Structure-function relationships in glucoamylases encoded by variant Saccharomycopsis fibuligera genes.
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The mutation Gly467-->Ser in Glu glucoamylase was designed to investigate differences between two highly homologous wild-type Saccharomycopsis fibuligera Gla and Glu glucoamylases. Gly467, localized in the conserved active site region, S5, is replaced by Ser in the Gla glucoamylase. These amino acid residues are the only two known to occupy this position in the elucidated glucoamylase sequences. The data from the kinetic analysis revealed that replacement of Gly467 with Ser in Glu glucoamylase decreased the kcat towards all substrates tested to values comparable with those of the Gla enzyme. Moreover, the mutant glucoamylase appeared to be less stable compared to the wild-type Glu glucoamylase with respect to thermal unfolding. Microcalorimetric titration studies of the interaction with the inhibitor acarbose indicated differences in the binding between Gla and Glu enzymes. The Gla glucoamylase, although less active, binds acarbose stronger (Ka congruent with 10(13).M(-1)) than the Glu enzyme (Ka congruent with 10(12).M(-1)). In all enzymes studied, the binding of acarbose was clearly driven by enthalpy, with a slightly favorable entropic contribution. The binding of another glucoamylase inhibitor, 1-deoxynojirimycin, was about 8-9 orders of magnitude weaker (Ka congruent with 10(4).M(-1)) than that of acarbose. From comparison of kinetic parameters for the nonglycosylated and glycosylated enzymes it can be deduced that the glycosylation does not play a critical role in enzymatic activity. However, results from differential scanning calorimetry demonstrate an important role of the carbohydrate moiety in the thermal stability of glucoamylase. (+info)
Mutant of the yeast Saccharomycopsis lipolytica that accumulates and excretes protorphyrin IX.
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The red, water-insoluble pigment excreted by a mutant strain of the yeast Saccharomycopsis lipolytica is show to be protoporphyrin IX. In genetic crosses the red phenotype has the properties characteristic of a defect in a single, recessive nuclear gene. The yield and ease of harvest of protoporphyrin IX from the yeast mutant indicate that this strain or its derivatives may be a valuable source of this substance. (+info)
Mutants of Saccharomycopsis lipolytica defective in lysine catabolism.
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Wild-type strains of Saccharomycopsis lipolytica are able to use lysine as a carbon or a nitrogen source, but not as a unique source for both. Mutants were selected that could not use lysine either as a nitrogen or as a carbon source. Some of them, however, utilized N-6-acetyllysine or 5-aminovaleric acid. Many of the mutants appeared to be blocked in both utilizations, suggesting a unique pathway for lysine degradation (either as a carbon or as a nitrogen source). Genetic characterization of these mutants was achieved by complementation and recombination tests. (+info)
The enzymatic and molecular characteristics of Saccharomycopsis alpha-amylase secreted from Saccharomyces cerevisiae.
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The Saccharomycopsis fibuligera alpha-amylase (Sfamy) gene was expressed in Saccharomyces cerevisiae. The highest productivity of Sfamy was 70 mg per liter of culture broth. We purified Sfamy from the culture broth and identified the NH2 terminal primary sequence. This sequence suggests that the Sfamy gene product is synthesized as a pre-pro-precursor, and the pro-sequence is cleaved after a Lys-Arg sequence with the calpain-like endopeptidase encode by the KEX2 gene, resulting in mature Sfamy protein composed of 468 amino acids. Furthermore, the enzyme Sfamy is a glycoprotein in which one N-linked sugar chain containing mannose residues is attached to the Asn residue at the 198 position. The Km and kcat values were 1.1 x 10(-4) M and 1.4 x 10(2) sec-1, respectively, using amylose (the degree of polymerization n = 18) as a substrate. Moreover, the secondary structure, the location of the secondary elements including alpha-helix, beta-sheet, and loop, and tertiary structure were predicted theoretically on the basis of the molecular structure of Aspergillus oryzae alpha-amylase. Taka-amylase A (TAA). These results indicate that Sfamy protein is composed of main (M) and C-terminal (C) domains. The molecular structure of M domain closely resembles that of TAA, but the C domain appears to be more compact than that of TAA because of deletions at three regions forming turns and one region forming alpha-helix. (+info)
Production of benzoylformic acid from phenylglycine by Saccharomycopsis lipolytica.
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Microbial production of benzoylformic acid (BF), which can be used as a substrate of enzymatic synthesis of (R)-(-)-mandelic acid, was investigated. Among 145 strains of yeasts and actinomycetes, Saccharomycopsis lipolytica (IAM 4964) was the best producer of BF from DL-phenylglycine (DL-PG). Culture conditions for BF production by the organism were optimized. When 0.2% fructose as a carbon source and 0.7% Bacto-tryptone as a nitrogen source were used in the presence of 4% DL-PG, 14.5 mg/ml of BF was produced (about 37% molar yield) in 4 days of cultivation. BF was synthesized from the L-form of PG, but not from the D-form. The BF was isolated from culture broth in a crystalline form and physicochemically identified. (+info)
A mutant alpha-amylase with enhanced activity specific for short substrates.
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The 210th lysine (K210) at the active site in Saccharomycopsis fibuligera alpha-amylase was altered to arginine (R) or asparagine (N) by site-directed mutagenesis. Replacement of K210 by R strengthened the 7th and weakened the 8th subsite affinities. K210 was found to contribute to both the 8th and the 7th subsites. The catalytic activity of the K210R enzyme for the hydrolysis of maltose (G2) was three-times higher than that of the native enzyme due to an increase in the affinity of the 7th subsite adjacent to the catalytic site, whereas the activity of the K210N enzyme for G2 was decreased to 1% of that of the native enzyme by a reduction in the 7th subsite affinity. (+info)
Purification and characterization of a neutral protease from Saccharomycopsis lipolytica.
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Saccharomycopsis lipolytica 37-1 produced two inducible extracellular proteases, one under neutral or alkaline growth conditions and the second under acid conditions. Secretion of the neutral protease was repressed in the presence of glycerol or glucose, both of which supported rapid growth of the organism. Ammonium ions also repressed the secretion of the enzyme. The neutral protease activity copurified with esterase activity during ammonium sulfate fractionation, chromatography on diethylaminoethyl-cellulose, and gel filtration on Sephadex G-150. The molecular weight of the enzyme was estimated to be 42,000 by sucrose density gradient centrifugation and 38,500 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme had a pH optimum of 6.8. Phenylmethylsulfonylfluoride inhibited both protease and esterase activities, indicating the presence of a serine residue in the active center. Protease, but not esterase, activity was sensitive to ethylenediaminetetraacetate and was significantly activated by divalent ions. Dithiothreitol inhibited both protease and esterase activities, indicating the presence of a critical disulfide bridge. The enzyme hydrolyzed casein (K(m) = 25.6 muM) and hemoglobin as well as the nitrophenyl esters of tyrosine (K(m) = 2.4 mM), glycine, tryptophan, and phenylalanine. (+info)
Subsite structure of Saccharomycopsis alpha-amylase secreted from Saccharomyces cerevisiae.
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The kinetic parameters (kcat/Km) and the cleaved-bond distributions for the hydrolysis of linear maltooligosaccharides Gn (3 less than or equal to n less than or equal to 9) by Saccharomycopsis alpha-amylase (Sfamy) secreted from Saccharomyces cerevisiae were determined at pH 5.25 and 25 degrees C. The subsite affinities of Sfamy were also evaluated from these data. The subsite structure of Sfamy is characteristic of the active site of an endo-cleavage type enzyme, consisting of internal repulsive sites with the catalytic residues and external attractive sites. Moreover, the pKa values of the catalytic residues were calculated from the pH dependence plot of the kinetic parameter (kcat/Km). The amino acid residues which contribute to the subsite affinities and the catalytic activity of Sfamy are proposed and compared with those of Taka-amylase A. (+info)