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1.
Masaki K Tsuchioka H Hirano T Kato M Ikeda H Iefuji H 《Applied microbiology and biotechnology》2012,93(4):1627-1636
Yeast host–vector systems have been very successful in expressing recombinant proteins. However, because there are some proteins
that cannot be expressed with existing systems, there is a need for new yeast expression systems. Here we describe a new host–vector
system based on the basidiomycetous yeast Cryptococcus sp. strain S-2 (S-2). Two advantages of S-2 are that it naturally produces some very useful enzymes, so it would be a good
system for expressing multiple copies of some of its genes, and that, it is a nonhazardous species. The orotate phosphoribosyltransferase
(OPRTase, EC 2.4.2.10) gene (URA5) was selected as a selectable marker for transformation in the new host–vector system. URA5 was isolated and introduced into a uracil auxotroph of S-2 by electroporation. To demonstrate the S-2 system, we selected
one of its unique enzymes, a plastic-degrading cutinase-like enzyme (CLE). We were able to insert multiple copies of the CLE
gene (CLE1) into the chromosomes in a high fraction of the targeted cells. Under optimal conditions, one transformant exhibited 3.5
times higher CLE activity than the wild type. Expression vectors, including an inducible promoter (the promoter for the xylanase
or α-amylase gene), were constructed for recombinant protein production, and green fluorescent protein was expressed under
the control of these promoters. The xylanase promoter was more tightly controlled. Furthermore, putting CLE1 under the control of the xylanase promoter, which is induced by xylose, increased CLE activity of the culture medium to approximately
15 times greater than that of the wild type. 相似文献
2.
A purified lipase from the yeast Cryptococcus sp. strain S-2 exhibited remote homology to proteins belonging to the cutinase family rather than to lipases. This enzyme could effectively degrade the high-molecular-weight compound polylactic acid, as well as other biodegradable plastics, including polybutylene succinate, poly (epsilon-caprolactone), and poly(3-hydroxybutyrate). 相似文献
3.
Heterologous production of a laccase from the basidiomycete Pycnoporus cinnabarinus in the dimorphic yeast Yarrowia lipolytica 总被引:1,自引:0,他引:1
Madzak C Otterbein L Chamkha M Moukha S Asther M Gaillardin C Beckerich JM 《FEMS yeast research》2005,5(6-7):635-646
Pycnoporus cinnabarinus lac1 gene was expressed in Yarrowia lipolytica. Different secretion signals and culture media were tested. Production was correlated to both culture growth rate and cell morphology (highest at low growth rate, without mycelium). Recombinant laccase was characterized (immunodetection, N-terminal sequencing) and purified. Production was estimated to 20 mgl(-1) in a bioreactor. Thus, complex metalloenzymes can be produced in Yarrowia, assuming some control of host physiology. Lac1p production was compared in Yarrowia, Pichia and Aspergillus: recombinant proteins were active, but host systems differed in transformation efficiency, production, and glycosylation. If not the best producer, Yarrowia offers very high transformation efficiencies, allowing the genetic engineering of laccases for industrial applications. 相似文献
4.
Matsui T Tabayashi A Iwano M Shinmyo A Kato K Nakayama H 《Plant & cell physiology》2011,52(2):413-420
Plant class III peroxidase (PRX) catalyzes the oxidation and oxidative polymerization of a variety of phenolic compounds while reducing hydrogen peroxide. PRX proteins are classified into apoplast type and vacuole type based on the absence or the presence of C-terminal propeptides, which probably function as vacuolar sorting signals (VSSs). In this study, in order to improve our understanding of vacuole-type PRX, we analyzed regulatory mechanisms of vacuolar sorting of a model vacuole-type PRX, the C1a isozyme of horseradish (Armoracia rusticana) (HRP C1a). Using cultured transgenic tobacco cells and protoplasts derived from horseradish leaves, we characterized HRP C1a's VSS, which is a 15 amino acid C-terminal propeptide (C15). We found that the C-terminal hexapeptide of C15 (C6), which is well conserved among vacuole-type PRX proteins, forms the core of the C-terminal-dependent VSS. We also found that the function of C6 is enhanced by the remaining N-terminal part of C15 which probably folds into an amphiphilic α-helix. 相似文献
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Peroxidases catalyze many reactions, the most common being the utilization of H2O2 to oxidize numerous substrates (peroxidative mode). Peroxidases have also been proposed to produce H2O2 via utilization of NAD(P)H, thus providing oxidant either for the first step of lignification or for the "oxidative burst" associated with plant-pathogen interactions. The current study with horseradish peroxidase characterizes a third type of peroxidase activity that mimics the action of catalase; molecular oxygen is produced at the expense of H2O2 in the absence of other reactants. The oxygen production and H2O2-scavenging activities had temperature coefficients, Q10, of nearly 3 and 2, which is consistent with enzymatic reactions. Both activities were inhibited by autoclaving the enzyme and both activities had fairly broad pH optima in the neutral-to-alkaline region. The apparent Km values for the oxygen production and H2O2-scavenging reactions were near 1.0 mM H2O2. Irreversible inactivation of horseradish peroxidase by exposure to high concentrations of H2O2 coincided with the formation of an absorbance peak at 670 nm. Addition of superoxide dismutase (SOD) to reaction mixtures accelerated the reaction, suggesting that superoxide intermediates were involved. It appears that horseradish peroxidase is capable of using H2O2 both as an oxidant and as a reductant. A model is proposed and the relevance of the mechanism in plant-bacterial systems is discussed. 相似文献
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The extracellular carboxymethyl cellulase (CSCMCase) from the yeast, Cryptococcus sp. S-2, was produced when grown on cellobiose. It was purified to homogeneity from the supernatant by ultrafiltration, DEAE-5PW anion exchange column and TSK-Gel G3000SW gel filtration. The purified enzyme was monomeric protein with molecular mass of approximately 34 kDa. The optimum temperature and pH for the action of the enzyme were at 40–50 °C and 3.5, respectively. It was stable at pH range of 5.5–7.5 and retained approximately 50% of its maximum activity after incubating at 90 °C for 1 h. Moreover, it could able to hydrolyze carboxymethyl cellulose sodium salt higher than insoluble cellulose substrate such as Avicel, SIGMACELL® and CM cellulose. Due to its action at acidic pH and moderately stable at high temperature, the gene encoding carboxymethyl cellulase (CSCMCase) was isolated and improved the enzyme yield by high cell-density fermentation of Pichia pastoris. The CSCMCase cDNA contains 1023 nucleotides and encodes a 341-amino acid. It was successfully expressed under the control of alcohol oxidase I promoter using methanol induction of P. pastoris fermentation in a 2L ABLE bioreactor. The production of the recombinant carboxymethyl cellulases was higher than that from Cryptococcus sp. S-2 of 657-fold (2.75 and 4.2 × 10−3 mg protein L−1, respectively) indicating that the leader sequence of CSCMCase has been recognized and processed as efficiently by P. pastoris. Furthermore, the recombinant enzyme was purified in two-step of ultrafiltration and hydrophobic interaction chromatography which would be much more convenient for large-scale purification for successful industrial application. 相似文献
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Five yeast strains belonging to the genus Cryptococcus Vuillemin were isolated from steppe plants and turf collected in the Prioksko-terrasny biosphere reserve (Moscow region, Russia). Sequence analyses of the D1/D2 domains of the 26S rDNA and of the internal transcribed spacer region revealed that these yeast strains and strain CBS 8016 have almost identical sequences and belong to the Holtermannia clade of the Tremellomycetidae (Basidiomycota, Hymenomycetes). A novel species named Cryptococcus festucosus (type strain VKM Y-2930) is proposed to accommodate these strains. Physiological characteristics and mycocin sensitivity patterns distinguishing Cryptococcus festucosus from the other species of this clade are presented. 相似文献
11.
The hyperfine shifted proton NMR spectrum of isoenzyme c of horseradish peroxidase indicated that one calcium ion is essential to the enzyme in maintaining the protein structure in the heme vicinity. 相似文献
12.
The optimisation of cellulase and beta-glucosidase production by a basidiomycete species was studied and cellulase and cellobiase production by this and Trichoderma viride (and its mutants) in shake flasks were compared. The former produced an active cellulase comparable to that of T. viride when tested on filter paper, carboxymethylcellulose, and cotton; however, it produced 20 to 26 times larger amounts of cellobiase. Both cellulase and beta-glucosidase were obtained in good yield only when cellulose was the carbon source. The production of these enzymes was not repressed by readily assimilated carbon sources in the presence of cellulose. Only traces of cellulase and beta-glucosidase were formed on glucose, fructose, maltose, and cellobiose although good growth was obtained on these substrates. These enzymes were not induced on sophorose, lactose, mannitol, or glycerol and growth was poor on these substrates. Cellobiose octaacetate was a less effective inducer of cellulase and beta-glucosidase than was cellulose. 相似文献
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Cutinase-Like Enzyme from the Yeast Cryptococcus sp. Strain S-2 Hydrolyzes Polylactic Acid and Other Biodegradable Plastics 总被引:2,自引:0,他引:2
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Kazuo Masaki Numbi Ramudu Kamini Hiroko Ikeda Haruyuki Iefuji 《Applied microbiology》2005,71(11):7548-7550
A purified lipase from the yeast Cryptococcus sp. strain S-2 exhibited remote homology to proteins belonging to the cutinase family rather than to lipases. This enzyme could effectively degrade the high-molecular-weight compound polylactic acid, as well as other biodegradable plastics, including polybutylene succinate, poly (-caprolactone), and poly(3-hydroxybutyrate). 相似文献
15.
The purity of horseradish peroxidase isoenzyme C was demonstrated using isoelectric focusing, polyacrylamide gel electrophoresis at two pH values and cellulose acetate electrophoresis at two pH values. The glycopeptides obtained upon trypsin digestion were isolated using the plant lectin, concanavalin A, and were resolved using paper electrophoresis. The carbohydrate content of the native peroxidase was 86% accounted for by the carbohydrate content of the glycopeptides thus suggesting little loss of carbohydrate during glycopeptide isolation and purification. In each of the seven glycopeptides isolated glucosamine was associated with asparagine, thus suggesting the carbohydrate chains are covalently bound to the peptide chain through N-glycosidic linkages. The purity of each glycopeptide was demonstrated by the sequential release of single amino acid residues by Edman degradation. As six glycopeptides had unique amino acid sequences, it was concluded that the carbohydrate prosthetic group was distributed in at least six units along the protein backbone. Five glycopeptides possessed the amino acid sequence about the point of carbohydrate attachment of Asn-X-(Ser, Thr) where X is any amino acid. The size of the carbohydrate units ranged from 1600 to 3000 daltons. The predominant carbohydrate residues in each glycopeptide were mannose and glucosamine with lesser and varying amounts of fucose, xylose, and arabinose. There was no apparent correlation of the carbohydrate composition with the amino acid sequence. 相似文献
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Chunlei Lin Kazuo Masaki Haruyuki Iefuji Katsuo Ohkata 《Biocatalysis and Biotransformation》2013,31(5):390-395
The effect of temperature on enantioselectivity and desymmetrization in the acetylation of 2-phenyl-1,3-propanediol (1a), 2-benzyl-1,3-propanediol (1b), 2-methyl-2-phenyl-1,3-propanediol (1c) and 2-benzyl-2-methyl-1,3-propanediol (1d) by a novel lipase (CSL) isolated from the yeast Cryptococcus spp. S-2 was studied. Desymmetrization of 1a, 1c and 1d by CSL-catalyzed acetylation was observed in the temperature range of ?20°C to 40°C, while diacetylation of 1b occurred considerably even at 0°C. The kinetic parameters of the selectivity indicated that the acetylation of 1a is an entropy controlled process whereas the reaction of 1c and 1d is mainly controlled by the enthalpy term. In the monoacetylation of the diol 1d, the preferred configuration in the enantiomeric induction by CSL was opposite to that of immobilized porcine pancreatic lipase (PPL). 相似文献
18.
Chunlei Lin Yoshikazu Hiraga Kazuo Masaki Haruyuki Iefuji Katsuo Ohkata 《Biocatalysis and Biotransformation》2006,24(5):390-395
The effect of temperature on enantioselectivity and desymmetrization in the acetylation of 2-phenyl-1,3-propanediol (1a), 2-benzyl-1,3-propanediol (1b), 2-methyl-2-phenyl-1,3-propanediol (1c) and 2-benzyl-2-methyl-1,3-propanediol (1d) by a novel lipase (CSL) isolated from the yeast Cryptococcus spp. S-2 was studied. Desymmetrization of 1a, 1c and 1d by CSL-catalyzed acetylation was observed in the temperature range of -20°C to 40°C, while diacetylation of 1b occurred considerably even at 0°C. The kinetic parameters of the selectivity indicated that the acetylation of 1a is an entropy controlled process whereas the reaction of 1c and 1d is mainly controlled by the enthalpy term. In the monoacetylation of the diol 1d, the preferred configuration in the enantiomeric induction by CSL was opposite to that of immobilized porcine pancreatic lipase (PPL). 相似文献
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M Yagi 《Cancer biochemistry biophysics》1979,4(2):105-117
Tryptophan-p-1 (trp-p-1) and p-2, which have high mutagenic and carcinogenic potential, are oxidized by catalase- and horseradish peroxidase (HRP)-H2O2 intermediates with optimum pH 5.9 (0.2 M-acetate) in catalase and pH 5.0 (0.2 M-acetate), 8.0 (0.01 M-phosphate) in HRP. The rate constants (k4) of the oxidation in the catalase at pH 5.9 (0.2 M-acetate) were 2965 M-1 x sec-1 for trp-p-1 and 576 M-1 x sec-1 for trp-p-2. In the case of HRP, 1894 M-1 x sec-1, (pH 5.0, 0.2 M-acetate) for trp-p-1 and 705 M-1 x sec-1 (pH 8.0, 0.001 M-phosphate) for trp-p-2 under each optimum condition. The oxidation products of trp-p-1 and p-2 by catalase or HRP lost completely their mutagenic potential in the mutation assay. 相似文献