Saccharomycopsis Fibuligera Research Articles

Concanavalin A-agarose gel system capable of accumulating extracellular glucoamylase produced by immobilized Saccharomycopsis fibuligera

Saccharomycopsis fibuligera immobilized within agarose gel beads extracellularly produces glucoamylase, with which concanavalin A can aggregate to form a concanavalin A-glucoamylase complex. The complex increasingly accumulates in the gel beads with increasing glucoamylase production in the presence of concanavalin A. The formation or degradation of the complex is controlled in response to the presence or absence of methyl-α- d-mannopyranoside, which has a higher affinity toward concanavalin A. Glucoamylase was harvested with high recovery and purity by adding methyl-α- d-mannopyranoside.

Crystallization and preliminary X-ray analysis of the Saccharomycopsis fibuligera glucoamylase expressed from the GLU1 gene in Escherichia coli.

The active non-glycosylated glucoamylase, overexpressed from the Saccharomycopsis fibuligera GLU1 gene in Escherichia coli BL21(DE3), has been purified from the solubilized inclusion bodies and then renatured in vitro. Crystals of the recombinant glucoamylase were obtained by vapour diffusion using PEG as precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell dimensions of a = 58.1, b = 87.8 and c = 99.9 A, and diffract to 1.7 A resolution. This is the first report of the crystallization of the full-length glucoamylase corresponding to the mature enzyme.

Purification and characterization of an extracellular β-glucosidase from the filamentous fungus Acremonium persicinum and its probable role in β-glucan degradation

A β-glucosidase from the culture filtrates of the filamentous fungus Acremonium persicinum has been purified by (NH 4) 2SO 4 precipitation followed by anion-exchange and gel filtration chromatography. SDS-PAGE of the purified enzyme gave a single band with an apparent molecular mass of 128 kDa. The enzyme is a monomeric protein with an isoelectric point of 4.3 and a pH optimum of 5.5. Comparison of the n-terminal amino acid sequence revealed similarities between the A. persicinum enzyme and several other extracellular fungal β-glucosidases including those from Trichoderma reesei, Aspergillus aculeatus, Saccharomycopsis fibuligera, and Pichia anomala. In addition to the hydrolysis of p-nitrophenyl-β-glucoside, the enzyme was also active against several other aryl-β-glucosides as well as a range of β-linked oligoglucosides including laminaribiose, gentiobiose, cellobiose, and sophorose. d-Glucono-1,5-lactone and glucose are competitive inhibitors while the enzyme was also inhibited by n-bromosuccinimide, n-acetylimidazole, dicyclohexyl carbodiimide, Woodward's Reagent K, 2-hydroxy-5-nitrobenzyl bromide, KMnO 4, and some metal ions. Possible roles for this enzyme in the noncellulolytic fungus A. persicinum are discussed in light of the increase in the rate of reducing sugar release from β-glucans by (1 → 3)- and (1 → 6)-β-glucanases when the β-glucosidase is also present in the reaction mixtures.

Predacious yeasts.

Haustorium-mediated predation was observed in seven yeast species. Arthroascus javanensis, Botryoascus synnaedendrus, Guilliermondella selenospora, Saccharomycopsis fibuligera, and three hitherto unknown species penetrate and kill other yeasts. These yeasts share an unusual requirement for organic sulphur. One isolate recovered from Australian Hibiscus was studied in detail and found to attack a broad range of prey species, including ascomycetous and basidiomycetous yeasts as well as moulds. Predation was most effective when growth was on a solid surface and the medium was poor in complex nutrients. Organic sulphur (exemplified by methionine) was identified as a key factor. It serves as a nutritional benefit to the predator and, depending on the concentration, acts as either an inhibitor of predation or possibly a signal for detection of prey. Sampling of a yeast habitat with a medium selective for selenium-resistant yeasts indicated that auxotrophic and predacious yeasts might be more widespread than anticipated.

Cloning and expression of an Arxula adeninivorans glucoamylase gene in Saccharomyces cerevisiae

The glucoamylase gene of the yeast Arxula adeninivorans Ls3 has been cloned from a genomic library and sequenced. The gene could be localized on chromosome 2 from A. adeninivorans and comprises 1875 bp. The first 16 N-terminal amino acids represent the signal sequence for entering the endomembrane system. Comparing the amino acid sequence from this glucoamylase with those of other fungal glucoamylases shows that the glucoamylase of strain Ls3 has a homology to the glucoamylases from Rhizopus oryzae (32.6%), Saccharomycopsis fibuligera (23.1%), Aspergillus niger (22.1%), and Saccharomyces diastaticus (15.4%). No homology could be detected to the glucoamylase of Schwanniomyces occidentalis. By using the GAL1 promoter from Saccharomyces cerevisiae within an autonomously replicating plasmid it was possible to express the isolated Arxula glucoamylase gene in Saccharomyces cerevisiae. The transformants secreted 95% of the enzyme into the culture medium. The N termini of glucoamylases synthesized in A. adeninivorans and S. cerevisiae transformants are identical, which means that the signal sequences were cleaved at the same positions during maturation of the proteins. The highest glucoamylase activities were reached in the culture medium of S. cerevisiae transformants after 36 h of fermentation. Northern hybridization showed that the glucoamylase transcripts were formed continuously for up to 70 h. These results reveal that the glucoamylase is expressed and secreted more rapidly in the S. cerevisiae transformants than in A. adeninivorans Ls3.

Cloning, sequence analysis and expression in yeasts of a cDNA containing a Lipomyces kononenkoae α-amylase-encoding gene

The yeast Lipomyces kononenkoae (Lk) secretes a highly active raw starch-degrading α-amylase (αAmy) that liberates reducing groups from glucose polymers containing both α-1,4 and α-1,6 bonds. The LKA1 gene encoding this industrially important αAmy was cloned as a 2261-bp cDNA fragment from a glucose-derepressed mutant (IGC4052B) of Lk and characterized. The nucleotide (nt) sequence of the cDNA fragment was determined, revealing an open reading frame of 1872 bp, encoding a 596 amino-acid (aa) mature protein (LKA1) with a calculated M r of 65706. The similarity between the aa sequence of LKA1 and those of other αAmy showed four common conserved regions characteristic of the αAmy protein family: (A) 264DIVVNH 269, (B) 349GLRIDTVKH 357, (B') 376GEVFD 380 and (C) 439FLENQD 444. The deduced aa sequence revealed significant homology to the aa sequences of the Aspergillus oryzae, Schwanniomyces occidentalis and Saccharomycopsis fibuligera αAmy, various bacterial cyclomaltodextrin glucanotransferases, a β-amylase and the 5′-region of a glucoamylase. LKA1 was expressed in Saccharomyces cerevisiae (Sc) under the control of the phosphoglycerate kinase (PGK1) promoter and Northern blot analysis showed the presence of a single 2.3-kb transcript. The 28-aa signal peptide of the LKA1 protein efficiently directed its secretion into the medium when expressed in Sc.

Secretion of the glucoamylase-β-lactamase chimera protein by Saccharomyces cerevisiae

Abstract Saccharomycopsis fibuligera glucoamylase ( GLU1 ) and Escherichia coli β-lactamase ( bla ) fusion gene was constructed and expressed in Saccharomyces cerevisiae. S. cerevisiae transformants with the plasmid harboring the GLU1-bla fusion gene produced in the culture medium a 90 Kd protein with both glucoamylase and β-lactamase activities, in agreement with the size of the chimera protein expected from the gene construct.

Funcelase — An efficient preparation for the isolation of reversion competent protoplasts from yeasts

The lytic preparation Funcelase was shown to be capable of releasing protoplasts from exponential phase cells ofCandida albicans, Kluyveromyces lactis, Saccharomyces cerevisiae, Saccharomycopsis fibuligera andSchizosaccharomyces pombe. The protoplasts so produced displayed reversion frequencies far superior to those isolated by treatment with Novozym 234 or Suc d'Helix pomatia.

DNA sequences fromSaccharomycopsis fibuligera capable of autonomous replication inSaccharomyces cerevisiae

A Sau3AI digest of total DNA from Saccharomycopsis fibuligera was cloned with the yeast-integrating plasmid YEp24 delta EcoRI and the capacity for autonomous replication (ARS) was assayed in yeast. From eighty clones, five mitoticaly unstable yeast transformants were picked up, recombinant plasmids from these clones were recovered with Escherichia coli, mapped, hybridized with total DNA of S. fibuligera and tested to mitotical stability in Saccharomyces cerevisiae. Experiments suggested the existence of DNA sequences from dimorphic Saccharomycopsis fibuligera with ARS activity in Saccharomyces cerevisiae.

The enzymatic and molecular characteristics of Saccharomycopsis .ALPHA.-amylase secreted from Saccharomyces cerevisiae.

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.

Analysis of the alpha-amylase gene of Schwanniomyces occidentalis and the secretion of its gene product in transformants of different yeast genera.

We have cloned and characterized the alpha-amylase gene (AMY1) of the yeast Schwanniomyces occidentalis. A cosmid gene library of S. occidentalis DNA was screened in Saccharomyces cerevisiae for alpha-amylase secretion. The positive clone contained a DNA fragment harbouring an open reading frame of 1536 nucleotides coding for a 512-amino-acid polypeptide with a calculated Mr of 56,500. The deduced amino acid sequence reveals significant similarity to the sequence of the Saccharomycopsis fibuligera and Aspergillus oryzae alpha-amylases. The AMY l gene was found to be expressed from its original promoter in S. cerevisiae, Kluyveromyces lactis and Schizo-saccharomyces pombe leading to an active secreted gene product and thus enabling the different yeast transformants to grow on starch as a sole carbon source.

A model for continuous fermentations with amylolytic yeasts

A two-stage, associative fermentation process is more effective for continuous yeast biomass production from starch than a single-stage mixed culture fermentation process. By operating two stages, competition for the same growth limiting substrate is reduced leading to efficient starch utilization. In this article, a mathematical model has been proposed for continuous, two-stage fermentation with a pure culture, amylolytic yeast in the first stage and a mixed culture second stage with a faster growing, nonamylolytic yeast. The model parameters were determined for Saccharomycopsis fibuligera and Candida utilis in continuous, single-stage, pure cultures. In the two-stage model, the effects of changes in dilution rate on biomass, amylase, reducing sugar, and starch concentration, and ratio of stage volumes on microbial composition are discussed and compared with experimental data.

Construction and characterization of mutant glucoamylases from the yeast Saccharomycopsis fibuligera.

To find amino acid residues which are required for glucoamylase activity, mutant glucoamylase genes were constructed by in vitro mutations of GLU1 DNA encoding Saccharomycopsis fibuligera glucoamylase and introduced into Saccharomyces cerevisiae, and the resulting mutant proteins were assayed for enzymatic activities. Eighteen mutant proteins were obtained by random insertions of a BamHX linker DNA. Six out of 7 proteins with mutations in conserved regions among divergent glucoamylases showed no activities, while 8 out of 11 proteins with mutations in unconserved regions had similar specific activities to a wild-type value, suggesting that the conserved regions are important to the activity. A series of amino-terminal deletion mutants were also constructed. A mutant protein with a deletion of only two amino acid residues from the amino terminus had a significant reduction in the activity, suggesting an essential role for the amino-terminal peptide. Ten mutant proteins with single amino acid replacements w...

Fermentation of Mevalonate by Saccharomycopsis fibuligera IFO 0107

Saccharomycopsis fibuligera IFO 0107 was found to overproduce R-mevalonate. In a typical experiment this strain produced 8.3 mg/ml of mevalonate. Mevalonate is synthesized by 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase and converted to 5-phosphomevalonate by mevalonate kinase. The yeast strain showed 3 ∼ 20 times higher HMG-CoA reductase and ∼4 times lower mevalonate kinase activities, as compared with several yeast strains which produced no detectable amounts of mevalonate. The high ratio of specific activity of HMG-CoA reductase/mevalonate kinase seems to be responsible for the accumulation of mevalonate by S. fibuligera IFO 0107.

Production, purification and characterization of an α‐amylase produced by Saccharomycopsis fibuligera

Saccharomycopsis fibuligera ST 2 produced high levels of extracellular amylase during the stationary phase of growth. Glucose or other low molecular weight metabolizable sugars did not repress the synthesis of the amylase, indicating the lack of catabolite repression in this organism. Of the nitrogen sources examined, yeast extract and corn steep liquor stimulated the highest yield of amylase. Ammonium sulphate inhibited α‐amylase synthesis. The enzyme was purified 118‐fold from the culture supernatant fluid by isopropanol precipitation and DEAE‐Sephadex A50 chromatography. The purified enzyme was characterized as an α‐amylase. The α‐amylase had the following properties: molecular weight, 40900 ± 500; optimum temperature, 60°C; activation energy, 1600 cal/mol; optimum pH, 4·8–6·0; range of pH stability, pH 4·0–9·4; Km (50°C, pH 5·5) for soluble starch, 0·572 mg/ml; final products of starch hydrolysis—glucose, maltose, maltotriose and maltotetraose.

Diversity and the role of yeast in spontaneous cocoa bean fermentation from Southeast Sulawesi, Indonesia

Jamili, Yanti NA, Susilowati PE. 2016. Diversity and the role of yeast in spontaneous cocoa bean fermentation from Southeast Sulawesi, Indonesia. Biodiversitas 17: 90-95. Yeast is one of the microbial group which is role in the process of cocoa spontaneously fermentation. The objective of this study was to determinate and to know the diversity of yeast that role on cocoa bean fermentation. Yeast was isolated by pour plate method from cocoa bean that was naturally fermented by a cocoa farmer in Kolaka District, Southeast Sulawesi using yeast mannitol agar (YMA) media. Yeast was characterized and identified using phenotypic characters based on numeric-phenetic analysis. Yeast isolates applied to cocoa bean to determine its role in cocoa bean fermentation. The result was obtained seven isolates the dominant yeast during cocoa bean fermentation in Kolaka District, Southeast Sulawesi. The result of numerical-phenetic analysis based on phenotypic characters to seven yeast isolates showed that 1 isolates (Klk1) identical with Candida krusei. Three isolates (Klk4, Klk5 and Klk7) identical with Candida tropicalis, one isolate (Klk2) identical with Saccharomycopsis fibuligera, one isolate (Klk3) identical with Kloeckera sp. and one isolate (Klk6) identical with Saccharomyces cerevisiae. The result also showed that fermentation of cocoa with seeding of yeast inoculums served to increase the quality of cocoa beans than spontaneous fermentation. Therefore, the seven yeast isolates potentially be used as an inoculum to improve the cocoa quality.