the influence of cultivation conditions on the myrosinase activity
TRANSCRIPT
THE INFLUENCE OF CULTIVATION CONDITIONS ON THE MYROSINASE ACTIVITY
AND GLUCOSINOLATE CONTENT IN WHITE CABBAGE
Anna Piekarska, Tadeusz Pilipczuk, Barbara Kusznierewicz, Jacek Namienik, Agnieszka Bartoszek
Faculty of Chemistry, Gdansk University of Technology
“Utilizing white cabbage for the phytoremediation and biofumigation of soils (AGROBIOKAP)”, co-financed by the European Union from the European Fund for Regional Development within the framework of the Operational Program for an Innovative Economy 2007-2013
METHODS
DETERMINATION OF MYROSINASE ACTIVITY Activity of enzyme myrosinase was determined using pH-stat method. The reaction mixture consisted of 15 mL 1% NaCl (pH 6.5) and 0,5 g of lyophilized leaves of cabbage containing myrosinase. After hydrolysis of cabbage internal glucosinolates and stabilisation of pH of mixture, 0,15 mL of external glucosinolate (40 mM glukotropaeolin) was added. During GLS hydrolysis pH decreases. The aim of this method is to keep constant pH level (6.5) by adding NaOH solution. Myrosinase activity was determined by measuring the acid release rate by titrating with 1 mM NaOH with T70 titrator (Mettler Toledo). The reaction time was 1 hour, the solution was kept at 37oC and gently stirred.
DETERMINATION OF GLUCOSINOLATE CONTENT Glucosinolates were extracted from cabbage lyophilisates with hot methanol, then extracts were poured into ion-exchange columns and washed with 1 mL of deionized water. Next, a solution of purified sulphatase (0.2 mL) was added into the columns, which were then left to incubate at room temperature for 12 h. The desulphoglucosinolates obtained were eluted twice with deionized water (1 mL). The glucosinolates were determined by reversed-phase HPLC-DAD and gradient elution. The mobile phases were water (A) and 20% acetonitrile (B); the elution programme was: 0 min – 100 % A, 20 min –100 % B, 25min – 100 % B, with a mobile phase flow rate of 1 mL/min. The target analytes were determined qualitatively by comparing their retention parameters with those of available standards and quantitatively using an internal standard (glucotropaeolin).
INTRODUCTION
In the process of biofumigation typically natural compounds present in Brassicaceae family are used to combat pests and protect crops. Glucosinolates, sulfur-containing secondary metabolites found in Brassica plants, are hydrolized by the enzyme myrosinase (β-thioglucosidase, EC 3.2.3.1) with the liberation of degradation products such as isothiocyanates, nitriles, thiocyanates and epithionitriles (fig. 1). Isothiocyanates are the most bioactive among these compounds and accordingly most efficient in the suppression of many kinds of herbivores and soil born pathogens. Other glucosinolates degradation products are much less effective, therefore it is desirable to direct the glucosinolate hydrolysis in such a way so as to obtain only isothiocyanates as the end products. Many factors like environmental conditions may influence myrosinase activity in cabbage as well as the presence of other specifier proteins, which promote formation of different products. All these factors determine the yield of isothiocyanates and in consequence, the efficacy of the biofumigation process.
CONCLUSIONS
Cultivation conditions and cabbage cultivars determine the content of glucosinolates and myrosinase activity in Brassica plants
Cultivar of cabbage Kamienna Gowa exhibits the highest content of glucosinolates, as well as myrosinase activity, irrespective of the place of cultivation
Even in the same cabbage cultivar grown in various conditions there are significant differences in glucosinolates profile
The goal of our research is to investigate the influence of a type of cultivar and cultivation condition on the content of glucosinolates and myrosinase activity in white cabbage (B.oleracea var.capitata) grown in the Northern and Southern regions of Poland (fig. 2). Places of cultivation varied as regards agricultural aspects: organic and industrial cultivation, soil class, intensity of sunlight and risk of pest attack.
Fig. 1. Products of GLS hydrolysis
Fig. 3. The content of glucosinolates in cabbages grown in different localisations
0
2
4
6
8
10
12
0
2
4
6
8
10
DN KG Kraków Mydlnik
TC Strzelno
GLUCOSINOLATE CONTENT
Am- Amager, DN- Ditmarska Najwczeniejsza, KG- Kamienna Gowa, TC- Tucana, AB- Ambrozja, GR- Gregorian
Fig. 2. Places of cabbage cultivation
ecological cultivation, field is surrouded by forest, away from roads; soil derived from former greenhouse contaminated with Hg
conventional cabbage cultivation, surrounded by forests
very polluted area in the middle of Upper Silesian Industrial Basin
area possibly polluted by industry
CZAPIELSK
STRZELNO
KRAKÓW
SOSNOWIEC
DN KG Kraków Mydlnik
TC Strzelno
Fig. 5. The activity of myrosinase present in cabbages grown in different localisations
MYROSINASE ACTIVITY
GLUCOSINOLATE PROFILE
Fig. 4. Profile of glucosinolates in cabbages Kamienna Gowa grown in different localisations
1- glucoiberin 2- progoitrin
5- glucoerucin 6- glucobrassicin
7- metoksyglucobrassicin 8- neoglucobrassicin
Aliphatic GLS
Indolyl GLS
Sosnowiec O!arowice
Anna Piekarska, Tadeusz Pilipczuk, Barbara Kusznierewicz, Jacek Namienik, Agnieszka Bartoszek
Faculty of Chemistry, Gdansk University of Technology
“Utilizing white cabbage for the phytoremediation and biofumigation of soils (AGROBIOKAP)”, co-financed by the European Union from the European Fund for Regional Development within the framework of the Operational Program for an Innovative Economy 2007-2013
METHODS
DETERMINATION OF MYROSINASE ACTIVITY Activity of enzyme myrosinase was determined using pH-stat method. The reaction mixture consisted of 15 mL 1% NaCl (pH 6.5) and 0,5 g of lyophilized leaves of cabbage containing myrosinase. After hydrolysis of cabbage internal glucosinolates and stabilisation of pH of mixture, 0,15 mL of external glucosinolate (40 mM glukotropaeolin) was added. During GLS hydrolysis pH decreases. The aim of this method is to keep constant pH level (6.5) by adding NaOH solution. Myrosinase activity was determined by measuring the acid release rate by titrating with 1 mM NaOH with T70 titrator (Mettler Toledo). The reaction time was 1 hour, the solution was kept at 37oC and gently stirred.
DETERMINATION OF GLUCOSINOLATE CONTENT Glucosinolates were extracted from cabbage lyophilisates with hot methanol, then extracts were poured into ion-exchange columns and washed with 1 mL of deionized water. Next, a solution of purified sulphatase (0.2 mL) was added into the columns, which were then left to incubate at room temperature for 12 h. The desulphoglucosinolates obtained were eluted twice with deionized water (1 mL). The glucosinolates were determined by reversed-phase HPLC-DAD and gradient elution. The mobile phases were water (A) and 20% acetonitrile (B); the elution programme was: 0 min – 100 % A, 20 min –100 % B, 25min – 100 % B, with a mobile phase flow rate of 1 mL/min. The target analytes were determined qualitatively by comparing their retention parameters with those of available standards and quantitatively using an internal standard (glucotropaeolin).
INTRODUCTION
In the process of biofumigation typically natural compounds present in Brassicaceae family are used to combat pests and protect crops. Glucosinolates, sulfur-containing secondary metabolites found in Brassica plants, are hydrolized by the enzyme myrosinase (β-thioglucosidase, EC 3.2.3.1) with the liberation of degradation products such as isothiocyanates, nitriles, thiocyanates and epithionitriles (fig. 1). Isothiocyanates are the most bioactive among these compounds and accordingly most efficient in the suppression of many kinds of herbivores and soil born pathogens. Other glucosinolates degradation products are much less effective, therefore it is desirable to direct the glucosinolate hydrolysis in such a way so as to obtain only isothiocyanates as the end products. Many factors like environmental conditions may influence myrosinase activity in cabbage as well as the presence of other specifier proteins, which promote formation of different products. All these factors determine the yield of isothiocyanates and in consequence, the efficacy of the biofumigation process.
CONCLUSIONS
Cultivation conditions and cabbage cultivars determine the content of glucosinolates and myrosinase activity in Brassica plants
Cultivar of cabbage Kamienna Gowa exhibits the highest content of glucosinolates, as well as myrosinase activity, irrespective of the place of cultivation
Even in the same cabbage cultivar grown in various conditions there are significant differences in glucosinolates profile
The goal of our research is to investigate the influence of a type of cultivar and cultivation condition on the content of glucosinolates and myrosinase activity in white cabbage (B.oleracea var.capitata) grown in the Northern and Southern regions of Poland (fig. 2). Places of cultivation varied as regards agricultural aspects: organic and industrial cultivation, soil class, intensity of sunlight and risk of pest attack.
Fig. 1. Products of GLS hydrolysis
Fig. 3. The content of glucosinolates in cabbages grown in different localisations
0
2
4
6
8
10
12
0
2
4
6
8
10
DN KG Kraków Mydlnik
TC Strzelno
GLUCOSINOLATE CONTENT
Am- Amager, DN- Ditmarska Najwczeniejsza, KG- Kamienna Gowa, TC- Tucana, AB- Ambrozja, GR- Gregorian
Fig. 2. Places of cabbage cultivation
ecological cultivation, field is surrouded by forest, away from roads; soil derived from former greenhouse contaminated with Hg
conventional cabbage cultivation, surrounded by forests
very polluted area in the middle of Upper Silesian Industrial Basin
area possibly polluted by industry
CZAPIELSK
STRZELNO
KRAKÓW
SOSNOWIEC
DN KG Kraków Mydlnik
TC Strzelno
Fig. 5. The activity of myrosinase present in cabbages grown in different localisations
MYROSINASE ACTIVITY
GLUCOSINOLATE PROFILE
Fig. 4. Profile of glucosinolates in cabbages Kamienna Gowa grown in different localisations
1- glucoiberin 2- progoitrin
5- glucoerucin 6- glucobrassicin
7- metoksyglucobrassicin 8- neoglucobrassicin
Aliphatic GLS
Indolyl GLS
Sosnowiec O!arowice