Biodiesel fuel production from plant oil catalyzed by Rhizopus oryzae lipase in a water-containing system without an organic solvent

A new enzymatic method of synthesizing methyl esters from plant oil and methanol in a solvent-free reaction system was developed. It is anticipated that such plant oil methyl esters can be used as a biodiesel fuel in the future. Lipase from Rhizopus oryzae efficiently catalyzed the methanolysis of soybean oil in the presence of 4–30 wt% water in the starting materials; however the lipase was nearly inactive in the absence of water. The methyl ester (ME) content in the reaction mixture reached 80–90 wt% by stepwise additions of methanol to the reaction mixture. The kinetics of the reaction appears to be in accordance with the successive reaction mechanism. That is, the oil is first hydrolyzed to free fatty acids and partial glycerides, and the fatty acids produced are then esterified with methanol. Although R. oryzae lipase is considered to exhibit 1(3)-regiospecificity, a certain amount of 1,3-diglyceride was obtained during the methanolysis and hydrolysis of soybean oil by R. oryzae lipase solution. Therefore, the high ME content in the reaction mixture is probably attributable to the acyl migration from the sn-2 position to the sn-1 or sn-3 position in partial glycerides.     

预处理固定化脂肪酶催化合成生物柴油

探讨了预处理固定化Candida antarctica脂肪酶催化餐饮废油合成生物柴油的过程。将固定化Candida antarctica脂肪酶用叔丁醇处理3h后再用废油浸泡4h，用于催化酯交换反应，酯交换反应速率明显加快。研究发现，固定化Candida antarctica脂肪酶预处理后，过量甲醇对酶的抑制作用仍然存在。采用分步添加甲醇工艺，按总醇油摩尔比3：1，分别在0、0．5、1．0、1．5、2．0、2．5h等量加入甲醇，反应4h后，体系中甲酯含量达到97．86％，反应：效率是未处理固定化酶催化合成生物柴油体系的6倍。固定化酶重复使用仍具有较高活性。     

高酸值麻疯树籽油制备生物柴油的杂多酸催化预酯化研究

以探索生物柴油制备中预酯化的绿色技术为目的,研究了杂多酸H3PW12O40催化预酯化高酸值麻疯树籽油。以单因素实验考察了酯化反应中各因素对酯化率的影响,得到H3PW12O40催化预酯化的最佳条件为:反应温度65℃,反应时间3 h,醇油物质的量比9∶1,催化剂H3PW12O40用量1%。在最佳条件下,麻疯树籽油的酯化率为96.1%,酸值(KOH)降至0.61 mg/g。提出了高酸值麻疯树籽油制备生物柴油中杂多酸催化预酯化的工艺路线,分析显示工艺具有一定优势。     

Stepwise ethanolysis of tuna oil using immobilized Candida antarctica lipase

Ethanolysis of fish oil under mild conditions has been strongly desired for preparing the starting materials for the purification of ethyl docosahexaenoate. Thus, we attempted ethanolysis of tuna oil using immobilized Candida antarctica lipase. The immobilized lipase was inactivated in the presence of 2/3 molar equivalent of ethanol against the total fatty acids in tuna oil. To avoid such inactivation, the first step of ethanolysis was conducted at 40°C in a mixture of tuna oil and 1/3 molar equivalent of ethanol using 4% immobilized lipase. After a 10-h reaction, ethanol was consumed and 33% of tuna oil was converted to its corresponding ethyl esters (E-FAs). The reactant is named Gly/E-FA33. The lipase was not inactivated in the presence of 2/3 molar equivalent of ethanol against the total fatty acids in Gly/E-FA33. These findings and the consideration of several factors affecting ethanolysis of tuna oil led to the development of the two- and three-step ethanolyses. The two-step reaction was performed as follows: the first step was carried out at 40°C for 12 h in a mixture of tuna oil and 1/3 molar equivalent of ethanol with 4% immobilized lipase; the second step was performed for 36 h (total reaction period, 48 h) after adding 2/3 molar equivalent of ethanol. On the other hand, the three-step reaction was conducted as follows: the first step was conducted under the same conditions as those in the two-step ethanolysis; in the second and third steps, 1/3 molar equivalent of ethanol was added after 12 and 24 h, respectively; and in the third step, the mixture was shaken for 24 h (total, 48 h). Both types of ethanolyses achieved the conversion of 95% or more of tuna oil to its corresponding E-FAs. To investigate the lipase stability, the two- and three-step ethanolyses were repeated by transferring the enzyme to a fresh substrate mixture of the first step after finishing one cycle of reaction. The two- and three-step reactions maintained over 95% of the conversion for 70 d and over 100 d, respectively.     

Modulator-mediated synthesis of active lipase of Pseudomonas sp. 109 by Escherichia coli cell-free coupled transcription/translation system

Catalytically active lipase was synthesized using Escherichia coli S30 extract from the signal-deleted lipL gene (lipL) in the presence of its N-terminal hydrophobic fragment-truncated modulator (rLimL) that was purified from the overexpressing E. coli cells. The specific activity of the lipase thus synthesized was 125 times higher than that of the purified one from Pseudomonas sp. 109. No lipase activity was detected in the absence of rLimL, even though the lipase protein itself was synthesized. Active lipase was also produced in vitro by coexpression of rlipL and the modulator gene (rlimL), although a much smaller amount of the lipase was formed. In the absence of rLimL, aggregates of the lipase were formed during its folding process. The addition of rLimL proportionally raised both lipase solubility and enzyme activity. An unstable but high activity peak of the lipase was found during its folding process.     

响应面法优化固定化脂肪酶催化棉籽油转化生物柴油的研究

利用响应面法对固定化脂肪酶催化棉籽油合成生物柴油的条件进行优化。以脂肪酸甲酯转化率为指标,考察了固定化脂肪酶用量（占棉籽油质量）、甲醇与棉籽油摩尔比、叔丁醇与棉籽油体积比、反应温度和反应时间对转化率的影响,确定最佳反应条件为：反应时间19.128 h,反应温度37.801℃,固定化脂肪酶用量12.070%,甲醇与棉籽油摩尔比4.949,叔丁醇与棉籽油体积比0.323。验证实验结果表明,转化率达到92.9%,与响应面法预测值94.3%的吻合程度较高。     

Increased riboflavin production from activated bleaching earth by a mutant strain of Ashbya gossypii

The production of riboflavin from vegetable oil was increased using a mutant strain of Ashbya gossypii. This mutant was generated by treating the wild-type strain with N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). Riboflavin production was 10-fold higher in the mutant compared to the wild-type strain. The specific intracellular catalase activity after 3 d of culture was 6-fold higher in the mutant than in the wild-type strain. For the mutant, riboflavin production in the presence of 40 mM hydrogen peroxide was 16% less than that in the absence of hydrogen peroxide, whereas it was 56% less for the wild-type strain. The isocitrate lyase (ICL) activity of the mutant was 0.26 mU/mg of protein during the active riboflavin production phase, which was 2.6-fold higher than the wild-type strain. These data indicate that the mutant utilizes the carbon flux from the TCA cycle to the glyoxylate cycle more efficiently than the wild-type strain, resulting in enhanced riboflavin production. This novel mutant has the potential to be of use for industrial-scale riboflavin production from waste-activated bleaching earth (ABE), thereby transforming a useless material into a valuable bioproduct.     

Effect of a global regulatory gene, afsR2, from Streptomyces lividans on avermectin production in Streptomyces avermitilis

The global regulatory gene, afsR2, from Streptomyces lividans was previously reported to highly stimulate two structurally unrelated antibiotics, actinorhodin and undecylprodigiosin, in both S. lividans and its close relative S. coelicolor. Production of eight avermectin components was also improved in S. avermitilis: the use of wild-type S. avermitilis and its high-producing mutant, transformed by introduction of multiple copies of afsR2, increased the total avermectin productions by 2.3-fold and 1.5-fold, respectively.     

Burkholderia sp.JXJ-16低温耐有机溶剂脂肪酶产酶条件优化及粗酶酶学性质

以Burkholderia sp.JXJ-16为出发菌株,对其低温耐有机溶剂脂肪酶的产酶条件进行单因素实验并研究其粗酶酶学性质。该菌株的最佳产酶条件为:蔗糖3.75 g/L、尿素11.25 g/L、K₂HPO₄2 g/L、（NH4）2SO₄1 g/L、Mn SO₄0.25 g/L、猪油乳化液体积分数2.5%,初始p H9.0、培养温度30℃、装样量20 m L/250 m L、接种量3%、发酵时间20 h。JXJ-16脂肪酶粗酶对中链对硝基苯酚酯有最大水解活力,最适底物为对硝基苯酚辛酸酯;该粗酶在35℃、p H8.5⁹.0时酶活力最高,且具有较好的温度（30⁶0℃）和p H（5.0¹0.5）稳定性;Na⁺、Mg²⁺、Ca²⁺、Mn²⁺、EDTA对粗酶活力具有激活作用,Zn²⁺、Cu²⁺、Fe³⁺对粗酶活力具有抑制作用,K⁺对粗酶活力没有显著性影响;除乙醇和乙腈外,该粗酶在一定体积分数的异丙醇、甲醇、丙酮、乙酸乙酯、三氯甲烷、二甲苯和正己烷中具有良好耐受性,且处于激活状态。综上,该菌株能利用廉价易得的培养基原料达到最佳产酶效果,其所产脂肪酶为低温碱性脂肪酶,具有较好的温度和p H稳定性,对多数供试有机溶剂具有良好耐受性。      

餐饮废油与甲醇/乙醇混合液制备生物柴油的工艺条件研究

以餐饮废油为原料,甲醇/乙醇混合液为酯交换剂,对甲苯磺酸为催化剂制备生物柴油的工艺及机理。该试验表明制备生物柴油的最适宜工艺条件为醇油摩尔比10/1,甲醇与乙醇摩尔比为7/3,催化剂用量占餐饮废油质量的7%,反应时间2 h,反应温度80℃,在此条件下通过甘油含量的测定得出生物柴油产率达到97.2%。研究发现甲醇/乙醇混合液作为酯交换剂具有良好的协同效应,乙醇的加入解决了甲醇在油中溶解性差的问题,而甲醇减轻了乙醇在反应过程中易乳化的缺陷,从而可显著提高生物柴油的产率。对自制生物柴油的性能指标进行检测,发现其具有较高纯度,并且主要性能指标符合生物柴油国家标准。     

一株酸性脂肪酶高产菌株的筛选与鉴定

目的分离筛选出一株酸性脂肪酶高产菌株并对其进行鉴定。方法通过溴甲酚紫酸碱指示剂进行平板初筛,甘油三丁酸酯透明圈法以及摇瓶培养复筛,筛选获得了一株酸性脂肪酶产生菌株;利用平板透明圈法、橄榄油乳化液滴定法和对硝基苯酚比色法3种方法对所筛菌株进行酶活力测定。结果分离获得一株具有高产酸性脂肪酶活力的菌株,将其命名为菌株WY19。经过检测,其粗酶活力达到11000 U/L。通过对菌株WY19进行形态学鉴定、生理生化实验以及分子生物学鉴定,结合系统发育树分析,确定本研究获得的酸性脂肪酶产生菌为克雷伯氏菌。结论菌株WY19所产脂肪酶为酸性脂肪酶,在食品、医药、油脂加工等领域方面具有较好的应用前景。     

Fumigant properties of physical preparations from mountain big sagebrush, Artemisia tridentata Nutt. ssp. vaseyana (Rydb.) beetle for stored grain insects

Vapors released from foliage of mountain big sagebrush, Artemisia tridentata Nutt. ssp. vaseyana (Rydb.) Beetle, through a patented process, were hypothesized to have an insecticidal time of action (24 h or less after time of exposure) similar to the fumigant methyl bromide. Patented preparations were more effective from plants harvested from a relatively wet site in mid to late summer (5 July to 11 September). Bioassays with the lesser grain borer, Rhyzopertha dominica (F.), 0–3 days after adult emergence indicated an LT₅₀ of 7.0±1.2 h for the volatiles generated from only 30 mg dry processed plant material (=0.56 mg active ingredients) per ml headspace. Hatching of eggs of the Indian meal moth, Plodia interpunctella (Hübner), was completely suppressed when exposed 4–20 h after oviposition to a concentration of 7 mg processed plant material per ml headspace (=0.14 mg active ingredients) in a container that allowed passive diffusion and from which the terpenes disappeared by 48 h. Adult red flour beetle, Tribolium castaneum (Herbst), had an LT₅₀ of 40.7±1.2 h when exposed to 29 mg processed plant material per ml headspace. Gas chromatography/mass spectrometry (GC/MS) analyses of the headspace above this processed plant material revealed five major peaks, all non-chlorinated and non-brominated. The two main volatiles, 1,8-cineole and camphor, occurred initially in a mean ratio of 1:3.2, gradually shifting to 1:2.4 over 24 h. The μg/ml headspace of each detectable compound in a sealed container was followed intensely (0.25, 1, 2, 12, 24, 48, and 72 h) for 72 h and at less frequent intervals for 60 days. The active compounds released by the plant material in a closed, but not airtight container, were no longer detectable after 24 h based on GC/MS analysis. Fumigative studies with the same ratio of the two main compounds generated synthetically indicated that embryos of P. interpunctella and adults of R. dominica were as sensitive to the synthetic mixture as they were to the processed plant material. Although one could apply the precise commercial terpenes in the same ratio, the plant material provides a natural formulation that is conveniently diluted (formulated) to levels safe for handling. Therefore, this preparation method and plant material shows good potential as an alternative to methyl bromide for protection of stored grain, commodity, and space fumigations. No residues are detectable in the headspace of aerated commodity, milled product, or in fumigated space.     

Efficient production of a heterologous peroxidase, DyP from Geotrichum candidum Dec 1, on solid-state culture of Aspergillus oryzae RD005

The productivity of a peroxidase (DyP) originating from Geotrichum candidum Dec 1 was enhanced in the solid-state culture using Aspergillus oryzae RD005. When the humidity, water content, and temperature were adjusted to 60%, 50% and 27°C, respectively, the productivity of DyP reached 5.3 g per kilogram wheat bran, which was used as the solid medium. The yield of 5.3 g per kg wheat bran corresponded to the yield of a 56 kg submerged culture. The productivity per gram carbon of the medium in the solid-state culture was 4.1-fold that in the submerged culture.     

Bacteriocin production by Lactobacillus pentosus B96 can be expressed as a function of temperature and NaCl concentration

Lactobacillus pentosus B96 is a bacteriocin-producing strain that was isolated from fermenting olive brines. The aim of the present work was the optimization of bacteriocin production, using response surface methodology (RS). A two-level screening Plackett–Burman design was used to select influencing factors. Then, a central composite design, with three repetitions in the centre, for pH, NaCl concentration, and temperature was carried out. Finally, an RS, which included the region of maximum accumulated bioactivity, was built as a function of NaCl concentration and temperature. Bioactivity accumulation was always observed during the exponential growth-phase, although no apparent correlation between maximum accumulated bioactivity and biomass formation was found. L. pentosus B96 is known to grow better at about 30 °C, neutral pH, and by the absence of NaCl; however, a suboptimal temperature (22 °C) and a moderate NaCl stress (0.65 mol l⁻¹) stimulated bacteriocin production. The research led to environmental conditions that maximized bacteriocin activity, which can be expressed as a polynomial function of temperature and NaCl concentration. The suboptimal growth conditions, which were found to produce the highest bacteriocin titres, resembled those prevailing during green table olive fermentation. This model can be used to improve “in situ” bacteriocin production thus contributing to the microbiological control of the process.     

Rheological properties of Lepidium sativum seed extract as a function of concentration, temperature and time

The seeds of Lepidium sativum (Garden Cress) were selected as a new source of hydrocolloid and its chemical composition and molecular parameters were determined. The macromolecular component of the extract had a molecular weight of 540 kDa, and was nearly as rigid as xanthan with regard to chain conformation. The main rheological features were investigated as a function of shear rate, concentration and temperature. The extract exhibited strong shear-thinning behaviour, which was even more pronounced than for xanthan. An increase in concentration or temperature led to an increase in pseudoplasticity. The Arrhenius model was applied to the temperature dependence of viscosity, and the activation energy (E_a) was found to decrease with increasing concentration. The extract solutions showed thixotropic behaviour at all the concentrations and temperatures studied, and the first-order stress decay model with a non-zero equilibrium stress fairly described the time-dependent behaviour. The rheological characteristics found indicated a potential application of the extract as a novel thickener.     

微拟球藻油脂提取动力学研究

对不同条件下微拟球藻油脂提取过程进行了动力学分析,以动力学方程C=C_e×(1-exp~(-kt))进行数据拟合,研究了溶剂、料液比、提取温度、振荡和超声波处理对提油率的影响。结果表明:该过程符合Fick第二定律,该方程能够较为准确地模拟微拟球藻的油脂提取过程,总提油率C_e和传质系数k随着提取条件不同而表现出差异;以石油醚为溶剂,在料液比1∶5、提取温度50℃、超声波辅助条件下,微拟球藻的提油率达到24.68%,比对照组(静置处理)提高了1.44倍,最大有效扩散率D_(eff)达到1.158 6×10~(-16)m~2/s。     

Effect of Zataria multiflora Boiss. essential oil and nisin on Salmonella typhimurium and Staphylococcus aureus in a food model system and on the bacterial cell membranes

The effects of different concentrations of Zataria multiflora Boiss. essential oil (EO: 0, 5, 15 and 30 μl 100 ml⁻¹) and nisin (N: 0, 0.25 and 0.5 μg ml⁻¹), temperatures (T: 25 and 8 °C), and storage times (up to 21 days) on growth of Salmonella typhimurium and Staphylococcus aureus in a commercial barley soup were evaluated in a factorial design study. The growth of S. typhimurium was significantly (P < 0.05) decreased by EO concentrations and their combinations with N concentrations at 8 °C. For S. aureus, the viable count was significantly (P < 0.05) inhibited by EO and N concentrations and their combinations, incubated at both storage temperatures. The mechanism of the antimicrobial action of EO, N, and their combinations against cell membranes of the tested organisms were also studied by measurement of the release of cell constituents and by the electronic microscopy observations of the cells. The significant increase of the cell constituents’ release of both organisms was observed as a result of treatments with EO and EO in combination with N. Electronic microscopy observations revealed that the cell membranes of S. typhimurium treated by EO and EO in combination with N were significantly damaged, while cells treated with only N looked similar to untreated cells. The electron micrographs of treated cells of S. aureus with EO, N, and their combination also showed important morphological damages and disrupted membranes.     

Effect of oxygen concentration on nitrogen removal by Nitrosomonas europaea and Paracoccus denitrificans immobilized within tubular polymeric gel

Tubular gel reactors containing Nitrosomonas europaea and Paracoccus denitrificans, which remove nitrogen from solutions through a process of nitrification and denitrification, require oxygen for ammonia oxidation, the first and rate-limiting step in the process. To accelerate ammonia oxidation, high concentrations of oxygen were applied to the reactors instead of air. Although a 50% O₂:N₂ gas mixture and pure oxygen were both toxic to free N. europaea cells, they actually accelerated ammonia oxidation by N. europaea immobilized within the tubular gel. Indeed, the rate of ammonia oxidation by a tube exposed to pure oxygen was twice that of one exposed to 20% O₂. When the distribution of N. europaea cells within the tubes was investigated using a fluorescently-labeled antibody, colonies were found on the external surface of the tube exposed to 20% O₂, but were located at a depth of 120–300 μm from the external surface in the case of the tube exposed to pure oxygen. The region between the external surface of the gel and the colonies apparently acted as a barrier, reducing the diffusion of oxygen and thus protecting the cells from oxygen cytotoxicity.     

鸡源大肠埃希氏菌mcr-1基因携带及分析

目的 了解在农业农村部禁止使用多黏菌素作为动物促生长使用后四川部分地区鸡源大肠埃希氏菌（E.coli）mcr-1基因的携带情况,为制定进一步防控措施提供依据。方法 采集四川部分地区市场售卖点肉鸡直肠拭子,用含有多黏菌素（终浓度4 μg/mL）的EC肉汤增菌接种含多黏菌素（终浓度4 μg/mL）的麦康凯平板,挑取可疑菌落,采用PCR方法鉴定菌株并检测mcr-1基因;微量肉汤稀释法测定mcr-1基因阳性菌株对临床常见抗菌药物耐药情况。脉冲场凝胶电泳（PFGE）对mcr-1基因阳性菌株进行同源分析。耐药基因质粒结合实验验证mcr-1基因传播途径。结果 从70份肉鸡样本中的13份检出mcr-1基因阳性大肠埃希氏菌,检出率18.57%（13/70）,对实验的13种抗生素,除13株mcr-1阳性菌株对头孢西丁有12株敏感以外,对其他抗生素都表现出不同程度的耐药,其中四环素和甲氧苄啶/磺胺甲恶唑耐药率最高,达到了100%（13/13）;其次是氨苄西林和氯霉素,耐药率为84.62%（11/13）。PFGE显示13株mcr-1阳性大肠埃希氏菌分属13个不同的型别;质粒结合实验显示mcr-1基因能够通过质粒传播。结论 mcr-1基因在鸡大肠内大肠杆菌中检测率比较高,且鸡大肠中mcr-1阳性大肠埃希氏菌的耐药情况比较严重。