Study On Isolation Of Bacterial Strain, Cloning And Expression The Key Enzymes In Degration Of Formaldehyde And The Characteristics Of Immobilized Cells

As a native toxin, formaldehyde has strong toxic effect on the organism and is one of the main pollutants of indoor air pollution. How to control formaldehyde pollution in the air has caused great concern, and degradation of formaldehyde by microorganism will be an effective way for the removal of formaldehyde.The aim of the research was to isolate a strain which can metabolize formaldehyde, then investigate the key enzymes in degradation of formaldehyde and the characteristics of immobilized cells by gel beads. The research provided a basic study for constructing a high tolerance and efficient engineering bacteria for formaldehyde degradation and the application for biological treatment technology about the indoor formaldehyde pollution.One bacterial strain which was capable to use formaldehyde as a sole carbon source was isolated from soil. Based on morphological, physiological, biochemical properties and 16S rDNA homology analysis, the strain was characterized as Methylobacterium and named as Methylobacterium sp. XJLW. The concentration of formaldehyde-tolerant by the strain was 0.1g/L in the initial acclimation period, and the strain grown slowly in liquid medium. The concentration of formaldehyde-tolerant of Pseudomonas putida xyz-zjut which was as isolated by our laboratory up to 6 g/L, and the degradation rate of formaldehyde was up to 0.114 g/L·h.This article has selected Pseudomonas putida xyz-zjut as the strain to study the key enzymes in degradation of formaldehyde, which was isolated by our laboratory and also proved to effectively degrade formaldehyde. Two pairs of specific primers was designed to amplify serine hydroxymethyltransferase (SHMT) and formaldehyde dehydrogenase (FDH) genes from Pseudomonas putida xyz-zjut, according to the genome of the strain from GenBank. The result showed serine hydroxymethyltransferase gene obtained from Pseudomonas putida xyz-zjut was 1254 bp in size and encoded 417 amino acids, and formaldehyde dehydrogenase gene was 1206 bp in size and encoded 401 amino acds. The DNA fragments had high homology with the key enzyme genes form Pseudomonas putida F1 and Pseudomonas putida KT2440. The engineering strain E. coli BL21(DE3)with recombinant vector pET-28b-fdh was obtained by introducing formaldehyde dehydrogenase gene into the expression vector pET-28b. The results from PCR, restriction enzyme digestion and SDS-PAGE analysis revealed that the recombinant expression vector pET-28b-fdh had been constructed and expressed in the host of E. coli BL21(DE3) successfully. Unfortunately, the efficiency of formaldehyde degradation of engineering strain was improved insignificantly. It was speculated that the target protein present in the inclusion body in the host, and does not have the activity of formaldehyde dehydrogenase.Using alginate as immobilized supporter, the immobilized cells of Pseudomonas putida xyz-zjut could degrade formaldehyde effectively. The optimum conditions that the immobilized cells metabolize formaldehyde was: 1.0 10⁸ cells/mL, MgSO₄·7H₂O 0.2 g/L,(NH₄)2SO₄ 2.4g/L,trace elements 0.1 mL/L, temperature 30℃, adjust pH to 9. Under these conditions, the effect of formaldehyde degradation was 0.107 g/L·h. The immobilized cells could applied to degradation of formaldehyde in bioreactor, and lay a foundation for the further research of application of biological formaldehyde degradation.