Improved Production of Recombinant Myrosinase in Pichia pastoris

Rosenbergová, Zuzana; Hegyi, Zuzana; Ferko, M.; Andelova, Natalia; Rebroš, Martin

doi:10.3390/ijms222111889

Cited by 6 publications

(3 citation statements)

References 40 publications

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“…The catalytic ability of crude Myr preparation to decompose GRN and form SFN was confirmed by an RP-HPLC analysis. We observed almost a 100% conversion of GRN to SFN under the used experimental conditions (Figure 3) at a reaction rate comparable to that measured with a purified enzyme [63,89,90]. Based on this observation, it can be concluded that the isolated GRN and the crude Myr preparation represent a suitable GLS-Myr system for the generation of SFN in biological samples under in situ conditions.…”

Section: Discussionsupporting

confidence: 79%

“…However, SFN shows an effect on microbial eukaryotes at 0.1 mM (Table 3) and on mammalian cells at ten times lower concentrations [42]. Recently, the effect of enzymatically generated SFN on murine leukemia cells was studied by means of recombinant Arabidopsis thaliana Myr [39], which was prepared and characterized by the group of Dr. Rebroš [89,90]. This study showed that the mixture of GRN with recombinant Myr in the presence of 10 µM L-ascorbic acid is able to produce the sufficient level of SFN to be cytotoxic against L1210 cells and induce cell death via autophagy [39,42].…”

Section: Discussionmentioning

confidence: 99%

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The Antimicrobial Effects of Myrosinase Hydrolysis Products Derived from Glucosinolates Isolated from Lepidium draba

Polozsányi,

Galádová,

Kaliňák

et al. 2024

Plants

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Lepidium draba (hoary cress) is a perennial plant belonging to the Brassicaceae family that produces two dominant glucosinolates (GLSs): glucoraphanin (GRN) and sinalbin (SBN). They represent the stored form, which is converted upon the myrosinase (Myr) hydrolysis activity to active compounds, mainly isothiocyanates (ITCs) such as sulforaphane (SFN) or p-hydroxybenzyl isothiocyanate (pHBITC). Research on ITCs that have proven anticancer, antimicrobial, and chemoprotective properties is usually conducted with pure commercially available compounds. However, these are chemically reactive, making it difficult to use them directly for preventive purposes in dietary supplements. Efforts are currently being made to prepare dietary supplements enriched with GLS and/or Myr. In this study, we report a simple but efficient chromatographic procedure for the isolation and purification of GLSs from MeOH extract from hoary cress based on a combination of ion exchange and gel permeation chromatography on DEAE-Sephadex A-25 and Sephadex LH-20. To obtain the Myr required for efficient hydrolysis of GLSs into antibacterial ITCs, we developed a rapid method for its extraction from the seeds of Lepidium sativum (garden cress). The yields of GLSs were 22.9 ± 1.2 mg GRN (purity 96%) and 10.4 ± 1.1 mg SBN (purity 92%) from 1 g of dry plant material. Both purified GLSs were used as substrates for the Myr. Analysis of the composition of hydrolysis products (HPs) revealed differences in their hydrolysis rates and in the degree of conversion from GLSs to individual ITCs catalyzed by Myr. When GRNs were cleaved, SFNs were formed in an equimolar ratio, but the formation of pHBITCs was only half that of cleaved SBNs. The decrease in pHBITC content is due to its instability compared to SFN. While SFN is stable in aqueous media during the measurement, pHBITC undergoes non-enzymatic hydrolysis to p-hydroxybenzyl alcohol and thiocyanate ions. Testing of the antimicrobial effects of the HPs formed from GRN by Myr under premix or in situ conditions showed inhibition of the growth of model prokaryotic and eukaryotic microorganisms. This observation could serve as the jumping-off point for the design of a two-component mixture, based on purified GLSs and Myr that is, usable in food or the pharmaceutical industry in the future.

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Section: Discussionsupporting

confidence: 79%

Section: Discussionmentioning

confidence: 99%

The Antimicrobial Effects of Myrosinase Hydrolysis Products Derived from Glucosinolates Isolated from Lepidium draba

Polozsányi,

Galádová,

Kaliňák

et al. 2024

Plants

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“…Therefore, both approaches are currently relevant. In the last two decades, heterologous expressions of the myrosinase (TGG1-TGG5) genes from Arabidopsis thaliana [19,44,49,60,61] and from Carica papaya [46] have been successfully performed in yeast (Pichia pastoris and Yarrowia lipolytica). Broccoli myrosinase genes were expressed in E. coli and S. cerevisiae [59].…”

Section: Enzymatic Properties Of Purified Myrosinasementioning

confidence: 99%

Sulphoraphane Affinity-Based Chromatography for the Purification of Myrosinase from Lepidium sativum Seeds

et al. 2022

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Sulforaphane and other natural isothiocyanates released from the respective plant glucosinolates by the plant enzyme myrosinase (β-thioglucoside glucohydrolase) show extensive anticancer and antimicrobial effects. In this study, myrosinase from garden cress (Lepidium sativum) seeds was purified to electrophoretic homogeneity by a fast and easy strategy consisting of fractionation by isoelectric precipitation with ammonium sulphate (AS) and affinity chromatography using sulforaphane (SFN) attached to cellulose resin. The overall purification of enzyme with respect to crude extract was 169-fold and recovery of 37%. Under non-reducing conditions, two protein bands exhibiting myrosinase activity with masses of about 114 and 122 kDa, respectively, and a 58 kDa protein band with no activity were detected by SDS-PAGE and zymography on polyacrylamide gel. MALDI-Tof/Tof of tryptic fragments obtained from the respective protein bands detected sequence motifs homologous to the regions responsible for glycoside-substrate binding and similarities to members of the enzyme subfamilies β-glucosidases and myrosinases GH. The enzyme hydrolyzed both the natural (sinigrin, sinalbin, glucoraphanin) and the synthetic (p-nitrophenol-β-D-glucopyranoside (pNPG)) substrates. The highest catalytic activity of purified enzyme was achieved against sinigrin. The KM and Vmax values of the enzyme for sinigrin were found to be 0.57 mM, and 1.3 mM/s, respectively. The enzyme was strongly activated by 30 μM ascorbic acid. The optimum temperature and pH for enzyme was 50 °C and pH 6.0, respectively. The purified enzyme could be stored at 4 °C and slightly acidic pH for at least 45 days without a significant decrease in specific activity.

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Alternative secretory signal sequences for recombinant protein production in Pichia pastoris

Karaoglan

2023

Enzyme and Microbial Technology

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Improved Production of Recombinant Myrosinase in Pichia pastoris

Cited by 6 publications

References 40 publications

The Antimicrobial Effects of Myrosinase Hydrolysis Products Derived from Glucosinolates Isolated from Lepidium draba

The Antimicrobial Effects of Myrosinase Hydrolysis Products Derived from Glucosinolates Isolated from Lepidium draba

Sulphoraphane Affinity-Based Chromatography for the Purification of Myrosinase from Lepidium sativum Seeds

Alternative secretory signal sequences for recombinant protein production in Pichia pastoris

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