Directed Expression Of Marine Aspergillus Niger Cellulases And Their Mechanisms Of Salt-tolerance And Thermostability

Cellulase plays an important role in utilization of lignocellulosic biomass,and development of cellulase with excellent properties can greatly promote the utilization efficiency.Aspergillus niger ZJUBE-1,which was screened and isolated from the sludge of the East China Sea,has a characteristic of salt-tolerant growth,and its cellulase cocktail possesses a property of salt-tolerance.Salt-tolerant cellulase has potential application in treatment of pulping wastewater,utilization of marine cellulose resources and improvement of saline-alkali soil.In this study,the marine A niger ZJUBE-1 was studied to mine the single cellulase components with salt-tolerant property from its cellulase cocktail.Firstly,the target cellulase components were directly expressed by introducing the separation factor,i.e.,homologous constitutive expression,in upstream process,which was convenient for separation and purification of cellulases in downstream process.Then the efficient obtainment of single cellulase components was achieved.Secondly,the mechanisms on special properties of the purified cellulase components were elucidated by analysis on amino acid composition,key residues and molecular dynamics simulation.The main results are as follows:(1)The genetic manipulation method on Agrobacterium tumefaciens-mediated transformation(AMT)of marine A.niger was established and optimized.Some recombinant marine A.niger constitutively expressing inherent cellobiohydrolases(Cel6,Cel7A and Cel7B),endoglucanase(EGL)and ?-glucosidases(BGL1 and BGL2)were constructed with AMT.Homologous constitutive expression was introduced in upstream process as a separation factor for efficient obtainment of single cellulase components.The recombinant with high expression of BGL1 was employed for optimization of medium compositions and culture conditions in homologous constitutive expression.As a result,the concentration of the target cellulase components in fermentation broth was distinctly increased,and the concentration of the heteroproteins was further decreased.When the fermentation was carried out without cellulase inducer,the constitutively expressed cellulases were predominant in fermentation broth.Subsequently,single cellulase components with high purity were obtained by anion exchange chromatography,demonstrating the feasibility on efficient obtainment of single cellulase components from cellulase cocktail by introducing the separation factor in upstream process.(2)Study on the enzymatic properties of the six purified cellulases(Cel6,Cel7A,Ce17B,EGL,BGL1 and BGL2)revealed that Ce17B exhibited an outstanding thermostability at pH 5.0 or higher,which was comparable with the thermostable cellulases from thermophiles.Besides,EGL exhibited a salt-tolerant property,and its activity with 4.5 M NaCl was increased by 29%compared with that in the absence of NaCl.Moreover,its thermostability was significantly enhanced in high salt concentration.With the increase of NaCl concentration,its half-life of enzyme activity at 65? increased from 20 min to 180 min.In addition,BGL2 also exhibited a salt-tolerant property,and its activity with 4 M NaCl was 40%higher than that in the absence of NaCl.(3)The mechanisms of salt-tolerance and thermostability were proposed by means of static analysis(amino acid composition and residues involved in substrate binding and catalysis)and dynamic analysis(molecular dynamics simulation)on the salt-tolerant and thermostable cellulases.Among them,Ce17B has a higher proportion of charged residues,which can form more salt bridges at high temperature.As a result,drastic changes in protein structure are avoided owing to the complicated salt bridge network and then Ce17B is imparted with the thermostable property.At room temperature,the salt ions interact with the loops of EGL,which increases the activity of the loops.It is speculated that the more active loops promote the binding between enzyme and substrate,thereby imparting EGL with the salt-tolerance.At high temperature,a type of salt bridges,-COO-…Na+...-OOC-,are formed between Na+and substrate binding pocket.The high salt concnetration provides the concentration difference that prevents Na+from escaping the pocket,thereby keeping the pocket from further expanding because of the excessive negative charges.Besides,the electrostatic shielding resulted from high salt concentration makes the force on loops more simple,then enhancing the stability of the loops.Stability improvement of pocket and loops in high salt concentration confers EGL on the distinct thermostability.The excess Glu492 in the substrate binding pocket of BGL2 and the negatively charged residues on the loops effectively attenuate the electrostatic shielding resulted from high salt concentration.The salt bridges formed between Na+and loops make the loops more stable.The weakened electrostatic shielding and the relatively stable loops impart BGL2 with a salt-tolerant property.(4)The cellulase production performance of the six recombinant marine A.niger was studied under inductive condition.After overexpression of the cellobiohydrolases,Ce16,Ce17A and Ce17B,the total cellulase activity of recombinants increased by 80%,70%and 63%,respectively,compared with that of the original strain.After overexpression of the endoglucanase,EGL,the total cellulase activity increased by 68%.However,after overexpression of the ?-glucosidases,BGL1 and BGL2,the total cellulase activity decreased.Besides,by mixing ce16,ce17a and ce17b recombinants with egl recombinant,the total cellulase activity of the mixed strains was increased by 114%,102%and 91%,respectively,compared with that of the original strain.These results revealed that cellobiohydrolase and endoglucanase were the key enzyme conponents for the improvement of total cellulase activity.Moreover,by adding salt-tolerant cellulase components into the original strain cellulase cocktail,the salt-tolerance of mixed cellulases were enhanced,which proved that the salt-tolerant property of marine A.niger cellulase cocktail was closely related to its salt-tolerant cellulase components.