Screening Of Poly (vinyl) Alcohol Degrading Bacteria And Secondary Alcohol Oxidase Metagenome

Poly (vinyl alcohol)(PVA), the only water soluble and non-toxic composite material, haslots of excellent physical properties such as high viscosity, film property, emulsifying propertyand resistance to water, grease and organic solvent, etc. Based on these characteristics, PVA iswidely applied to many industries like adhesive, coating, emulsifier, paper and textile etc.Nowadays, PVA pollution problem was more and more serious because of its deficiency inbiodegradation. Therefore, it is of great significance to carry out deep research on PVAdegrading bacteria and PVA degrading enzymes for reducing environmental pollution.In this work, a mixed PVA degradation culture SH-TD was isolated and the biodegradationcharacteristics of PVA by SH-TD were investigated. The production of PVA degrading enzymehas been significantly improved through the Response Surface Methodology (RSM) ofoptimization of culture medium. A function-based screening strategy was designed to screenthe gene of SAO from Fosmid and sub-cloning libraries. The main results are listed as follows:1. A PVA-degrading mixed culture was obtained from the activated sludge sample oftextile finishing operations. When0.1%PVA was used as a sole carbon source in culturemedium, it could be completely degraded in only4days. Three strains with PVA-degradingability were isolated from the mixed culture: SH1(Aeromonas sp.), TD5(Pseudomonas sp.),and TD33(Acinetobacter sp.). Over99%of PVA, at an initial concentration of0.1%, wasdegraded within220and160hours cultivation with the resting cells of TD5and TD33alone,respectively. However, SH1could only utilize low molecular weight PVA. The enzyme activityof SAO was only detectedin the fermentation supernatant of SH-TD.2. The initial fermentation culture medium consisting of PVA, Yeast Extract,(NH4)2HPO4,KH2PO4, K2HPO4, MgSO4·7H2O, FeSO4·7H2O, CaCl2·2H2O, NaCl, Phe, Trp and Thr, wasdetermined based on the single factor experiments about growth factors, nitrogen source andmetal ions. On the basis of Plackett-Burman experiments, three constituents from the startingculture medium were found to have significant influence on PVA degrading rate which wereYeast Extract,(NH4)2HPO4, BaCl2. Through ascent and Box-Behnken experiments, a RSMmodel was constructed. Data was analyzed using Design-Expert software. The optimalconcentrations (g·L-1) of the variables were determined as: Yeast Extract0.04g·L-1,(NH4)2HPO448.42g·L-1, BaCl20.06g·L-1. Under the optimal conditions, the PVA degradationrate after24h increased from30%to78.76%.3. By using metagenomic technology, a Fosmid library containing the whole genes of SH-TD was constructed. Among about150,000clones,42clones expressing the SAO activity wasobtained which were named FTD1-FTD42. They can form clear halos on the PVA-OPHscreening plate. The SAO enzyme activity of FTD2reached57mU mL-1. Several sub-clonelibraries whose genetic source was from the positive FTD strains were constructed and screenedas described.