Cloning, Expression And Hydrolysis Characteristics Of Protease Genes From Fungi

Some fungi strains were the predominant strains used in the production of traditionalfermentation food of soybean in china. Because of a long time of acclimatization on the highprotein medium, these strains could synthesize and secrete several proteinases, which hadgood suitability to plant protein and showed high hydrolysis efficiency to plant protein,especially for soybean protein. At present, researches of both domestic and abroad werefoucused on improving yields of proteases by optimizing solid or liquid fermentation andenzymic properties, while neglected cloning and expression of protease genes from fungi. Forthis reason, our research focused on clone and expression of protease genes from severalfungi, site-directed mutagenesis, and hydrolysis efficiency to soybean protein.Three new aminopeptidase genes from fungi, such as Actinomucor elegans, Rhizopuschinensis and Mucor racemosus, etc. were achieved, and respectively submitted to GenBank（access number HQ825158, JQ657815and JQ657814, the below is same）, and two newserine protease genes （GU356536and JF922913）. Further, other protease genes were gainedby RT-PCR techniques, these genes were as follow: four serine protease genes （L19059,XM₀01391433, XM₀01824768and XM₀01820092）, five acid proteases genes（XM₀01399818, XM₀01401056, D13894, AB090877and AB044079）, and one neutralprotease genes （AF099904）. In a word, fifteen proteases genes were obtain in our research.These protease genes were predicted and analyzed by in detail based on thebioinformatics theory and technology. The results showed all protease genes, which haddifferent preference for codon usage, coded different length of amino acid residues. Six kindsof proteases belonged to Proteinase K family and exhibited the closest genetic relationshipeach other. Moreover, according to the six proteases structure modeled via homologymodeling with Proteinase K （PDB code:1IC6） as the crystal coordinate, it was found that sixproteases had no disulfide bonds and possessed a Ca2+binding site, and that the differences insubstrate-binding region, hydrogen bond, salt bridge and disulfide bond between proteaseswere probably responsible for the special hydrolysis ability.Five kinds of proteases belonged to acid protease Asp family, and were extracellularproteases, and exhibited the closest genetic relationship each other. Moreover, according to the five proteases structure modeled via homology modeling with Aspergillopepsin I （PDBcode:1IBQ B） as the crystal coordinate, it was found that five proteases respectively had adisulfide bond and possessed a Zn2+binding site. The differences in the solvent accessiblesurface area of active sites, hydrogen bond, salt bridge, substrate-binding region and ψ-loopstructure between five proteases were probably responsible for the special enzymaticcharacteristics.Active sites, motif structure and evolutionary relationship of the neutral protease and theaminopeptidase genes respectively were predicted and analyzed. Because these proteases hadno suitable homology models, the secondary structure and the tertiary structure were notpredicted.The fragments containing protease genes, which respectively were from A.elegans, R.chinensis, A.niger, A.oryzae and A.elegans, were cloned into pET-22b（+） expression vector,then were transformed into E. coli Bl21and expressed under the induction of IPTG. Theresults showed the expressed recombinant proteases were inactive proteases. These proteasegenes were cloned into the secreted expression pPIC9K vector, and the recombinant vectorswere transferred into Pachia pastoris KM71strain, then the recombinant KM71strains wereinduced using methyl hydrate. The results showed only three protease genes could beexpressed sucessfuly, and the three protease genes respectively was AlpI gene of A.niger,AlpII gene of A.oryzae and NpI gene of A.oryzae. Furtherly, the characters of the threerecombinant proteases separated and purified respectively were studied. The results ofrecombinant protease（rAlpI） of A.niger showed that （1） when the protease were induced toexpress in a10-L fermentor，the protease activity of the fermentation broth was up to4050U/mL;（2） the optimum pH value and temperature of the protease were respectively8.0-9.0and50℃，and the protease was stable at a pH value of6.0-9.0below40℃;（3） PMSFcould absolutely inhibite the activity of the protease, and the the activity of recombinantprotease was respectively activated by Ca2+, Mn2+and K+, and slightly inhibited by Zn2+、Fe2+and Mg2+.The results of recombinant protease（rAlpII） of A.oryzae showed that （1） when theprotease was induced to express in a10-L fermentor，the protease activity of the fermentationbroth was up to4100U/mL;（2） the optimum pH value and temperature of the protease were respectively8.5-9.5and50℃, and the protease was stable at a pH value of6.0to10.0below40℃;（3） PMSF could completely inhibite the activity of protease, and the activity ofrecombinant protease was respectively activated by Ca2+and Mg2+, and slightly inhibited byZn2+,Fe2+and Mn2+.The results of recombinant protease（rNpI） of A.oryzae showed （1） when the proteasewas induced to express in a10-L fermentor，the protease activity of the fermentation brothwas up to43101U/mL;（2）the optimum pH value and temperature of the protease wererespectively8.0and55℃, and the protease was stable at a pH value of5.0to9.0below45℃;（2）EDTA could completely inhibited the activity of protease, and the activity of recombinantprotease was inhibited by Cu2+and Zn2+.In order to improve the thermal stability of the recombinant proteases for industrialexploitation and utilization, the site directed mutagenesis have been used to add the disulfidebonds into the recombinant proteases. The results showed that the thermal stability of themutant enzyme AlpII G33C-S126C of A.oryzae, which indicated that amino acids residuals ofsite33and site126were mutated into C, were improved, while the mutant enzymeI179C-T253C was reduced, and the mutant enzyme G33C-S126C-179C-T253C could not beexpressed in P. pastoris. The three mutant enzymes of A.niger, G34C-S127C, I180C-T254Cand G34C-S127C-180C-T254C, could not be expressed in P. pastoris, too. A series ofmutations to “HExxH¹9aa^E” Motif of the rNpI of A.oryzae were constructed, the resultsshowed the three active sites were important to the the rNpI, and further was confirmed theprotease NpI as a gluzincin.Using isolated soybean proteins as substrate, the hydrolysis efficiency of the threerecombinant proteinases was studied. The results indicated （1） that the rApII of A.oryzae hadthe highest hydrolysis degree （15.8%） at45℃, pH10.0,1-1.5h and1000U/g;（2） the rApI ofA.niger had the highest hydrolysis degree （15.6%） at40℃, pH10.0,1h and1000U/g;（3） therNpI of A.oryzae had the highest hydrolysis degree （16.4%） at45℃, pH8.0,1-1.5h and1500U/g.