Isolation and characterization of a phytase with improved properties from Citrobacter braakii

Citrobacter braakii YH-15 produced an intracellular phytase which was purified 12800 fold to homogeneity with the specific activity of 3457 units mg^–1, which is 1.9 times higher than E. coli phytase previously recorded as having the highest specific activity. Its molecular weight was 47 kDa by SDS-PAGE gel. Enzyme activity was optimal at pH 4 and at 50 °C. The K _m value for sodium phytate was 0.46 mM with a V _max 6027 U mg^–1. The phytase was resistant to proteases such as trypsin, pepsin, papain, pancreatin, and elastase.     

海水养殖生境中柠檬酸杆菌降解半胱氨酸产生硫化物的机制研究

【目的】含硫氨基酸的生物降解是造成海水养殖环境中毒性硫化物上升的重要因素,而微生物降解含硫氨基酸的机制和影响因素解析是控制该系统中硫化物浓度的关键环节。【方法】本研究利用稀释涂布-叠皿夹法自本实验室海水养殖环境的沉积物中分离得到一株产生硫化物的厌氧菌株,并通过代谢组学研究其以半胱氨酸为底物产生硫化物的机制和途径。【结果】经鉴定,该菌株为弗氏柠檬酸杆菌(Citrobacterfreundii),能够在厌氧条件下还原硫酸盐,能够降解半胱氨酸产生硫化物,投加L-半胱氨酸可提高其还原硫酸盐的能力。该菌株以1g/L半胱氨酸为底物,在35℃、盐度为10、pH 8.0的条件下,硫化物的最高积累量可达302.4 mg/L。对菌株中硫化物产生具有重要贡献的半胱氨酸脱巯基酶研究表明,该酶最适温度为35℃,在pH6.0–8.0有较高的活性,能够快速降解半胱氨酸产生硫化物。结合代谢组学研究发现,该菌株中含有的3-巯基丙酮酸硫酸转移酶、胱硫醚-γ-裂解酶、半胱氨酸脱巯基酶催化半胱氨酸降解是产生硫化氢的主要途径;亚硫酸盐还原酶还原硫酸盐、亚硫酸盐是其产生硫化氢的次要途径。【结论】通过揭示柠檬酸杆菌降解半胱氨酸产生...     

The bacterial flora of the midgut of two Danish populations of healthy fifth instar larvae of the turnip moth, Scotia segetum

Examination of the midgut bacteria of two Danish populations of healthy fifth instar turnip moth larvae, Scotia (=Agrotis) segetum, living on potatoes and celery gave the following results. The total number of living microorganisms in the midgut varied between 1.0 × 10⁴ and 4.0 × 10⁵. Larvae from celery in N. W. Zeeland always contained Streptococcus faecalis and six members of Enterobacteriaceae, viz., Citrobacter freundii, Klebsiella pneumoniae, Hafnia alvei, Proteus mirabilis, P. vulgaris, and Erwinia amylovora. In larvae from potatoes in E. Jutland, the species consistently present were Streptococcus faecalis and four species of Enterobacteriaceae, viz., Escherichia coli, Erwinia amylovora, E. carotovora var. atroseptica, and one other, probably a member of the E. carotovora group. Streptococcus faecalis is supposed to occur as a mutualist in the alimentary tract, suppressing Gram-positive bacteria.     

Development of a process for biodetoxification of metal cyanides from waste waters

A bacterial consortium capable of utilising metal cyanides as a source of nitrogen was used to develop a microbiological process for the detoxification of metal cyanides (viz. copper cyanide and zinc cyanide) from electroplating waste water. Optimal conditions biodegradation of both the metal-cyanide compounds were pH 7.5, temperature 35°C, inoculum size 10⁹ cells per ml and glucose or sugarcane molasses requirement of 5 mM or 0.6 ml/l, respectively. Metal precipitates obtained during metal-cyanide biodegradation were identified as metal-hydroxides. When the treatment was carried out in a 27 l rotating biological contactor (RBC) in continuous mode, the system could achieve >99.9% removal of 0.5 mM metal cyanide (ca. 52 mg/l cyanide and 30–40 mg/l copper/zinc) in 15 h with sugarcane molasses as carbon source. The RBC treated effluent was found to be safe for discharge in the environment as confirmed by chemical analysis and fish bioassay studies.     

Acetylene reduction (N2-fixation) by Enterobacteriaceae isolated from industrial wastewaters and biological treatment systems

Summary Acetylene reducing (N₂-fixing) Entero-bacteriaceae have been isolated from activated sludge plants treating waste from the paper and food industries (10³ to 10⁶ cells per ml) and from composting plants handling forest waste (10⁵ to 10⁶ cells per g wet weight). Detailed studies on se-lected strains of all taxa showed that: (1) pure cul-tures were able to utilize a range of carbohy-drates, polyols, amino acids and carboxylic acids as sole sources of carbon (2) high levels of nitro-genase were attained during growth with a range of carbon substrates: highest levels (12—66 n mole C₂H₄.min⁻¹.mg protein⁻¹) were found for glucose and sucrose, variable levels for polyols, and lower levels for citrate and fumarate (1—23 n mole C₂H₄.min⁻¹.mg protein⁻¹) (3) organic ni-trogen compounds which were utilized as sole sources of nitrogen did not generally repress the synthesis of nitrogenase, although low levels were found for some strains during growth with glu-cosamine. Samples from a laboratory model acti-vated sludge system showed a mean rate of acety-lene reduction corresponding to the fixation of 26 μg N.h⁻¹.1⁻¹, and direct analysis of the in-fluent and effluent waters and sludge showed a net increase in nitrogen. These observations corre-lated with the presence of a population of N₂-fix-ing Enterobacteriaceae of ca. 10⁵ cells per ml and pure strains isolated from the system had a mean nitrogenase specific activity of 88 n mole C₂H₄.min⁻¹.mg protein⁻¹. It is therefore con-cluded that endogenous N₂-fixing Enterobacteria-ceae contained in some kinds of industrial waste-waters could successfully be used to diminish the addition of combined nitrogen to activated sludge treatment plants.     

弗氏柠檬酸菌1,3-丙二醇合成的代谢工程研究

【目的】研究弗氏柠檬酸菌(Citrobacter freundii) 1,3-丙二醇合成的代谢过程。【方法】构建甘油脱氢酶基因GSR-lacZ、1,3-丙二醇氧化还原酶基因PDO-lacZ和甘油脱水酶基因GL-lacZ等报告基因。在此基础上,构建3个相应的转座子突变文库。【结果】筛选到6株突变子,其相应关键酶表达水平提高1?11倍,1,3-丙二醇产量提高幅度为3%?50%。对转座子插入位点分析显示,5株突变子插入位点均为β-内酰胺酶(CKO_02592)编码基因,1株突变子插入位点为二氢硫辛酰胺基转移酶(CKO_02433)编码基因。进一步分析发现,β-内酰胺酶基因突变显著提高甘油脱水酶和甘油脱氢酶的表达水平,而1,3-丙二醇氧化还原酶表达水平没有变化;二氢硫辛酰胺基转移酶基因突变显著提高1,3-丙二醇氧化还原酶表达水平,其他两种关键酶基因表达水平不变。【结论】β-内酰胺酶和二氢硫辛酰胺基转移酶基因能够分别影响1,3-丙二醇合成代谢途径关键酶的表达,为构建工程菌株打下基础。     

The effects of bee (Apis mellifera) venom phospholipase A2 on Trypanosoma brucei brucei and enterobacteria

The potential role of phospholipases in trypanosomiasis was investigated using bee venom phospholipase A2 (bvPLA2) as a model. The effects of bvPLA2 on the survival of Trypanosoma brucei brucei, 2 h and 12 h cultures of Enterobacter cloacae, Escherichia coli, Citrobacter freundii were studied. About 1 mg ml⁻¹ bvPLA2 was trypanocidal after 30 min. Some growth occurred at lower concentrations up to 2 h after treatment but viability decreased up to 8 h. Even very low concentrations of bvPLA2 (10⁻¹² mg ml⁻¹) had some trypanocidal activity. Bee venom PLA2 was bactericidal to 2 h bacterial cultures but bacteriostatic to 12 h ones. Minimum bactericidal concentrations were 10⁻⁵-10⁻⁶ mg ml⁻¹. The results showed that bvPLA2 had significant trypanocidal and antibacterial effects on Gram-negative bacteria. The relationship to events occurring during infection is discussed. Phospholipases may play a role in increased endotoxin levels in trypanosomiasis.     

弗氏柠檬酸杆菌对河触致病性的研究

首次报道了弗氏柠檬酸杆菌对河蟹的病原性、并导致其病理变化,药对物该菌的敏感试验及生产中药物治疗效果。从垂死的病蟹肝胰脏中分离得到可疑病菌,经人工感染试验证实此菌导致健康蟹发病死亡。该菌可导致河触的肝胰腺、鳃等发生病理变化,最终引起败血症而死亡。通过16种药物对该菌的敏感试验,筛选出有效药物,应用于生产中取得了较好疗效。