificity, under conditions favouring hydrolysis, yielding besides the desired products, monosaccharides and traces of trisaccharides. The products were purified to >95% by gel filtration, with a final yield of 12% of 1 and 17% of 2, based on added acceptor. In a separate experiment Gal1-6GlcNAc-SEt (3) was synthesized by the transglycosylation reaction using -galactosidase fromEscherichia coli. No other isomers were detected. Compound 3 was purified by HPLC.     

Cloning and expression of β-galactosidase from psychotrophic Bacillus subtilis KL88 into Escherichia coli

Summary The -galactosidase gene from Bacillus subtilis KL88 was cloned into Escherichia coli and the gene product characterized for its potential use in the dairy industry. The two recombinant plasmids that we obtanied encoded a -galactosidase with the same catalytic and thermal characteristics as the native -galactosidase from B. subtilis. The recombinant -galactosidases exhibited high activity at low temperature (10°C), with maximum activity at 50°C and an optimum pH of 6.0. Its molecular weight was estimated to be 90 Kd. The restriction maps of the recombinant plasmids were constructed. The -galactosidase gene was located in a 2.3 Kb fragment.     

Cloning and expression of β-galactosidase gene from Streptococcus thermophilus in Saccharomyces cerevisiae

Summary The -galactosidase gene ofStreptococcus thermophilus was cloned into plasmid vector, pVT100-U, and used to transform a strain ofEscherichia coli andSaccharomyces cerevisiae. Transformants which expressed -galactosidase activity were obtained in bothE. coli andSaccharomyces cerevisiae, the highest activity found in a yeast recombinant. The expression and thermostability of the cloned -galactosidase genes from different plasmid constructions were compared with the streptococcal -galactosidase. The recombinant protein was equivalent to the specific activity and thermostability ofS. thermophilus.     

Identification of new enzyme activities of several strains of Thermus species

New thermostable enzyme activities of seven Thermus strains were compared using the API ZYM system. All the strains exhibited high levels of - and -glycosidases, esterase (C₄) and esterase-lipase (C₈) activities intracellularly. Only T. thermophilus HB8 (ATCC 27634) showed -glucosidase and esterase activities in the supernatant. According to the intensity of -galactosidase activity, Thermus strains were divided in three groups. Group 0, which showed a weak -galactosidase activity, included Thermus spp. ATCC 31674 (T351) and 27978 (X-1) as well as T. thermophilus ATCC 27634 (HB8). Group I which consisted of T. aquaticus ATCC 25104 (YT-1), ATCC 25105 (Y-VII-51B) and Thermus sp. ATCC 27737 (T2), had a specific activity of approximately 40.0 U mg^–1 and galactose as inducer. T. aquaticus ATCC 31558 (group 2) was particularly effective for -galactosidase production (2840 U) with a specific activity of 98 U mg^–1. For each strain, galactose (0.5%) was a better inducer of -galactosidase production than lactose (1%). The detection of -galactosidase activity was dependent on the derivative chromogenic substrates used (naphthyl or nitrophenol coupled to sugar). Oligosaccharides were synthesized from cellobiose, lactulose, maltose or lactose as substrates at high temperature in some strains of Thermus.