| Poly(β-hydroxybutyrate) (PHB) is a structurally simple macromolecule synthesized by many gram-positive and gram-negative bacteria as inclusion bodies. The polymer functions as both carbon and energy reserves, and usually accumulates under unbalanced growth conditions. The unique feature of PHB is that it is completely biodegradable, PHB has gained much attention as potential replacement polymer in the plastic industry. This is especially important since plastic-waste accumulation in landfills has become an environmental concern. An extracellular PHB depolymerase was separated and purified from the culture supernatant of a fungus which can degrade PHB efficiently. Investigate its enzyme and protein properties. The main results obtained from this work are as follows: 1,Using the fungus (Penicillium sp. DS9701-09a) determined its depolymerase activity every day. And it showed the most activity was in 96h. The PHB depolymerase was separated and purified by centrifugation, ultrafiltration, (NH4)2SO4 salting out and gel filtration chromatography. The specific activity of the purified enzyme was increased by 37.9 folds over crude extract, and the recovery yield was 11.81%. The SDS- polyacrylamide gel electrophoresis, high performance liquid chromatography (HPLC) and mass spectrometer had been used to test the protein. 2,Enzyme properties: The molecular mass of the purified enzyme was estimated to be about 44.8KDa by MALDI TOF Mass Spectrometer, and pI was 6.7 by two-dimensional electrophoresis. The kinetics of enzyme-catalyzed reactions Km was 0.128 mg/ml. The optimum activity of enzyme was observed at the temperature 50℃ and at pH 5.0. Some of the chemicals such as ethylene diaminetertracetic acid (EDTA), sodium do decyl sulfate (SDS) and mercaptoethanol inhibited the depolymerase activity strongly. 3,The protein was hydrolyzed at 110℃ by HCl, using Agilent Zorbax HPLC C18 chromatographic column to determine the amino acid composition. It contained 16 kinds of normal amino acids and did nothing with Trp. The N-terminal amino acid sequence of the purified enzyme was determined by using a sequence analyzer (model 491A; ABI) with the method of Edman's degradation. The first amino acid was alanine (Ala) or asparagine (Asn), the second was threonine (Thr) or valine (Val), the third was threonine (Thr). 4,The secondary structure of the protein was determined by circular dichroism(CD) spectrum. The α-Helix and β-Turn were 65.80% and 34.20% when without (NH4)2SO4. The α-Helix, β-Turn and Random were 7.90%, 29.30% and 62.80% with (NH4)2SO4. |
