【作者】 曾化伟；

【导师】 廖祥儒；

【作者基本信息】 江南大学 ， 发酵工程， 2011， 博士

【摘要】 过氧化氢酶（Catalase, CAT）通过催化一对电子的转移最终将H₂O₂分解为H2O和O2。其广泛应用于食品、纺织、造纸、环保等行业。因此,研究微生物发酵生产CAT具有一定的理论价值和潜在的应用前景。论文研究了产CAT菌株的筛选,经透射电镜观察、脂肪酸等生理生化观测和16S rRNA基因序列分析鉴定了一株高产CAT的细菌。考察了其发酵产酶条件,研究了发酵产酶调控方法、产酶生理、进行了酶的纯化与性质研究,克隆了该酶基因并进行了序列分析,取得了如下主要结果:分别采用H₂O₂富集生长法和H₂O₂起泡检测法,从37份土壤和污泥样品中,共筛选获得141株潜在的CAT高产菌。用H₂O₂起泡检测法从纺织厂漂洗车间含H₂O₂污泥中筛选获得一株CAT产量最高的菌株。在LB培养基平板培养48 h后,菌落为紫红色、圆形、光滑、湿润,表面隆起呈金属光泽,边缘整齐或不整齐,有轻微异味,色素不扩散到培养基中。该菌株有丙酮酸脱羧酶、赖氨酸脱羧酶,无精氨酸双水解酶。菌株能利用蔗糖、木糖、阿拉伯糖、乳糖、山梨醇,但不能利用L-鼠李糖、纤维二糖、蜜二糖。经透射电镜观察,这株菌能产圆形或棒形芽孢。主要的脂肪酸含量为C17:0 CYCLO （17.3 %）,C_16:0 （14.8 %）,C_10:03OH （10.0 %）,C_17:0 CYCLO （17.3 %）,C_14:0 （6.0 %）。经16S rRNA基因序列鉴定,将菌株命名为粘质沙雷菌（Serratia marcescens SYBC08）。研究还发现该菌株在碳源利用、主要脂肪酸含量等生理生化特征和其它的粘质沙雷菌有差异。该菌株CAT比活力较高,是生长于无H₂O₂环境S. marcescens SYBC-01的10.6倍。高的CAT比活力和产芽孢可能是其能够生长在H₂O₂胁迫环境的机制。通过对硫酸铵沉淀得到了粗CAT,进行主要应用酶学性质的研究表明,初步纯化的CAT在高温强碱和低温条件有一定的应用潜力。研究了S. marcescens SYBC08摇瓶发酵产CAT的最佳条件。以合成NADH主要途径TCA循环的中间产物柠檬酸为碳源发酵产CAT,较其它碳源提高了产量。推测CAT产量的提高与NADH的合成有密切的关系。再经响应面方法对发酵条件进一步优化。优化后的发酵条件:柠檬酸30 g/L、玉米浆33.8 g/L、初始pH 5.91、接种量4 % （v/v）、装液量50 mL/250 mL、转速250 r/min、温度32.8℃、培养36 h。酶产量从1470 U/mL提高到12165 U/mL,提高了7.27倍。在5 L小型发酵罐下,以摇瓶发酵的最佳培养基为发酵罐条件下的发酵培养基,研究了S. marcescens SYBC08产CAT的分批发酵条件。最佳的培养条件:通气量1.5 vvm、pH 7.0、转速400 r/min、相对溶氧控制在5 %、发酵时间22 h。在此条件下,CAT最高产量为22422 U/mL。进一步添加100 mg/L的烟酸以调控NADH的合成,CAT产量提高了15.1 %,达到26442 U/mL。分别以葡萄糖和柠檬酸为发酵碳源,研究了S. marcesces SYBC08产CAT过程的生理特征,分析了柠檬酸促进CAT产量的机制。CAT产量提高的过程中,AhpC活力及胞内VC含量提高较明显,MDA含量下降明显。发现两种碳源发酵的CAT产量均与抗氧化指标有显著或极显著的相关性,说明CAT产量的提高与氧化胁迫有关,柠檬酸为发酵碳源较葡萄糖为发酵碳源有更高的CAT产量,是增强了氧化胁迫。S. marcescens SYBC08的粗酶液经过硫酸铵沉淀、离子交换层析、凝胶过滤层析纯化得到纯的CAT。该过程的纯化倍数为13.8,回收率为22 %。CAT的分子量为230 kDa,由4个分子量为58 kDa同源亚基组成。该酶的比活力（199584 U/mg蛋白）是Halomonas sp. Sk1的3.44倍。该酶在405 nm和280 nm有吸收峰,A405/A280比值为0.42。K_m和V_max分别为78 mmol/L和188212 U/mg。1.0 mmol/L的Mg²⁺和Ca²⁺对CAT活力有显著的激活作用,Mn²⁺、EDTA和邻苯二酚对CAT活力有显著的抑制作用。CAT有较宽的pH最佳活力（pH 7.0-9.0）和pH稳定性范围（pH 5.0-11.0）。最适的温度大约为20℃,在0℃依然有78.5 %的活力,这个结果证明其为常温菌产的低温单功能CAT。经LC-MS/MS氨基酸序列分析,该酶与S. proteamaculans 568的CAT氨基酸序列（gi|157371515 ）最为匹配。由S. proteamaculans 568 CAT的基因序列设计引物并获得该CAT基因。该酶基因长度为1437 bp,Genbank上的登录号为HM068611。经该基因推导的氨基酸序列与相关的23个CAT比对并构建系统进化树。结果表明,虽然大部分具有重要功能的位点在24个相关的CAT中是相当保守,S. marcescens SYBC08 CAT推导的氨基酸序列残基（M53、S93、H341、H284和F194）在相关CAT中并不完全保守,说明不同CAT之间的催化行为有一定的多样性。该高比活CAT在肠杆菌科中具有最高的相似性,说明一些肠杆菌科微生物中的该类酶在抵御H₂O₂对细胞的伤害中发挥了重要的生理功能。该基因推导的氨基酸序列预测的二级、三级结构表明,二级结构中含的螺旋结构、折叠片状结构、环形及其它结构含量分别为27.8 %、16.7 %和55.4 %,预测的NORS区在343-423。预测的酶亚基三维结构与已知的1e93A（2.00 ?）有85.169 %的相似度。更多还原

【Abstract】 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 %）.更多还原