Isolation And Identification Of Serratia Marcescens Sybc08 And Its Fermentation For Catalase Production

Catalase (CAT) can decompose H₂O₂ to H2O and O2 by two-electron transfer. The enzyme is widely applied in the food, textiles, paper, environmental protection industries, etc. Therefore, the investigation of CAT production by microbial fermentation has theoretical value and potential applications.In the thesis, screen and identification of a high-CAT-producing bacterium by using transmission electron microscopy, physiological and biochemical analysis and 16S rRNA gene sequence analysis were carried out. Its conditions and control strategies for CAT production, physiological properties during CAT production, purification and properties, gene cloning and the deduced amino acid sequence analysis of the cloned CAT gene were investigated. The results shown as follows:Two methods (including H₂O₂ concentration growth method and H₂O₂ foaming test) were applied in screening high-CAT -producing strains from 37 soil and sludge samples. Only 37 strains were obtained by using H₂O₂ concentration growth method, and 104 strains were obtained by using H₂O₂ foaming test. A high-CAT-producing strain, which was isolated by using H₂O₂ foaming test from sludge containing hydrogen peroxide in a bleaching workshop of a textile factory, showed the highest CAT production(1,401 U/mL)among all strains examined, was identified as S. marcescens SYBC08 by 16S rRNA gene sequence analysis and electron microscopic observation. Physiological and biochemical characteristics, fatty acid content were not identical to those of S. marcescens and the spore-forming S. marcescens spp. sakuensis. Its activity was 10.6-fold higher than that of S. marcescens SYBC-01 in an H₂O₂-free environment (294 U/mg of protein). The relatively high CAT activity and the spore structures may enable the strain to survive in a hydrogen peroxide environment. Properties of the CAT after purification by ammonium sulfate precipitation were studied, these results suggest that the enzyme has potential applications in relatively high temperature and alkali, and low-temperature conditions.The medium and condition of liquid fermentation for the yield of CAT by S.marcescens SYBC08 were optimized with single factor experiment and response surface method. CAT yield obtained in the presence of citric acid was higher than that obtained in the presence of other carbon source, and speculates that CAT yield have relation with biosystenm of NADH. The optimized medium and conditions were as follows: 30 g/L citric acid,33.8 g/L corn steep liquor powder, initial pH5.91, liquid volume 50 mL/250 mL flask, 4 % inoculation, 32.8°C , 250 r/min, for 36 hours. Under these conditions, the CAT yield was increased from1, 470 U/mL to12, 165 U/mL and has increasement of 7.28-fold.The optimizated batch fermentation conditions of S. marcescens SYBC08 was studied in 5.0 L bioreactor with the opitmized medium. The optimized conditions were as follows: pH 7.0, temperature 32.8°C, agitation speed 400 r/min, aeration 1.5 vvm, relatively dissioved oxygen 5 % and fermentation time 22 h. When S. marcescens SYBC08 was cultured in the optimized conditions, the CAT production reached 22,422 U/mL.When regulation of NADH was carried out by addition of 100 mg/L nicotinic acid at the optimizaed condition, the CAT production increased 15.1 % and reached 26,442 U/mL.During fermentatal process for CAT production, the physiological metabolic characteriza- tion of S. marcescens SYBC08 obtained in the presence of glucose or citric acid was studied, and the mechanism of enhancing CAT production obtained in the presence of citric acid was analyzed. It was found that when the yield of CAT was enhaced in the fermental process, activity of AhpC and the content of VC were obviously increased, and the content of MDA was obviously deduced. CAT production was significant positively correlated with partial antioxidant indexs, indicating improve oxidative stress enhanced CAT production.The isolation and purification of CAT from the crude extract of S. marcescens SYBC08 was carried out by ammonium sulfate precipitation, ion exchange chromatography, and gel filtration, and a single band from ion exchange was revealed by SDS-PAGE. During those processes, the enzyme was purified 62.5-fold in a 32 % yield. The purified CAT had an estimated molecular mass of 230 kDa, consisting of four identical subunits of 58 kDa. High specific activity of the CAT(199,584 U/mg protein) was 3.44 times higher than that of Halomonas sp. Sk1 CAT (57,900 U/mg protein). The two maxima at 405 nm (Soret peak) and 280 nm (protein maximum) were obviously appeared, and A405/A280 of 0.42 was calculated. The enzyme without peroxidase activity was found to be a monofunctional CAT. The apparent K_m and V_max were 78 mmol/L and 188, 212 U/mg, respectivly.The enzyme was significent activated by 1.0 mmol/L Mg²⁺ and Ca²⁺. However, it was significent inhibited by Mn²⁺, EDTA and catechol. The enzyme displayed a broad pH activity range (pH 5.0–11.0), with optimal pH range of 7.0–9.0. It was most active at 20°C and had 78 % activity at 0°C. Its thermo stability was slightly higher compared to that of commercial CAT from bovine liver. The result suggested that the CAT is a cold-adapted enzyme from mesophilic bacterium.The peptide sequences from S. marcescens SYBC08 CAT have the highly match CAT sequences from S. proteamaculans 568 (gi|157371515 ) by LC–MS/MS analysis. A encoding gene was cloned by using two PCR primers which was designed according to matching gene sequence from S. proteamaculans 568 CAT under LC-MS/MS analysis, and Its gene sequence of 1437bp was deposited in the GenBank under the accession number (HM068611).The sequence was compared with that of 23 related CATs. Although most of important founctional residues in the enzyme were well conserved in 23 related CATs, but some other important residues (such as M53, S93, H341, H284 and F194) are weakly conserved. It might further supported some degree of specificity in their catalysis behaviors.Its sequence was closely related with that of CAT from pathogenic bacterium in the family Enterobacteriaceae. These results imply that the enzyme with high specific activity plays a significant role in preventing those microorganisms of the family Enterobacteriaceae against hydrogen peroxide resulted in cellular damage.The analysis of predicted secondary and tertiary structure indicated that the content of helical structure, bundle structure and ring structure are 27.8 %, 16.7 % and 55.4 %, NORS region is located in 343-423.The three-dimensional structure from S. marcescens SYBC08 showed the highest similarity to the known 1e93A (2.00 ?) (85.169 %).