Myrosinase (thioglucoside glucohydrolase EC 3.2.3.147) is found in all glucosinolate containing plants such as Brassicaseae in some bacteria and fungi (Rask et al. 2000). Myrosinase is normally segregated from its substrates, glucosinolates, sugar anionic thioesters containing beta-thioglucolate glycoside bonds, in plant tissues (Brown and Morra, 1996). When plant cells are damaged or decomposed, myrosinase is released and catalyzes the hydrolysis of glucosinolates. The products of glucosinolate hydrolysis include glucose, sulphate, and a number of active allelochemicals such as isothiocyanates, nitriles, thiocyanates, cyanides, and others depending on substrates and reaction conditions used (Gil and Macleod, 1980). These allelochemicals have been found to inhibit weed seed germination and some pathogens in soil. Recently, several studies have proposed the use of glucosinolate containing plants as a cover crop to reduce the use of synthetic pesticides (Boydston and Hang, 1995; Yenish et al. 1996). Glucosinolates are useful, not only for their activity against bacteria, fungi, nematodes, tumour cell growth and in cancer prevention (Fenwick et al. 1983; Lazzeri et al. 1993; Leoni et al. 1997) but also their potential to be used as intermediates in chemical synthesis (Gueyrade et al. 2000). Myrosinase is, thus, the key enzyme for allelochemical production derived from glucosinolates.

Myrosinase and glucosinolates are beneficial to human and animals and have been studied extensively. Several reports have described the isolation and characterization of myrosinase, especially from white mustard and oilseed rape. A significant amount of work has been put into the recent cloning of plant myrosinase genes (Xue et al. 1992). Over the past 40 years a number of microorganisms including bacteria and fungi have been reported for their glucosinolate-degradation properties. Our laboratory has been interested in studying myrosinase from Aspergillus sp. in many aspects including biodegradation in liquid and solid culture (Sakornet al. 1999; Rakariyatham and Sakorn 2002), and new techniques for detectionof microorganisms that produce myrosinase by using sinigrin-bariumagar plate (Sakorn et al. 2002). Our most recent work was on chemical mutagenesis of strains of Aspergillus sp. to improvemyrosinase production. Strains with higher myrosinase activity will offer potential application in allylisothiocyanate (AIT) production or degradation of glucosinolate in rapeseed and mustard seed for feed industry.

A myrosinase producing fungus, Aspergillus sp. NR4617 (wild type), was newly isolated from decayed soil sample obtained in Lamphun province, Thailand. Primary myrosinase screening of soil samples was performed on sinigrin-barium agar plates (Sakorn et al. 2002). The positive strains exhibited opaque zone on sinigrin-barium agar plates. Incubation was then carried out at 30ºC and growths were observed daily. Each distinct growing colony was picked and then plated onto nutrient agar plates or potato dextrose agar plates. Re-plating was performed until pure isolate was obtained. Although growth was observed for several strains, a decrease in the sinigrin content was observed for only eight chosen strains with high activity. The highest fungal strains of the eight, Aspergillus sp. NR4617 (wild type), produced myrosinase only intracellularly, thus was selected for further experiments.

Fungal isolates of the wild type were subjected to single exposure to two selected chemical mutagens, ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) which alter their genetic materials randomly. The procedures were adapted from that of Al-Aidross and Seifert (1980) and Chadha et al. (1999), respectively. Four survival spores with myrosinase production from each chemical mutagenesis include NR4617E1-E4 (EMS) and NR4617MG1-MG4 (MNNG). Our result suggested that mutant strains, NR4617E1, NR4617E4 and NR4617MG3 produced myrosinase higher than the wild-type. The enhancement of myrosinase activity might be due to structural change of the enzyme, higher amount of the enzyme, increased level of modulator proteins or cofactors promoting the enzyme activity or decreased level of inhibitory proteins.

Studies of myrosinase production using mustard extract medium revealed that Aspergillus sp. NR4617E1 produced myrosinase the most at 1.902 U ml^-1 at 36 h of the cultivation, equivalent to 171% of the enzyme production in wild-type, followed by NR4617MG3 at 1.626 U ml^-1 (148%). To our knowledge, myrosinase improving chemical mutants characterized by such high levels of enzymatic activities have not been previously reported. The advantages of using Aspergillus sp. NR4617 and its mutants were possession of higher myrosinase activity and ability to grow in the low cost medium.

The stability of enzyme present in the cell free extracts was subsequently determined. Myrosinase from the wild-type Aspergillus sp. NR4617 was inactive after 2 hrs at 30ºC. The stability of the wild-type enzyme was similar to those of enzymes from other microorganisms suggesting myrosinase from microorganisms were not stable at high temperature (Lazzeri et al. 1993). Chemical mutagenesis produced NR4617E1 and NR4617MG3 strains with higher myrosinase production and similar stability.

The product analysis of myrosinase from wild-type Aspergillus sp. NR4617 and all mutant strains revealed that glucosinolate was degraded to allylisothiocyanate (AIT) in neutral conditions, while allylcyanide, a possible toxic product, could not be detected. The growth profile of mutant strains suggested that all the mutants gave the same time course of growth and optimal conditions as wild type strain. Maximum myrosinase activity was detected in extract from mutant Aspergillus sp. NR4617E1 which degraded 10 mM of glucosinolate completely in 36 hrs. This growth profile was better than wild-type and from that reported in previous work (Sakorn et al. 1999; Rakariyatham and Sakorn, 2002). NR4617E1 and NR4617MG3 degraded higher glucosinolate concentration (10 mM) at the same time course of growth in liquid media. These results are useful for seed detoxification and animal feed production.

In conclusion, an attempt was made to mutate Aspergillus sp. NR4617 to increase myrosinase production. Two of myrosinase production strains (NR4617E1 and NR4617MG3) were identified and NR4617E1 was found to offer better yield of myrosinase with the same stability of the wild-type. Overall results indicated that NR4617E1 mutant can be a potential strain for myrosinase production. Hence, the enzyme from Aspergillus sp. NR4617E1 mutant offers a great potential for industrial applications for example feed detoxification, yield enhancement of flavor production (allylisothiocyanate production) and development for a bioreactor for hydrolyzing large amount of glucosinolates for use as coproducts of aqueous-enzyme biorefining of cruciferous oil-bearing seeds in the future.

The authors wish to thank Graduated school Chiang Mai University. We also thank the Department of Chemistry, Faculty of Science, Chiang Mai University for chemical support. This research was financially supported by the Thailand Research Fund (TRF).

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