Identification Of 17 Strains Of Lentinula Edodes And Relative Analysis Of Laccase In Lentinula Edodes Strain Cultivated On JUNCAO Grass Variety

Background:The Lentinula edodes expands years of application in the oriental world which are both traditional and advancing due to the wide-spread attention as a result of their array of important properties.The shiitake or xianggu as it is known in the oriental world is a white rot fungus which is biologically versatile and hence serves as a good basis for a variety of biotechnological and environmental applications.The initial attraction to this particular mushroom was its culinary delight and medicinal properties.The pharmaceutical properties of this fungus are underlined in the bioactive substances(i.e.polysaccharides,sterols and lipids)and these factors have subsequently led the mushroom to becoming the second most cultivated mushroom in the world.This edible white rot fungus possesses a variety of extracellular lignin degrading enzymes such as the lignin peroxidases(LiP,EC 1.11.1.14),manganese peroxidases(MnP,EC 1.11.1.13),and laccases(EC 1.10.3.2)as well as versatile peroxidases(VP EC 1.11.1.16).These aforementioned enzymes are reported to be involved in the degradation of environmentally persistent xenobiotics,endocrine-disrupting chemicals such as pentachlorophenol,polychlorinated biphenyls and dioxins.In particular,the laccases belong to the multicopper oxidases(MCOs),which possess a wide array of catalytic activities involved in processes such as polyphenol degradation as well as synthesis,lignin degradation,and melanin establishment.Currently,the exact number of laccases in L.edodes is not known,and there is no comprehensive list of properties and biological functions.Based on the extensive number of reports,fungal laccases are implicated in cell wall formation,pathogenicity,and fruiting body coloration.With recent findings,it was observed that majority of basidiomycete fungi are said to possess at least 10 genes related to encoding different laccases in their genomes.The function of these laccases are not limited to the biological needs of the organisms that possesses them but recent reports also suggest that due to the high reduction potential of these versatile oxidases,they potentially can be used in the biotechnological sectors such as in food processing,wood pulp industry and bioremediation which can consequently prove to be more economically and environmentally friendly.Methods:Firstly,we aimed to identify and study the genetic diversity of L.edodes strains utilizing DNA sequencing technology.Two varieties of DNA-based approaches such as the Internal transcribed spacer sequencing and Random amplified polymorphic DNA were employed to identity the individual strain from the collection obtained from National Engineering Research Center of Juncao Technology and as well as study the genetic diversity of the population of strains.Secondly,once our strain of interest was chosen,two different substrate formulations were employed to test for their effectivity to provide sufficient nutrition for the strain to grow sufficiently into maturity.Thirdly,the chosen strain(LC3031)was cultivated and we extracted laccase enzyme from eight separate samples of the L.edodes strain,namely:mycelium colonization which was divided into a 10 day interval over a total duration of 30 days;during the browning stage of the mushroom,from the substrate utilized to cultivate the mature mushroom;and from the mature harvested mushroom biological parts such as the pileus;the lamella and the stipe in both the control sample(sawdust based sample)and the treated sample(Juncao grass variety sample).We analyzed the laccase enzyme activity by measuring the oxidation of ABTS through determining the increase in absorbance at 420mn with an extinction coefficient of 36mM-1 cm.To further understand and correlate the relative laccase enzyme activity,RNA sequences representing the 4 selected multi-copper oxidases,lcc1:lcc13;lcc6;lcc12 were isolated and relative transcription pattern analysis was conducted in 8 separate samples such as mycelium colonization which was divided into a 10 day interval over a total duration of 30 days;during the browning stage of the mushroom;from the substrate utilized to cultivate the mature mushroom;and from the mature harvested mushroom biological parts such as the pileus;the lamella and the stipe in both the control sample(sawdust based sample)and the treated sample(Juncao grass variety sample).Results:It was observed that from using the DNA-based approaches such as the internal transcribed spacer region markers,our sample fungus was confirmed to be Lentinula edodes.The random amplified polymorphic DNA pattern profiles were utilized to study the genetic variation in our population of Lentinula edodes.Based on the findings of our study,it was observed that there was little or no relationship between the RAPD genetic distance and geographic location.In the second portion of the study,two varieties of substrate formulations were employed to study the effectivity of the substrate via the mycelium running rate of the strain before the utilizations of the formulae.It was observed that there was no statistical significance in the rate of mycelium running between the utilized formulae and that Lentinula edodes is a versatile macro-fungus which can be cultivated on a series of substrate formulations.In the third portion of the study.The overall activity of laccase was observed to be higher in the Juncao treated sample which was suggestive of the treatment having a inductive effect on the laccase,but it cannot be assumed that the laccase product produced in the different treatment is the same,as the type of laccase isoenzymes production can be influenced by environmental conditions and substrate composition.Lastly,in the analysis of the transcription of the multicopper oxidase encoding genes,it was observed that the laccase gene activity was proportional to the mycelial growth during the early phases of cultivation and development.The lcc1 and lcc6 were highly transcribed in the mycelia relative to the fruiting body.The Lcc13 and lcc12 genes were most active during the early stages of mycelium development.Furthermore,the transcription of these genes both during mycelium development(together with lignin degradation)and in fruiting body,further cements the notion that laccase function is diverse.Conclusion:The findings support that Juncao grass variety treatment has a stimulating effect on the laccase activity during developmental stages of the mushroom.Moreover,the multi-copper oxidase encoding genes show a proportional activity relative to the mycelial growth during the early stages of cultivation.Together this highlights the imperative roles of function of laccase activity throughout development which can be related to morphogenesis and lignin degradation.Significance:The importance of this study is to thoroughly study the molecular behavior of the laccase enzyme and its related multi-copper oxidase encoding genes in response to variation in substrate formulation.In an attempt to replace an already valuable source such as wood(sawdust);the study intends on using environmentally friendly grass formulation to investigate the effect of the substrate on production of the laccase enzyme,consequent activity and study the transcription patterns of the several multi-copper oxidase encoding genes throughout its different developmental stages.Hence this will provide comprehensive details about the influence of substrate on laccase behavior and related genes,which have immensely important applications in both the biotechnology field and major biological functions related to development of the mushroom and lignin degradation.