Effective Stimulation of the Biotechnological Pote

Authors: Magdalena Jaszek Katarzyna Kos Anna Matuszewska Marcin Grąz Dawid Stefaniuk Monika OsińskaJaroszuk Monika Prendecka Ewa Jóźwik Krzysztof Grzywnowicz

Publish Date: 2014/08/03

Volume: 174, Issue: 2, Pages: 644-656

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Abstract

The effect of menadione MQ 2methyl14naphtoquinone a superoxidegenerating agent on the natural biodegradation system in the medicinal white rot fungus Phellinus pini was determined While measuring the activities of extracellular manganesedependent peroxidase MnP and intracellular chitinase it was found that the application of MQ 075 mM distinctly stimulated the activities of these enzymes in comparison to the control values without MQ Using the capillary electrophoresis CE method an increase in the extracellular oxalic acid OXA concentration was detected during the first days after the addition of MQ It was observed that the rate of intracellular proteolysis at pH 35 evidently decreased under oxidative stress conditions Contrary to these results the activities of serine proteases at pH 95 measured against fluorogenic peptide substrates distinctly increased in stressed cultures The MQ treatment also caused an evident increase in the catalase CAT activity as well as the levels of superoxide anion radicals SORs formaldehyde FA and phenolic compounds PHC in the experimental cultures The results obtained confirm that prooxidants may find application as an effective way to stimulate biotechnological production of MnP and chitinase by white rot fungiRecently the white rot Basidiomycota have received increasing attention from researchers as an applicable source of natural components including low molecular compounds proteins polysaccharides or polysaccharide–protein complexes for industry food processing or manufacture of drugs The production of natural bioactive constituents is a growing field of the contemporary biotechnology 1 2 Phellinus pini from the family Hymenochaetacae is a widespread red color mushroom that frutifies over the stem of Pinaceae Cupressaceae etc 3 It can be an interesting source of potentially medicinal substances such as enzymes polysaccharides or ceramides 4 The extract obtained from the P pini fruiting body has been shown to possess antitumor immunostimulating antiviral antibacterial and hypocholesterolemic activities Additionally two resinous natural compounds that inhibit NO production were detected in the P pini fruit body 5 6 7 8Development and reproduction of organisms in the natural environment can be regulated by various types of stressful conditions 9 The stresstolerant Sselected strategies evolved by fungi allow them to colonize the niches of some extreme environmental conditions 10 This type of life way is observed in a majority of wooddecaying white rot Basidiomycota Normally the characteristic course of their life cycle is strictly connected with a relatively high concentration of their selfproduced reactive oxygen species ROS which might be a key factor eg initiating wood degradation processes 11 12 The level of ROS generated during the cellular metabolism can be elevated by external stress factors such as an increase or decrease in culturing temperature treatment with redoxcycling compounds or presence of metal ions When the internal balance between the enzymatic and nonenzymatic antioxidants is disrupted a phenomenon of oxidative stress can be observedIn response to surrounding environmental factors including excess of ROS these fungi secrete a broad spectrum of the abovementioned ligninolytic hydrolytic or antioxidative enzymes and low molecular derivatives like phenolic compounds oxalic acid formaldehyde or ROS 12 13 14 Apart from their synergistic action in the degradation of biopolymers the specific ligninolytic enzymes ie peroxidases manganesedependent peroxidase MnP EC 111113 lignin peroxidase LiP EC 111114 or laccase LAC EC 11032 are often described in the literature as being very interesting for modern industry and biotechnology eg biotransformation of environmental pollutants decolorization of textile dye wastes pulping and bleaching processes food processing medicine 15 16 17 18 According to the available data LAC and MnP may also act as an important element of the general fungal response to changes in temperature presence of cadmium or proxidant treatment 12 13 19 20 21 22Hydrolytic enzymes are an important part of the fungal metabolism These include chitinases described as defense against chitincontaining pathogens important for nutrition morphogenesis and interspecific mycelial interactions between different fungal strains These enzymes are produced by a number of organisms including bacteria fungi plants and yeasts Chitinases EC 32114 catalyze the hydrolysis of chitin into smaller chitooligosaccharides Some of them for example GlcNAc 6 and GlcNAc 7 have been reported to possess possible antitumor activity Due to the variety of physiological functions chitinases have found several applications 12 23Contrary to the biodegradation processes conducted by the white rot fungi only few reports described their oxidative stress response system including the possible role of ligninolytic and hydrolytic enzymes or low molecular weight compounds in this type of defense 24 This is the reason why the main question of the present study was whether the stress stimulation like other inducers could be an effective way to increase the biosynthesis of fungal biotechnologically applicable and bioactive compounds and to stimulate their natural potential of biodegradation Because of its specific quinone structure a superoxidegenerating agent—menadione MQ—was considered as a very interesting prooxidant especially for white rot fungi 25 Intermediate metabolites produced during lignin biodegradation may include phenolic compounds which are enzymatically transformed to quinine forms The quinoneredox cycle catalyzed by LAC is one of the most important sources of ROS eg the superoxide anion radical SOR hydrogen peroxide and hydroxyl radical byproduct of Fentontype reactions 26 The prooxidants or heat shock conditions can also control the biosynthesis of MnP an enzyme that catalyzes the Mnmediated oxidation of phenolic lignin compounds 12 13 27 28Based on the above information our first aim was to determine the extracellular activities of LAC and MnP in the P pini cultures treated with MQ The next step of the experimental cycle was to determine whether the addition of MQ really caused overproduction of SORs and the other low molecular stress marker formaldehyde FA under these experimental conditions The level of phenolic compounds PHC and oxalic acid OXA as targets employed in ligninolytic enzyme reactions and fungal stress response mechanisms was also measured Furthermore in the experiments described above the activities of SOD and CAT the wellknown enzymatic antioxidants were measured in the stressed and control cultures The effect of MQ addition on the activities of proteases and chitinase was also determined in the present workThe P pini Brot Fr Ames strain was obtained from the culture collection of the Department of Biochemistry M CurieSkłodowska University in Lublin Poland 29 The fungal cultures were stored on 2  w/v malt agar slants For inoculation fungal agar plugs approximately 05 cm2 were cut and put into the mineral medium prepared according to Fahreus and Reinhammar FR 30 The fungal cultures were performed as described previously 13 The mycelium was collected and homogenized in a Warning blender Experimental stationary cultures were inoculated with 25  v/v of homogenized fungal material and incubated in 25ml Erlenmeyer glass flasks with 10 ml of the FR media at 25 °C for 14 days Since the ligninmodifying enzymes and many biotechnologically important compounds are expressed mainly during the secondary metabolic phase of fungal growth 31 14dayold stationary cultivated idiophasic cultures of P pini were treated with different concentrations of menadione solution 05–25 mM The beginning of the idiophase was determined according to Jennings and Lysek 32 The proper experimental concentration of MQ was established based on the MnP LAC and chitinase activities The most significant changes were observed in the presence of 075 mM of MQ final concentration in the medium In the case of stationary cultivated fungal cultures the portions of MQ must be added immediately to the culture fluid

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