海洋细菌对柠檬烯的生物转化及萜类产物鉴定

从南海大亚湾的海洋微生物中筛选出3株对单萜D-柠檬烯有显著生物催化转化作用的海洋细菌(Vibrio cholerae、Listonella dam sela和Vibrio alginolyticus)。以2216E为培养基,添加200mg/L的柠檬烯,在28℃,以120 r/m in摇瓶培养5 d,用乙酸乙酯提取培养液,经GC-MS分析其转化产物,结果显示,这些细菌能在柠檬烯的不同位置进行羟基化、羰基化等,并伴随有还原、水解、酯化、开环等反应,但转化能力和转化程度不同;在产物中,还检测到系列结构不同的其它萜类:包括倍半萜、二萜以及三萜等,这些萜类化合物的产生跟柠檬烯的加入有关,说明柠檬烯能影响细菌代谢产物的产生。单萜微生物转化反应操作简单、条件温和、高效率和高选择性、无毒、环境友好,能够分离发现系列新的在医药、有机合成、精细化工等领域有用的化合物,为丰富的天然单萜资源的开发与利用提供新的途径和方法。     

中国风化煤和泥炭的微生物转化

用瓦克青霉菌和铜绿假单孢菌对两种中国风化煤,一种泥炭极它们的预处理样品进行了微生物转化研究,两种微生物能很好地把ATQ,HAQ,HGC,ATHG和HAP样品转化成液态产物,经过23d的表面固体溶煤实验和17d的液体摇瓶溶煤实验,瓦克青霉菌对样品ATQ,HAQ,ATHG和HAP中有机部分的转化率是100％,溶煤产物与原样相比,醇羟基和羧基含量减少,酚羟基和C－O－C官能团的含量增加,表明微生物溶煤过程中存在着氧化水解作用及酯化作用。微生物溶煤主要是酶的作用,并伴随碱性物质及生物螯合作用。－COOH和－OH含量高的样品易于被微生物所溶解。     

肝脏中的生物转化与人体健康*

许多疾病是非营养物质作用于人体的结果。生物转化是清除体内非营养物质的主要方式,肝脏是生物转化的主要场所。在各类生物转化和若干转化反应实例基础上提出了生物转化的基本方向是非营养物质灭活和增溶的观点。指出多数生物转化对机体起到了净化和保护作用,而少数偏离基本方向的转化反应一般对人体健康是有害的。阐明了肝脏健康、生物转化与人体健康3者的关系。     

高分子支撑液膜固定化细胞甾体△1-脱氢转化的研究

研究了高分子支撑液膜固定化细胞的制备、性质以及影响其催化氢化可的松→去氢氢化可的松生物转化的各种因素,并与经典珠状包埋法固定细胞的转化作用进行了比较。结果表明前者的△¹-脱氢速率和产物转化率均高于后者。在所使用的条件下二者未见机械强度方面明显差别。     

柠檬烯和柠檬烯氧化物与硫酸的非均相反应动力学

主要对天然挥发性有机物柠檬烯和柠檬烯氧化物在30%-80% (w)硫酸表面的非均相吸收反应进行了研究, 借以评估天然挥发性有机物与大气环境中的酸性气溶胶的反应活性. 采用自行搭建的配以单光子激光电离飞行时间质谱的湿壁流动反应管的设备对柠檬烯及其氧化物在硫酸表面的非均相吸收动力学进行了测定,计算了稳态摄取系数(γ). 实验结果表明, 柠檬烯氧化物在硫酸表面比只含有双键的柠檬烯的反应活性高, 室温下柠檬烯氧化物在30%-50% (w)硫酸上对应的稳态摄取系数是(7.100±0.023)×10^-5-(8.150±0.162)×10^-3. 此外, 还利用气相色谱-质谱(GC-MS)联用和电喷雾电离质谱(ESI-MS)对柠檬烯氧化物与硫酸的体相反应产物进行了研究, 产物包括单萜烯、松油醇、水合萜二醇和水合萜二醇二硫酸酯. 其中, 水合萜二醇二硫酸酯作为有机硫酸酯的一种, 能够改变气溶胶的吸湿性, 增强云凝结核的活性, 对于大气中灰霾的形成可能有明显的促进作用.     

大环酮的微生物转化

微生物转化在有机合成上有着独特的地位。虽然甾族化合物的微生物转化应用得非常广泛,但有关大环化合物的微生物转化很少报导,而大环酮又是名贵的麝香系列香料。因此本实验意欲筛选使大环酮发生转化的微生物,同时也希望这些反应能在麝香酮的合成上代替有关化学反应。我们选择了三组典型的催化不同类型反应的微生物。     

生物质水热液化水相产物形成机理及资源回收

生物质水热液化转化为生物原油是极具潜力的可再生液体燃料制备途径。但水热液化水相产物产量大、成分复杂,具有较高的环境风险,限制了水热液化的绿色发展。论文以本课题组近10年(2012—2021)的研究积累为核心,综述了水热液化水相产物的形成机理、理化特性和资源回收路径。本文介绍了水相副产物形成的影响因素,总结了不同反应变量下水热转化和元素迁移途径;综述了水相生物转化的途径和研究进展,包括好氧微生物降解、微藻养殖、厌氧处理和微生物电化学等,介绍了膜分离和吸附等物理方法用于水相物质分离、获得高价值组分的研究,论述了利用水相制备农业杀菌剂的潜力。最后对水相的处理原则和研究方向进行展望,以期为水热液化水相的处理和资源回收提供参考。     

灰色链霉菌ATCC 13273 位点特异性转化商陆皂苷元羟基化反应研究

为增加商陆皂苷元的结构多样性,本研究采用两步培养法,利用灰色链霉菌ATCC 13273对其进行生物转化,得到一个极性增大的羟基化产物。通过二维核磁、高分辨质谱等方法鉴定该转化产物为2β, 3β, 23, 29-四羟基-齐墩果烷-12-烯-28, 30-二酸-30-甲酯。     

细菌介导重金属转化过程及金属组学研究方法

重金属污染依然是我国严重的环境问题之一。在重金属胁迫下，微生物可通过复杂的过程，对重金属进行转化，降低重金属的毒性，其中转化涉及的分子机制广受关注。目前，在上述机制相关研究中，对转化过程中重金属的衡量多局限于总量的测定，而对其赋存形态的研究不足，导致难以取得有效进展。对细菌介导重金属的转化过程及金属在其中的赋存形态进行综述，探讨了金属组学研究方法在其中的应用，重点针对重金属的颗粒态与蛋白质结合态进行分析、表征和鉴定，为微生物介导重金属的转化研究提供新视角。     

3-氰基吡啶微生物转化产物的高效液相色谱分析

]本文研究了3-氰基吡啶微生物转化产物的反相高效液相色谱RP-HPLC）分析方法。采用径向加压C18分离柱和甲醇-H2O2080为流动相,在研究选定的流动相速度梯度色谱条件下,底物3-氰基吡啶和产物尼克酰胺、尼克酸得到了全分离,其最低检出量分别为110-9、l.510-10和1.010-10g。此法分析周期短,重现性好,操作简便,可同时完成底物和产物的分离分析,适用于批量微生物样品的分析。     

A novel method for spectrophotometric determination of pregabalin in pure form and in capsules

Background

Dihydrolinalool and terpineol are sources of fragrances that provide a unique volatile terpenoid alcohol of low toxicity and thus are widely used in the perfumery industry, in folk medicine, and in aromatherapy. They are important chemical constituents of the essential oil of many plants. Previous studies have concerned the biotransformation of limonene by Pseudomonas putida. The objective of this research was to study biotransformation of myrcene by Pseudomonas aeruginosa. The culture preparation was done using such variables as different microbial methods and incubation periods to obtain maximum cells of P. aeruginosa for myrcene biotransformation.

Results

It was found that myrcene was converted to dihydrolinalool and 2,6-dimethyloctane in high percentages. The biotransformation products were identified by Fourier-transform infrared spectroscopy (FT-IR), ultraviolet (UV) analysis, gas chromatography (GC), and gas chromatography-mass spectroscopy (GC-MS). Comparison of the different incubation times showed that 3 days was more effective, the major products being 2,6-dimethyloctane (90.0%) and α-terpineol (7.7%) and comprising 97.7%. In contrast, the main compounds derived for an incubation time of 1.5 days were dihydrolinalool (79.5%) and 2,6-dimethyloctane (9.3%), with a total yield of 88.8%.     

Screening a Strain of Klebsiella sp. O852 and the Optimization of Fermentation Conditions for Trans-Dihydrocarvone Production

Flavors and fragrances have high commercial value in the food, cosmetic, chemical and pharmaceutical industries. It is interesting to investigate the isolation and characterization of new microorganisms with the ability to produce flavor compounds. In this study, a new strain of Klebsiella sp. O852 (accession number CCTCC M2020509) was isolated from decayed navel orange (Citrus sinensis (L.) Osbeck), which was proved to be capable of converting limonene to trans-dihydrocarvone. Besides, the optimization of various reaction parameters to enhance the trans-dihydrocarvone production in shake flask was performed for Klebsiella sp. O852. The results showed that the yield of trans-dihydrocarvone reached up to 1 058 mg/L when Klebsiella sp. O852 was incubated using LB-M medium for 4 h at 36 °C and 150 rpm, and the biotransformation process was monitored for 36 h after adding 1680 mg/L limonene/ethanol (final ethanol concentration of 0.8% (v/v)). The content of trans-dihydrocarvone increased 16 times after optimization. This study provided a basis and reference for producing trans-dihydrocarvone by biotransformation.     

Secondary organic aerosol from limona ketone: insights into terpene ozonolysis via synthesis of key intermediates

Limona ketone was synthesized to explore the secondary organic aerosol (SOA) formation mechanism from limonene ozonolysis and also to test group-additivity concepts describing the volatility distribution of ozonolysis products from similar precursors. Limona ketone SOA production is indistinguishable from alpha-pinene, confirming the expected similarity. However, limona ketone SOA production is significantly less intense than limonene SOA production. The very low vapor pressure of limonene ozonolysis products is consistent with full oxidation of both double bonds in limonene and furthermore with production of products other than ketones after oxidation of the exo double bond in limonene. Mass-balance constraints confirm that ketone products from exo double-bond ozonolysis have a minimal contribution to the ultimate product yield. These results serve as the foundation for an emerging framework to describe the effect on volatility of successive generations of organic compounds in the atmosphere.     

Determination of the disproportionation products of limonene used for the catalytic hydrogenation of castor oil

The direct determination by GC-MS analysis of the products from the limonene disproportionation obtained from the catalytic hydrogenation of castor oil is not possible since a transesterification of the oil is required before injection. Therefore, a method for the determination of those products is proposed here: a solid-phase microextraction (SPME) followed by GC-MS. With the optimization of SPME it was possible to isolate a great number of products of the disproportionation of limonene used as hydrogen donor in the catalytic hydrogenation of castor oil by Pd/C. This system also proved to be appropriate to monitor castor oil hydrogenation by following and identifying the limonene disproportionated products.     

Chemoenzymatic epoxidation of alkenes based on peracid formation by a Rhizomucor miehei lipase-catalyzed perhydrolysis reaction

A chemoenzymatic and selective method for the epoxidation of a series of cyclic and linear alkenes is described. Epoxides have been obtained in moderate to excellent conversions under mild reaction conditions through a two-step sequence, carried out in one-pot. This chemoenzymatic approach is based on a Rhizomucor miehei lipase-catalyzed perhydrolysis reaction to form the corresponding peracid, and subsequent epoxidation of the corresponding alkenes. Reaction parameters with influence in the biotransformation have been optimized specially focusing in the efficient enzymatic peracid formation by means of the correct choice of solvent, oxidant, and peracid precursor. This chemoenzymatic approach has been efficiently applied for the first time, in the regioselective chemical oxidation of (S)-carvone and limonene, both showing an opposite behavior for the oxidation of the internal and external C–C double bond, respectively.     

Microorganism screening for limonene bioconversion and correlation with RAPD markers

The use of microorganisms for biotransformations of monoterpenes has stimulated the biotechnological market. Aiming at the highest efficiency in the process of strains screening, the application of molecular biology techniques have been proposed. Based on these aspects, the objective of this work was to select different strains able to convert limonene using fermentative process and random amplified polymorphic DNA (RAPD) markers. The results obtained in the fermentative screening, from 17 strains tested, pointed out that four microorganisms were able to convert limonene into oxygenated derivatives. The RAPD study showed a polymorphism of 96.02% and a similarity from 16.02 to 51.51%. Based on this it was possible to observe a high genetic diversity, even among strains of same species, concluding that the RAPD was not able to correlate the genetic characteristics of the microorganism with the results obtained from the biotransformation process.     

Biotransformation of (R)-(+)- and (S)-(-)-citronellol by Aspergillus sp. and Penicillium sp., and the use of solid-phase microextraction for screening

The biotransformation of (R)-(+)- and (S)-(-)-citronellol by fungi was studied. For screening experiments, solid-phase microextraction (SPME) was used as analytical sampling technique. It was found that sporulated surface cultures of Aspergillus niger were able to convert the substrate into cis- and trans-rose oxides and nerol oxide. The relative contents in the headspace SPME extract of the three bioconversion products cis- and trans-rose oxide and nerol oxide were up to 54, 21 and 12%, respectively. Rose oxide is found in minor amounts in some essential oils, such as Bulgarian rose oil and geranium oil and contributes to its unique odor. It is one of the most important fragrance materials in perfumery in creating rosy notes. Other bioconversion products were 6-methyl-5-hepten-2-one, 6-methyl-5-hepten-2-ol, limonene, terpinolene, linalool and alpha-terpineol. These bioconversion reactions were confirmed by sporulated surface cultures on larger scale and sampling by dynamic headspace sweep and steam distillation solvent extraction. The same conversions were noticed with A. tubingensis and Penicillium roqueforti. This bioconversion was enantioselective since more of the chiral cis- than trans-rose oxide was obtained (cisitrans ratio up to 95/5). Submerged liquid cultures of P. roqueforti yielded two unidentified metabolites after conversion of citronellol (yield up to 5%). The stability and acid-catalyzed conversion of citronellol was also investigated. No chemical oxidation or auto-oxidation products were detected in acidified liquid control broths up to pH 3.5. However, when control tests were run with solid media, acid-catalyzed conversion of the substrate to small amounts of cis- and trans-rose oxides, nerol oxide, linalool and alpha-terpineol was observed at pH 3.5 and when heat treatment (steam distillation solvent extraction) was applied.     

Cloning and expression of the limonene hydroxylase of Bacillus stearothermophilus BR388 and utilization in two-phase limonene conversions

A 3.6-kb fragment of Bacillus stearo thermophilus Br388 chromosomal DNA that confers growth on limonene to Escherichia coli has been sequenced, revealing a single open reading frame encoding a single subunit limonene hydroxylase containing 444 amino acid residues. This enzyme proved capable of limonene hydroxylation to a mixture of carveol and perillyl alcohol as well as dehydrogenation of these products to carvone and perillyl aldehyde. Oxygen, FAD, and NADH were found to stimulate the hydroxylation reaction in cell extracts, and NAD⁺ stimulated the dehydrogenase reaction. In two-phase bioconversionsusing viable E. coli cells overexpressing the limonene hydroxylase, perillyl alcohol and carvone were the principal products observed.     

Biotransformation of antifungal ilicicolin H

The biotransformation of natural product is a powerful method for the functionalization of small molecules, particularly oxidation of unactivated carbons. Ilicicolin H is a tetracyclic antifungal agent consisting of a decalin moiety and a phenyl-pyridone unit. The lipophilic decalin unit is devoid of any heteroatoms. The biotransformation of ilicicolin H led to the formation of eight new oxidized products. The predominant product formed was the result of selective oxidation of the C-19 methyl group of the decalin ring leading to the C-19 hydroxy methyl analog. While these compounds showed poorer activity than ilicicolin H, the new functional groups did provide the opportunity for further chemical modifications and optimization.