Genetic control of maltase synthesis in yeast

Summary A new series of maltase negative mutants have been isolated from yeast strains carrying the MAL4 gene. These mutants are allelic to the MAL4 gene and fail to ferment maltose, sucrose, and alphamethylglucoside. Most revertants isolated from these mutants restore the ability to ferment above sugars, and also produce the same levels of maltase as the parental strains. One of the revertants (NA-520-R1), however, ferments maltose slowly, and produces 24 fold less enzyme than the parental strain. Genetic studies revealed that revertant (NA-520-R1), is not a truc back mutation but is carrying an extra-genic suppressor, which suppresses the mal4 allele in mutant (NA-520). Since several lines of published evidence indicate that the MAL4 gene is a regulatory gene, it is suggested that the MAL4 gene codes for a regulatory protein, which acts as positive regulatory element in maltase synthesis.     

Genetic and biochemical evidence of sucrose fermentation by maltase in yeast

Summary Strain 1403-7A, which carries the MAL4 gene responsible for constitutive maltase synthesis, can ferment sucrose in the absence of sucrose genes. Sucrose fermentation cannot be separated from maltose fermentation either by genetic recombination or by mutation. Crude extracts of strain 1403-7A also lack the classical invertase, and fractionation of such extracts by gel filtration results in a peak of maltase activity which corresponds exactly to the activity with respect to sucrose hydrolysis. Moreover, in vitro, both of these disaccharides are hydrolyzed maximally at pH 6.4 to 6.8. It is suggested that, as long as sucrose can penetrate the cell, maltase, if present at high level in any strain, should be able to hydrolyze sucrose and therefore permit its fermentation. We have, however, identified in one of our yeast stocks a single recessive gene (ssf gene) which specifically interferes with sucrose fermentation in strain 1403-7A, probably by limiting the penetration of sucrose.     

Characterisation of maltase from enteropathogenic Escherichia coli

Abstract Enteropathogenic strains of faecal Escherichia coli produced significantly ( P < 0.01) more maltase than the non-pathogenic strains of the organism. The enzyme was induced by maltose but repressed by glucose and fructose. The maltase was partially purified by ammonium sulphate precipitation, followed by dialysis and gel permeation chromatography. The partially purified maltase had an M _r of 144500 and an apparent K _m of approx. 7.6 mM for maltose. The enzyme was stimulated by Ca²⁺, inhibited by Cu²⁺, Hg²⁺, Uo²⁺, IAA and EDTA, and exhibited optimum activity at pH 6.5 at 30°C.     

Effect of the petite mutation on maltose and alpha-methylgucoside fermentation inSaccharomyces cerevisiae.

Summary Several hundred petite mutants were isolated from yeast strains of different genotype to examine the effect of the petite mutation on maltose and alpha-methylglucoside fermentation. In most cases petite mutants isolated retain the ability to ferment maltose and alpha-methylglucoside, although at a slower rate. In one strain (1403-7A), however, the ability to ferment alpha-methylglucoside is completely lost in all petite mutants isolated from this strain. It is suggested that mitochondrial factors may be involved in the utilization of alpha-methylglucoside in strain 1403-7A.     

Glucose repression of maltase and methanol-oxidizing enzymes in the methylotrophic yeastHansenula polymorpha: Isolation and study of regulatory mutants

Regulation of the synthesis of maltase and methanol-oxidizing enzymes by the carbon source has been analyzed in the methylotrophic yeastHansenula polymorpha. Maltase was shown to be responsible for the growth ofH. polymorpha not only on maltose, but also on sucrose. The affinity of maltase towards maltase substrates decreased in the order: 4-nitrophenyl glucoside (pNPG) <sucrose <maltose. Mutants with glucose repression-insensitive synthesis of alcohol oxidase and maltase were obtained fromH. polymorpha by mutagenesis and subsequent selection on methanol medium in the presence of 2-deoxy-d-glucose. One of the isolated mutants, L63, was studied in more detail. Mutant L63 was recessive and monogenic and it was not deficient in hexokinase. Its analysis revealed thatH. polymorpha most probably has a repressor protein that in the presence of glucose can down-regulate expression of both maltase and enzymes of methanol oxidation.     

Production of an extracellular maltase by thermophilic Bacillus sp. KP 1035.

Production of extracellular maltase was studied with thermophilic Bacillus sp. KP 1035, which was selected as the organism producing the highest levels of maltase. The final enzyme yield was increased by maltose, peptone, and yeast extract but reduced by succinate and fumarate. Maximum enzyme production was achieved at 55 degrees C and at an initial pH of 6.2 to 7.0 on a medium containing 0.3% maltose, 1% peptone, 0.1% meat extract, 0.3% yeast extract, 0.3% KH2PO4, and 0.1% KH2PO4. Maltase was synthesized in cytoplasm and accumulated as a large pool during the logarithmic growth phase, which preceded sporulation. At the end of this phase, the enzyme appeared in the culture broth, and its accumulation increased in parallel with a rise in the extracellular protein level. Maltase was stable for 24 h at 60 degrees C over a pH range of 5.6 to 9.0 and retained 95% of the original activity after treatment for 20 min at 70 degrees C at pH 6.8.     

Production of an extracellular maltase by thermophilic Bacillus sp. KP 1035.

Production of extracellular maltase was studied with thermophilic Bacillus sp. KP 1035, which was selected as the organism producing the highest levels of maltase. The final enzyme yield was increased by maltose, peptone, and yeast extract but reduced by succinate and fumarate. Maximum enzyme production was achieved at 55 degrees C and at an initial pH of 6.2 to 7.0 on a medium containing 0.3% maltose, 1% peptone, 0.1% meat extract, 0.3% yeast extract, 0.3% KH2PO4, and 0.1% KH2PO4. Maltase was synthesized in cytoplasm and accumulated as a large pool during the logarithmic growth phase, which preceded sporulation. At the end of this phase, the enzyme appeared in the culture broth, and its accumulation increased in parallel with a rise in the extracellular protein level. Maltase was stable for 24 h at 60 degrees C over a pH range of 5.6 to 9.0 and retained 95% of the original activity after treatment for 20 min at 70 degrees C at pH 6.8.     

Neutral maltase of human granulocytes: localization on the extracytoplasmic side of the plasma membrane and some properties

Neutral maltase is an alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) which is present in human granulocytes and B-lymphocytes but not in T-lymphocytes. These cells have been reported to contain a renal-type neutral maltase which cross-reacts with an antiserum raised against kidney brush-border enzyme. No study has been performed to assess the subcellular localization of the enzyme. Molecular properties of leukocyte neutral maltase from any species are unknown. We report in this paper that neutral maltase is present on the extracytoplasmic side of human granulocyte plasma membrane. These results are supported by subcellular fractionation on Percoll gradient and by papain digestion of intact granulocytes. The enzyme is probably an integral membrane protein. The anchorage to the lipid bilayer may be similar to that of the stalked brush-border hydrolases. Some properties of granulocyte neutral maltase were also determined on a plasma membrane-enriched fraction. The enzyme cleaves maltose and nigerose but not other glucosides disaccharides and oligosaccharides. The Km for maltose is (+/- SD) 0.78 (+/- 0.06) mM, that for nigerose 21.05 (+/- 1.43) mM. The Vmax for nigerose is 0.83-fold that for maltose. Tris, maltotriose, maltotetraose, and maltopentaose were inhibitors of granulocyte neutral maltase.     

Identification of new genes involved in disaccharide fermentation in yeast

Summary Maltose non-fermenting mutants were obtained from strains carrying a MAL4 allele which permits constitutive synthesis of maltase. Cells carrying this allele are able to utilize sucrose in the absence of the classical sucrose genes. All maltose non-fermenting mutants were also sucrose non-fermenters. Eight mutants had become maltase negative; 19 mutants could still form maltase constitutively.In crosses with segregational maltose and sucrose non-fermenting strains, enzyme negative mutants gave diploids unable to ferment maltose and sucrose. Enzyme positive, non-fermenting mutants gave diploids which readily fermented maltose and sucrose. This latter type of mutants was designated dsf (disaccharide fermentation) mutants.The diploids derived from crossing non-fermenting mutants with segregational non-fermenters were subjected to tetrad analysis. Enzyme negative non-fermenters gave only non-fermenting progeny. The dsf mutants segregated both fermenting and non-fermenting progeny, some of which showed the dsf phenotype. This indicated that none of the dsf mutants had a defect in a gene closely linked to MAL4. Crosses between dsf mutants and strains carrying the maltose genes MAL2 and MAL3 showed that the mutations affected maltose fermentation in general. Sucrose fermentation in the presence of the classical sucrose gene SUC3 was not affected, nor were fermentation of glucose, fructose and galactose.The uptake of radioactivity from uniformly labeled maltose appeared to be blocked in mutants of at least four of the dsf genes. Only one non-leaky and a leaky mutant showed a significant uptake.These results suggest that there is an extremely complex transport system for maltose and sucrose or that the utilization of these disaccharides requires a complex series of metabolic reactions.     

Clustering of MAL genes in Hansenula polymorpha: cloning of the maltose permease gene and expression from the divergent intergenic region between the maltose permease and maltase genes

Hansenula polymorpha uses maltase to grow on maltose and sucrose. Inspection of genomic clones of H. polymorpha showed that the maltase gene HPMAL1 is clustered with genes corresponding to Saccharomyces cerevisiae maltose permeases and MAL activator genes orthologues. We sequenced the H. polymorpha maltose permease gene HPMAL2 of the cluster. The protein (582 amino acids) deduced from the HPMAL2 gene is predicted to have eleven transmembrane domains and shows 39-57% identity with yeast maltose permeases. The identity of the protein is highest with maltose permeases of Debaryomyces hansenii and Candida albicans. Expression of the HPMAL2 in a S. cerevisiae maltose permease-negative mutant CMY1050 proved functionality of the permease protein encoded by the gene. HPMAL1 and HPMAL2 genes are divergently positioned similarly to maltase and maltose permease genes in many yeasts. A two-reporter assay of the expression from the HPMAL1-HPMAL2 intergenic region showed that expression of both genes is coordinately regulated, repressed by glucose, induced by maltose, and that basal expression is higher in the direction of the permease gene.     

Non- pleiotropic nature of the gal 3 mutation in yeast.

The pleiotropic nature of three independant isolates of yeast galactose long-term adaptation mutants (termed $\text{gal}$ 3) were investigated with respect to the effect of the mutation on the adaptation time of the mutants to their utilization of maltose. Enzyme induction and growth studies with homozygous diploids of the isolates and heterozygous diploid of two of the isolates demonstrate that the mutants readily induce for maltase and grow without delay on maltose as the sole source of carbon and energy, yet still exhibit the long-term adaptation phenotype with respect to the utilization of galactose.     

Mutational analysis of the MAL1 locus of Saccharomyces: identification and functional characterization of three genes

Summary Fermentation of maltose by Saccharomyces strains depends on the presence of any one of five unlinked MAL loci (MAL1, MAL2, MAL3, MAL4 or MAL6). Earlier mutational analyses of MAL2 and MAL6 containing strains have identified a single complementation group at each of these two loci. However complementation analysis between naturally occurring Mal^– Saccharomyces strains isolated from the wild demonstrated the presence of two complementation groups (designated MALp and MALg) at the MAL1, MAL3 and MAL6 loci. The available evidence suggests that the MALp gene is functionally equivalent to the complementation group identified by mutational analysis at the MAL6 locus and that this gene encodes a protein involved in the regulation of the coordinate induction of both maltase and maltose permease synthesis.In this paper we report the isolation, in a well characterized MAL1 strain, of 47 mutants unable to ferment maltose. All the mutants, with one exception, map at the MAL1 locus. These mal1 mutants, except for one, are recessive to MAL1 and fall into two major complementation groups. Evidence is presented that these two classes of mutants identify both a gene involved in the regulation of maltose fermentation (MAL1R) and a gene involved in maltose transport (MAL1T). We also report here the isolation of a temperature sensitive maltose nonfermenting mutant mapping at the MAL1 locus identifying a third gene (MAL1S) at this locus. The maltase synthesized by this mutant, when assayed in cell-free extracts, is significantly more thermolabile than the wild type enzyme. Our findings demonstrate that MAL1 is a complex locus comprising at least three genes: MAL1R, a gene involved in the coordinate regulation of the synthesis of maltase and maltose transport; MAL1T, a gene encoding a component of the maltose transport system; and MAL1S, a likely candidate for the structural gene for maltase.     

Purification and some properties of an extracellular maltase from Bacillus subtilis.

Bacillus subtilis P-11, capable of producing extracellular maltase, was isolated from soil. Maximum enzyme production was obtained on a medium containing 2.0% methyl-alpha-D-glucose, 0.5% phytone, and 0.2% yeast extract. After the removal of cells, extracellular maltase was precipitated by ammonium sulfate (85% saturation). The enzyme was purified by using the following procedures: Sephadex G-200 column chromatography, diethylaminoethyl-Sephadex A-50 ion-exchange column chromatography, and a second Sephadex G-200 column chromatography. A highly purified maltase without amylase or proteinase activities was obtained. Some properties of the extracellular maltase were determined: optimum pH, 6.0; optimum temperature, 45 C, when the incubation time was 30 min; pH stability, within 5.5 to 6.5; heat stability, stable up to 45 C; isoelectric point, pH 6.0 (by gel-isoelectric focusing); molecular weight, 33,000 (by gel filtration with Sephadex G-200); substrate specificity: the relative rates of hydrolysis of maltose, maltotriose, isomaltose, and maltotetraose were 100:15:14:4, respectively, and there was no activity toward alkyl or aryl-alpha-D-glucosides, amylose, or other higher polymers. Transglucosylase activity was present. Glucose and tris(hydroxymethyl)aminomethane were competitive inhibitors with Ki values of 4.54 and 75.08 mM, respectively; cysteine was a noncompetitive inhibitor. Michaelis constants were 5 mM for maltose, 1 mM for maltoriose, and 10 mM for isomaltose. A plot of pKm (-log Km) versus pH revealed two deflection points, one each at 5.5 and 6.5; these probably corresponded to an imidazole group of a histidine residue in or near the active center; this assumption was supported by the strong inhibition of enzyme activity by rose bengal.     

Purification and some properties of an extracellular maltase from Bacillus subtilis.

Bacillus subtilis P-11, capable of producing extracellular maltase, was isolated from soil. Maximum enzyme production was obtained on a medium containing 2.0% methyl-alpha-D-glucose, 0.5% phytone, and 0.2% yeast extract. After the removal of cells, extracellular maltase was precipitated by ammonium sulfate (85% saturation). The enzyme was purified by using the following procedures: Sephadex G-200 column chromatography, diethylaminoethyl-Sephadex A-50 ion-exchange column chromatography, and a second Sephadex G-200 column chromatography. A highly purified maltase without amylase or proteinase activities was obtained. Some properties of the extracellular maltase were determined: optimum pH, 6.0; optimum temperature, 45 C, when the incubation time was 30 min; pH stability, within 5.5 to 6.5; heat stability, stable up to 45 C; isoelectric point, pH 6.0 (by gel-isoelectric focusing); molecular weight, 33,000 (by gel filtration with Sephadex G-200); substrate specificity: the relative rates of hydrolysis of maltose, maltotriose, isomaltose, and maltotetraose were 100:15:14:4, respectively, and there was no activity toward alkyl or aryl-alpha-D-glucosides, amylose, or other higher polymers. Transglucosylase activity was present. Glucose and tris(hydroxymethyl)aminomethane were competitive inhibitors with Ki values of 4.54 and 75.08 mM, respectively; cysteine was a noncompetitive inhibitor. Michaelis constants were 5 mM for maltose, 1 mM for maltoriose, and 10 mM for isomaltose. A plot of pKm (-log Km) versus pH revealed two deflection points, one each at 5.5 and 6.5; these probably corresponded to an imidazole group of a histidine residue in or near the active center; this assumption was supported by the strong inhibition of enzyme activity by rose bengal.     

Inactivation of maltose permease and maltase in sporulating Saccharomyces cerevisiae

Maltose transport and maltase activities were inactivated during sporulation of a MAL constitutive yeast strain harboring different MAL loci. Both activities were reduced to almost zero after 5 h of incubation in sporulation medium. The inactivation of maltase and maltose permease seems to be related to optimal sporulation conditions such as a suitable supply of oxygen and cell concentration in the sporulating cultures, and occurs in the fully derepressed conditions of incubation in the sporulation acetate medium. The inactivation of maltase and maltose permease under sporulation conditions in MAL constitutive strains suggests an alternative mechanism for the regulation of the MAL gene expression during the sporulation process.