Identification Of Paecilomyces Sp.DS1407and Characterization Of Its Extracellular Depolymerase

poly-3-hydroxybutyrate-co-4-hydroxybutyrate(P34HB) is the fourth generation product of the polyhydroxyalkanoates (PHAs) family.It has the best biodegradation and biocompatibility. Furthermore,as the second monomer successfully introduced, P34HB is very similar with Mechanics(PE)and polypropylene (PP) in mechanical property and it can be processed with traditional plastic equipment. so it has wide application prospect in industry,agriculture, medicine and other fields.This paper screened a biodegradable P34HB strain of fungal from nature environments. Through the18S rDNA identification, founding that the homology between the strain and Paecilomyces nostocoides was99%. Combining the observation of the colony characteristics and morphological, The strain was identified as a strain of Paecilomyces sp. DS1407.The optimum fermentation conditions for the production of P34HB depolymerase by strain DS1407were investigated by the single factor experiment and orthogonal test. Single factor optimization results show that the optimal fermentation conditions for temperature28℃, pH7.0, carbon content is0.25%(w/v), with fluid amount is75/250ml, training time is4days.Accoding to the single factor optimization results, the orthogonal test was designed in four factors and three levels.Finally the optimal conditions of enzyme production of strain DS1407were showed as follows:the temperature was28℃, pH was7.0, the carbon content was0.3%(w/v), the ventilation was50/250mL. The verification experiment showed enzyme activity increased by41%after optimization.P34HB depolymerase was purified by centrifugation, lyophilization, cation exchange chromatography and hydrophobic interaction chromatography,with relative molecular weight of about55kDa.By calculating the purification factor was8.86and the recovery rate was21.33%. The optimum reaction temperature and pH of the P34HB depolymerase were30℃and6.0, respectively.The enzyme was stable in a temperatue range of4-50℃and a pH range of4.0-7.0. The depolymerase activity was promoted by Mg2+,but inhibited by Ca2+, Zn2+, Fe2+,Fe3+, Cu2+,and Mn2+in different degree; Several organic solvents and inhibitors (EDTA, PMSF) also inhibited the activity. In terms of substrate specificity of the enzyme degradation, the enzyme degraded p-nitrophenyl esters with different carbon chain length,so it had esterase activity; The enzyme degraded other members of PHAs family,but not polycaprolactone (PCL) and polylactic acid (PLA). The purified P34HB depolymerase substrate catalytic efficiency was highest for C4. The main product of the P34HB depolymerase was hydroxbutyrate monomer (HB) via mass spectrometry detection,suggesting that PHB depolymerase acted as an exotype hydrose, releasing one monmer unit at a time. It had no difference in identifying ester bond of3HB-3HB,4HB-4HB and3HB-4HB.