Heterologous Expression And Analysis Of Degradation Specificity Of Polyvinyl Alcohol Dehydrogenase From Bacillus Cereus. DG01

Polyvinyl alcohol（PVA）is a biodegradable polymer material that has been widely used in food,textile and biological medicine.However,it has caused serious environmental pollution problems due to its slow degradation under natural conditions and easy aggregation in water bodies.At present,most of the PVA degrading bacteria obtained in the study,or because of the difficulty of their screening,or their cultivation conditions,harsh degradation conditions,and low degradation efficiency,are not the best choice for digesting and treating PVA.In this study,the gene encoding the protein of polyvinyl alcohol dehydrogenase（PVADH）,a key enzyme in the degradation of PVA,was amplified by RT-PCR from Bacillus cereus.DG01 strain,which was newly selected by our previous group and has the ability to degrade PVA.The main findings obtained are as follows:1.The full-length 1965 bp PVADH encoding gene was amplified from Bacillus cereus.DG01 and processed to yield the c PVADH gene.The recombinant prokaryotic expression strain E.coli BL21（DE3）-p ET32a（+）-c PVADH was constructed.2.E.coli BL21（DE3）-p ET32a（+）-c PVADH was induced for 3h at 30℃with 1 mmol/L IPTG to express 84.2 k Da PVADH fusion protein.After cleavage by enterokinase r EK,the 67.1 k Da purified PVADH was obtained,and the specific activity of the enzyme was 32.90 U/mg.3.Construction of the P.pastoris expression plasmid p PIC9K p PVADH and obtaining a high copy of the recombinant strain P.pastoris GS115-p PIC9K-p PVADH.The recombinant strains were induced at 30°C with the addition of 1.0%methanol for 120 h,and the enzyme activity of the fermented supernatant was obtained to be 30.07 U/m L.4.Through factor optimization of fermentation process conditions of recombinant strains P. pastoris GS115-p PIC9K-p PVADH,the optimal fermentation conditions were determined to be 25.5℃with an initial concentration of 1.1%added dropwise to methanol,a final concentration of 3.3%with an initial concentration of OD₆₀₀ of 1.85,and an initial p H of 6.0 in culture medium PVADH enzyme activity in the fermented supernatant could reach 54.55 U/m L after 120 h incubation.After chromatography purification,the specific activity of the enzyme protein was 173.42 U/mg with a purification factor of 2.7.5.The K_m values of PVADH for the three substrates,PVA1799,PVA1788,as well as PVA2488,were 1.49±0.024 mg/m L,1.17±0.015 mg/m L,1.21±0.011 mg/m L respectively,which indicates that PVADH is more affinity for PVA with low alcoholysis degree.However,PVA is not sensitive to the degree of PVA affinity with different degrees of polymerization.According to the hydrophilicity analysis of the protein,it is speculated that PVADH is hydrophilic protein,more and accommodates a neutral weak alkaline environment,and easily degrades low alcohol solubility PVA which has good solubility and low acidity.