Isolation,Identification Of An Extremely Halophilic Fungus And Cloning And Expression Of Its Cellobiohydrolase(cbh?) Gene

Halophilic fungi,which could grow in high salt environments,are a kind of extreme environmental eukaryotic organism.The halophilic fungui may have unique characteristics in cell structures,physiological functions or metabolites which are different from common organism for its particular growth environment.In recent years,researchers have tried to isolate new halophilic fungi from seawater or other high salt environments,expecting to discover some special metabolites or take advantage of the extreme fungi.Cellulases are a group of complexes which are capable of degrading cellulose which are the most abundant renewable resource on earth.With the decreaseing of non-renewable resources such as oil and the increasingly serious environmental problems,using cellulases to degrade lignocellulose or prouducing bio-ethanol fuel through fermentation are of great significance to energy crisis,which have became a hot research flied in recent decades.Exoglucanase can function on cellulose's crystalline region which is difficult to degrade in the cellulose's degredation process.Therefore,it is important to study the exoglucanase gene from extreme microorganisms.In this study,a halophilic fungus MF1 which was screened from dried sea fish was indentified systematically by the methods of morphological characteristics analysis and polygenic phylogenetic analysis.Since the strain can produce cellulase in high concentration salt environment,its exoglucanase gene was further cloned and heterologous expression successfully.This study mainly contains the following contents and conclusions:1.Identification of strain MF1: a fungus was screened from dried sea fish using Czapek's medium containing 10% NaCl,In order to characterize the fungi,the morphological characteristics on different kinds of medium,micro-morphological characteristics,salinity tolerance abilities and substrate degration abilities under the condition of 15% NaCl were discussed using the Phialosimplex salinarum DSM 27530 as standard strain.Our results displayed that the NaCl tolerance of the strain is 0%-33%,optimum growth at 15%;the strain can grow using starch,lipid,lignin,protein and cellulose as the sole carbon source under 15% NaCl.Moreover,the multivariate phylogenetic analysis was performed bycombing 18 S rDNA,ITS,Tsr1,CaM,BenA,Cct8,PBR1 and PBR2 genes,and the isolated strain was identified as a strain of Phialosimplex and named as Phialosimplex halophila –MF1.2.The coloning of exonglucanase(cbh II)gene from the halophilic fungus MF1.The isolated strain could grow in medium palte containg 15% NaCl and CMC-Na,which demonstrated that it could produce cellulase enzymes in high salt environment.Thus,we attempted furtherly to clone the exonglucanase gene cbh II from the isolate strain.The amino acid sequences of the GH6 family were downloaded from NCBI and its conserved region was found according to the conserved region and codon preferences.The full-length nucleotide sequence of the cbhII gene was obtained by genomic walking PCR,and then furtherly analyzed by bioinformatics method.The specific primers were designed and the cDNA sequences of the cbh II gene were cloned by RT-PCR.The CBHII showed the higest indentity of 70% with these sequences from GeneBank,so it has a certain degree of novelty.The three-dimensional spatial structure model of CBHII was construted.3.Expression cbhII gene from MF1 in Pichia pastoris.The pPIC9k-cbh II vector was constructed by ligating cbh II gene encoding mature protein with the pPIC9 k.Then it was transformed into Pichia pastoris GS115.Subsequently,the recombinant strains were identified by PCR method.The recombinant strain with relatively high activity was selected and induced with 0.5% methanol for scale-up fermentation.The enzyme activity was determined by DNS method and the protein detected by SDS-PAGE.The results showed that a recombinant Pichia pastoris strain with an exogastatinase activity of 1.27 U/L was obtained.The activity of the recombinant enzyme was 0.44 U/mg.The enzyme activity of the recombinant enzyme was the highest at 50?,and the relative activity of the recombinase retained more than 80% at 20-50 ?.These resuts indicate that recombinant enzyme can remain a certain activity at lower temperatures.It is the first report of a functional gene cloning and heterologous expression from phialosimplex.