恶臭假单胞菌NA_1菌株烟酸羟基化酶活性的诱导和转化条件的研究

恶臭假单胞菌NA_1菌株的培养和产酶特性与已报道的产酶菌株粘质沙雷氏菌(Serratia marcescens) IFO 12648和荧光假单胞菌(Psudomonas fluorescens) TN5有所不同, 主要反映在最适碳源及浓度、最适诱导剂浓度和最适培养温度等方面。最适的转化条件是温度为30℃,pH为7.0, 烟酸的浓度为3％。采用初步优化后的条件和流加底物的方式进行4L上罐生产,恶臭假单胞菌NA_1菌株的6_羟基烟酸产率可达到108.39g/L。     

恶臭假单胞菌NA-1菌体培养转化和静息细胞转化联合工艺生产6-羟基烟酸研究

现有微生物羟基化烟酸采用的是静息细胞转化工艺。但研究揭示,恶臭假单胞菌NA-1(Pseudomonas putidaNA-1)在培养过程中不降解发酵液中由诱导剂烟酸转化形成的6-羟基烟酸,这是由于烟酸的存在抑制了羟基烟酸降解酶的作用,而不是因为细胞停止生长不利用羟基烟酸的缘故。因而尝试利用菌体诱导培养过程进行烟酸转化生产,建立了一种新的生产工艺,即菌体培养转化和静息细胞转化联合工艺。该工艺在恶臭假单胞菌NA-1培养过程中持续补充烟酸以维持1%(W/V)浓度,使烟酸被生长细胞转化为羟基化烟酸并在发酵液中线性积累,而不被进一步降解;培养转化结束后,发酵液中的静息细胞依然拥有很高的羟基化酶活力,能够再次用于转化反应。该联合转化工艺与传统的静息细胞转化工艺相比,不仅节约了诱导剂烟酸,而且6-羟基烟酸的产量提高了65%。     

一株烟酸羟基化转化菌株的筛选和鉴定

从南京地区的土壤中筛选到一株高效转化烟酸为 6_羟基烟酸的菌株NA_1。形态及生理生化特征测定结果表明 ,NA_1菌株与假单胞菌属 (Pseudomonas)中的恶臭假单胞菌 (P .putida)种的特征基本一致。测定了该菌株的16SrDNA序列并根据 16SrDNA构建了系统发育树 ;在系统发育树中 ,NA_1菌株与恶臭假单胞菌形成一个类群 ,序列同源性为 99%。因此将NA_1菌株鉴定为恶臭假单胞菌     

产芳香腈水解酶的恶臭假单胞菌Pseudomonas putida CGMCC3830的筛选、鉴定及发酵优化(英文)

近年来微生物腈水解酶水解腈类化合物制备有机酸已逐步受到关注。本研究分离到一株表现出较高腈水解酶活力的细菌菌株,通过形态学、生理生化实验以及16S rRNA基因序列分析将其鉴定为恶臭假单胞菌Pseudomonas putida CGMCC3830。结合单因素及响应面法对该菌株产腈水解酶的发酵条件进行了优化,获得最适培养条件为:甘油13.54 g/L,胰蛋白胨11.59 g/L,酵母粉5.21 g/L,KH2PO4 1 g/L,NaCl 1 g/L,脲1 g/L,初始pH 6.0及培养温度30℃。通过优化,酶活由2.02 U/mL提升至36.12 U/mL。对该菌株底物特异性的考察结果表明,恶臭假单胞菌腈水解酶对芳香族腈类化合物具有较高的水解活力。将其应用于烟酸的生物合成中,2 mg/mL游离细胞能90 min内将20.8 g/L 3-氰基吡啶彻底转化,制备得到相应烟酸。这些结果表明恶臭假单胞菌P.putida CGMCC3830在烟酸的规模化生产中具有一定的应用潜力。     

腈水解酶psn基因工程菌的发酵及产酶条件优化

来自恶臭假单胞菌的腈水解酶具有高效催化3-氰基吡啶产烟酸的能力,对表达该酶的基因psn进行发酵和产酶条件优化,通过对C源、N源、磷酸盐、金属离子、温度、诱导剂浓度和诱导时间进行单因素考察,获得最适培养基条件(g/L):葡萄糖5、蛋白胨15、酵母粉5、(NH4)2SO45、K2HPO424.5、KH2PO45.76、MgSO40.48;最佳诱导条件:培养2.5 h后添加IPTG诱导,浓度0.2 mmol/L,诱导温度30℃。在该条件下培养,重组大肠杆菌的腈水解酶比酶活可达到45.67 U/mL,比优化前提高了2.26倍。在此基础上,于5 L发酵罐上进行C、N源的补料研究,获得最适分批补料策略,发现其腈水解酶活力可达到75.40 U/mL,是优化前的3.74倍。     

铜绿假单胞菌产蛋白酶的发酵条件优化

【目的】鉴定一株来源于酱油曲能够分泌蛋白酶的铜绿假单胞菌CAU342A,优化其产蛋白酶的发酵条件。【方法】采用形态学观察、16S r RNA基因序列比对和生理生化方法鉴定菌株CAU342A;通过碳源、氮源、初始pH、温度、表面活性剂及发酵时间的单因素优化和正交试验获得最适发酵条件。【结果】菌株CAU342A被鉴定为铜绿假单胞菌(Pseudomonas aeruginosa),其最适发酵产酶条件为(质量体积比):3%酒糟,1.5%酵母浸提物,0.05%吐温-80,0.5%NaCl,0.7%K_2HPO_4,0.3%KH_2PO_4,0.04%MnSO_4,培养基初始pH 7.5,30°C培养72 h。在最适发酵条件下,该菌株最大产酶水平达到2 653.5 U/m L。蛋白酶酶谱分析表明该菌株能够产生至少4种具有蛋白酶活性的同工酶,其中两个主要酶谱带对应分子量分别为32 k D和50 k D。【结论】铜绿假单胞菌CAU342A高产蛋白酶,具有很大的工业应用潜力。     

交替假单胞菌LP621菌株产右旋糖苷酶的培养条件优化

从江苏连云港海域分离和筛选到1株产右旋糖苷酶的海洋细菌交替假单胞菌Pseudoalteromonas tetraodonis LP621,通过单因素试验和正交试验对该菌株产右旋糖苷酶培养条件进行优化。单因素试验结果表明,最佳培养时间为24 h,最适产酶温度为25℃;产酶pH范围为5.0~11.0,最适产酶pH为6.0;产酶NaCl浓度范围为1%~10%,NaCl浓度为4%时产酶较高;装液量在25%。麦芽糖、胰蛋白胨和酵母膏促进产酶。利用响应面方法对LP621产右旋糖苷酶的发酵条件进行优化。选择培养基pH、时间、麦芽糖浓度和装液量4因素进行优化,结果为pH 7.07,发酵时间21.94 h,麦芽糖浓度0.42%,装液量为21.88%,酶活为270.1U/mL。     

睾酮丛毛单胞菌JA1菌株羟基化烟酸产生6-羟基烟酸的发酵产酶条件研究

睾酮丛毛单胞菌Comamonas testosteroni JA1是一株具有氰基吡啶羟基化酶活性的菌株,研究表明它还具有很高的烟酸羟基化酶的活性,是未报道过的具有烟酸羟基化酶活性的新菌种。该菌最适生长和产酶的碳源为1%葡萄糖、氮源为1%蛋白胨、诱导物为1%烟酸,此外,它的发酵条件优越,在温度25~37℃、初始pH6.5~7.0、装液量(100mL锥形瓶)10mL~40mL范围内,产酶能力均保持很高水平,很具有工业化生产应用的前景。     

假单胞菌产生铁氧化酶条件的研究

以氧化铁锰假单胞菌FEl3-26为实验菌株,对其铁氧化酶的产酶条件进行研究,结果表明柠檬酸铁铵和硝酸钠分别是该菌产酶的最佳碳氮源.最适产酶条件为温度28～30℃.起始pH值6.5～7.5,接种量2%,150 r/min振荡培养72 h.酶的定位研究结果显示该菌产生的铁氧化酶是一种胞外酶.     

产脂肪酶菌株C7828-5的筛选、鉴定以及产酶条件的优化

以花生油为唯一碳源,从海口市各地被油脂污染土样中分离筛选出1株中温碱性脂肪酶菌株C7828-5。形态学、生理生化特征和分子生物学鉴定结果表明,该菌株为铜绿假单胞菌(Pseudomonas aeruginosa)。该菌所产脂肪酶的最适温度为37℃,最适pH为8.0。优化了菌株的产酶条件,最适产酶培养基(g/L)为:蔗糖5、牛肉膏20、(NH_4)_2SO_41、MgSO_4·7H_2O 0.5、CaCl_20.5,聚乙烯醇花生油乳化液120 mL,发酵72 h,获得高达8.08 U/mL的脂肪酶表达量。     

Molybdenum-dependent degradation of nicotinic acid by Bacillus sp. DSM 2923

Abstract Growth of Bacillus sp. DSM 2923 on nicotinic acid in mineral medium was dependent on the concentration of sodium molybdate added. Addition of increasing amounts of tungstate to the medium resulted in an inhibition of growth on nicotinic acid or 6-hydroxynicotinic acid as sole source of carbon and energy. Chlorate-resistant mutants were isolated which were not able to degrade nicotinic acid and 6-hydroxynicotinic acid nor to reduce nitrate. Additionally, enzyme activities of nicotinic acid dehydrogenase and 6-hydroxynicotinic acid dehydrogenase increased with increasing concentrations of molybdate (10⁻⁸ to 10⁻⁶ M) added to the medium, and decreased with increasing amounts of tungstate (10⁻⁶ to 10⁻⁵ M) in the medium.     

基于微孔板的高通量筛选6-羟基烟酸转化菌方法的建立

建立了一种基于96孔板-酶标仪的双波长紫外分光光度法高通量筛选6-羟基烟酸转化菌的方法.实验以251nm为测定波长、231nm为参比波长测定转化样品的6-羟基烟酸含量,6-羟基烟酸与△A251-231在0.5～11 μg/mL浓度范围内有良好的线性关系,服从朗伯-比尔定律,平均回收率为99.11%～100.81%.利用96孔板-酶标仪,每天筛选量可达到2000～5000个反应,达到高通量筛选要求.     

Studies on the production of enantioselective nitrilase in a stirred tank bioreactor by Pseudomonas putida MTCC 5110

Nitrilases constitute an important class of hydrolases, having numerous industrial applications. The present work aims to address the production of nitrile hydrolyzing enzymes from Pseudomonas putida MTCC 5110 in a 6l bioreactor. Effect of various physico-chemical conditions and process parameters like pH, temperature, aeration and agitation rates and inducer concentration was studied. Further, the enzyme activity was enhanced by adopting the inducer feeding strategy. Various biochemical engineering parameters pertaining to the cultivation of P. putida in different physico-chemical conditions were reported. Finally, segregation of growth phase from the enzyme production phase allowed significant reduction in total fermentation time.     

Efficient production of D-glucosaminic acid from D-glucosamine by Pseudomonas putida GNA5

D-glucosaminic acid was produced efficiently from glucosamine by oxidative fermentation using a newly isolated strain, Pseudomonas putida GNA5. After optimization of the fermentation process, 51.5 g L(-1) D-glucosaminic acid was produced from an initial concentration of 60 g L(-1) D-glucosamine-HCl after 72 h of oxidative fermentation, which corresponded to a molar yield of 95.4%. This production process is potentially of considerable economic significance because very few by-products were detected. Furthermore, D-glucosaminic acid was accumulated stably during the oxidative fermentation process without the addition of an inhibitor of D-glucosaminic acid breakdown, even though D-glucosamine was exhausted. These results suggest that the mechanisms of D-glucosaminic acid-related metabolism differ between Pseudomonas putida GNA5 and the strain Pseudomonas genera, which was previously reported to produce D-glucosaminic acid.     

Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase

Enzymatic production of L-tryptophan from DL-serine and indole by a coupled reaction of tryptophan synthase and amino acid racemase was studied. The tryptophan synthase (EC 4.2.1.20) of Escherichia coli catalyzed beta-substitution reaction of L-serine into L-tryptophan and the amino acid racemase (EC 5.1.1.10) of Pseudomonas putida catalyzed the racemization of D-serine simultaneously in one reactor. Under optimal conditions established for L-tryptophan production, a large-scale production of L-tryptophan was carried out in a 200-liter reactor using intact cells of E. coli and P. putida. After 24 h of incubation with intermittent indole feeding, 110 g liter-1 of L-tryptophan was formed in molar yields of 91 and 100% for added DL-serine and indole, respectively. Continuous production of L-tryptophan was also carried out using immobilized cells of E. coli and P. putida. The maximum concentration of L-tryptophan formed was 5.2 g liter-1 (99% molar yield for indole), and the concentration decreased to 4.2 g liter-1 after continuous operation for 20 days.     

Identification of the prosthetic group of urocanase. The mode of its reaction with sodium borohydride and of its photochemical reactivation.

Urocanase from Pseudomonas putida and from beef liver were isolated by modifying described procedures. Both enzymes were inactivated and labeled on treatment with tritiated sodium borohydride and gave, upon subsequent hydrolysis, a radioactive acid. The previously reported identity of this acid as 2-hydroxybutanoic acid was disproved by several criteria. Other hydroxy acids were also proved to be different from the radioactive acid derived from urocanase. A large portion of the radioactive material from P. putida was found to be nicotinic acid by 1H NMR spectroscopy, gas-liquid chromatography of its methyl ester, and co-crystallization with authentic reference compounds both as the acid and as the hydrazide. A significant portion of the radioactive material derived from beef liver urocanase also co-crystallized with nicotinic acid. Sodium borohydride-treated inactive urocanase was partially reactivated by light. The action spectrum of the photoreactivation showed a maximum at 330 nm. Treatment of urocanase with sodium borodeuteride followed by hydrolysis afforded a sample of nicotinic acid which carried deuterium mainly in position 6. Both the reversible reducibility of urocanase and its action spectrum of photoreactivation suggest that urocanase contains an enzyme-bound nicotinamide nucleotide molecule which is essential for enzymic activity.