| In this study the work focused on three points:study on adsorption and hydrolysis reaction of cellulase during cellulose hydrolysis process; directed evolution of cellulase;isolation of novel cellulase from environment.First,study on adsorption and hydrolysis of cellulase during hydrolysis process.Protein adsorption onto solid substrates usually takes place in an irreversible fashion and this irreversible adsorption also occurs in some enzymatic reactions.In fact,the adsorption and hydrolysis of cellulose by cellulases occurred at the solid-liquid interface in the biphasic system on account of the insolublecellulose in liquid.In this study the adsorption characteristic of cellobiohydrolaseâ… onto cellulose was monitored by surface plasmon response.The results show that once cellulase adsorbes onto cellulose,it's desorption is not easy and partially irrevisible adsorption can be observed.Taking into account the irreversible adsorption,the developed model properly exhibits the actual adsorption behavior of cellulase.Several simulated parameters in the equation such as Kads(0.26L·cm-2·s-1)and Ktra 0.41 (s-1)were given.To evaluate the influence of adsorption on cellulose enzymatic hydrolysis,the reaction dynamics on pure cellulose were determined.A plot of the hydrolysis rate against the surface density of irreversibly adsorbed cellobiohydrolaseâ… ,revealed an inverse relationship in which an apparent decrease in the hydrolysis rate was observed with increasing surface density.Taken together,results presented here should be useful for modifying the binding characteristics of CBHâ… and making them more effective in cellulose hydrolysis.Second,directed evolution of cellulase.To test whether the phage display technology could be applied in cellulase engineering,the phagemids harboring the genes encoding the mature forms of CBHâ… and endoglucanaseâ… from filamentous fungus Trichoderma reesei, respectively,were constructed.CBHâ… and EGâ… fused to the phage coat protein encoded by the g3 gene were displayed on phage M13.The phage-bound cellulases retained their activities as determined by hydrolysis of the corresponding substrates.Also,their binding abilities to insoluble cellulose substrate were confirmed by an ELISA method. Overall,these results demonstrate that cellulases can be displayed on phage surface while maintaining their biological function,implicating phage display to be a potentially useful technology platform for directed evolution of improved cellulases.The cbhâ… DNA can be properly obtained with the addition of 3% DMSO in the random mutant PCR system.The mutant frequency of PCR production per Kb fell into 4.5～5.1/Kb,which is optimum to make the corresponding mutant protein have a variety with several amnion acids, was controlled by optimizing parameters of random mutant PCR.The optimal molar ratio of DNA and vector is 5 to 1 used in the ligation system.Transformation efficiency of 3×108～9/μg supercoiled pUCmt- 18 with E.coli TG1 can be obtained,when electrocompetent cell prepared by developed KOB medium.The primary library size of CBHâ… and EGâ… approximately reached 106 members,respectively,when used the above mentioned optimal mutant PCR system,molar ratio in ligation and elcetrocomepent TG1.Subsequently the phage libraries were subjected to denaturing conditions to exert selection pressure on protein stability before panning in the primary library treated by low concentration of denatured reagent GdnHC1.Third,isolation of novel cellulases from environment.A bacterial strain,ZY-1,which was isolated from acid stream of gold mine in Zhao Yuan city,was subjected to a polyphasic taxonomic study using phenotypic characterization and phylogenetic and genetic methods. Phylogenetic analysis based on 16S rDNA sequences showed that strain ZY-1 forms an evolutionary lineage within the radiation enclosing Acidiphilium species and,in particular,a coherent cluster with Acidiphilium cryptum and Acidiphilium multivorum.Strain ZY-1 had a cellular fatty acid profile containing straight-chain,branched, unsaturated and 9-cyclo fatty acids.The major fatty acid was saturated fatty acid.Moderate amounts of branched-chain fatty acids(i16:0)and fatty acid with a cyclopropane(19:0 cycloω9c)were present. Importantly,the unsaturated fatty acid(18:1ω9t)existed substantially with lower amounts.The 16S rDNA of strain ZY-1 has a high similarity to that of the relative strains of A.cryptum and A.multivorum.However, there exist obvious differences in physiological characteristics and morphological distinctiveness.The range of growth temperature of strain ZY-1 was from 28 to 42℃,which reveals that the strain is a mesophilic bacterium,and the optimal growth temperature is around 37℃.Strain ZY-1 is an acidophile growing optimally at approx,pH 2～4 and the optimum growth pH value is around 3.0.Colonies of ZY-1 are smooth,shiny,convex,and white and have a maximum diameter of 1 mm.The cells display short rods and Gram-negative.The organisms grew heterotrophically under aerobic conditions and they did use a broad range of organic substrate as the sole carbonsource.Phenotypic distinctiveness was shown when strain ZY-1 was cultured in medium with various nutrients as the sole carbon source.The surface of the strain ZY-1 cells cultured with glucose as the sole carbon source was smooth observed by transmission electron microscope,while protrusions can be observed on the cell surface when cured with the sole carbon source of cellulose.This is the first report that the strain in Acidiphilium genus has the ability to use of cellulose.Importantly, these appendixes of strain ZY-1 cultured in cellulose medium were similar to the feature of cellosome.In addition,a fungal strain named ZY-E1,which can digest cellulose,was isolated and identified.Strain ZY-E1 belongs to Aspergillus fumigatus based on the phenotypic characteristic and the sequence of 18S rDNA.The bacterial and the fungal strains were isolated which also displayed the diversity of microbe community inhabiting mostly acid environment.
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