Secretion, purification and activity of two recombinant pepper endo‐β‐1,4‐glucanases expressed in the yeast <i>Pichia pastoris</i>

Ferrarese, Luca; Trainotti, Livio; Gattolin, Stefano; Casadoro, Giorgio

doi:10.1016/s0014-5793(97)01592-5

Cited by 23 publications

(4 citation statements)

References 21 publications

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“…This expression system has recently been used for the recombinant expression of secreted plant EGases (Ferrarese et al, 1998). We initially tried to express ⌬ 1-90 Cel16 in Escherichia coli as a fusion protein to thioredoxin but could not detect any enzymatic activity using CMC4M as substrate.…”

Section: Discussionmentioning

confidence: 99%

Characterization of a Functional Soluble Form of a Brassica napus Membrane-Anchored Endo-1,4-beta -Glucanase Heterologously Expressed in Pichia pastoris

Molhoj

2001

Plant Physiology

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The Brassica napus gene, Cel16, encodes a membrane-anchored endo-1,4-beta-glucanase with a deduced molecular mass of 69 kD. As for other membrane-anchored endo-1,4-beta-glucanases, Cel16 consists of a predicted intracellular, charged N terminus (methionine(1)-lysine(70)), a hydrophobic transmembrane domain (isoleucine(71)-valine(93)), and a periplasmic catalytic core (lysine(94)-proline(621)). Here, we report the functional analysis of Delta(1-90)Cel16, the N terminally truncated Cel16, missing residues 1 through 90 and comprising the catalytic domain of Cel16 expressed recombinantly in the methylotrophic yeast Pichia pastoris as a soluble protein. A two-step purification protocol yielded Delta(1-90)Cel16 in a pure form. The molecular mass of Delta(1-90)Cel16, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was about 130 kD and about 60 kD after enzymatic removal of N-glycans, fitting the expected molecular mass of 59 kD. Delta(1-90)Cel16 was highly N glycosylated as compared with the native B. napus Cel16 protein. Delta(1-90)Cel16 had a pH optimum of 6.0. The activity of Delta(1-90)Cel16 was inhibited by EDTA and exhibited a strong dependence on calcium. Delta(1-90)Cel16 showed substrate specificity for low substituted carboxymethyl-cellulose and amorphous cellulose. It did not hydrolyze crystalline cellulose, xyloglycan, xylan, (1-->3),(1-->4)-beta-D-glucan, the highly substituted hydroxyethylcellulose, or the oligosaccharides cellotriose, cellotetraose, cellopentaose, or xylopentaose. Size exclusion analysis of Delta(1-90)Cel16-hydrolyzed carboxymethylcellulose showed that Delta(1-90)Cel16 is a true endo-acting glucanase.

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Section: Discussionmentioning

confidence: 99%

Characterization of a Functional Soluble Form of a Brassica napus Membrane-Anchored Endo-1,4-beta -Glucanase Heterologously Expressed in Pichia pastoris

Molhoj

2001

Plant Physiology

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“…Over the past decade, a number of cellulases have been discovered from animal guts, forest fungi and plants, some of which were successfully expressed in Pichia pastoris [5-22]. Several patented or commercial extracellular cellulases were derived from Trichoderma species [23-26].…”

Section: Introductionmentioning

confidence: 99%

Systematic screening of glycosylation- and trafficking-associated gene knockouts in Saccharomyces cerevisiaeidentifies mutants with improved heterologous exocellulase activity and host secretion

et al. 2013

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BackgroundAs a strong fermentator, Saccharomyces cerevisiae has the potential to be an excellent host for ethanol production by consolidated bioprocessing. For this purpose, it is necessary to transform cellulose genes into the yeast genome because it contains no cellulose genes. However, heterologous protein expression in S. cerevisiae often suffers from hyper-glycosylation and/or poor secretion. Thus, there is a need to genetically engineer the yeast to reduce its glycosylation strength and to increase its secretion ability.ResultsSaccharomyces cerevisiae gene-knockout strains were screened for improved extracellular activity of a recombinant exocellulase (PCX) from the cellulose digesting fungus Phanerochaete chrysosporium. Knockout mutants of 47 glycosylation-related genes and 10 protein-trafficking-related genes were transformed with a PCX expression construct and screened for extracellular cellulase activity. Twelve of the screened mutants were found to have a more than 2-fold increase in extracellular PCX activity in comparison with the wild type. The extracellular PCX activities in the glycosylation-related mnn10 and pmt5 null mutants were, respectively, 6 and 4 times higher than that of the wild type; and the extracellular PCX activities in 9 protein-trafficking-related mutants, especially in the chc1, clc1 and vps21 null mutants, were at least 1.5 times higher than the parental strains. Site-directed mutagenesis studies further revealed that the degree of N-glycosylation also plays an important role in heterologous cellulase activity in S. cerevisiae.ConclusionsSystematic screening of knockout mutants of glycosylation- and protein trafficking-associated genes in S. cerevisiae revealed that: (1) blocking Golgi-to-endosome transport may force S. cerevisiae to export cellulases; and (2) both over- and under-glycosylation may alter the enzyme activity of cellulases. This systematic gene-knockout screening approach may serve as a convenient means for increasing the extracellular activities of recombinant proteins expressed in S. cerevisiae.

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“…A large number of EGase family members have been described in plants (Fig. 1), but only in a few cases has the CMCase or HECase activity been verified experimentally (Ferrarese et al 1998, Loopstra et al 1998. It is therefore not excluded that some odd family members have different substrate specificities.…”

Section: Introductionmentioning

confidence: 99%

Towards Understanding the Role of Membrane-bound Endo-β-1,4-glucanases in Cellulose Biosynthesis

Mølhøj¹,

Pagant²,

Höfte³

2002

Plant and Cell Physiology

116

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Recent studies have highlighted the involvement of membrane-anchored endo-beta-1,4-glucanases in cellulose biosynthesis in plants, suggesting that there are parallels with Agrobacterium tumefaciens and other bacteria which also require endo-beta-1,4-glucanases for cellulose synthesis. This review summarises recent literature on endo-beta-1,4-glucanases and their role in plant development and addresses the possible functions of membrane-anchored isoforms in the synthesis of cellulose.

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Secretion, purification and activity of two recombinant pepper endo‐β‐1,4‐glucanases expressed in the yeast Pichia pastoris

Cited by 23 publications

References 21 publications

Characterization of a Functional Soluble Form of a Brassica napus Membrane-Anchored Endo-1,4-beta -Glucanase Heterologously Expressed in Pichia pastoris

Characterization of a Functional Soluble Form of a Brassica napus Membrane-Anchored Endo-1,4-beta -Glucanase Heterologously Expressed in Pichia pastoris

Systematic screening of glycosylation- and trafficking-associated gene knockouts in Saccharomyces cerevisiaeidentifies mutants with improved heterologous exocellulase activity and host secretion

Towards Understanding the Role of Membrane-bound Endo-β-1,4-glucanases in Cellulose Biosynthesis

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