Characterization of ATPase activity in the mycelial form cells of yeast Saccharomycopsis fibuligera

1. The properties of ATPase activity were studied with the mycelial form cells of Saccharomycopsis fibuligera. 2. Optimal pH for the activity was about 9.5. 3. The activity was stimulated by Mg2+. 4. The activity was inhibited by DCCD, NaF and oligomycin, but not inhibited by ouabain.     

Nucleotide sequence of the glucoamylase gene GLU1 in the yeast Saccharomycopsis fibuligera.

The complete nucleotide sequence of the glucoamylase gene GLU1 from the yeast Saccharomycopsis fibuligera has been determined. The GLU1 DNA hybridized to a polyadenylated RNA of 2.1 kilobases. A single open reading frame codes for a 519-amino-acid protein which contains four potential N-glycosylation sites. The putative precursor begins with a hydrophobic segment that presumably acts as a signal sequence for secretion. Glucoamylase was purified from a culture fluid of the yeast Saccharomyces cerevisiae which had been transformed with a plasmid carrying GLU1. The molecular weight of the protein was 57,000 by both gel filtration and acrylamide gel electrophoresis. The protein was glycosylated with asparagine-linked glycosides whose molecular weight was 2,000. The amino-terminal sequence of the protein began from the 28th amino acid residue from the first methionine of the putative precursor. The amino acid composition of the purified protein matched the predicted amino acid composition. These results confirmed that GLU1 encodes glucoamylase. A comparison of the amino acid sequence of glucoamylases from several fungi and yeast shows five highly conserved regions. One homology region is absent from the yeast enzyme and so may not be essential to glucoamylase function.     

Properties of ATPase activity in intact vegetative cells and sporulating cells of yeast Saccharomyces cerevisiae

The properties of ATPase activity were examined in the intact cells of yeast. The activity was stimulated by Mg2+, Mn2+ and Co2+. The activity was inhibited by NaN3 and by high concentrations of NaF, NaVO3 and PCMB. Optimal pH for the activity was approximately 8. The maximum value of the activity was obtained in the cells at the early stationary phase and it decreased in 3 hr after transfer to sporulation medium.     

Growth of Saccharomycopsis fibuligera in a continuous-stirred-tank fermentor

Growth rates and amylase production rates were determined for the yeast Saccharomycopsis fibuligera grown on a simulated potato processing waste in a continuous-stirred-tank fermentor. S. fibuligera formed large multicellular flocs in the fermentor, and cell growth was reduced at low dilution rates because of mass-transfer resistance. The average Thiele modulus, which is the measure of extent of substrate diffusion, had a value ranging from ?_av = 2.2 for D = 0.10 to 0.3 for D = 0.40. Growth rates were described by the Monod equation modified to include mass-transfer effects. This modified Monod equation was used to predict growth rates from measured floc-size distribution. Experimentally determined growth rates were in close agreement with these predicted values.     

Purification and characterization of the amylolytic enzymes of Saccharomycopsis fibuligera

• 1.1. A complex of extracellular amylolytic enzymes produced by Saccharomycopsis fibuligera KZ, grown on fine fibre (waste product from corn starch production) and corn-steep liquor, has been studied.
• 2.2. α-Amylases and glucoamylases, as the main representatives of this complex, were separated by hydrophobic chromatography on Spheron 300 LC.
• 3.3. Individual isoenzymes of one type were separated on FPLC-Mono Q.
• 4.4. The relative molecular weight of α-amylases is 54,000, glucoamylases 62,000, maximal activity is reached by both enzymes between pH 5.0 and 6.2 at a temperature of 40–50°C.
• 5.5. Glucoamylases have a higher stability of the native structure than α-amylases, they retain 55% of their original activity, even after 10 min of incubation at 100°C.

     

扣囊复膜孢酵母b-葡萄糖苷酶基因在工业酿酒酵母中的表达

本文以工业酿酒酵母菌株( Saccharomyces cerevisiae Y )为研究对象,针对其复杂的生理生化遗传特性,建立了相对应的转化体系。以pRS41H质粒为基础载体,构建了含有工业酿酒酵母自身的gpd2启动子、终止子和扣囊复膜孢酵母的b-葡萄糖苷酶基因bgl的重组质粒pRS-gb。电击转化进入工业酿酒酵母细胞,潮霉素抗性筛选,获得重组菌。该重组菌可以在以纤维二糖为唯一碳源的培养基中生长,培养36 h,b-葡萄糖苷酶酶活达到0.967 u/ml。以纤维二糖为唯一碳源的酒精发酵中,酒精度可以达到0.92 g/l。这对工业生产中利用纤维素为原料发酵生产酒精具有重要意义。     

Biochemical characterization of a glucoamylase from Saccharomycopsis fibuligera R64

Glucoamylase from the yeast Saccharomycopsis fibuligera R64 (GLL1) has successfully been purified and characterized. The molecular mass of the enzyme was 56,583 Da as determined by mass spectrometry. The purified enzyme demonstrated optimum activity in the pH range of 5.6–6.4 and at 50°C. The activity of the enzyme was inhibited by acarbose with the IC₅₀ value of 5 μM. GLL1 shares high amino acid sequence identity with GLU1 and GLA1, which are Saccharomycopsis fibuligera glucoamylases from the strains HUT7212 and KZ, respectively. The properties of GLL1, however, resemble that of GLU1. The elucidation of the primary structure of GLL1 contributes to the explanation of this finding.     

A kinetic study of homocitrate synthetase activity in the yeast Saccharomycopsis lipolytica

• 1.1.|A rapid method for estimating the activity of the first enzyme of lysine biosynthesis in yeasts (acetyl-coenzyme A: 2-ketoglutarate C-acetyl transferase, EC 4.1.3.21) is described.
• 2.2.|In the wild type strain, the fixation of one substrate, S-acetyl coenzyme A, shows sigmoidal saturation kinetics. The initial rate experiments indicate that the reaction obeys an ordered mechanism, 2-ketoglutaric acid binding before S-acetyl coenzyme A.
• 3.3.|The activity is completely inhibited in vitro by lysine and by some lysine analogs, which all show cooperative binding and have an heterotrophic effect on 2-ketoglutaric binding sites. A second class of effectors is found, including 2-aminoadipic acid, pipecolid acid and dipicolinic acid, which all affect the cooperativity of S-acetyl coenzyme A binding sites.
• 4.4.|Two types of mutations which modify these inhibition patterns without affecting the catalytic activity are described. One results in a desensitization towards lysine and lysine analogs only. The other entirely abolishes the susceptibility towards the second type of inhibitors, without affecting the susceptibility to lysine.
• 5.5.|No variations of the specific activity could be detected in the wild type strain at all; mutants showing an increased or a reduced activity were isolated.
• 6.6.|Our results do not support the existence of isoenzymes at the level of homocitrate synthetase in this yeast.

     

Structure-function relationships in glucoamylases encoded by variant Saccharomycopsis fibuligera genes.

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.     

An investigation into the pectolytic activity of the yeast Saccharomycopsis fibuliger

Saccharomycopsis fibuliger cells produce an inducible hydrolase, tentatively characterized as a polygalacturonase [poly(1,4-α-d-galacturonide) glycanohydrolase, EC 3.2.1.15], which is associated with the yeast cells and which causes the partial hydrolysis of pectin or poly-d-galacturonic acid. No evidence of pectinesterase (pectin pectyl hydrolyase, EC 3.1.1.11) or pectate lyase [poly(1,4-α-d-galacturonide) lyase, EC 4.1.1.1] activity has been found. Enzyme production took place at an optimum temperature of 28°C, whereas optimum activity was at ～45°C. The optimum pH for pectolytic activity was similar to the optimum pH for cell growth. A reduction in the concentration of dissolved oxygen in the culture medium and an increase in cell age caused an increase in the rate of pectin decomposition within the limits employed. Products of pectin decomposition consisted of a mixture of uronides including d-galacturonic acid.     

The nucleotide sequence of the glucoamylase gene GLA1 from Saccharomycopsis fibuligera KZ

The nucleotide sequence of the 2544-bp PstI fragment carrying the glucoamylase gene of Saccharomycopsis fibuligera KZ, designated as GLA1, has been determined. When compared with the nucleotide sequence of the GLU1 gene one nucleotide substitution was found in the 321- bp of the 5'-flanking region: 24 nucleotides were altered within the 1557 bp of the structural gene causing the deduced protein products of both genes to differ in three amino acids in the signal-peptide region and in eight amino acids of the mature protein. Six nucleotide insertions and 27 substitutions were in the 663 bp of the 3'-flanking region. The gene product expressed and secreted in Saccharomyces cerevisiae into the functional enzyme was not homogeneous. In situ detection of the enzyme in a polyacrylamide gel revealed two dominant and three minor bands.     

Assay of the Ca pump ATPase activity of intact red blood cells.

An assay for the Ca pump ATPase of intact human red blood cells (RBCs) was developed. The assay utilized a small volume (typically 10 microliters) of packed RBCs in 1 ml of a buffer of known composition. The assay was based on the exposure of intact RBCs to the ionophore, A23187, in the presence of Ca. Such exposure caused a rapid degradation of ATP in RBCs. This degradation process is modeled in a numerical simulation in a companion paper (Vincenzi, F. F. and Hinds, T. R. (1992) Biochim. Biophys. Acta 1105, 63-70). The loss of ATP followed pseudo-first-order kinetics, and the rate constants for ATP degradation was taken as a measure of the capacity of the Ca pump ATPase. A number of variables were examined to optimize the activity of the ATPase. These variables included the concentrations of Ca and A23187. Because A23187 can promote loss of cellular Mg, it was necessary to include MgCl2 in the incubation medium to optimize ATPase activity. Likewise, it was determined that inclusion of iodoacetic acid optimized the rate of ATP loss, presumably by preventing the resynthesis of ATP from ADP and inorganic phosphate. Cobalt inhibited the ionophore-dependent loss of ATP by apparent competition with Ca for binding to A23187. Results of many assays demonstrated substantial differences in the rate constant for ATP loss in RBCs from different individuals. RBCs were selected according to density. Density associated loss of Ca pump ATPase activity was observed both by the intact RBC assay, and by assay of Ca pump ATPase activity in saponin lysates of RBCs. The correlation coefficient between the two assays was 0.93. It is suggested that the rate constant for ATP loss in intact RBCs exposed to A23187 and Ca can be taken as a measure of the Ca pump ATPase activity. This may be useful when isolated membrane ATPase assays fail (e.g., dog RBCs). The intact cell assay can also be carried out on very small volumes of cells and may be of particular value when RBC volumes are limited.     

Cloning and expression of a gene for an alpha-glucosidase from Saccharomycopsis fibuligera homologous to family GH31 of yeast glucoamylases

Cloning of cDNA encoding an α-glucosidase from the dimorphous yeast Saccharomycopsis fibuligera and characterization of the gene product were performed. The cDNA of the putative α-glucosidase gene consists of 2,886 bp, which includes an open reading frame encoding a 19 amino acid signal peptide at the N-terminal end and a 944 amino acid mature protein with a predicted molecular mass of 105.4 kDa and pI value of 4.52. The deduced amino acid sequence shows a high degree of identity (70%) with two yeast glucoamylases, namely, the extracellular glucoamylase Gam from Schwanniomyces occidentalis and the cell surface glucoamylase Gca from Candida albicans. The recombinant product, synthesized in Saccharomyces cerevisiae, is localized on the cell surface and hydrolyses maltooligosaccharides exclusively without the ability to digest soluble starch, which is consistent with the specificity characteristic of α-glucosidase, EC. 3.2.1.20.     

A kinetic study of homocitrate synthetase activity in the yeast Saccharomycopsis lipolytica.

1. A rapid method for estimating the activity of the first enzyme of lysine biosynthesis in yeasts (acetyl-coenzyme A: 2-ketoglutarate C-acetyl transferase, EC 4.1.3.21) is described. 2. In the wild type strain, the fixation of one substrate, S-acetyl coenzyme A, shows sigmoidal saturation kinetics. The initial rate experiments indicate that the reaction obeys an ordered mechanism, 2-ketoglutaric acid binding before S-acetyl coenzyme A. 3. The activity is completely inhibited in vitro by lysine and by some lysine analogs, which all show cooperative binding and have an heterotropic effect on 2-ketoglutaric binding sites. A second class of affectors is found, including 2-aminoadipic acid, pipecolic acid and dipicolinic acid, which all affect the cooperativity of S-acetyl coenzyme A binding sites. 4. Two types of mutations which modify these inhibition patterns without affecting the catalytic activity are described. One results in a desensitization towards lysine and lysine analogs only. The other entirely abolishes the susceptibility towards the second type of inhibitors, without affecting the susceptibility to lysine. 5. No variations of the specific activity could be detected in the wild type strain at all; mutants showing an increased or a reduced activity were isolated. 6. Our results do not support the existence of isoenzymes at the level of homocitrate synthetase in this yeast.     

扣囊复膜孢酵母α-淀粉酶基因在乳酸克鲁维酵母中的表达与重组酶性质

通过PCR方法从扣囊复膜孢酵母基因组DNA中克隆获得α-淀粉酶基因成熟肽编码区（SfA）,插入乳酸克鲁维酵母表达载体pKLACl的d因子信号肽下游,构建重组表达载体pKLACl-SfA。重组载体转化乳酸克鲁维酵母GG799,筛选获得表达α-淀粉酶SfA水平较高的重组茵。酶活检测和SDS．PAGE电泳检测均显示,重组茵分泌重组酶SfA到发酵液中。酶学性质研究表明：SfA最适温度为45℃,最适pH5．0,在pH4．5～5．5、50℃条件下保持稳定。Ca2＋等二价金属离子对SfA酶活有激活作用,EDTA强烈的抑制SfA活性。HPLC分析显示SfA水解糊精获得麦芽寡糖和少量葡萄糖,其中麦芽三糖是主要产物,占水解产物总量的52％。     

扣囊复膜孢酵母BGL1基因在工业酒精酵母中的整合表达

构建了含有工业酿酒酵母自身GPD2启动子和终止子、扣囊复膜孢酵母b-葡萄糖苷酶基因(BGL1)和潮霉素选择性标记hyg的重组质粒pPIC-gpd-bgl-hyg, 通过酵母染色体同源重组, 将BGL1基因整合进入工业酒精酵母的染色体上。重组酵母可以在以纤维二糖为唯一碳源的培养基上生长, 48 h时b-葡萄糖苷酶酶活达到0.764 U/mL。在玉米浓醪酒精发酵实验中, 与宿主菌株相比, 重组酵母醪液中纤维二糖含量减少约80％, 达到了消耗醪液中纤维二糖含量的目的。     

Some properties of coupled hepatoma mitochondria exhibiting uncoupler-insensitive ATPase activity

Ribulose-1,5-diphosphate car?ylase from the photosynthetic bacterium Chromatium catalyses the oxidative formation of phosphoglycolate and 3-phosphoglycerate from ribulose-1,5-diphosphate at an alkaline pH (9.3) in an atmosphere of oxygen. The catalytically active oligomeric form of the large subunit of the car?ylase molecule, A_m, was proved to be functionally active in the ribulose-1,5-diphosphate oxygenase reaction without the presence of the smaller subunit.