Characterization of a Brassica napus Myrosinase Expressed and Secreted by Pichia pastoris

Härtel, Frauke V.; Brandt, Anders

doi:10.1006/prep.2001.1562

Cited by 25 publications

(14 citation statements)

References 36 publications

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“…However, the cell-free supernatant after fermentation was contaminated with many proteins ( Figure 2 ). Most of them were successfully removed by anion-exchange chromatography [modified from Härtel and Brandt [ 22 ]] and subsequent desalting on a 30 kDa cut-off membrane ( Figure 3 ). The purification of myr was also attempted, however, only partially purified myrosinase was obtained.…”

Section: Resultsmentioning

confidence: 99%

“…Previously, Härtel and Brandt [ 21 ] reported the extracellular production of MYR1 myrosinase from Brassica napus in P. pastoris GS115 (Mut + ). The size of the produced myrosinase corresponded well with the size of its glycosylated form, although a micro-heterogeneity of the pI was observed [ 22 ]. However, a difference in glycosylation patterns between different P. pastoris strains has been previously reported [ 25 ], suggesting that the production strain itself could have affected myrosinase glycosylation.…”

Section: Resultsmentioning

confidence: 99%

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Improved Production of Recombinant Myrosinase in Pichia pastoris

Rosenbergová

Hegyi

Ferko

et al. 2021

IJMS

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The effect of the deletion of a 57 bp native signal sequence, which transports the nascent protein through the endoplasmic reticulum membrane in plants, on improved AtTGG1 plant myrosinase production in Pichia pastoris was studied. Myrosinase was extracellularly produced in a 3-liter laboratory fermenter using α-mating factor as the secretion signal. After the deletion of the native signal sequence, both the specific productivity (164.8 U/L/h) and volumetric activity (27 U/mL) increased more than 40-fold compared to the expression of myrosinase containing its native signal sequence in combination with α-mating factor. The deletion of the native signal sequence resulted in slight changes in myrosinase properties: the optimum pH shifted from 6.5 to 7.0 and the maximal activating concentration of ascorbic acid increased from 1 mM to 1.5 mM. Kinetic parameters toward sinigrin were determined: 0.249 mM (Km) and 435.7 U/mg (Vmax). These results could be applied to the expression of other plant enzymes.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Improved Production of Recombinant Myrosinase in Pichia pastoris

Rosenbergová

Hegyi

Ferko

et al. 2021

IJMS

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“…The latter system has found wide usage, because this yeast utilizes most of the posttranslational modification pathways typically associated with higher eukaryotes and is easy to use (Cregg et al, 1993). Furthermore, it has been successfully employed to express several b-glucosidases, including the Prunus serotina cyanogenic b-glucosidases amygdalin hydrolase and prunasin hydrolase (Zhou et al, 2002), Brassica napus myrosinase (Hartel and Brandt, 2002), and the Arabidopsis b-mannosidase/b-glucosidase BGLU44 (Xu et al, 2004).…”

Section: Heterologous Expression Of Bglu45 and Bglu46mentioning

confidence: 98%

Arabidopsis thaliana β-Glucosidases BGLU45 and BGLU46 hydrolyse monolignol glucosides

et al. 2006

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“…Anticipating that most members will be glycosylated (see Table 2) and therefore not expressed in active and soluble form in E. coli, we chose the P. pastoris expression system, because this yeast utilizes most of the post-translational modification pathways typically associated with higher eukaryotes and is easy to use (Cregg et al, 1993). Furthermore, this system has been successfully employed to express several b-glucosidases, including the Prunus serotina cyanogenic b-glucosidases amygdalin hydrolase and prunasin hydrolase (Zhou et al, 2002) and B. napus myrosinase (Hartel and Brandt, 2002). As proof of concept, BGLU44 was selected as the first target hydrolase for purification and characterization.…”

Section: Heterologous Expression and Purification Of Bglu44mentioning

confidence: 99%

Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 1

et al. 2004

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In plants, Glycoside Hydrolase (GH) Family 1 beta -glycosidases are believed to play important roles in many diverse processes including chemical defense against herbivory, lignification, hydrolysis of cell wall-derived oligosaccharides during germination, and control of active phytohormone levels. Completion of the Arabidopsis thaliana genome sequencing project has enabled us, for the first time, to determine the total number of Family 1 members in a higher plant. Reiterative database searches revealed a multigene family of 48 members that includes eight probable pseudogenes. Manual reannotation and analysis of the entire family were undertaken to rectify existing misannotations and identify phylogenetic relationships among family members. Forty-seven members (designated BGLU1 through BGLU47 ) share a common evolutionary origin and were subdivided into approximately 10 subfamilies based on phylogenetic analysis and consideration of intron-exon organizations. The forty-eighth member of this family ( At3g06510; sfr2 ) is a beta -glucosidase-like gene that belongs to a distinct lineage. Information pertaining to expression patterns and potential functions of Arabidopsis GH Family 1 members is presented. To determine the biological function of all family members, we intend to investigate the substrate specificity of each mature hydrolase after its heterologous expression in the Pichia pastoris expression system. To test the validity of this approach, the BGLU44 -encoded hydrolase was expressed in P. pastoris and purified to homogeneity. When tested against a wide range of natural and synthetic substrates, this enzyme showed a preference for beta -mannosides including 1,4- beta -D-mannooligosaccharides, suggesting that it may be involved in A. thaliana in degradation of mannans, galactomannans, or glucogalactomannans. Supporting this notion, BGLU44 shared high sequence identity and similar gene organization with tomato endosperm beta -mannosidase and barley seed beta -glucosidase/ beta -mannosidase BGQ60.

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Characterization of a Brassica napus Myrosinase Expressed and Secreted by Pichia pastoris

Cited by 25 publications

References 36 publications

Improved Production of Recombinant Myrosinase in Pichia pastoris

Improved Production of Recombinant Myrosinase in Pichia pastoris

Arabidopsis thaliana β-Glucosidases BGLU45 and BGLU46 hydrolyse monolignol glucosides

Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 1

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