Insoluble Active ELPs-xylanase Aggregates: From The Characteristics Tokinetic Properties In Batch Reactor

We fused the genes of elastin-like polypeptides (ELPs) and xylanase and thenexpressed them in Escherichia coli. Unexpectedly, the fusion proteins self-assembledinto insoluble active particles as the ELPs underwent a hardly reversible phasetransition. The specific activity of the particles was92%of the native counterparts,which means it can act as a pull-down handler for converting soluble proteins intoactive aggregates.Firstly,we evaluated the characterizations of the insoluble activeELPs-xylanaseaggregrates(IAEXA) in detail and the results were encouraging. The pH optimum (6.0)of IAEXA was the same as the free one, but the optimum pH range was5～7, whilethe free xylanase was6～7. The free xylanase had an optimum temperature of50℃,whereas the IAEXA shifted to70℃. The pH stability, thermostability and storagestability of the xylanase particles increased significantly when compared with the freexylanase. We also observed an increase of the Kmvalues of the free xylanase from0.374g L-1to0.980g L-1at the insoluble state. The considerable higher activity andstability of the xylanase particles were much like immobilized xylanases and could bevaluable for its industrial application.Secondly,we regard IAEXA as a immoblilized xylanase. The kinetic properties inbatch reactor wasinvestigated and the entire process was optimized. The resultsshowed that the value of KmandVmaxwas2.50g L-1and2.64×10-4mol L-1min-1,respectively.The maximum reaction rate constant K+2was0.475min-1by using Enzyme Kinetics Module softwar.No substrate inhibitionphenomena was observed,but the product inhibition constant Kpwas0.09g L-1.Thewhole process of the optimization showed that the objective function for eachidentified DH was only related to the initial enzyme concentration and substrateconcentration. In theory, the highest amount of IAEXA that optimal process wasobtained, but the cost of hydrolytic enzyme and time of the whole process need to beconsidered in the practical production.The optimalconcentrations of substrate wasobtainedby the the function diagram of h(S0) verus S0.Finally,we investigated IAEXA stability under different salt and bufferconditions, the results showed that IAEXA was relatively stable under different saltsand buffer conditions, The activities of the fusion proteins in IAEXA were in the range of78.01～97.32%. Which was83%to97%in different buffers. Then IAEXAwas dealed with ultrasonic and high pressure homogenization,the activity of IAEXAwas almost all restored to the soluble state, but also it was more stable after dealingwith1000bar high pressure homogenization process and the average size was thesmallest. The fourier transform infrared spectrum showed IAEXA contained β-sheetstructure, random coil structure and α-helical structure,which indicated thatsomesecondary bondsbetween ELPs and xylanase might leadtothe formation of IAEXA.