m
EHOOPrAHH^ECKAa XHMH3, 2014, moM 40, № 3, c. 315-319
ROLE OF LACCASE FROM CORIOLUS VERSICOLOR MTCC-138 IN SELECTIVE OXIDATION OF AROMATIC METHYL GROUP
© 2014 Pankaj Kumar Chaurasia, Sunil Kumar Singh, Shashi Lata Bharati#
Department of Chemistry, D.D.U. Gorakhpur University, Gorakhpur, Uttar Pradesh, 273009 India Received August 30, 2013; in final form September 23, 2013
Now a day, laccases are the most promising enzymes in the area of biotechnology and synthesis. One of the best applications of laccases is the selective oxidation of aromatic methyl group to aldehyde group. Such transformations are valuable because it is difficult to stop the reaction at aldehyde stage. Chemical methods used for such biotransformations are expensive and give poor yields. But, the laccase-catalyzed biotransformations of such type are non-expensive and yield is excellent. Authors have used crude laccase obtained from the liquid culture growth medium of fungal strain Coriolus versicolor MTCC-138 for the biotransformations of toluene, 3-nitrotoluene, and 4-chlorotoluene to benzaldehyde, 3-nitrobenzaldehyde, and 4-chlorobenz-aldehyde, respectively, instead of purified laccase because purification process requires much time and cost. This communication reports that crude laccase can also be used in the place of purified laccase as effective biocatalyst.
Keywords: laccase, Coriolus versicolor, mediator, HPLC, biotransformation
DOI: 10.7868/S0132342314020031
INTRODUCTION
Laccase [EC 1.10.3.2] belongs to a group of polyphenol oxidases containing copper atoms in the catalytic center, which catalyze the reduction of molecular oxygen to water [1—3]. It was first reported in Japanese lacquer tree Rhus vernicifera [4]. Little is known about higher plant laccases, probably due to their presence in cell walls. Laccases are the lygnolytic enzymes and abundantly occur in the fungal systems [5], mainly in ascomycetes, deuteromycetes, and ba-sidiomycetes; its production in lower fungi has never been demonstrated. They occur in fungal causative agents of the soft rot, in most, white rot causing fungi, soil saprophytes, and edible fungi. These laccase producing fungi are generally called wood degrading fungi. White rot fungi are the highest producers of the lac-cases, but litter decomposing and ectomicorrhizal fungi also secrete laccases. Almost all white rot fungi are laccase producers [5—7], except for Phanerochaete chrysosporium. Some examples of the fungi which produce laccases are Pleurotus ostreatus, Coriolus sanguineus, Trametes hirsuta, T. vercicolor, T. villosa, Corio-lopsis polyzona, Phlebia radiate, Podospora anserine, Lentinus tigrinus, Pleurotus eryngii, Fomes durrisimus MTCC-1173, Pleurotus sajor caju MTCC-141, and Trametes trogii. Laccase is also reported in bacteria, e.g. Azospirillum lipoferum [8], as well as in wasp venom [9].
# Corresponding author (e-mail: shashilatachem@gmail.com).
However, it should not be generalized that only the fungal system has ability to produce the laccases. Several reports can be referred to in the literature on production of laccase in ascomycetes, such as Gaeuman-nomyces graminis [10], Magnaporthe grisea [11], Ophiostoma novo-ulmi [12], Mauginella [13], Melano-carpus albomyces [14], Monocillium indicum [15], Neurospora crassa [16], and Podospora anserine [17]. In addition to plant pathogenic species, laccase production was also reported for some soil ascomycete species from the genera Aspergillus, Curvularia, and Penicillium [18—20] and in some freshwater ascomycetes [21].
The ability of laccases to catalyze oxidation of various phenolic, as well as non-phenolic compounds, coupled to the reduction of molecular oxygen to water makes, it valuable from the point of view of their commercial applications [2, 22—24]. The importance of laccases has increased after the discovery of mediators [25, 26]. During the last two decades, laccases have turned out to be the most promising enzymes for industrial uses [23, 24] having applications in food, pulps, paper, textile, cosmetics industries, and in synthetic organic chemistry [27—30].
Selective oxidation of aromatic methyl group to its aldehyde group has been done previously [31—33] using purified laccase. Purification of enzyme is a long process that needs high-cost chemical materials and takes much time. In the present communication, crude laccase obtained from liquid culture growth me-
Activity, IU/mL
Days
Fig. 1. Secretion of laccase by Coriolus versicolor MTCC-138 in the liquid culture medium supplemented with different natural lignin containing substrates saw dust (x), corn cob (■), wheat straw (A), coir dust (♦), bagasse (*), and control (•).
dium containing natural lignin substrate, coir dust, of C. versicolor MTCC-138 was used to demonstrate selective oxidation of aromatic methyl group in the presence of ABTS as mediator molecule. The purpose of using crude laccase for such biotransformations was to prove that the crude laccase is also an effective biocat-alyst like purified laccase and there is no need of high-cost materials in order to obtain the crude laccase. Thus, the novelty of this communication is in the use of crude laccase obtained from C. versicolor MTCC-138, which was not reported so far, for such selective oxidation reactions.
RESULTS AND DISCUSSION
The experiment to define the maximum secretion of the laccase in the liquid culture growth medium amended with various lignin containing natural substrates, like corn cob, coir dust, saw dust, wheat straw, and bagasse particles, was performed with Coriolus versicolor MTCC-138. The control experiment had similar medium composition except for the natural lignin containing substrate being absent. The extracellular secretion of laccase was maximum in the case of the growth medium containing coir dust (Fig. 1). In order to optimize secretion of laccase, it was studied in the presence of different amounts of coir dust. The maximum level of laccase was secreted in the liquid culture medium containing 500 mg of the coir dust per 25 mL of the culture medium (Fig. 2). The crude laccase obtained from liquid culture growth medium with
a natural lignin substrate, coir dust, as described above, was used for selective biotransformation of aromatic methyl group to the corresponding aldehyde group.
Selective transformations of aromatic methyl group to its aldehyde group are, generally, a difficult task in organic synthesis. The chemical routes of this transformation are inconvenient because once a methyl group is attacked, it is likely to be oxidized to the carboxylic acid and it is very difficult to stop the reaction at the aldehyde stage. Transformation of aromatic methyl group to aldehyde group is one of the best applications of the laccases, which do it very sharply within 1—3 hrs. The conversion done with crude laccase occurs under milder conditions, yield is >85% and the process is ecofriendly. The use of laccases for this purpose has been studied in the presence of a mediator molecule 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS) [31]. Toluene, 3-nitrotoluene, and 4-chlorotoluene were successfully converted to benzaldehyde, 3-nitrobenzaldehyde, and 4-chlorobenzaldehyde, respectively, in the presence of ABTS as a mediator molecule.
High performance liquid chromatography (HPLC).
In above enzyme catalyzed biotransformations, all products formed are easily available and simple. So, authors have used HPLC technique to confirm the actual product formation by comparing the HPLC chro-matograms of standard aldehyde compounds with the enzymatically transformed compounds. The retention time of the standard sample of toluene was 4.21 min
Activity, IU/mL
Days
Fig. 2. Optimization of laccase secretion by Coriolus versicolor MTCC-138 in liquid culture medium supplemented with different amount of coir dust: 100 mg (♦), 200 mg (□), 400 mg (A), 500mg (x), 600 mg (*), 800 mg (O), and 1000 mg (+).
and the retention time of the standard sample of benzaldehyde was 3.52 min. The retention time of the product of the enzyme-catalyzed reaction (3.50 min) coincided with the retention time of benzaldehyde (3.52 min), confirming that the product of enzyme catalyzed reaction was benzaldehyde. The yield of the product was found to be 100%.
The similar type of reactions were done for 3-ni-trotoluene bioconversion to 3-nitrobenzaldehyde and 4-chlorotoluene bioconversion to 4-chlorobenzaldehyde in the presence of ABTS as a mediator molecule. The retention time of the standard samples of3-nitrotoluene, 4-chlorotoluene, 3-nitrobenzaldehyde and 4-chlo-robenzaldehyde were 6.58, 7.33, 5.91, and 6.25 min, respectively. Thus, the retention time of the products of the enzyme catalyzed reaction (5.96 and 6.23 min) revealed that the products of enzyme catalyzed reaction were 3-nitrobenzaldehyde and 4-chlorobenzaldehyde. In these cases, yield of the extracted 3-nitrobenzaldehyde and 4-chlorobenzaldehyde were 100 and 89%, respectively.
Infrared (IR) spectroscopy. Identification and characterization of products obtained during enzymatic reaction were analyzed on the basis of IR-re-sults. IR results obtained for the expected benzaldehyde are the following: band at vC—H = 3008 cm-1 is due to the aromatic C-H stretching, aldehydic C-H stretching band comes at vC-H = 2760 cm-1, while conjugated aldehydic C=O stretching band comes at vC=O = 1708 cm-1. A peak at 1345 cm-1 is due to the
aldehydic C—H bending. The results demonstrate that the product is benzaldehyde.
For expected product 3-nitrobenzaldehyde, following spectral datas are obtained. Band at vC—H = = 3010 cm-1 is due to the aromatic C—H stretching while band at vC—H = 2900 cm-1 is due to the aldehydic C—H stretching. Conjugated aldehydic C=O band appears at vC=O = 1740 cm-1. Two bands obtained at vN=O = 1500 cm-1, 1305 cm-1 are due to the asymmetric N=O stretching and symmetric N=O stretching, respectively. A peak at 1390 cm-1 is d
Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.