固定化脂肪酶催化高酸废油脂酯交换生产生物柴油

 探讨了固定化脂肪酶Novozym 435催化高酸废油脂与乙酸甲酯酯交换生产生物柴油. Novozym 435能催化高酸废油脂与乙酸甲酯的酯交换反应,反应24 h后甲酯产率为77.5%,但该值大大低于以精制玉米油为原料时的甲酯产率(86.2%). 系统研究了反应体系中的水、游离脂肪酸和乙酸对反应的影响. 当反应体系中的水含量低于0.05%时,水对酶反应速率和甲酯产率影响甚小,而水含量高于0.05%时,酶反应速率和甲酯产率随着水含量的增加而降低. 游离脂肪酸对反应有较大影响,甲酯产率随着游离脂肪酸含量的增加而急剧下降. 乙酸甲酯与游离脂肪酸反应产生的副产物乙酸是导致甲酯产率显著下降的原因. 在反应体系中添加适量(油重的10%)的有机碱三羟甲基氨基甲烷或三乙胺可有效提高酶促高酸废油脂的酯交换反应速率和甲酯产率,使反应12 h后的甲酯产率分别达到85.9%和80.8%; 碱的加入还提高了酶的操作稳定性,添加有机碱三羟甲基氨基甲烷或三乙胺可使反应10批次后Novozym 435的相对酶活力分别由对照值86%提高到97%和93%.     

New eutectic ionic liquids for lipase activation and enzymatic preparation of biodiesel

The enzymatic preparation of biodiesel has been hampered by the lack of suitable solvents with desirable properties such as high lipase compatibility, low cost, low viscosity, high biodegradability, and ease of product separation. Recent interest in using ionic liquids (ILs) as advanced reaction media has led to fast reaction rates and high yields in the enzymatic synthesis of biodiesel. However, conventional (i.e., cation-anion paired) ILs based on imidazolium and other quaternary ammonium salts remain too expensive for wide application at industrial scales. In this study, we report on newly-synthesized eutectic ILs derived from choline acetate or choline chloride coupled with biocompatible hydrogen-bond donors, such as glycerol. These eutectic solvents have favorable properties including low viscosity, high biodegradability, and excellent compatibility with Novozym(?) 435, a commercial immobilized Candida antarctica lipase B. Furthermore, in a model biodiesel synthesis system, we demonstrate high reaction rates for the enzymatic transesterification of Miglyol(?) oil 812 with methanol, catalyzed by Novozym(?) 435 in choline acetate/glycerol (1:1.5 molar ratio). The high conversion (97%) of the triglyceride obtained within 3 h, under optimal conditions, suggests that these novel eutectic solvents warrant further exploration as potential media in the enzymatic production of biodiesel.     

有机相酶催化拆分制备(S)-2-氯-1-(2-噻吩)-乙醇

首次在有机相中对酶催化条件下的2-氯-1-(2-噻吩)-乙醇的反应进行了研究. 通过对不同来源酶的筛选, 找到了Novozym 435和Alcaligenes sp两种选择性较好的酶, 它们均对该反应具有较高的选择性和较快的反应速度, 在此基础上进一步通过对溶剂、温度、摇床转速以及酶用量的筛选, 确定了能够有效拆分2-氯-1-(2-噻吩)-乙醇的较佳反应条件. 当温度35 ℃, 酶量10 mg/mL, 反应72.5 h, 产物的ee值为98.5%时收率为48.6%.     

华根霉全细胞脂肪酶催化合成生物柴油

比较了5种不同商品化脂肪酶和自制的华根霉CCTCCM201021全细胞脂肪酶(RCL)催化油脂合成生物柴油的转化效果,结果表明,RCL能有效应用于无溶剂体系催化合成生物柴油.在无溶剂体系中对该酶催化生物柴油的转酯化反应工艺进行优化,考察了甲醇用量、体系含水量、酶的添加量和反应温度对生物柴油收率的影响,使生物柴油最终收率大于86.0%.在有机溶剂体系中选择不同有机溶剂作为助溶剂进行转酯化反应,发现logP值在4.0～4.5的有机溶剂具有较好的转化效果.其中以正庚烷为助溶剂的转酯化反应具有最高的生物柴油收率86.7%.在无溶剂体系中RCL催化转化油酸和模拟高酸价油脂合成脂肪酸甲酯的研究表明,该酶具有很好的催化合成生物柴油的潜力.     

Preparation of Biodiesel with Liquid Synergetic Lipases from Rapeseed Oil Deodorizer Distillate

To reduce industrial production cost, cheap and easily available rapeseed oil deodorizer distillates were used as feedstock to prepare biodiesel in this study. As a result, liquid forms of Candida rugosa lipase and Rhizopus oryzae lipase (ROL) were functioned as new and effective catalysts with biodiesel yield of 92.63% for 30 h and 94.36% for 9 h, respectively. Furthermore, the synergetic effect between the two lipases was employed to enhance biodiesel yield with a result of 98.16% in 6 h under optimized conditions via response surface methodology. The obtained conversion rate surpassed both yields of the individual two lipases and markedly shortened the reaction time. The resultant optimal conditions were ROL ratio 0.84, water content 46 wt% (w/w), reaction temperature 34 °C, and reaction time 6 h.     

基于氯化镁饱和溶液中固定化脂肪酶Lipozyme TL IM催化光皮树油脂合成生物柴油

基于氯化镁饱和溶液反应体系中,对采用固定化脂肪酶Lipozyme TL IM催化光皮树油脂转化为生物柴油的工艺进行了研究。考察了固定化脂肪酶Lipozyme TL IM催化光皮树油转酯化的工艺中甲醇的用量、固定化脂肪酶的添加量、摇床的转速和反应时间对生物柴油产率的影响。实验结果表明,采用氯化镁饱和溶液反应体系,在醇油摩尔比为3∶1,固定化酶Lipozyme TL IM用量为光皮树油质量的20%,摇床转速为150 r/min,反应8 h时,生物柴油产率最高,达到86.5%。与传统的三步甲醇醇解或者有机溶剂反应体系比较,采用的氯化镁饱和溶液体系的酶稳定性更好,反应效率更高,有效地解决了酶在甲醇中失活的问题,生产成本低,可成为生产生物柴油的新工艺。     

Optimization of enzymatic production of biodiesel from castor oil in organic solvent medium

We studied the production of fatty acid ethyl esters from castor oil using n-hexane as solvent and two commercial lipases, Novozym 435 and Lipozyme IM, as catalysts. For this purpose, a Taguchi experimental design was adopted considering the following variables: temperature (35–65°C), water (0–10 wt/wt%), and enzyme (5–20 wt/wt%) concentrations and oil-to-ethanol molar ratio (1∶3 to 1∶10). An empirical model was then built so as to assess the main and cross-variable effects on the reaction conversion and also to maximize biodiesel production for each enzyme. For the system containing Novozym 435 as tatalyst the maximum conversion obtained was 81.4% at 65°C, enzyme concentration of 20 wt/wt%, water concentration of 0 wt/wt%, and oil-to-ethanol molar ratio of 1∶10. When the catalyst was Lipozyme IM, a conversion as high as 98% was obtained at 65°C, enzyme concentration of 20 wt/wt%, water concentration of 0 wt/wt%, and oil-to-ethanol molar ratio of 1∶3.     

The Purification and Characterization of Lipases from <Emphasis Type="Italic">Lasiodiplodia theobromae</Emphasis>, and Their Immobilization and Use for Biodiesel Production from Coconut Oil

The coconut kernel-associated fungus, Lasiodiplodia theobromae VBE1, was grown on coconut cake with added coconut oil as lipase inducer under solid-state fermentation conditions. The extracellular-produced lipases were purified and resulted in two enzymes: lipase A (68,000 Da)—purified 25.41-fold, recovery of 47.1%—and lipase B (32,000 Da)—purified 18.47-fold, recovery of 8.2%. Both lipases showed optimal activity at pH 8.0 and 35 °C, were activated by Ca²⁺, exhibited highest specificity towards coconut oil and p-nitrophenyl palmitate, and were stable in iso-octane and hexane. Ethanol supported higher lipase activity than methanol, and n-butanol inactivated both lipases. Crude lipase immobilized by entrapment within 4% (w/v) calcium alginate beads was more stable than the crude-free lipase preparation within the range pH 2.5–10.0 and 20–80 °C. The immobilized lipase preparation was used to catalyze the transesterification/methanolysis of coconut oil to biodiesel (fatty acyl methyl esters (FAMEs)) and was quantified by gas chromatography. The principal FAMEs were laurate (46.1%), myristate (22.3%), palmitate (9.9%), and oleate (7.2%), with minor amounts of caprylate, caprate, and stearate also present. The FAME profile was comparatively similar to NaOH-mediated transesterified biodiesel from coconut oil, but distinctly different to petroleum-derived diesel. This study concluded that Lasiodiplodia theobromae VBE1 lipases have potential for biodiesel production from coconut oil.     

Biodiesel fuel production by the transesterification reaction of soybean oil using immobilized lipase

The enzymatic alcoholysis of soybean oil with methanol and ethanol was investigated using a commercial, immobilized lipase (Lipozyme RMIM). The effect of alcohol (methanol or ethanol), enzyme concentration, molar ratio of alcohol to soybean oil, solvent, and temperature on biodiesel production was determined. The best conditions were obtained in a solvent-free system with ethanol/oil molar ratio of 3.0, temperature of 50 degrees C, and enzyme concentration of 7.0% (w/w). Three-step batch ethanolysis was most effective for the production of biodiesel. Ethyl esters yield was about 60% after 4 h of reaction.     

Biodiesel production and comparison using commercial lipase and chemical catalyst from Cassia auriculata oil

In this present investigation, Cassia auriculata was explored as a feedstock for production of biodiesel. Transesterification reaction was performed by both enzyme (lipase) and chemical (potassium hydroxide) catalyst with diverse acyl acceptors such as methanol, ethanol, propanol, n-propanol, butanol, n- butanol, and finally their biodiesel yield were also recorded. Process optimization was performed by both one factor at a time method and response surface method. The maximal biodiesel yield of 92% (weight/weight) was obtained at the following optimal conditions: Oil:Methanol molar ratio of 1:6 (moles/moles), the lipase concentration of 2% (weight/weight), at 35 ?°C and 120 ?min. The highest biodiesel yield from Cassia auriculata oil was occurred with excess methanol that aids the equilibrium shift in the forward direction. The kinetics of the transesterification reaction was investigated under optimal conditions and the activation energy was found to be 14.91 ?kJ/mol. Simultaneously Gas Chromatography – Mass Spectroscopy was also carried out for the biodiesel produced from Cassia auriculata and the same has been reported. The GC analysis declares the existence of fatty acid esters like hexadecanoic acid methyl ester, methyl stearate and the peak with retention time 12.8 ?min signifies the evidence of hexadecanoic acid methyl ester with 28% of yield content. This investigation also evaluated the biodiesel quality produced from lipase-transesterified Cassia auriculata oil based on fuel properties. Biodiesel properties Flash Point (FC), Pour Point (PP) and kinematic viscosity were compared with American (ASTM 6751) and European (EN 14214) Standards. Cassia auriculata oil had PP 6.7 ?°C and Kinematic viscosity (813 ?kg/m³) that agreed with both the standards. Thus this study showed that Cassia auriculata oil could be a better fuel alternative with further improvement of fuel properties.     

有机相酶催化氨解反应拆分制备(R)-4-氯-3-羟基丁酸乙酯

通过脂肪酶催化的氨解反应拆分4-氯-3-羟基丁酸乙酯. 经过对脂肪酶及反应溶剂的筛选, 确定最佳脂肪酶及溶剂分别为Novozym435和二氧六环; 并在该反应体系中考察了温度、底物浓度、酶浓度与摇床转速对反应的影响. 综合考虑反应的反应速度和对映体选择率, 确定较佳的反应条件为: 温度30 ℃、底物浓度0.5 mol/L、酶量10 mg/mL、摇床转速200 n/min. 反应35 h后, ee可以达到99%以上, 此时转化率为57.7%.     

Enzyme catalyzed production of biodiesel from olive oil

Biodiesel (fatty acid methyl esters) was produced by transesterification of triglycerides (triolein) present in olive oil with methanol and Novozym435. The effect of the molar ratio of methanol to triolein, semibatch (stepwise addition of methanol) vs batch operation, enzyme activity, and reaction temperature on overall conversion was determined. Stepwise methanolysis with a 3:1 methanol to triolein molar ratio and an overall ratio of 8:1 gave the best results. The final conversion and yield of biodiesel were unaffected by initial enzyme concentrations greater than 500 U/mL olive oil. The optimum reaction temperature was 60 degrees C. Comparison of conversion data between a test-tube scale reactor and a 2-L batch reactor revealed that the difference in conversion was within 10%. Experiments were also carried out with used cooking oil; the conversion with used cooking oil was slightly lower but no major differences were observed. The efficacy of Novozym435 was determined by reusing the enzyme; although the enzyme's relative activity decreased with reuse, it still retained 95% of its activity after five batches and more than 70% after as many as eight batches.     

Enzymatic synthesis of sorbitan methacrylate according to acyl donors

Recently, sugar polymers have been considered for use as biomaterials in medical applications. These biomaterials are already used extensively in burn dressings, artificial membranes, and contact lenses. In this study, we investigated the optimum conditions under which the enzymatic synthesis of sorbitan methacrylate can be affected using Novozym 435 in t-butanol from sorbitan and several acyl donors (ethyl methacrylate, methyl methacrylate, and vinyl methacrylate). The enzymatic synthesis of sorbitan methacrylate, catalyzed by Novozym 435 in t-butanol, reached an approx 68% conversion yield at 50 g/L of 1,4-sorbitan, 5% (w/v) of enzyme content, and a 1∶5 molar ratio of sorbitan to ethyl methacrylate, with a reaction time of 36 h. Using methyl methacrylate as the acyl donor, we achieved a conversion yield of approx 78% at 50 g/L of 1,4-sorbitan, 7% (w/v) of enzyme content, at a 1∶5 molar ratio, with a reaction time of 36 h. Sorbitan methacrylate synthesis using vinyl methacrylate as the acyl donor was expected to result in a superior conversion yield at 3% (w/v) of enzyme content, and at a molar ratio greater than 1∶2.5. Higher molar ratios of acyl donor resulted in more rapid conversion rates. Vinyl methacrylate can be applied to obtain higher yields than are realized when using ethyl methacrylate or methyl methacrylate as acyl donors in esterification reactions catalyzed by Novozym 435 in organic solvents. Enzyme recycling resulted in a drastic reduction in conversion yields.     

Combining Enzymatic Esterification with Conventional Alkaline Transesterification in an Integrated Biodiesel Process

An integrated biodiesel process that combines enzymatic esterification and alkaline transesterification is suggested. With focus on the enzymatic step, the paper provides proof of concept and suggestions for further process development. Hence, palm fatty acid distillate (PFAD) has been enzymatically converted to fatty acid methyl esters in a two-step process using the immobilized lipase Novozym 435 in packed-bed columns. With only a small excess of methanol, the first reaction stage could reduce the free fatty acid (FFA) content from 85% to 5%. After removal of water by simple phase separation, it was possible to lower the FFA content to 2.5% in a second reaction stage. Both reaction stages are relatively fast with suggested reaction times of 15 min in column 1 (productivity 10 kg/kg/h) and 30 min in column 2 (productivity 5 kg/kg/h), resulting in 15% FFA after column 1 and 5% FFA after column 2. A lifetime study indicated that approximately 3,500 kg PFAD/kg Novozym 435 can be treated in the first reaction stage before the enzyme has become fully inactivated. With further optimization, the enzymatic process could be a real alternative to today’s sulfuric acid catalyzed process.     

固定化脂肪酶催化碳酸二甲酯制备生物柴油的研究

以介孔硅材料(MPS)为载体将脂肪酶固定化,以碳酸二甲酯为酰基受体,对固定化酶催化碳酸二甲酯进行了反应路径(原料油)、反应条件(反应温度、碳酸二甲酯的用量、加水量)的优化,在最佳的条件下对实验过程中所用的固定化酶进行重复使用性的考察.实验结果表明,不同种油与碳酸二甲酯反应在同定化酶的催化下制备生物柴油的产率以麻疯树油为最高,最佳反应条件是碳酸二甲酯的浓度为16mL/g、不加水,在50℃下反应24h,生物柴油得率达81.6％.     

Lipase-Catalyzed Transesterification of Rapeseed Oil for Biodiesel Production with tert-Butanol

Biodiesel is a fatty acid alkyl ester that can be derived from any vegetable oil or animal fat via the process of transesterification. It is a renewable, biodegradable, and nontoxic fuel. In this paper, we have evaluated the efficacy of a transesterification process for rapeseed oil with methanol in the presence of an enzyme and tert-butanol, which is added to ameliorate the negative effects associated with excess methanol. The application of Novozym 435 was determined to catalyze the transesterification process, and a conversion of 76.1% was achieved under selected conditions (reaction temperature 40 °C, methanol/oil molar ratio 3:1, 5% (w/w) Novozym 435 based on the oil weight, water content 1% (w/w), and reaction time of 24h). It has also been determined that rapeseed oil can be converted to fatty acid methyl ester using this system, and the results of this study contribute to the body of basic data relevant to the development of continuous enzymatic processes.     

新型酰基供体用于酶法动力学拆分制备(R)-1-(2-萘基)乙胺的研究

首次成功实现了光学纯(R)-1-(2-萘基)乙胺的高效酶法动力学拆分制备,考察了脂肪酶种类、溶剂、酰基供体、底物浓度、反应温度等对拆分效果的影响,发现新型酰基供体——正戊酸对氯苯酯能够很好地抑制非酶促自催化酰胺化效应.在甲苯溶剂中,底物浓度300 mmol/L,40℃条件下,采用该供体在脂肪酶Novozym 435催化下,动力学拆分反应8 h转化率达到理论最佳值50%,eep>99%.     

柴油溶剂中脂肪酶催化高酸值废油脂酯化制备生物柴油

采用0＃柴油作为反应溶剂,利用固定化脂肪酶催化高酸值废油脂与甲醇酯化反应制备生物柴油。来源于Candida antarctica的固定化脂肪酶Novozym435在0＃柴油溶剂中具有极高的催化活性。以酸价高达157×10-3的废油脂为原料,废油脂质量比10%的Novozym435,甲醇与废油脂初始摩尔比2∶1,0＃柴油与废油脂质量比5∶1,摇床摇速170r/min,50℃下反应2h甲酯化率可达95.10%。0＃柴油作为反应溶剂有效地溶解了高酸值废油脂和甲醇,降低了反应体系的黏度和消除了甲醇对Novozym435的负面影响,提高了Novozym435的稳定性。同时,0＃柴油溶剂对未脱胶废油脂中残留的对脂肪酶有害的磷脂等胶类物质具有一定的稀释作用。该工艺省却了溶剂蒸馏的繁琐工序,直接得到脂肪酸甲酯和石化柴油的混合燃料。     

Monitoring lipase-catalyzed methanolysis of sunflower oil by reversed-phase high-performance liquid chromatography: elucidation of the mechanisms of lipases

Reversed-phase high-performance liquid chromatography (RP-HPLC) with UV detection at 210 nm was used to monitor the formation of the major compounds during the lipase-catalyzed transesterification reaction of sunflower oil with methanol. Individual triacylglycerols, diacylglycerols, monoacylglycerols as well as fatty acids and their corresponding methyl esters were separated using acetonitrile/acetone as a mobile phase and a combined linear gradient-isocratic-step gradient-isocratic elution procedure. Another relatively short method consisting of a linear gradient elution followed by an isocratic elution gave similar results, yet with lower resolution. HPLC/mass spectrometry with an ion trap analyzer and atmospheric pressure chemical ionization source was used for the identification of the individual compounds. Individual calibration curves obtained with UV detection at 210 nm were found to be of use for quantitative analyses of double-bond containing methyl esters and acylglycerols. The use of the RP-HPLC methods in the elucidation of the mechanisms of three immobilized lipases, namely Lipozyme TL IM, Lipozyme RM IM and Novozym 435, in biodiesel production was described.     

Enzymatic Synthesis of Biodiesel via Alcoholysis of Palm Oil

The enzymatic alcoholysis of crude palm oil with methanol and ethanol was investigated using commercial immobilized lipases (Lipozyme RM IM, Lipozyme TL IM). The effect of alcohol (methanol or ethanol), molar ratio of alcohol to crude palm oil, and temperature on biodiesel production was determined. The best ethyl ester yield was about 25 wt.% and was obtained with ethanol/oil molar ratio of 3.0, temperature of 50 °C, enzyme concentration of 3.0 wt.%, and stepwise addition of the alcohol after 4 h of reaction. Experiments with 1 and 3 wt.% of KOH and 3 wt.% of MgO were carried out to compare their catalytic behavior with the enzymatic transesterification results. The commercial immobilized lipase, Lipozyme TL IM, showed the best catalytic performance.