Study on bacterial myrosidase. II. Purification and General characteristics of bacterial myrosinase produced by Enterobacter cloacae.

Tani, Naoki; Ohtsuru, Masaru; Hata, Toshiyuki

doi:10.1271/bbb1961.38.1623

Cited by 28 publications

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“…It is not known whether the insect myrosinase may actually be produced by and localized to the bacteria or if it is resident in insect cells. Myrosinase activity has been reported in certain bacteria [31,32].…”

Section: Discussionmentioning

confidence: 99%

Purification and characterization of myrosinase from the cabbage aphid (Brevicoryne brassicae), a brassica herbivore

Pontoppidan¹,

Ekbom

Eriksson³

et al. 2001

European Journal of Biochemistry

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Aphids are among the most serious insect pests of agricultural crops in the world. They often have specific hosts, and the cabbage aphid (Brevicoryne brassicae) is a specialist on Cruciferae. It has previously been described that certain insects contain the enzyme myrosinase (EC 3.2.3.1), which is considered an important defence enzyme of crucifers. Myrosinase was purified to homogeneity from cabbage aphid soluble extracts using anionexchange and phenyl-Sepharose chromatography. The protein has an apparent subunit molecular mass of 57±58 kDa and is a dimer. The isoelectric point is 4.9 and the enzyme has a temperature optimum around 40 8C. The enzyme was active towards the glucosinolates tested, sinigrin and glucotropaeolin, but was inhibited by ascorbate at concentrations that normally activate plant myrosinases.Using sinigrin as the substrate K m was determined as 0.41 mm, and the k cat as 36 s 21 . With glucotropaeolin the K m and k cat values were determined as 0.52 mm and 22.8 s 21 , respectively. The enzyme was stable upon storage at 4 8C for many months, but lost some activity upon freezing. The insect myrosinase did not cross-react with antibodies raised to plant myrosinase. Peptide sequencing of a tryptic digest of the protein showed homology to b-glucosidases. The presence of myrosinase in an insect pest specialist may be an example of a coevolution process that facilitates host specialization.

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Section: Discussionmentioning

confidence: 99%

Purification and characterization of myrosinase from the cabbage aphid (Brevicoryne brassicae), a brassica herbivore

Pontoppidan¹,

Ekbom

Eriksson³

et al. 2001

European Journal of Biochemistry

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“…, Fusarium sp. [ 18 ], Citrobacter [ 19 ], Enterobacter [ 20 ] as well as many other species. Strains capable of metabolizing glucosinolates are part of the human gut microbial community [ 21 – 23 ].…”

Section: Introductionmentioning

confidence: 99%

Endophytic fungi from the roots of horseradish (Armoracia rusticana) and their interactions with the defensive metabolites of the glucosinolate - myrosinase - isothiocyanate system

et al. 2018

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BackgroundThe health of plants is heavily influenced by the intensively researched plant microbiome. The microbiome has to cope with the plant’s defensive secondary metabolites to survive and develop, but studies that describe this interaction are rare. In the current study, we describe interactions of endophytic fungi with a widely researched chemical defense system, the glucosinolate - myrosinase - isothiocyanate system. The antifungal isothiocyanates are also of special interest because of their beneficial effects on human consumers.ResultsSeven endophytic fungi were isolated from horseradish roots (Armoracia rusticana), from the genera Fusarium, Macrophomina, Setophoma, Paraphoma and Oidiodendron. LC-ESI-MS analysis of the horseradish extract incubated with these fungi showed that six of seven strains could decompose different classes of glucosinolates. Aliphatic, aromatic, thiomethylalkyl and indolic glucosinolates were decomposed by different strains at different rates. SPME-GC-MS measurements showed that two strains released significant amounts of allyl isothiocyanate into the surrounding air, but allyl nitrile was not detected. The LC-ESI-MS analysis of many strains’ media showed the presence of allyl isothiocyanate - glutathione conjugate during the decomposition of sinigrin. Four endophytic strains also accepted sinigrin as the sole carbon source. Isothiocyanates inhibited the growth of fungi at various concentrations, phenylethyl isothiocyanate was more potent than allyl isothiocyanate (mean IC50 was 2.30-fold lower).As a control group, ten soil fungi from the same soil were used. They decomposed glucosinolates with lower overall efficiency: six of ten strains had insignificant or weak activities and only three could use sinigrin as a carbon source. The soil fungi also showed lower AITC tolerance in the growth inhibition assay: the median IC50 values were 0.1925 mM for endophytes and 0.0899 mM for soil fungi.ConclusionsThe host’s glucosinolates can be used by the tested endophytic fungi as nutrients or to gain competitive advantage over less tolerant species. These activities were much less apparent among the soil fungi. This suggests that the endophytes show adaptation to the host plant’s secondary metabolites and that host metabolite specific activities are enriched in the root microbiome. The results present background mechanisms enabling an understanding of how plants shape their microbiome.Electronic supplementary materialThe online version of this article (10.1186/s12870-018-1295-4) contains supplementary material, which is available to authorized users.

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“…The default products are isothiocyanates, or, in vivo in the presence of so called specifier proteins, other less toxic volatiles can be formed. The activity itself is shown to be present in various Brassicaceae plants [ 3 , 4 ], microorganisms [ 5 , 6 , 7 ], and organisms associated with such plants, like endophytes from horseradish roots [ 8 ] or some insects feeding on host plants with such metabolites [ 9 , 10 , 11 ]. A simple plant can contain various myrosinase isoenzymes, as shown in Arabidopsis thaliana [ 12 ] and other Brassicaceae plants [ 13 , 14 , 15 , 16 ].…”

Section: Introductionmentioning

confidence: 99%

“…Full characterization of enzymes requires purification during which activity is usually monitored by a routine, specific assay. Purification of enzymes with this activity was successful from several sources, including various Brassicaceae plants [ 4 , 13 , 17 , 18 ], microorganisms [ 6 , 7 ] or insects [ 11 , 19 ]. Myrosinase assays usually detect either the decomposition of the substrate (glucosinolates), or release of one of the product compounds.…”

Section: Introductionmentioning

confidence: 99%

A Simple Method for On-Gel Detection of Myrosinase Activity

et al. 2018

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Myrosinase is an enzyme present in many functional foods and spices, particularly in Cruciferous vegetables. It hydrolyses glucosinolates which thereafter rearrange into bioactive volatile constituents (isothiocyanates, nitriles). We aimed to develop a simple reversible method for on-gel detection of myrosinase. Reagent composition and application parameters for native PAGE and SDS-PAGE gels were optimized. The proposed method was successfully applied to detect myrosinase (or sulfatase) on-gel: the detection solution contains methyl red which gives intensive red bands where the HSO4− is enzymatically released from the glucosinolates. Subsequently, myrosinase was successfully distinguished from sulfatase by incubating gel bands in a derivatization solution and examination by LC-ESI-MS: myrosinase produced allyl isothiocyanate (detected in conjugate form) while desulfo-sinigrin was released by sulfatase, as expected. After separation of 80 µg protein of crude extracts of Cruciferous vegetables, intensive color develops within 10 min. On-gel detection was found to be linear between 0.031–0.25 U (pure Sinapis alba myrosinase, R2 = 0.997). The method was successfully applied to detection of myrosinase isoenzymes from horseradish, Cruciferous vegetables and endophytic fungi of horseradish as well. The method was shown to be very simple, rapid and efficient. It enables detection and partial characterization of glucosinolate decomposing enzymes without protein purification.

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Study on bacterial myrosidase. II. Purification and General characteristics of bacterial myrosinase produced by Enterobacter cloacae.

Abstract: Myrosinase in cell-free extract of Enterobacter cloacae, no. 506, was purified about 1,000 fold by precipitation with ammonium sulfate, chromatography on CM-Sephadex and gel filtration on Sephadex G-200 and Sephadex G-100. The enzyme was shown to be homo

Cited by 28 publications

References 0 publications

Purification and characterization of myrosinase from the cabbage aphid (Brevicoryne brassicae), a brassica herbivore

Purification and characterization of myrosinase from the cabbage aphid (Brevicoryne brassicae), a brassica herbivore

Endophytic fungi from the roots of horseradish (Armoracia rusticana) and their interactions with the defensive metabolites of the glucosinolate - myrosinase - isothiocyanate system

A Simple Method for On-Gel Detection of Myrosinase Activity

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