液相色谱-串联质谱法定量分析莱茵衣藻1,3-二油酸-2-棕榈酸甘油三酯

研究建立了基于超高效液相色谱串联三重四级杆质谱技术的1,3-二油酸-2-棕榈酸甘油三酯(1,3-Dioleoyl-2-palmitoylglycerol, OPO)分析方法。在正离子模式下, 以[876>577]为定量离子对, 通过多反应监测模式(Multiple Reaction Monitoring, MRM)扫描, 采用内标法对OPO进行定量分析。对缺氮胁迫条件下一株野生型莱茵衣藻(Chlamydomonas reinhardtii)cc-5325及其半乳糖基水解酶基因敲降突变体M08的OPO进行了定量分析, 研究结果发现莱茵衣藻中OPO在缺氮胁迫期间显著积累, 且不同遗传背景的莱茵衣藻OPO积累量不同。在缺氮第1至第3天, M08的OPO含量相较于cc-5325分别提高了3.70、3.04和2.74倍, M08的OPO产量相较于cc-5325分别提高了1.13、1.53和1.33倍, 说明通过遗传改造莱茵衣藻的某些脂质代谢途径相关基因可以增加其OPO含量, 因此莱茵衣藻具有改成为商业化油脂新食品原料的潜在应用价值。研究建立的功能脂质OPO的定量分析方法对进一步研究不同遗传改造的莱茵衣藻中OPO结构脂的检测提供了技术支持和分析基础。     

2-溴棕榈酸调节背根神经节中ASIC3蛋白的膜定位缓解大鼠骨癌痛

骨癌痛（BCP）是恶性肿瘤患者最常见的疼痛之一,严重影响患者的生活质量。BCP的分子作用机制和新药研发都迫在眉睫。2-溴棕榈酸（2-BP）作为一种蛋白质棕榈化抑制剂在病理性疼痛中有镇痛效果,而在骨癌痛中作用仍不清楚。酸敏感离子通道3型（ASIC3）,作为一个重要的疼痛因子能否受到2-BP的调控也未知。为了检测2-BP在骨癌痛中的作用,并研究其对背根神经节（DRG）中ASIC3的调控,本文开展了相关工作。1）首先建立BCP大鼠模型,将大鼠乳腺癌细胞（MRMT-1）注射入雌大鼠胫骨骨髓腔内,21 d后通过X射线和机械痛检测,发现与假性手术组相比,BCP模型大鼠的胫骨被破坏;同时,BCP组大鼠的机械疼痛值明显上升（假性手术组PWT vs. BCP PWT:16.1 ± 1.5 vs. 5.3 ± 1.5; P<0.01）;表明大鼠乳腺癌骨转移疼痛模型成功构建。2）蛋白质免疫印迹检测结果显示,与正常和假性手术组相比,BCP大鼠L4-L6 DRG中酸敏感离子通道3蛋白表达上调（0.63 ± 0.03, 0.64 ± 0.1 和 1.07 ± 0.05）。3）在术后第21 d,给BCP大鼠腹腔注射2-BP,发现给药组BCP大鼠的机械疼痛值下调 （6 h后,PWT 对照 vs. PWT 2-BP: 6.9 ± 2.0 vs. 10.8 ± 1.6, P<0.01）,表明2-BP在骨癌痛模型大鼠中具有镇痛作用。4）蛋白质免疫印迹结果显示,与给药前相比,2-BP处理后降低了BCP大鼠L4-L6 DRG中膜上ASIC3蛋白的表达（1.05 ± 0.13, 0.66 ± 0.12）。同时,在ASIC3介导的酸痛模型中,2-BP给药降低大鼠震颤的次数（对照组为27 ± 1.8次,2-BP组为10 ± 1.5次）,表明2-BP给药阻断ASIC3介导的酸痛。5）在ASIC3转染的SH-SY5Y细胞中,与对照相比,2-BP给药后明显降低膜上ASIC3蛋白表达量（1.0 ± 0.2, 0.58 ± 0.10）。这些结果表明,2-BP在骨癌痛中具有镇痛作用,其镇痛机制涉及到调控背根神经节中膜上酸敏感离子通道3的表达。     

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）。     

节杆菌82菌株降解胆甾醇生成3-氧代-联原胆烷一1，4-二烯-22-酸

从19株有降解胆甾醇能力的微生物中,筛选出一株节秆菌(Arithrobacter)82菌株,它能在含硫酸钴的培养基中转化胆甾醇和积累3-氧代-联原胆烷-1,4-二烯-22酸(BNc)。转化过程中形成的主要中间体为胆甾烯酮(Cholestenone)。降低培养基中葡萄糖的浓度并增加玉米浆的量,可促进胆甾烯酮侧链的降解,有利于BNC的积累。BNC可在酸性溶液中形成结晶并沉淀下来,故适宜于离心收集。用传统的理化方法及光谱分析技术测定了产物的结构及特性。     

羰基还原酶产生菌Candida ontarioensis制备(R)-2-氯-1-(3-氯苯基)乙醇

从实验室保藏的菌株中筛选获得Candida sp.PT2A,并通过18S rRNA鉴定为安大略假单胞菌Candida on-tarioensis。对C.ontarioensis不对称还原合成(R)-2-氯-1-(3-氯苯基)乙醇的发酵产酶条件和转化条件进行优化,确定了最适的发酵产酶条件和转化条件:温度30℃,初始pH 6.5,摇床转速180 r/min,菌体质量浓度200 g/L。采用2-氯-1-(3-氯苯基)乙酮质量浓度为10 g/L时,还原反应72 h,(R)-2-氯-1-(3-氯苯基)乙醇的e.e.值为99.9%,产率为99%;底物质量浓度提高至30 g/L时,产率下降为84.3%。采用十六烷基三甲基溴化铵(CTAB)对C.ontarioensis细胞进行通透性处理(CTAB g/L,4℃下处理20 min),在30 g/L底物下反应24 h,产物的e.e.和产率分别达到99.9%和97.5%。     

高选择性不对称还原N,N-二甲基-3-酮-3-(2-噻吩)-1-丙胺的重组氧化还原酶催化性质及其酶促转化

【目的】通过表达多种重组立体选择性氧化还原酶,分析其催化不对称还原N,N-二甲基-3-酮-3-(2-噻吩)-1-丙胺(DKTP)的性质,从而构建酶促合成(S)-N,N-二甲基-3-羟基-3-(2-噻吩)-1-丙胺(DHTP)的反应体系。【方法】基于已有立体选择性氧化还原酶重组大肠杆菌,通过Ni离子亲和层析法纯化得到重组氧化还原酶,以DKTP为底物,考察不同重组氧化还原酶对DKTP的催化活性和选择性,进一步对高选择性酶促合成(S)-DHTP的重组酶CR2进行性质分析,并考察其在最适条件下不对称还原DKTP的过程。【结果】筛选获得产物构型为(S)-型的催化活性最高的酶为CR2,该酶米氏常数Km为0.135 mmol/L,kcat/Km为3.689 L/(mmol·s),最适p H 8.4(0.1 mol/L三乙醇胺缓冲液),最适反应温度为35°C,在10-45°C条件下和p H 7.5-8.5较为稳定,Zn2+离子对酶活有促进作用。CR2催化DKTP不对称还原反应6 h后,DHTP的产率达92.1%、光学纯度达99.9%。【结论】基于活性和选择性分析,获得不对称还原DKTP的目标酶CR2,其催化特性有利于高立体选择性还原DKTP生成度洛西汀中间体(S)-DHTP,从而为进一步提高酶促不对称还原DKTP的转化效率提供研究基础。     

犁头霉11α-羟基化制备16β-甲基-11α，17α，21 三羟基孕甾-1，4 二烯 3，20 二酮

选育到一株对16β-甲基-17α,21-二羟基孕甾-1,4-二烯3,20-二酮(Ⅱa)11α-羟基化活性强的犁头霉A28菌株,并发现底物21乙酰化(Ⅱb)可明显提高11α-羟基化的能力。在适宜的转化条件下,Ⅱb投料浓度0.5%,产物16β-甲基-11α,17α,21-三羟基孕甾-1,4-二烯3,20-二酮(Ⅲ)收率为73%,结构经波谱分析确认。     

N-（4-甲基-2-氨基苯并噻唑）α-氨基-α-（3-三氟甲基苯基）-0，0-二（2-烷氧基乙基）亚膦酸酯对小麦赤霉病原菌的抑制活性及作用机制的初步探究

实验室条件下采用生长速率法测定化合物N-(4-甲基-2-氨基苯并噻唑)α-氨基-α-(3-三氟甲基苯基)-O,O-二(2-烷氧基乙基)亚膦酸酯对小麦赤霉病原菌(Fusarium graminearum)的离体抑制效果,并初步研究了其抑制小麦赤霉病原菌作用机制.实验结果表明,该化合物对小麦赤霉病原菌的EC_(50)为46.05 μg/mL,当化合物浓度为50 μg/mL时,对该病原菌的抑制率就达到了60.5 %.以浓度为250 μg/mL的该供试化合物处理小麦赤霉病原菌菌丝24 h后,其细胞膜通透性增强,菌体内还原糖、几丁糖和可溶性蛋白含量及几丁质酶活性在短时间内均出现先升高然后下降的趋势.     

Enzymatic synthesis of 2-ethylhexyl esters of fatty acids by immobilized lipase from Candida sp. 99–125

The 2-ethylhexyl esters of fatty acids were synthesized by immobilized lipase from Candida sp. 99–125. The reuse stability of immobilized lipase was at least four batches. The conditions of enzymatic synthesis of 2-ethylhexyl palmitate were optimized. In the system of petroleum ether, 10% (w/w) immobilized lipase was used in the esterfication of 2-ethyl hexanol (7.8 mmol) and palmitic acid (7.8 mmol) at 40 °C with silica gel as the water absorbent. The esterification degree was 91% under these conditions. The purity of 2-ethylhexyl palmitate was 98% after purification consisting washing by water and evaporation to remove the organic solvent.     

假丝酵母99-125脂肪酶的发酵工艺研究

对假丝酵母99-125脂肪酶的发酵工艺条件进行了一系列研究。选择了合适的培养基成分并进行优化 ,获得了最优的摇瓶培养基配方 (% ,W/V) :豆油 4.0 ,全脂豆粉 4.0 ,K₂HPO₄0.1,KH₂PO₄ 0.1。产酶水平能达到 5000IU/mL。在 30L发酵罐上进行初步放大实验 ,其产酶水平能达到 8100IU/mL。在1m³发酵罐上进行中试放大 ,产酶水平可达到 8000IU/mL。     

假丝酵母99-125脂肪酶喷雾干燥工艺及催化性质的研究

利用响应面法对假丝酵母脂肪酶喷雾干燥工艺条件进行优化,考察进口温度、雾化速度、保护剂含量对脂肪酶活力收率的影响。确定了最佳喷雾条件:保护剂为10～15 g/L的阿拉伯胶,进口温度115～120℃,雾化速度0.4 L/h,可得到收率最高为60.5%的脂肪酶酶粉。制得的固定化酶用于手性拆分(R,S)-1-苯乙醇,光学产率最高可达到53.6%;用于催化合成生物柴油,转化率最高可达到90.2%。在4、30℃下密封保存,半衰期可分别达到15个月、3个月。     

D301树脂固定化假丝酵母脂肪酶

本研究选择7种吸附和离子交换树脂进行了假丝酵母脂肪酶(Candida sp.lipase)的固定化试验,通过测定固定化后各脂肪酶的酶活,筛选出固定化效果较好的弱碱性阴离子交换树脂D301;并通过扫描电镜将D301与脂肪酶Novozym 435的表面形貌做比较,进一步选定D301树脂作为载体,并对其采用戊二醛交联固定化,研究并优化了其固定化条件。结果表明,5%戊二醛溶液的加入量为8mL,处理时间为5h,酶液浓度为1.0g/L,磷酸缓冲盐溶液pH6.0,固定化处理10h效果最好,获得的固定化酶活力可达35U/mg,酶的固定化效率约为3.5U/(mg·h)。     

Lipase catalyzed methanolysis to produce biodiesel: Optimization of the biodiesel production

A lipase from Candida sp., suitable for transesterification of fats and oils to produce fatty acid methyl ester (FAME), was immobilized on a cheap cotton membrane, in this paper. The conversion ratio of salad oil to biodiesel could reach up to 96% with the optimal reaction conditions. Continuous reaction in a fixed bed reactor was also investigated. A three-step transesterification with methanol (methanolysis) of oil was conducted by using a series of nine columns packed with immobilized Candida sp. 99–125 lipase. As substrate of the first reaction step, plant or waste oil was used together with 1/3 molar equivalent of methanol against total fatty acids in the oil. Mixtures of the first- and second-step eluates and 1/3 molar equivalent of methanol were used for the second- and third-reaction steps. A hydrocyclone was used in order to on-line separate the by-product glycerol after every 1/3 molar equivalent of methanol was added. Petroleum ether was used as solvent (3/2, v/v of oil) and the pump was operated with a flow rate of 15 L/h giving an annual throughput of 100 t. The final conversion ratio of the FAME from plant oil and waste oil under the optimal condition was 90% and 92%, respectively. The life of the immobilized lipase was more than 10 days. This new technique has many strongpoints such as low pollution, environmentally friendly, and low energy costs.     

Enzymatic synthesis of fatty acid methyl esters from lard with immobilized Candida sp. 99-125

In this paper, immobilized lipase catalyzed biodiesel production from lard was studied. Using Candida sp. 99-125, the effect of temperature, water content, enzyme amount, solvent and three-step methanolysis were investigated. The optimal conditions for processing 1 g of lard were: 0.2 g immobilized lipase, 8 ml n-hexane as solvent, 20% water based on the fat weight, temperature 40 °C, and three-step addition of methanol. As a result, the fatty acid methyl esters (FAMEs) yield was 87.4%. The lipase was proved to be stable when used repeatedly for 180 h.     

Enzymatic resolution of 2-phenoxy-1-propanols through the enantioselective acylation mediated by Achromobacter sp. lipase

2-(Substituted phenoxy)-1-propanols, e.g. 2-(4-chlorophenoxy)-1-propanol, belonging to primary alcohols with an oxygen atom at the stereocenter, were resolved with moderate to good enantioselectivity, as judged by the value of enantiomeric ratio E (up to 27), through the enantioselective acylation with vinyl butanoate mediated by the little-known lipase from Achromobacter sp. in diisopropyl ether, after the examination of potential factors affecting the reaction such as organic solvents and acyl donors.     

Aminolysis of 2-hydroxy esters catalyzed by Candida antarctica lipase

Candida antarctica lipase catalyzed the aminolysis of 2-hydroxy esters with amines in organic solvents to yield the corresponding 2-hydroxy amides. The reactions proceeded at 28–30 °C in dioxane for 6 h with 3 mM substrates with yields ranging between 45% (w/w) (for branched substrates) to 88% (w/w) (for linear substrates). Although the reaction was not enantioselective, because of its simplicity it represents an alternative method for the synthesis of functionalised amides.     

Pretreatment of immobilized Candida sp. 99-125 lipase to improve its methanol tolerance for biodiesel production

Candida sp. 99-125 lipase immobilized on textile membrane was pretreated with several methods to improve its activity and methanol tolerance for biodiesel production. Lipase pretreatments with short chain alcohols from n-propyl alcohol to isobutyl alcohol did not have any positive effect on the lipase activity and methanol tolerance. While lipase treated with methanol solutions from 10 to 20% volume concentrations did enhance the enzyme activity and methanol tolerance, and this lipase activation effect did not exist when methanol volume concentration was 40%. 1 mM salt solutions of (NH₄)₂SO₄, CaCl₂, KCl, K₂SO₄ and MgCl₂ pretreatments were the useful tools to improve the lipase activity and methanol tolerance. The reason might be that salts could incorporate with the protein molecular to form a more stable molecular to resist conformation change induced by high methanol concentration. The operational stability of pretreated lipase was improved dramatically for biodiesel production during batch reactions.     

Candida ruelliae sp. nov., a novel yeast species isolated from flowers of Ruellia sp. (Acanthaceae)

Two novel yeast strains designated as 16Q1 and 16Q3 were isolated from flowers of the Ruellia species of the Acanthaceae family. The D1/D2 domain and ITS sequences of these two strains were identical. Sequence analysis of the D1/D2 domain of large-subunit rRNA gene indicated their relationship to species of the Candida haemulonii cluster. However, they differ from C. haemulonii by 14% nucleotide sequence divergence, from Candida pseudohaemulonii by 16.1% and from C. haemulonii type II by 16.5%. These strains also differ in 18 physiological tests from the type strain of C. haemulonii, and 12 and 16 tests, respectively, from C. pseudohaemulonii and C. haemulonii type II. They also differ from C. haemulonii and other related species by more than 13% sequence divergence in the internal transcribed spacer region. In the SSU rRNA gene sequences, strain 16Q1 differs by 1.7% nucleotide divergence from C. haemulonii. Sporulation was not observed in pure or mixed cultures on several media examined. All these data support the assignment of these strains to a novel species; we have named them as Candida ruelliae sp. nov., and designate strain 16Q1(T)=MTCC 7739(T)=CBS10815(T) as type strain of the novel species.