scholarly journals A Methylotrophic Pathway Participates in Pectin Utilization by Candida boidinii

2000 ◽  
Vol 66 (10) ◽  
pp. 4253-4257 ◽  
Author(s):  
Tomoyuki Nakagawa ◽  
Tatsuro Miyaji ◽  
Hiroya Yurimoto ◽  
Yasuyoshi Sakai ◽  
Nobuo Kato ◽  
...  
Keyword(s):  
Methyl Ester ◽  
Alcohol Oxidase ◽  
Pectate Lyase ◽  
Methylotrophic Yeast ◽  
Pectin Methylesterase ◽  
Candida Boidinii ◽  
Methanol Metabolism ◽  
Genes Encoding ◽  
Dihydroxyacetone Synthase ◽  
In Cells

ABSTRACT The methylotrophic yeast Candida boidinii S2 was found to be able to grow on pectin or polygalacturonate as a carbon source. When cells were grown on 1% (wt/vol) pectin, C. boidinii exhibited induced levels of the pectin-depolymerizing enzymes pectin methylesterase (208 mU/mg of protein), pectin lyase (673 mU/mg), pectate lyase (673 mU/mg), and polygalacturonase (3.45 U/mg) and two methanol-metabolizing peroxisomal enzymes, alcohol oxidase (0.26 U/mg) and dihydroxyacetone synthase (94 mU/mg). The numbers of peroxisomes also increased ca. two- to threefold in cells grown on these pectic compounds (3.34 and 2.76 peroxisomes/cell for cells grown on pectin and polygalacturonate, respectively) compared to the numbers in cells grown on glucose (1.29 peroxisomes/cell). The cell density obtained with pectin increased as the degree of methyl esterification of pectic compounds increased, and it decreased in strains from which genes encoding alcohol oxidase and dihydroxyacetone synthase were deleted and in a peroxisome assembly mutant. Our study showed that methanol metabolism and peroxisome assembly play important roles in the degradation of pectin, especially in the utilization of its methyl ester moieties.

scholarly journals Peroxisomal assembly: membrane proliferation precedes the induction of the abundant matrix proteins in the methylotrophic yeast Candida boidinii

1990 ◽  
Vol 96 (4) ◽  
pp. 583-590
Author(s):  
M. Veenhuis ◽  
J.M. Goodman
Keyword(s):  
Early Stage ◽  
Morphological Changes ◽  
Polyclonal Antibodies ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Candida Boidinii ◽  
Peroxisomal Membrane ◽  
Cell Induction ◽  
The Matrix ◽  
Dihydroxyacetone Synthase

Peroxisomes are massively induced when methylotrophic yeasts are cultured in medium containing methanol. These organelles contain enzymes that catalyze the initial steps of methanol assimilation. In Candida boidinii, a methylotrophic yeast, the peroxisomal matrix (internal compartment) is composed almost exclusively of two proteins, alcohol oxidase and dihydroxyacetone synthase; catalase is present in much lower abundance. Monoclonal and polyclonal antibodies are available against peroxisomal matrix and membrane proteins. These were utilized to correlate the induction of specific proteins with the morphological changes occurring during peroxisomal proliferation. Cells cultured in glucose-containing medium contain two to five small microbodies, which are identifiable by catalase staining and immunoreactivity with a monoclonal antibody against PMP47, an integral peroxisomal membrane protein. Three stages of proliferation can be distinguished when cells are switched to methanol as the carbon source. (1) There is an early stage (within 1 h) in which several peroxisomes develop from a preexisting organelle. This is accompanied by an increase in catalase activity and an induction of PMP47, but no detectable induction of alcohol oxidase or dihydroxyacetone synthase is observed. (2) From 1 to 2.5 h there is further division of these microbodies until up to 30 small peroxisomes generally are present in each of one or two clusters per cell. Induction of alcohol oxidase, dihydroxyacetone synthase and PMP20, a protein that is distributed in the matrix and membrane, is detectable during this time. Serial sections reveal that some peroxisomes remain uninduced while others undergo proliferation. Such sections also show no obvious connections between peroxisomes within clusters.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals High-CO2 Treatment Prolongs the Postharvest Shelf Life of Strawberry Fruits by Reducing Decay and Cell Wall Degradation

Foods ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1649
Author(s):  
Hyang-Lan Eum ◽  
Seung-Hyun Han ◽  
Eun-Jin Lee
Keyword(s):  
Cell Wall ◽  
Shelf Life ◽  
Physiological Mechanism ◽  
Pectate Lyase ◽  
Pectin Methylesterase ◽  
Cell Wall Degradation ◽  
Cell Wall Degrading Enzymes ◽  
High Co2 ◽  
Genes Encoding ◽  
Co2 Treatment

Improved methods are needed to extend the shelf life of strawberry fruits. The objective of this study was to determine the postharvest physiological mechanism of high-CO2 treatment in strawberries. Harvested strawberries were stored at 10 °C after 3 h of exposure to a treatment with 30% CO2 or air. Pectin and gene expression levels related to cell wall degradation were measured to assess the high-CO2 effects on the cell wall and lipid metabolism. Strawberries subjected to high-CO2 treatment presented higher pectin content and firmness and lower decay than those of control fruits. Genes encoding cell wall-degrading enzymes (pectin methylesterase, polygalacturonase, and pectate lyase) were downregulated after high-CO2 treatment. High-CO2 induced the expression of oligogalacturonides, thereby conferring defense against Botrytis cinerea in strawberry fruits, and lowering the decay incidence at seven days after its inoculation. Our findings suggest that high-CO2 treatment can maintain strawberry quality by reducing decay and cell wall degradation.

scholarly journals Green methods for methyl esters determination in foodstuffs

2013 ◽  
Author(s):  
Lucyna Marta Lekawska-Andrinopoulou
Keyword(s):  
Methyl Ester ◽  
Flow Rate ◽  
Methyl Esters ◽  
Alcohol Oxidase ◽  
Pectin Methylesterase ◽  
Enzymatic Reactions ◽  
Automated Method ◽  
Chemiluminescent Detection ◽  
Through Flow ◽  
Diet Drinks

In this thesis development of novel, green methods for methyl esters in foodstuffs is described. Methods are based on enzymatic reactions and fluidics. Study focuses on two methyl esters: pectin methyl ester and aspartame, a methyl ester of aspartic acid/phenylalanine dipeptide. Pectin and aspartame are enzymatically hydrolysed by pectin methylesterase or _- chymotrypsin, respectively. Methanol is released and quantified. Several methanol determination methods have been tested, with the method with 4-AAP and phenol showing the best prospects for automation. Method was optimized and its robustness was investigated. Ascorbic acid interference removal with 4-hydroxy TEMPO was tested. Development of two automated methods for methyl esters determination is described: spectrophotometric pectin methyl esters determination and chemiluminescent aspartame determination.The method for pectin methyl esters is the first work on pectin analysis through flow injection. Detection limit down to 1.47 mM was achieved at the analysis rate of 7 samples h-1. The method provides identical results with manual off-line method. The development of the aspartame analyzer was preceded by the development of a spectrophotometric method, which showed good results in samples containing higher aspartame concentrations than expected in beverages. In order to improve method for possible application in beverages chemiluminescent detection was selected for the automated method. The chemistry from kinetic study was modified to accommodate luminol chemiluminescent detection and optimization of the system was performed. Several manifolds were constructed and the effect of following parameters was tested: flow rate, mixing coils length, location and number, preincubation time, alcohol oxidase concentration, use of separate solutions of AOX and HRP. 0.8 ml/min/line flow rate in combination with one 100 cm mixing coil, 60 s preincubation and use of separated solutions of AOX an HRP resulted in sufficient sensitivity that allowed for construction of a calibration curve within the range of aspartame concentration found in diet drinks. Additionally, following side projects related to the main topic of the study are described: development of PME activity assay and galacturonic acid determination.

scholarly journals The absence of Pmp47, a putative yeast peroxisomal transporter, causes a defect in transport and folding of a specific matrix enzyme.

1996 ◽  
Vol 134 (1) ◽  
pp. 37-51 ◽  
Author(s):  
Y Sakai ◽  
A Saiganji ◽  
H Yurimoto ◽  
K Takabe ◽  
H Saiki ◽  
...  
Keyword(s):  
Single Gene ◽  
Alcohol Oxidase ◽  
Candida Boidinii ◽  
High Electron ◽  
Gene Encoding ◽  
Peroxisomal Membrane ◽  
Carboxyl Terminal ◽  
Dihydroxyacetone Synthase ◽  
Physiological And Biochemical ◽  
Solute Transporters

Candida boidinii Pmp47, an integral peroxisomal membrane protein, belongs to a family of mitochondrial solute transporters (e.g., ATP/ADP exchanger), and is the only known peroxisomal member of this family. However, its physiological and biochemical functions have been unrevealed because of the difficulties in the molecular genetics of C. boidinii. In this study, we first isolated the PMP47 gene, which was the single gene encoding for Pmp47 in a gene-engineerable strain S2 of C. boidinii. Sequence analysis revealed that it was very similar to PMP47A and PMP47B genes from a polyploidal C. Boidinii strain (ATCC32195). Next, the PMP47 gene was disrupted and the disruption strain (pmp47delta) was analyzed. Depletion of PMP47 from strain S2 resulted in a retarded growth on oleate and a complete loss of growth on methanol. Both growth substrates require peroxisomal metabolism. EM observations revealed the presence of peroxisomes in methanol- and oleate-induced cells of pmp47delta, but in reduced numbers, and the presence of material of high electron density in the cytoplasm in both cases. Methanol-induced cells of pmp47delta were investigated in detail. The activity of one of the methanol-induced peroxisome matrix enzymes, dihydroxyacetone synthase (DHAS), was not detected in pmp47delta. Further biochemical and immunocytochemical experiments revealed that the DHAS protein aggregated in the cytoplasm as an inclusion body, while two other peroxisome matrix enzymes, alcohol oxidase (AOD) and catalase, were active and found in peroxisomes. Two peroxisome-deficient mutants, strains M6 and M13 (described in previous studies), retained DHAS activity although it was mislocalized to the cytoplasm and the nucleus. We disrupted PMP47 in these peroxisome-deficient mutants. In both strains, M6-pmp47delta and M13-pmp47delta, DHAS was enzymatically active and was located in the cytoplasm and the nucleus. We suggest that an unknown small molecule, which PMP47 transports, is necessary for the folding or the translocation machinery of DHAS within peroxisomes. Pmp47 does not catalyze folding directly because active DHAS is observed in the M6-pmp47delta and M13-pmp47delta strains. Since both AOD and DHAS have the PTS1 motif sequences at their carboxyl terminal, our results first show that depletion of Pmp47 could dissect the peroxisomal import pathway (PTS1 pathway) of these proteins.

scholarly journals Establishment of Cyanophycin Biosynthesis in Pichia pastoris and Optimization by Use of Engineered Cyanophycin Synthetases

2009 ◽  
Vol 76 (4) ◽  
pp. 1062-1070 ◽  
Author(s):  
Anna Steinle ◽  
Sabrina Witthoff ◽  
Jens P. Krause ◽  
Alexander Steinbüchel
Keyword(s):  
Pichia Pastoris ◽  
Alcohol Oxidase ◽  
Methylotrophic Yeast ◽  
Medium Composition ◽  
Site Directed Mutagenesis ◽  
Soluble Form ◽  
C Terminus ◽  
Fermentation Parameters ◽  
Optimization Of Fermentation ◽  
In Cells

ABSTRACT Two strains of the methylotrophic yeast Pichia pastoris were used to establish cyanophycin (multi-l-arginyl-poly-l-aspartic acid [CGP]) synthesis and to explore the applicability of this industrially widely used microorganism for the production of this polyamide. Therefore, the CGP synthetase gene from the cyanobacterium Synechocystis sp. strain PCC 6308 (cphA 6308) was expressed under the control of the alcohol oxidase 1 promoter, yielding CGP contents of up to 10.4% (wt/wt), with the main fraction consisting of the soluble form of the polymer. To increase the polymer contents and to obtain further insights into the structural or catalytic properties of the enzyme, site-directed mutagenesis was applied to cphA 6308 and the mutated gene products were analyzed after expression in P. pastoris and Escherichia coli, respectively. CphA6308Δ1, which was truncated by one amino acid at the C terminus; point mutated CphA6308C595S; and the combined double-mutant CphA6308Δ1C595S protein were purified. They exhibited up to 2.5-fold higher enzyme activities of 4.95 U/mg, 3.20 U/mg, and 4.17 U/mg, respectively, than wild-type CphA6308 (2.01 U/mg). On the other hand, CphA proteins truncated by two (CphA6308Δ2) or three (CphA6308Δ3) amino acids at the C terminus showed similar or reduced CphA enzyme activity in comparison to CphA6308. In flask experiments, a maximum of 14.3% (wt/wt) CGP was detected after the expression of CphA6308Δ1 in P. pastoris. For stabilization of the expression plasmid, the his4 gene from Saccharomyces cerevisiae was cloned into the expression vector used and the constructs were transferred to histidine auxotrophic P. pastoris strain GS115. Parallel fermentations at a one-to-one scale revealed 26°C and 6.0 as the optimal temperature and pH, respectively, for CGP synthesis. After optimization of fermentation parameters, medium composition, and the length of the cultivation period, CGP contents could be increased from 3.2 to 13.0% (wt/wt) in cells of P. pastoris GS115 expressing CphA6308 and up to even 23.3% (wt/wt) in cells of P. pastoris GS115 expressing CphA6308Δ1.

Pectin utilization by the methylotrophic yeast Pichia methanolica

Microbiology ◽  
2005 ◽  
Vol 151 (6) ◽  
pp. 2047-2052 ◽  
Author(s):  
Tomoyuki Nakagawa ◽  
Kaichiro Yamada ◽  
Shuki Fujimura ◽  
Takashi Ito ◽  
Tatsuro Miyaji ◽  
...  
Keyword(s):  
Wild Type Strain ◽  
Carbon Sources ◽  
Alcohol Oxidase ◽  
Growth Yield ◽  
Methylotrophic Yeast ◽  
Pectin Methylesterase ◽  
Growth Conditions ◽  
Wild Type ◽  
Pichia Methanolica ◽  
Metabolizing Enzyme

The methylotrophic yeast Pichia methanolica was able to grow on pectic compounds, pectin and polygalacturonate, as sole carbon sources. Under the growth conditions used, P. methanolica exhibited increased levels of pectin methylesterase, and pectin-depolymerizing and methanol-metabolizing enzyme activities. On the other hand, P. methanolica has two alcohol oxidase (AOD) genes, MOD1 and MOD2. On growth on pectin, the P. methanolica mod1Δ and mod1Δmod2Δ strains showed a severe defect in the growth yield, although the mod2Δ strain could grow on polygalacturonate to the same extent as the wild-type strain. The expression of MOD1 was detected in pectin-grown cells, but the MOD2-gene expression detected by pectin was much lower than that of MOD1. Moreover, pectin could induce peroxisome proliferation in P. methanolica, like methanol and oleic acid. These findings showed that P. methanolica was able to utilize the methylester moiety of pectin by means of methanol-metabolic enzymes in peroxisomes, and that the functional AOD subunit for pectin utilization was Mod1p in P. methanolica.

scholarly journals Peroxisomes induced in Candida boidinii by methanol, oleic acid and D-alanine vary in metabolic function but share common integral membrane proteins

1990 ◽  
Vol 97 (1) ◽  
pp. 193-204 ◽  
Author(s):  
J.M. Goodman ◽  
S.B. Trapp ◽  
H. Hwang ◽  
M. Veenhuis
Keyword(s):  
Oleic Acid ◽  
Membrane Proteins ◽  
Carbon Sources ◽  
Methylotrophic Yeast ◽  
Candida Boidinii ◽  
Acid Oxidase ◽  
General Function ◽  
Amino Acid Oxidase ◽  
Peroxisomal Membrane ◽  
Methanol Metabolism

Peroxisomes massively proliferate in the methylotrophic yeast Candida boidinii when cultured on methanol as the only carbon and energy source. These organelles contain enzymes that catalyze the initial reactions of methanol utilization. The membranes contain abundant proteins of unknown function; their apparent molecular masses are 20, 31, 32 and 47 × 10(3) Mr and are termed PMP20, PMPs31-32 and PMP47. Recently, we reported that peroxisomes in this yeast are also induced by oleic acid and D-alanine as carbon sources, and that these peroxisomes contain increased concentrations of the enzymes of fatty acid beta-oxidation or D-amino acid oxidase, respectively. This report extends these findings and further compares the enzyme composition from peroxisomes induced by methanol, oleic acid and D-alanine. the patterns of matrix proteins represented on SDS-polyacrylamide gels from peroxisomes induced by oleic acid or D-alanine were found to be very different from those of peroxisomes induced by methanol. In order to differentiate between membrane proteins that have specific functions in pathways of substrate utilization from those with more generalized functions, peroxisomal membranes from cultures grown on methanol, oleic acid or D-alanine were purified. Analysis of these fractions demonstrated that while PMP20 is found only in peroxisomes induced by methanol, the PMPs31-32 and PMP47 were the abundant peroxisomal membrane proteins (PMP) regardless of inducing substrate. The data strongly suggest that the function of PMP20 is related to methanol metabolism. In contrast, the functions of PMPs31-32 and PMP47 are ‘substrate-nonspecific’. We speculate that they may relate to the structure, assembly or general function of the organelle.

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