Cloning, molecular characterization, and expression of an endo-polygalacturonase-encoding gene from Saccharomyces cerevisiae IM1-8b

Blanco, Pilar

doi:10.1016/s0378-1097(98)00220-1

Cited by 24 publications

(45 citation statements)

References 15 publications

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“…Maximum enzymatic activity (7,200 U/ml) was found at 35 h after derepression. The level of expression found in the culture medium was about 40-fold higher than that shown by the wild-type S. cerevisiae strains and 4-fold higher than that found when the same gene was overexpressed in S. cerevisiae (1). When the location of the enzyme was tested according to the procedure described by Villa et al (16), it was found that endopolygalacturonase activity occurred mainly in the growth medium, and only certain levels of intracellular activity (ca.…”

mentioning

confidence: 83%

“…At the end of the fermentation, only 20% of cells had retained the plasmid. However, the amount of recombinant product in the low-cost medium was three times higher than when the PGU1 gene was overexpressed in S. cerevisiae (1).…”

mentioning

confidence: 89%

“…In Saccharomyces cerevisiae, the PGU1 gene codes for an endopolygalacturonase (1). In the present paper, we report the cloning, expression, and efficient secretion of the PGU1 gene in Schizosaccharomyces pombe and the characterization of the recombinant enzyme as a product with better properties for industrial applications.…”

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confidence: 98%

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Heterologous Expression of the Saccharomyces cerevisiae PGU1 Gene in Schizosaccharomyces pombe Yields an Enzyme with More Desirable Properties for the Food Industry

Sieiro

Poza

Vilanova

et al. 2003

Appl Environ Microbiol

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The Saccharomyces cerevisiae PGU1 gene was successfully expressed in Schizosaccharomyces pombe. The optimum pH and temperature for the recombinant enzyme were 5 and 40°C, respectively, these being around 0.5 U higher and 5°C lower than those shown by the native enzyme. The K m value was about fourfold higher than that of the S. cerevisiae enzyme. The recombinant endopolygalacturonase was more efficient in reducing the viscosity of polygalacturonic acid and was also more stable at different pHs and temperatures than the native enzyme.Pectic substances are plant polysaccharides that contain a large proportion of galacturonic acid subunits linked by ␣-1,4-glycosidic bonds. Pectin and pectate are pectic substances that are either partially methyl esterified or free of methyl groups, respectively. The enzymes able to degrade pectic substances are pectin esterases, which release the methyl ester groups from pectin, and depolymerases, including pectate or pectin lyases and polygalacturonases (4).Pectinases have many applications in industry, particularly in fruit and vegetable processing. These applications include such procedures as extraction and clarification of fruit juices (e.g., grape and apple juice), as well as textile processing, paper making, coffee and tea fermentation, and pectic wastewater treatment. Additionally, pectolytic enzymes can be used in complex mixtures with other hydrolases in the formulation of animal feeds (7).To date, commercial pectinase preparations consist of enzymatic mixtures of fungal origin, where, in addition to having different pectinolytic activities, certain enzymes with undesirable properties (methylesterases, arabinofuranosidases, and amylases) may also be found. In view of this, it would be of advantage to produce the different pectic enzymes separately and then mix them in the downstream process as required. It is interesting to note that total hydrolysis of certain substrates often requires the concerted action of several enzymes in the correct proportions (2).In Saccharomyces cerevisiae, the PGU1 gene codes for an endopolygalacturonase (1). In the present paper, we report the cloning, expression, and efficient secretion of the PGU1 gene in Schizosaccharomyces pombe and the characterization of the recombinant enzyme as a product with better properties for industrial applications.Expression of the PGU1 gene in S. pombe. A XhoI-BamHI fragment containing the S. cerevisiae PGU1 gene was recovered from plasmid pBSK-PGU1 (1) and ligated to S. pombe pREP3X vector (9) by standard procedures (13), thus generating plasmid pREP3X-PGU1. In this plasmid, the PGU1 gene was cloned under the control of the thiamine-repressible nmt1 promoter (9). The recombinant plasmid was amplified in Escherichia coli DH5␣ and purified according to previously described methods (13). Cells of S. pombe (h ϩ ade6-M210 leu1-32 ura 40-18 PG Ϫ ) grown in YEPD medium (1% yeast extract, 1% peptone, 2% glucose) were transformed with the pREP3X-PGU1 plasmid by the lithium acetate protocol (6). After 4 days at 30°C, s...

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confidence: 89%

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Heterologous Expression of the Saccharomyces cerevisiae PGU1 Gene in Schizosaccharomyces pombe Yields an Enzyme with More Desirable Properties for the Food Industry

Sieiro

Poza

Vilanova

et al. 2003

Appl Environ Microbiol

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“…DocB7 gp5 was significantly related to the phage T7 gene 17 tail fiber (63). While lacking identifiable conserved domains, DocB7 gp7 was 47% identical to Gordonia terrae phage GTE5 gp1, predicted to have an endopolygalacturonase (PGU1) domain (7). The amino-terminal 80 residues of DocB7 gp7 were significantly related to the amino termini of hundreds of bacterial and fungal glycoside hydrolase family proteins, such as Lam55a from Phanerochaete chrysosporium (35).…”

Section: Vol 77 2011 Genomic and Functional Analyses Of R Equi Phamentioning

confidence: 99%

Genomic and Functional Analyses of Rhodococcus equi Phages ReqiPepy6, ReqiPoco6, ReqiPine5, and ReqiDocB7

Summer

Liu

Gill

et al. 2011

Appl Environ Microbiol

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The isolation and results of genomic and functional analyses of Rhodococcus equi phages ReqiPepy6, ReqiDocB7, ReqiPine5, and ReqiPoco6 (hereafter referred to as Pepy6, DocB7, Pine5, and Poco6, respectively) are reported. Two phages, Pepy6 and Poco6, more than 75% identical, exhibited genome organization and protein sequence likeness to Lactococcus lactis phage 1706 and clostridial prophage elements. An unusually high fraction, 27%, of Pepy6 and Poco6 proteins were predicted to possess at least one transmembrane domain, a value much higher than the average of 8.5% transmembrane domain-containing proteins determined from a data set of 36,324 phage protein entries. Genome organization and protein sequence comparisons place phage Pine5 as the first nonmycobacteriophage member of the large Rosebush cluster. DocB7, which had the broadest host range among the four isolates, was not closely related to any phage or prophage in the database, and only 23 of 105 predicted encoded proteins could be assigned a functional annotation. Because of the relationship of Rhodococcus to Mycobacterium, it was anticipated that these phages should exhibit some of the features characteristic of mycobacteriophages. Traits that were identified as shared by the Rhodococcus phages and mycobacteriophages include the prevalent long-tailed morphology and the presence of genes encoding LysBlike mycolate-hydrolyzing lysis proteins. Application of DocB7 lysates to soils amended with a host strain of R. equi reduced recoverable bacterial CFU, suggesting that phage may be useful in limiting R. equi load in the environment while foals are susceptible to infection.Although the "tailed phage," or members of the virus order Caudovirales, constitute the majority of DNA diversity on the planet, the genomes of only a few hundred are known. Moreover, the genomic data that are available for bacteriophages are extremely unevenly distributed in terms of host species, with two-thirds derived from phages of only eight host genera or closely related bacterial species, including those belonging to the Staphylococcus, Mycobacterium, Pseudomonas, Lactococcus, and Burkholderia genera and the Enterobacteriaceae family (28). Nevertheless, some important themes have emerged from analyzing multiple phages that infect closely related host genera. One theme emerging is that despite rampant lateral gene transfer, there are recognizable "phage types" spanning geography and host taxa. Casjens (11a) was able to group 73 tailed-enterophage genomes into 13 phage types, the members of which were more closely related to each other by a number of criteria than they were to any member of another type. It should be noted that the classic enterophages do not include representatives of all phage types. When groups of phages from other host clades are sequenced, new phages and new phage types are identified. For example, among the sequenced phages of Burkholderia, there are members of the established , Mu, and P2 temperate phage types, as well as entirely new virulent phage types, such a...

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“…The bacterial strains used were Escherichia coli DH5K (Life Technologies) and XL1-Blue (Stratagene). S. cerevisiae 1389-8b+pYES-PGU1 was as described previously [9].…”

Section: Strains and Plasmidsmentioning

confidence: 99%

Mutagenesis of key amino acids alters activity of a Saccharomyces cerevisiae endo-polygalacturonase expressed in Pichia pastoris

Blanco¹

2002

FEMS Microbiology Letters

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A polygalacturonase (PG)-encoding gene from Saccharomyces cerevisiae (PGU1) was successfully expressed in the methylotrophic yeast Pichia pastoris. PG secretion was efficiently directed by the S. cerevisiae K-factor signal sequence, while the native (PGU1) leader peptide was unable to direct protein export in P. pastoris. The level of PGU1 activity achieved in P. pastoris was significantly enhanced when compared to activity using the same gene in S. cerevisiae. Expression of PG proteins, engineered by site-directed mutagenesis, in P. pastoris showed that aspartic acid residues at positions 179, 200, and 201, and histidine 222 were essential for enzyme activity. Mutation of the two potential glycosylation sites in PGU1 showed that the two residues individually (N318D, N330D) did not affect secreted enzyme activity, but the double mutant caused a 50% reduction in enzyme activity when compared to the wild-type PGU1 transformant. ß

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Cloning, molecular characterization, and expression of an endo-polygalacturonase-encoding gene from Saccharomyces cerevisiae IM1-8b

Cited by 24 publications

References 15 publications

Heterologous Expression of the Saccharomyces cerevisiae PGU1 Gene in Schizosaccharomyces pombe Yields an Enzyme with More Desirable Properties for the Food Industry

Heterologous Expression of the Saccharomyces cerevisiae PGU1 Gene in Schizosaccharomyces pombe Yields an Enzyme with More Desirable Properties for the Food Industry

Genomic and Functional Analyses of Rhodococcus equi Phages ReqiPepy6, ReqiPoco6, ReqiPine5, and ReqiDocB7

Mutagenesis of key amino acids alters activity of a Saccharomyces cerevisiae endo-polygalacturonase expressed in Pichia pastoris

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