石油脱硫菌 Gordonia sp. WQ-01激光诱变选育及关键酶DszC克隆表达分析

利用脱硫菌株Gordonia sp. WQ-01为出发菌株进行激光诱变育种,考察了不同照射时间和输出功率对菌株的正突变率影响,根据DszC酶活检测、序列比对、空间结构分析以及在大肠杆菌的异源表达研究了突变菌株的关键性能。结果发现,在最适宜诱变条件下（激光照射15 min、输出功率20.0 mW）获得脱硫效率提高28％［0.15 mmol/（L·h）］、有效脱硫时间缩短（36 h）的脱硫菌株,该菌株关键酶DszC活性提高了33.3%。此外,突变菌株脱硫基因dszC的核酸和氨基酸序列、二级结构和三级结构均发生了改变,从而导致酶活显著提高。dszC基因在大肠杆菌BL21(DE3)中经IPTG诱导后可以表达出45 kDa大小的DszC蛋白。     

Enhancement of Palmarumycins C12 and C13 Production in Liquid Culture of Endophytic Fungus Berkleasmium sp. Dzf12 after Treatments with Metal Ions

The influences of eight metal ions (i.e., Na⁺, Ca²⁺, Ag⁺, Co²⁺, Cu²⁺, Al³⁺, Zn²⁺, and Mn⁴⁺) on mycelia growth and palmarumycins C₁₂ and C₁₃ production in liquid culture of the endophytic fungus Berkleasmium sp. Dzf12 were investigated. Three metal ions, Ca²⁺, Cu²⁺ and Al³⁺ were exhibited as the most effective to enhance mycelia growth and palmarumycin production. When calcium ion (Ca²⁺) was applied to the medium at 10.0 mmol/L on day 3, copper ion (Cu²⁺) to the medium at 1.0 mmol/L on day 3, aluminum ion (Al³⁺) to the medium at 2.0 mmol/L on day 6, the maximal yields of palmarumycins C₁₂ plus C₁₃ were obtained as 137.57 mg/L, 146.28 mg/L and 156.77 mg/L, which were 3.94-fold, 4.19-fold and 4.49-fold in comparison with that (34.91 mg/L) of the control, respectively. Al³⁺ favored palmarumycin C₁₂ production when its concentration was higher than 4 mmol/L. Ca²⁺ had an improving effect on mycelia growth of Berkleasmium sp. Dzf12. The combination effects of Ca²⁺, Cu²⁺ and Al³⁺ on palmarumycin C₁₃ production were further studied by employing a statistical method based on the central composite design (CCD) and response surface methodology (RSM). By solving the quadratic regression equation between palmarumycin C₁₃ and three metal ions, the optimal concentrations of Ca²⁺, Cu²⁺ and Al³⁺ in medium for palmarumycin C₁₃ production were determined as 7.58, 1.36 and 2.05 mmol/L, respectively. Under the optimum conditions, the predicted maximum palmarumycin C₁₃ yield reached 208.49 mg/L. By optimizing the combination of Ca²⁺, Cu²⁺ and Al³⁺ in medium, palmarumycin C₁₃ yield was increased to 203.85 mg/L, which was 6.00-fold in comparison with that (33.98 mg/L) in the original basal medium. The results indicate that appropriate metal ions (i.e., Ca²⁺, Cu²⁺ and Al³⁺) could enhance palmarumycin production. Application of the metal ions should be an effective strategy for palmarumycin production in liquid culture of the endophytic fungus Berkleasmium sp. Dzf12.     

Effect of Nanoencapsulated Vitamin B1 Derivative on Inhibition of Both Mycelial Growth and Spore Germination of Fusarium oxysporum f. sp. raphani

Nanoencapsulation of thiamine dilauryl sulfate (TDS), a vitamin B1 derivative, was proved to effectively inhibit the spore germination of Fusarium oxysporum f. sp. raphani (F. oxysporum), as well as mycelial growth. The average diameter of nanoparticles was measured as 136 nm by being encapsulated with an edible encapsulant, lecithin, whose encapsulation efficiency was about 55% in containing 200 ppm of TDS concentration: the 100 ppm TDS nanoparticle solution showed a mycelial growth inhibition rate of 59%. These results were about similar or even better than the cases of treating 100 ppm of dazomet, a positive antifungal control (64%). Moreover, kinetic analysis of inhibiting spore germination were estimated as 6.6% reduction of spore germination rates after 24 h treatment, which were 3.3% similar to the case of treating 100 ppm of a positive control (dazomet) for the same treatment time. It was also found that TDS itself could work as an antifungal agent by inhibiting both mycelial growth and spore germination, even though its efficacy was lower than those of nanoparticles. Nanoparticles especially played a more efficient role in limiting the spore germination, due to their easy penetration into hard cell membranes and long resident time on the surface of the spore shell walls. In this work, it was first demonstrated that the nanoparticle of TDS not a harmful chemical can control the growth of F. oxysporum by using a lower dosage than commercial herbicides, as well as the inhibiting mechanism of the TDS. However, field trials of the TDS nanoparticles encapsulated with lecithin should be further studied to be effectively used for field applications.     

鱼腥藻7120中异形胞发育的影响因子

以鱼腥藻7120为对象,考察了碳源浓度和种类、氮源浓度和种类、光强及NaCl浓度对鱼腥藻7120异形胞发育及发生频率的影响. 结果表明,NaNO3和NH4Cl都会抑制异形胞的分化,NaNO3对异形胞的抑制可以通过提高CO2浓度而解除,但NH4Cl的抑制作用是彻底的;而添加NaHCO3却不能在NO3-存在时诱导鱼腥藻产生异形胞;提高光强和NaCl浓度能够增加异形胞的比例. 证明了胞内碳氮比例的升高是异形胞分化的直接原因.     

细菌和真菌分解低品位磷矿

研究了枯草芽孢杆菌、假单孢菌和曲霉对低品位磷矿粉的分解作用. 结果表明,3种菌株均显著促进了磷矿粉中磷的浸出,磷的浸出率最高可达7%,其中曲霉的解磷能力远强于枯草芽孢杆菌和假单孢菌,进一步证实了真菌的解磷能力强于细菌. 磷矿粉用量越低,磷的浸出率越高. 随培养时间增加,磷的浸出率逐渐升高,但培养到15 d后磷的浸出几乎达到饱和. 另外,碳源浓度为3%时,枯草芽孢杆菌和假单孢菌培养液中磷的浸出率最高,而碳源浓度为2%时曲霉培养液中磷的浸出率达到最高,浓度过高或过低均抑制磷的浸出.     

一株青霉发酵产生木素过氧化物酶的工艺条件

研究了一株青霉菌P6 (Penicillium sp. P6)液体发酵产生木素过氧化物酶的工艺条件,考察了青霉菌P6在天然培养基稻米糠、玉米糠、麦糠和合成培养基GMY中的产酶情况,结果表明,麦糠是较理想的底物. 通过单因素方法研究了添加苯甲醇和MnSO4对酶活的影响;采用正交实验对MnSO4添加量、麦糠含量和培养基pH进行了研究,在实验范围内最佳酶活达到了2.69 U/mL. 进一步通过单双因素回归实验研究了麦糠和MnSO4的最佳含量,得到了最佳培养条件为孢子接种量1.1×106 mL-1,培养时间8 d,500 mL三角瓶装液量150 mL. 最佳培养基组成为麦糠50 g/L,初始pH 4.8, MnSO4 5.66 mmol/L.     

Lipase-catalyzed esterification of lactic acid with straight-chain alcohols

Enzymatic synthesis of esters of lactic acid and straight-chain alcohols with different chain lengths (C₆–C₁₈) were investigated in batch reactions with hexadecanol (C₁₆) as the model alcohol. Cyclohexane was the best solvent for higher ester yields, and the best biocatalyst was the immobilized Candida antarctica lipase B (Novozym 435) as well as the textile-immobilized Candida sp. lipase. A method was established to obtain ester yields in the range of 71 to 82% for the different alcohols, and the most favorable conditions for the esterification reaction using Novozym 435 were an equimolar ratio of lactic acid to alcohol, each at a concentration of 120 mM each; a 50°C reaction temperature; 190 rpm shaking speed; and the addition of 100 mg molecular sieves (4 Å) for drying. The ester yield increased with increasing lipase load, and a yield of 79.2% could be obtained after 24 h of reaction at 20 wt% of Novozym 435. The immobilized Candida sp. lipase prepared in the laboratory also could be used to produce esters of lactic acid and straight-chain alcohols, but it had a much lower activity than Novozym 435 with a temperature optimum of 40°C.     

Production of a halotolerant lipase from Halomonas sp. strain C2SS100: Optimization by response-surface methodology and application in detergent formulations

The aim of this work was to optimize the production of a new lipase by a halotolerant bacterial strain Halomonas sp. C2SS100, by means of the response-surface methodology (RSM). The process parameters having the most significant effect on lipase production were identified using the Plackett–Burman screening design-of-experiments. Then, Box–Behnken design was applied to optimize lipase activity and the quadratic regression model of the lipase production was built. Indeed, the lipase yield was increased, and the value obtained experimentally (39 ± 2 U/ml) was very close to the rate predicted by the model (40.3 U/ml). Likewise, optimization of parameters by RSM resulted in 2.78-fold increase in lipase activity. These findings provide the first report on lipase production and optimization by a halotolerant bacterial strain belonging to Halomonas genus. Afterward, the biochemical properties of the produced lipase were studied for apply in oil stains removal. The crude lipase showed a maximum activity at 60°C and at pH ranging from 7 to 10. It displayed an important stability at high temperature, pH, and NaCl. Interestingly, this bacterial lipase exhibited a prominent stability toward some commercial solid and liquid detergents after 30 min of incubation at 50°C. The capability of the crude lipase to eliminate stain was ascertained on polycotton fabric pieces stained with lubricating oil. Whether with the addition of hot water alone or of a commercially available detergent, lipase is able to considerably boost the elimination of oil stains. The actual findings highlight the capacity of Halomonas sp. lipase for energy-efficient biocatalytic application.     

Cloning, Expression, and Characterization of Thermotolerant Manganese Superoxide Dismutase from Bacillus sp. MHS47

A superoxide dismutase gene from thermotolerant Bacillus sp. MHS47 (MnSOD47) was cloned, sequenced, and expressed. The gene has an open reading frame of 612 bp, corresponding to 203 deduced amino acids, with high homology to the amino acid sequences of B. thuringiensis (accession no. EEN01322), B. anthracis (accession no. NP_846724), B. cereus (accession no. ZP_04187911), B. weihenstephanensis (accession no. YP_001646918), and B. pseudomycoides. The conserved manganese-binding sites (H28, H83, D165, and H169) show that MnSOD47 has the specific characteristics of the manganese superoxide dismutase (MnSOD) enzymes. MnSOD47 expressed an enzyme with a molecular weight of approximately 22.65 kDa and a specific activity of 3537.75 U/mg. The enzyme is active in the pH range 7-8.5, with an optimum pH of 7.5, and at temperatures in the range 30-45 °C, with an optimum temperature of 37 °C. Tests of inhibitors and metal ions indicated that the enzyme activity is inhibited by sodium azide, but not by hydrogen peroxide or potassium cyanide. These data should benefit future studies of MnSODs in other microorganisms and the biotechnological production of MnSOD47, and could also be used to develop a biosensor for the detection of antioxidants and free radical activity. In the future, this basic knowledge could be applicable to the detection of cancer risks in humans and therapeutic treatments.     

海洋来源真菌Fusarium sp.次级代谢产物的研究(Ⅱ)

采用多种色谱技术对海洋来源真菌Fusarium sp.的发酵物进行化学成分分离,通过波谱及理化性质分析确定了化合物的结构.从菌丝体的丙酮提取物中共分离得到10个化合物,分别为1,3,5-三羟基-7-甲基蒽醌(1)、6-羟基芦荟大黄素(2)、3-O-βD-吡喃葡萄糖基-豆甾-5,24(28)Z-二烯(3)、尿嘧啶核苷(4...