Fermentation of maltose and starch by Klebsiella oxytoca P2

Klebsiella oxytoca P2, which has genes from Zymomonas mobilis encoding the alcohol dehydrogenase and pyruvate decarboxylase integrated in its chromosome, fermented 50 g maltose/l to 25.4 g of ethanol/l. It also fermented 10, 20 and 40 g starch/l yielding 4, 8.4, and 17.7 g ethanol/l, respectively, representing 72, 75 and 78% of the theoretical yield. © Rapid Science Ltd. 1998     

Involvement of a plasmid in exopolysaccharide production by Klebsiella oxytoca

A Klebsiella oxytoca isolate which can produce significant levels of an exopolysaccharide using whey as a growth substrate has been reported. The plasmid profile of this isolate was shown to be different from that of the non-exopolysaccharide-producing K. oxytoca ATCC 43863. Irreversible curing of the single plasmid in the K. oxytoca isolate was achieved using 30 g acriflavin/ml. The ability to produce the exopolysaccharide was lost with the curing of the plasmid (parent strain produced a medium viscosity of 1260 cP at 1 s^–1 compared to 1.6 cP at 200 s^–1 produced by the cured strain). However, the ability to metabolize lactose was not significantly affected by curing, and both the parent and the cured strain produced similar levels of viable cells (~10⁹ cfu/ml) after 62 h growth on lactose-rich medium. The exopolysaccharide-producing ability of the isolate was stable for at least 139 generations.     

Biodegradation of Phenol by Actinobacillus Sp.: Mathematical Interpretation and Effect of Some Growth Conditions

Models of the cultivation process of Actinobacillus sp. cells in two media, rich (NB) and minimal (M9) that includes phenol as a sole carbon source, have not been described in the available literature. For these reasons, several single-substrate inhibition models (Monod, Andrew, and Tesseir) were investigated in order to determine the mathematical expression of Actinobacillus sp. growth rate. The experimental data for both nutrient broth and M9 media were fitted to the above models mentioning that Andrews' model best fits these data adequately for both media with regression coefficient of 0.973 and 0.962, respectively. The maximum predicted growth rate by this model is 0.37 h^{- 1} for both media obtained when the initial concentration of phenol is 100 mg/L. The half-saturation concentration constant, K_P, is 1.00 mg/L, which represents the phenol concentration when μ is equal to half μ_max. On the other hand, the inhibition constant, K_p is 13,000.00 mg/L for broth medium and 12,000 mg/L for M9 medium, which is a measure of sensitivity to inhibition by inhibitory substances. When cells are grown in nutrient broth and minimal media, the rate of cell production with time can be expressed by the Reccati and Voltera models. Voltera model better fits in the case of M9 minimal medium plus phenol as sole carbon source. The pH of 7, the incubation temperature of 35°C to 37°C, and the agitation rate of 150 rpm are the optimal conditions for achieving the higher percentage of phenol degradation by Actinobacillus sp. Succinic acid and glycine as carbon and nitrogen source, respectively, were the most efficient of the cosubstrates (out of 10 substrates tested) for removal of phenol on an mg/L basis.     

产酸克雷伯氏杆菌发酵产2，3-丁二醇的培养基优化

采用不同设计方法相结合的策略对耐高糖产酸克雷伯氏杆菌（Klebsiella oxytoca）ME—UD-3-4发酵产2,3-丁二醇的培养基进行优化。首先在单因素实验的基础上采用Plackett—Burrnan设计法对影响ME—UD-3-4发酵产2,3-丁二醇的相关因素进行研究,筛选到3种有显著效应的因素（P〈0．05）：葡萄糖、玉米浆和MgSO4·7H2O。然后利用响应曲面法（Response Surface Methodology,RSM）对这3种因素的最佳水平范围进一步探讨;对得到的回归模型进行分析,得最佳条件（g／L）：葡萄糖220、玉米浆19和MgSO4·7H2O 0．4;在最佳条件下,发酵80h,2,3-丁二醇产量从原来的57．3 g／L提高到86．1 g／L,生产强度由0．72g／（L·h）提高到1．08g/（L·h）。     

产酸克雷伯氏菌赖氨酸脱羧酶的异源表达及粗酶性质

赖氨酸脱羧酶,可以催化赖氨酸脱羧生成戊二胺。戊二胺是重要的平台化合物,可以合成新型聚酰胺材料、脂肪族异氰酸酯等新材料。本研究对来自于产酸克雷伯氏菌的赖氨酸脱羧酶进行异源表达。以pUC18质粒为载体,将来源于产酸克雷伯氏菌的赖氨酸脱羧酶基因ldc克隆到大肠杆菌,得到菌株LN18。在添加0.5 mmol/L IPTG的LB培养基中,对LN18进行摇瓶培养,发酵液酶活可达到35 U/g发酵液,从发酵液制备的赖氨酸脱羧酶粗酶蛋白的酶活可以达到30 000 U/g粗蛋白。产酸克雷伯氏菌赖氨酸脱羧粗酶蛋白大小约80 kDa,粗酶的最适温度和pH值分别为55℃和5.5,与文献中报道的大肠杆菌的赖氨酸脱羧酶Cad A在pH 8.0几乎没有酶活不同,产酸克雷伯氏菌的赖氨酸脱羧酶在pH 8.0的酶活达到最优pH下酶活的30%以上。金属离子对酶活有一定的影响,Mg~(2+)对酶活有促进作用,Fe~(2+)、Zn~(2+)、Ca~(2+)有一定的抑制作用。     

Short Communication: Optimization of bioprocess conditions for exopolysaccharide production by Klebsiella oxytoca

Optimization of bioprocess conditions increased exopolysaccharide production by a strain of Klebsiella oxytoca from 6g/l to 15g/l; this corresponded to an increase in medium viscosity from 36cP at 12s–1 to 20,000 cP at 0.6 s–1. A combination of equal proportions of tryptone nitrogen and urea nitrogen proved to be the best nitrogen source. Lactose was shown to be the preferred carbon source. At an optimum temperature of 25°C, a pH of 7 was found to be the best for exopolysaccharide production. The concentration of exopolysaccharide produced on whey, enriched whey, enriched whey permeate and lactose-rich medium was comparable.     

Identification of heat stable protease of Klebsiella oxytoca isolated from raw milk

AIMS: The aim of this study was to identify and characterize heat stable proteinases of psychrotrophic proteolytic bacteria isolated from raw milk. METHODS AND RESULTS: A strain of Klebsiella oxytoca producing a high proteolytic activity when cultured on milk was isolated. Maximum proteolytic activity was observed at the stationary phase during growth on milk or casein-peptone broth. The bacterium demonstrated the capability to grow at 7 degrees C, classified as psychrotrophic. The crude enzyme showed optimum activity at 37 degrees C, and pH 5.0 and 7.0. The proteinase was very resistant to heat, maintaining 74% of initial activity after incubation at 142 degrees C. CONCLUSIONS: A heat stable protease of a psychrotrophic strain of K. oxytoca was identified and partially characterized. SIGNIFICANCE AND IMPACT OF THE STUDY: Thermal stable proteases may constitute a serious problem to ultra-high temperature (UHT) processed milk, leading to undesirable physical and sensory alterations.     

产酸克雷伯氏菌M5al普鲁兰酶的异源表达及酶学性质研究

普鲁兰酶(EC 3.2.1.41)是一类淀粉脱支酶,能够特异性水解淀粉中的α-1,6-糖苷键,从而提高淀粉的利用率,在以淀粉为原料的食品、纺织、生物燃料和洗涤剂等行业中具有重要的应用价值。本研究以产酸克雷伯氏菌Klebsiella oxytoca M5al基因组DNA为模板,将PCR扩增得到的普鲁兰酶基因pul A克隆至表达载体p ET28a(+),构建好的重组质粒转化大肠杆菌Escherichia coli BL21(DE3),在培养基中添加0.5 mmol/L异丙基硫代半乳糖苷(IPTG)的条件下对该酶基因进行诱导表达,经镍柱纯化获得重组普鲁兰酶用于酶学性质研究。SDS-PAGE及Western Blot检测显示普鲁兰酶基因pul A在上述大肠杆菌宿主中成功获得了表达。该重组酶最适反应p H5.5,最适温度60℃。金属离子对酶活性有一定影响。Mn2+对酶活促进作用显著;Fe3+、Mg2+、Fe2+对酶活只有微弱的促进作用,而Cu2+对酶活造成强烈抑制。来源于Klebsiella oxytoca M5al的普鲁兰酶最适催化条件符合工业生产中淀粉糖化工艺的要求,具有应用于淀粉工业的潜力。     

Production of 2,3‐butanediol by Klebsiella oxytoca from various sugars in microalgal hydrolysate

A new fermentation process using a mixed sugar medium is proposed in this study for 2,3‐butanediol (2,3‐BDO) production. The medium contained seven different monosugars known to be present in Nannochloropsis oceanica hydrolysate. The performance of each sugar when existing alone or together with glucose was evaluated. All the sugars except fucose were successfully metabolized for 2,3‐BDO production. A 2,3‐BDO yield of 0.31g/g was achieved with the mixed sugar medium, which was very close to that with the glucose‐only medium. However, the 2,3‐BDO productivity (0.28 g L^?1 h^?1) was found to be about 30% lower than that with glucose, implying, as expected, the existence of glucose repression on the uptake of other sugars. Strain development is in need to remove such negative effect of glucose for improved process efficiency. Fucose with the lowest uptake rate and no contribution to 2,3‐BDO production can be a high value‐added byproduct, once recovered and purified. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1669–1675, 2015     

The respiratory responses to cyanide of a cyanide-resistant Klebsiella oxytoca bacterial strain

The respiratory system of a cyanide-resistant Klebsiella oxytoca was analyzed by monitoring the changes in the cytochrome contents in response to various inhibitors in the presence of various concentrations of cyanide. The cells grown in the medium without cyanide (KCN) have two terminal oxidases, cytochrome d (Ki = 10(-5) M KCN) and o (Ki = 10(-3) M KCN). When cells were grown on medium with 1 mM KCN, the expression of both b-type cytochrome and cytochrome d in the plasma membranes of the cell decreased by more than 50%, while cytochrome o increased by 70%, as compared with the cells grown in the absence of KCN. Two terminal oxidases with Ki values of about 10(-3) M and 1.7 x 10(-2) M KCN were observed in the plasma membrane fractions of the cells growing on KCN enriched medium. 2-n-Heptyl-4-hydroxyquinoline-N-oxide markedly inhibited the oxidation of NADH by the plasma membranes from the cells grown in the medium without KCN, but not in those plasma membranes from KCN-grown cells. The NADH oxidases in plasma membranes of K. oxytoca grown with and without KCN were equally sensitive to UV irradiation. Adding freshly isolated quinone to the UV-damaged plasma membranes restored the NADH oxidase activity from both types of plasma membranes. From these results, we propose the presence of a non-heme type of terminal oxidase to account for the KCN resistance in K. oxytoca.     

Klebsiella oxytoca: Resistance to aztreonam by overproduction of the chromosomally encoded β-lactamase

Abstract Aztreonam-resistant Klebsiella oxytoca strain SL7811 was selected on agar containing 1 μg of aztreonam per ml from a susceptible strain SL781. The MICs for the resistant mutant towards penicillins, aztreonam and ceftriaxone were much higher, to cefotaxime slightly higher and to ceftazidime unchanged. Synthesis of β-lactamase was 223-fold greater in the mutant compared with the susceptible strain. SL781 and its resistant mutant SL7811 produced β-lactamase with the same isoelectric point and substrate profile. The β-lactamase genes from SL781 and SL7811 were cloned in plasmid pBGS18 giving pBOF-1 and pBOF-4 respectively. The sequences of the two putative promoters indicated two modifications in the resistant plasmid pBOF-4: a transversion (G → T) in the first base of the − 10 consensus sequence and a deletion of one C residue four base pairs upstream of the − 10 hexamer.     

质子自杀法选育克雷伯氏菌产乳酸突变株

以产酸克雷伯氏菌(Klebsiella oxytoca) M5al为出发菌株, 经亚硝基胍诱变处理, 运用质子自杀法选育, 从含0.17 mol/L NaBr-NaBrO3的初筛平板上选出44个具有稳定遗传性的单菌落, 然后结合培养基优化后的摇瓶发酵复筛, 获得3个产乳酸突变株, 其乳酸脱氢酶活性分别为出发菌株的50.6%、58.8%、61.3%。对其中乳酸脱氢酶活性最低的菌株在5 L自动发酵罐上进行批式发酵, 结果显示: 突变株乳酸产量大幅降低, 而乙酸、1,3-丙二醇的产量则显著增加。     

阴沟肠杆菌基因nifA经转座子Tn5整入催娩克氏杆菌NG13的基因组

根际固氮细菌与禾谷类作物如水稻、小麦、玉米等进行联合固氮,能使作物有不同程度的增产（APp等1980,Watanable和hn1984）。水稻是世界上主要粮食作物之一,因此,研究增强水稻根际细菌的联合固氮作用,吸引着科学家们的兴趣。催娩克氏杆菌（KMFithemp）NG13与水稻能进行有效的联合固氮（Yoo等1986）。但是在有氨的生长条件下,细菌的固氮活力受到阻遏。ZhU等将带有nifA的重组质粒引进阴沟肠杆菌EZ6后,观察到有氨存在条件下固氮酶组成型生成。我们实验室构建了具有广泛接合转移特性的阴沟肠杆菌Tns－nifA嵌合质粒PBF101,基因nif…     

Endophytic colonization of Typha australis by a plant growth-promoting bacterium Klebsiella oxytoca strain GR-3

AIMS: To isolate and characterize endophytic diazotrophic bacteria from a semi-aquatic grass (Typha australis) which grows luxuriantly with no addition of any nitrogen source. METHODS AND RESULTS: Ten endophytic diazotrophic bacteria from surface-sterilized roots and culm of T. australis were isolated and screened for plant growth-promoting activities employing standard methods. Based on the rate of nitrogenase activity, indole acetic acid (IAA) production and phosphate (P) solubilization, one root isolate namely GR-3 was found to be the most efficient one. This isolate was identified as Klebsiella oxytoca on the basis of 16S rDNA sequence analysis. Amplification of nifH by polymerase chain reaction (PCR) and detection of dinitrogenase reductase by western blot confirmed the diazotrophic nature of GR-3. It was tagged with gusA fused to a constitutive promoter and the resulting transconjugant was inoculated onto endophyte-free rice variety Malviya dhan-36 seedlings to express cross-infection ability which resulted in a significant increase in root/shoot length and chlorophyll a content. CONCLUSIONS: Roots and culm of T. australis harbour several endophytic diazotrophic bacteria. One root isolate, identified as K. oxytoca GR-3, seems to be an efficient plant growth-promoting bacterium. SIGNIFICANCE AND IMPACT OF THE STUDY: Plant growth-promoting properties of GR-3 suggest that this promising isolate merits further investigations for potential application in agriculture.     

耐高糖高产2,3-丁二醇产酸克雷伯氏杆菌的选育

以产酸克雷伯氏杆菌(Klebsiella oxytoca) ME-UD-3为出发菌株,经紫外线及硫酸二乙酯复合诱变后分别在葡萄糖浓度逐渐提高的液体培养基中进行富集培养,筛选获得了一株耐高糖的2,3-丁二醇高产突变菌株K. oxytoca ME-UD-3-4;该菌株的初始葡萄糖耐受浓度从出发菌株的120g/L提高到300g/L以上,在初始葡萄糖浓度为95 g/L的条件下发酵培养,与出发菌株相比发酵时间缩短了8h,2,3-丁二醇的产量由原来的38.5g/L提高到43.0g/L,生产强度从0.80 g/L·h提高到1.08 g/L·h,转化率达到了理论值的91%。     

Phenol Biodegradation and Metal Removal by a Mixed Bacterial Consortium

This paper reports the tolerance and biodegradation of phenol by a heavy metal–adapted environmental bacterial consortium, known as consortium culture (CC). At the highest tolerable phenol concentration of 1200 mg/L, CC displayed specific growth rate of 0.04 h^?1, phenol degradation rate of 6.11 mg L^?1 h^?1 and biomass of 8.45 ± 0.35 (log₁₀ colony-forming units [CFU]/ml) at the end of incubation. Phenol was degraded via the ortho-cleavage pathway catalyzed by cathechol-1,2-dioxygenase with specific activity of 0.083 (µmol min^?1 mg^?1 protein). The different constituent bacterial isolates of CC preferentially grow on benzene, toluene, xylene, ethylbenzene, cresol, and catechol, suggesting a synergistic mechanism involved in the degradation process. Microtox assay showed that phenol degradation was achieved without producing toxic dead-end metabolites. Moreover, lead (Pb) and cadmium (Cd) at the highest tested concentration of 1.0 and 0.1 mg/L, respectively, did not inhibit phenol degradation by CC. Simultaneous metal removal during phenol degradation was achieved using CC. These findings confirmed the dual function of CC to degrade phenol and to remove heavy metals from a mixed-pollutant medium.     

乙酸、糠醛和5-羟甲基糠醛对产酸克雷伯氏菌发酵生产2,3-丁二醇的影响

为了解产酸克雷伯氏菌对木质纤维素水解液中主要抑制物的耐受和代谢,考察了产酸克雷伯氏菌发酵生产2,3-丁二醇(2,3-butanediol,2,3-BDO)过程中对3种发酵抑制物乙酸、糠醛和5-羟甲基糠醛(5-hydroxymethylfurfural HMF)的耐受以及抑制物浓度的变化,检测了糠醛和HMF的代谢产物.结果表明:产酸克雷伯氏菌对乙酸、糠醛和HMF的耐受浓度分别为30 g/L、4 g/L和5 g/L.并且部分乙酸可作为生产2,3-丁二醇的底物,在0～30 g/L浓度范围内可提高2,3-丁二醇的产量.发酵过程中产酸克雷伯氏菌可将HMF和糠醛全部转化,其中约70％HMF被转化为2,5-呋喃二甲醇,30％HMF和全部糠醛被菌体代谢.研究表明在木质纤维素水解液生产2,3-丁二醇的脱毒过程中可优先考虑脱除糠醛,一定浓度的乙酸可以不用脱除.     

含hup基因的质粒在催娩克氏杆菌中的稳定性及吸氢酶的表达

以质粒pKT230为栽体,亚克隆大豆根瘤菌吸氢酶结构基因(hupSL)片段,构建成嵌合质粒pKH1。将质粒分配系统基因(parDE)片段和吸氢酶结构基因(hup)片段插入载体质粒pRK415,构建成质粒pRKBH。质粒pKH1、pRKBH和载体pRK415经转化和三亲本杂交,得到DH5α/pHR11、DH5α/pRKBH、E1201/pKH1、NG13/pKH1(NGH999)、NG1390/pRK415、NG1390/pHR11、NG1390/pRKBH和NG1390/pKH1(NGH982)等接合子。稳定性分析发现,质粒pKH1在催娩克氏杆茵中传80代后仍有92％以上的菌株含此质粒,说明质粒pKH1有较高的稳定性。吸氢酶活性分析表明,H     

利用甲硝唑及外加氧方法筛选耐氧产氢Klebsiella oxytoca HP1突变菌株

摘氢酶是生物制氢的关键酶,大多数氢酶因对氧极敏感而易失活,因此提高氢酶的氧耐受性对生物制氢有重要意义。本研究利用1％甲基磺酸乙酯对Klebsiella oxytoca HPl进行了两轮诱变,经40mmol／L甲硝唑和21％氧联合处理1h（第一轮诱变）或2h（第二轮诱变）进行筛选。所得突变菌株经产氢测试,结果在15％氧浓度条件下,第一代突变菌株HPl-A15产氢活性为出发菌株Klebsiella oxytoca HPl的3.70倍,在21％氧浓度条件下第二代突变菌株HPAl5-37产氢活性为HPl-A15菌株的2.75倍,是出发菌株的11倍。突变菌株HPl-A15和HPAl5-37具有较好的遗传稳定性。本试验结果说明利用MNZ和外加氧的方法适用于兼性厌氧菌耐氧产氢突变菌株的筛选。     

利用甲硝唑及外加氧方法筛选耐氧产氢Klebsiella oxytoca HP1突变菌株

氢酶是生物制氢的关键酶, 大多数氢酶因对氧极敏感而易失活, 因此提高氢酶的氧耐受性对生物制氢有重要意义。本研究利用1%甲基磺酸乙酯对Klebsiella oxytoca HP1进行了两轮诱变, 经40 mmol/L 甲硝唑和21%氧联合处理1 h(第一轮诱变)或2 h(第二轮诱变)进行筛选。所得突变菌株经产氢测试, 结果在15%氧浓度条件下, 第一代突变菌株HP1-A15产氢活性为出发菌株Klebsiella oxytoca HP1的3.70倍, 在21%氧浓度条件下第二代突变菌株 HPA15-37产氢活性为HP1-A15菌株的2.75倍, 是出发菌株的11倍。突变菌株HP1-A15和 HPA15-37具有较好的遗传稳定性。本试验结果说明利用MNZ和外加氧的方法适用于兼性厌氧菌耐氧产氢突变菌株的筛选。