固定化细胞生产L—苹果酸动力学及两种酶反应器的比较

研究了产氨短杆菌ＭＡ－２,黄色短杆菌ＭＡ－３的固定化细胞在富马酸铵转化体系中生成Ｌ－苹果酸的动力学参数,同时比较了固定化细胞在填充床及连续机械搅拌反应器中酶转化反应的差异。研究结果表明：当转化率小于４０％时,酶反应在两种反应器所需的停留时间相当。随着转化率的提高,填充床反应器较连续机械搅拌反应器所需的停留时间短且不会因剪切力使固定化颗粒受到损伤,因此,在富马酸铵体系中用固定化酶生产Ｌ－苹果酸采用填     

固定化嗜热脂肪芽孢杆菌合成低聚半乳糖

利用海藻酸钙、明胶和壳聚糖为固定化载体包埋嗜热脂肪芽孢杆菌细胞合成低聚半乳糖 (GOS)。通过比较三种方法的酶活力回收、最适反应条件、GOS的得率和和载体机械强度 ,选择明胶作为固定化细胞的载体。反应体系的温度、pH、乳糖浓度、乳糖的转化率和载体的传质阻力对GOS合成有明显影响。在CSTR反应器中水解 60 %乳糖 ,GOS最大得率为31 2 % ,经过 96h( 8批反应 ) ,产物得率为原来的 88%。在空速 0 0 9h- 1条件下 ,利用填充床反应器连续水解乳糖 ,GOS的得率和反应器生产能力分别为 31 5%和 1 7 4g (L·h) ,连续反应1 40h,GOS得率下降 2 0 %。产物经过活性炭柱层柱分离纯化 ,通过13C NMR鉴定四糖的化学结构为 β D Gal ( 1→ 3) D Gal ( 1→ 6) D G ( 1→ 4) D Glu。     

酶法合成糖及糖醇酯

以脂肪酸为酰基供体,糖和糖醇为酰基受体,利用吸附到涤棉布上的假丝酵母脂肪酶作催化剂,在含叔丁醇的系统中,研究了酯化反应条件。酯化最适温度和pH值分别为40℃～45℃和50～75。在酰基供体中,以亚油酸和油酸最好,C8到C22的饱和脂肪酸的酯化程度相仿。在23种糖和糖醇中,果糖、木糖、海藻糖、山梨糖、木糖醇、甘露醇以及异丙基葡萄糖和甲基葡萄糖比其它酰基受体的酯化率高。糖醇的酯化程度明显高于相应的糖。此外,酰基供体与受体的摩尔比大于2∶1时,有利于酯化。在由30mmol(085g)油酸,02mmol山梨醇(0036g),3mL叔丁醇和30mg固定化酯肪酶(600u)组成的反应系统中,40℃震荡反应48h,以等摩尔的底物计算,酯化程度达到90%以上。反应产物经薄层色谱鉴定为单酯和双酯。     

D201—GM大孔树脂吸附交联固定菊粉酶的研究

克鲁维酵母Ｙ－８５产生的胞内菊粉酶,以Ｄ２０１－ＧＭ大孔径阴离子交换树脂吸附交联法固定化,其制备固定化酶（ＩＥ）的适宜条件：树脂吸附酶时ｐＨ６．５、温度３０℃、时间３ｈ;交联时戊二醛浓度０．０３％、温度４℃、时间３ｈ。上述条件下制得ＩＥ的活性产率可达６２％,水解菊粉底物的最适温度５５℃,对热的稳定性和贮存稳定性均有明显提高,用ＩＥ填充床反应器连续降解菊粉抽提液（总糖４．５％）的实验结果表明,进料空     

固定化假丝酵母1619脂肪酶催化油酸油醇酯的合成

比较了14种不同来源的脂肪酶催化油酸油醇酯的合成。其中,假丝酵母(Candidasp．)1619脂肪酶酯化能力最强,以硅藻土为载体,分别按0．1％添加椰子油、吐温80．按l％添加MgSO43种共固定物,醇化反应初速度提高了1．5倍。此固定化酶催化油酸油醇酯合成的最适温度为30℃,0～60℃下反应24h的酯化率均在90％以上,100℃下还有10．25％的酯化率。最适酯化pH6．0。反应中去水,可使终酯化率提高到99％。在添加的23种有机溶剂中,以异辛烷促进酯化的效果最好．正壬烷和正己烷次之。此固定化酶在28℃下批式重复反应的半衰期为990h,柱式固定床反应器中28℃连续运转1000h后酯化率为78％。     

大孔树脂共吸附固定葡聚糖-脂肪酶催化合成香茅酯

以大孔树脂为载体对脂肪酶和葡聚糖进行共吸附固定,考察葡聚糖的共吸附对脂肪酶固定化效果的影响,并应用所得固定化酶在无溶剂体系催化合成月桂酸香茅酯。结果表明:在固定化过程中添加终质量浓度为0.75mg/m L的葡聚糖可提高固定化酶酶活回收率,使用该固定化酶在无溶剂体系催化月桂酸与香茅醇酯化,酶的催化效率及操作稳定性均有提高。在底物月桂酸与香茅醇物质的量的比为1∶1,加入1 U的固定化脂肪酶,在50℃时无溶剂体系中反应10 h,反应的酯化率达95.3%。添加终质量浓度为0.75 mg/m L的T-20及T-40(葡聚糖相对分子质量为2×10~4和4×10~4)制备的固定化酶可将到达95%酯化率的反应时间缩短至6 h,其中添加T-40的固定化酶经10次连续催化后,仍保持75%以上的催化活性。     

螺旋纤维床固定化生物反应器同时产酶降解壳聚糖的研究

采用多孔聚酯泡沫固定里氏木霉,在鼓泡柱固定化反应器中同时产酶降解壳聚糖。结果表明通过控制降解时间可以得到不同平均聚合度的降解产物。在28℃,pH4.8,通气量3vvm条件下,利用固定化反应器,在30d内连续进行10批同时产酶降解试验,结果发现壳聚糖酶活力和壳聚糖降解率能保持稳定。每批产生的壳聚糖酶活力平均达到0.15u/mL以上,壳聚糖平均降解率为73%。     

二氧化硅纳米材料固定中性脂肪酶的条件优化及其特性

以二氧化硅纳米材料为载体,采用吸附法对脂肪酶进行固定化,研究了不同条件对固定化脂肪酶的催化活性的影响,得到最佳的固定化条件:给酶量为28300U/g,固定化温度为45oC,pH值为7.5,时间为10h,此时固定化酶的活力约为3867U/g载体。固定化酶的最适反应温度为45oC,比游离酶的反应温度高5oC,最适pH下降到5.5,低于游离酶的反应pH(pH7)。固定化酶的热稳定性和pH稳定性较游离酶有了很大的提高,其在70oC以下能保持70%以上的酶活力,而游离酶在50oC下残余酶活力仅为30%。在pH5～8的范围内,固定化酶的酶活力能保持50%以上,而游离酶只能保持20%左右。用固定化的中性脂肪酶催化不同的油品,即大豆油、菜籽油及泔水油生产生物柴油,菜籽油的酯化率最高。     

有机介质中脂肪酶催化合成亚麻酸甘油酯

研究固定化脂肪酶在有机溶剂中催化亚麻酸甘油酯的合成反应。考察了有机溶剂、酯化温度、物料比、催化剂用量、酯化时间等因素对酯化率的影响。结果表明:以正己烷为溶剂、酯化温度60℃、酸醇摩尔比1:2.5条件下、反应24h,亚麻酸甘油酯化率为90%以上。     

离子液体中固定化脂肪酶催化拆分（±）-薄荷醇

以自制的平均粒径为4．5um磁性高分子微球为载体,采用离子交换法固定化Candida rugosa脂肪酶,催化（±）-薄荷醇的酯化反应,以考察反应时间、pH、反应温度、水活度等因素对酶的固定化以及酯化反应的影响。在固定化反应150min、pH5．0、酯化反应温度30℃、固定化酶的水活度为0．78的条件下,所制备的固定化脂肪酶在离子液体[bmim]PF6中催化拆分（±）-薄荷醇的效果最佳,与游离酶相比固定化脂肪酶的立体选择性有很大的提高,对映体过量率可达93％,对映体选择值为35。     

Continuous enzymatic esterification of glycerol with (poly)unsaturated fatty acids in a packed-bed reactor

Enzymatic synthesis of mono-, di-, and triacyglycerols from (poly)unsaturated fatty acids (linoleic, oleic, and conjugated linoleic acids) has been studied as a solvent-free reaction in a packed-bed reactor containing an immobilized lipase from Mucor miehei. The extents of the esterification reactions of interest are primarily determined by the molar ratio of glycerol to fatty acid because the presence of excess glycerol as a immiscible phase is responsible for reducing the activity of the water produced by the esterification reactions. For molar ratios of fatty acid to glycerol of less than 1.5, the percentage of the fatty acid esterified decreases quasi-linearly with an increase in this molar ratio. By appropriate manipulation of the fluid-residence time, one can control the relative proportions of the various acylglycerols in the effluent stream. At the outlet of the reactor, one observes excellent spontaneous separation of the glycerol and acylglycerol/fatty acid phases. At 50 degrees C and a fluid residence time of 1 hour, as much as 90% of the fatty acid can be esterified when the molar ratio of fatty acid to glycerol is 0.33 or less.     

Green enzymatic production of glyceryl monoundecylenate using immobilized Candida antarctica lipase B

Enzymatic synthesis of glyceryl monoundecylenate (GMU) was performed using indigenously immobilized Candida anatarctica lipase B preparation (named as PyCal) using glycerol and undecylenic acid as substrates. The effect of molar ratio, enzyme load, reaction time, and organic solvent on the reaction conversion was determined. Both batch and continuous processes for GMU synthesis with shortened reaction time were developed. Under optimized batch reaction conditions such as 1:5 molar ratio of undecylenic acid and glycerol, 2?h of reaction time at 30% substrate concentration in tert-butyl alcohol, conversion of 82% in the absence of molecular sieve, and conversion of 93% in the presence of molecular sieve were achieved. Packed bed reactor studies resulted in high conversion of 86% in 10-min residence time. Characterization of formed GMU was performed by FTIR, MS/MS. Enzymatic process resulted in GMU as a predominant product in high yield and shorter reaction time periods with GMU content of 92% and DAG content of 8%. Optimized GMU synthesis in the present study can be used as a useful reference for industrial synthesis of fatty acid esters of glycerol by the enzymatic route.     

基于响应面设计脂肪酶Novo435催化合成甘油二酯的工艺优化

以甘油、油酸为原料,优化在无溶剂体系中以固定化脂肪酶Novo435催化合成甘油二酯（diglyceride,DAG）的工艺。系统考察底物摩尔比（油酸／甘油）、反应温度、时间和加酶量等因素对油酸转化率和甘油二酯含量影响的基础上,利用响应面试验设计优化各主效因子,并经回归分析获得最优的工艺条件。所得最优条件：油酸与甘油底物摩尔比2．27、反应温度48．14℃、反应时间6．3h、加酶量1．68％。在此条件下,实验测得油酸转化率为45．42％,甘油二酯质量分数为70．01％,与响应面模型预测值吻合。     

Response surface modeling of 1-stearoyl-3(2)-oleoyl glycerol production in a pilot packed-bed immobilized Rhizomucor miehei lipase reactor

A dual response approach using diacylglycerol (DAG) and triacylglycerol (TAG) as responses for optimization of 1-stearoyl-3(2)-oleoyl glycerol-enriched DAG synthesis using response surface methodology (RSM) was investigated. Four variables from a lipase-catalyzed esterification reaction were optimized using a central composite rotatable design. The following optimized conditions yielded 51 wt.% DAG and 22 wt.% TAG: reaction temperature of 55 °C, enzyme dosage of 9.5 wt.%, fatty acid/glycerol molar ratio of 2.1 and reaction time of 3 h. Results were repeatable at 10 kg production scale in a pilot packed-bed enzyme reactor. No significant losses in enzyme activity or changes in fatty acid selectivity on DAG synthesis were observed during the five pilot productions. Lipozyme RM IM showed selectivity towards the production of stearic acid enriched DAG. The purity of DAG oil after purification was 90 wt.%.     

Synthesis of monoacylglycerol containing pinolenic acid via stepwise esterification using a cold active lipase

High purity monoacylglycerol (MAG) containing pinolenic acid was synthesized via stepwise esterification of glycerol and fatty acids from pine nut oil using a cold active lipase from Penicillium camembertii as a biocatalyst. Effects of temperature, molar ratio, water content, enzyme loading, and vacuum on the synthesis of MAG by lipase‐catalyzed esterification of glycerol and fatty acid from pine nut oil were investigated. Diacylglycerol (DAG) as well as MAG increased significantly when temperature was increased from 20 to 40°C. At a molar ratio of 1:1, MAG content decreased because of the significant increase in DAG content. Water has a profound influence on both MAG and DAG content through the entire course of reaction. The reaction rate increased significantly as enzyme loading increased up to 600 units. Vacuum was an effective method to reduce DAG content. The optimum temperature, molar ratio, water content, enzyme loading, vacuum, and reaction time were 20°C, 1:5 (fatty acid to glycerol), 2%, 600 units, 5 torr, and 24 h, respectively. MAG content further increased via lipase‐catalyzed second step esterification at subzero temperature. P. camembertii lipase exhibited esterification activity up to ?30°C. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Determination of cis,cis-methylene interrupted polyunsaturated fatty acids in aqueous solutions by lipoxygenase chemiluminescence

The chemiluminescent reaction of luminol during lipoxygenase-catalyzed oxygenations was studied with the purpose of developing a specific luminometric assay for cis,cis-1,4-pentadiene fatty acids directly in aqueous solutions. The addition of picomole levels of either linoleic or arachidonic acids to reaction systems containing 0.04 mM luminol and 40 micrograms/ml of purified soybean lipoxygenase-1 gave light emission curves with a single sharp maximum. Under these conditions the peak heights were linearly dependent on the fatty acid concentration and the detection limit for both of the fatty acids was 2 pmol with a signal to noise ratio of 2. For maximum reproducibility of the assays a procedure for the proper quantitation of the enzyme was developed. The fact that the assay proved to be relatively interference-free was ascribed to the high molar enzyme/substrate ratio (above 1).     

Enhancing operational stability and exhibition of enzyme activity by removing water in the immobilized lipase‐catalyzed production of erythorbyl laurate

Erythorbyl laurate was continuously synthesized by esterification in a packed‐bed enzyme reactor with immobilized lipase from Candida antarctica. Response surface methodology based on a five‐level three‐factor central composite design was adopted to optimize conditions for the enzymatic esterification. The reaction variables, such as reaction temperature (10–70°C), substrate molar ratio ([lauric acid]/[erythorbic acid], 5–15), and residence time (8–40 min) were evaluated and their optimum conditions were found to be 56.2°C, 14.3, and 24.2 min, respectively. Under the optimum conditions, the molar conversion yield was 83.4%, which was not significantly different (P < 0.05) from the value predicted (84.4%). Especially, continuous water removal by adsorption on an ion‐exchange resin in a packed‐bed enzyme reactor improved operational stability, resulting in prolongation of half‐life (2.02 times longer compared to the control without water‐removal system). Furthermore, in the case of batch‐type reactor, it exhibited significant increase in initial velocity of molar conversion from 1.58% to 2.04%/min. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:882–889, 2013     

A practical approach to obtain high yield lipase-mediated synthesis of octyl caprylate with Novozym 435

Abstract

This study evaluated coupled effects of molar ratio of substrates and enzyme loading in a solvent-free system using a simple mathematical approach to obtain high conversions on octyl caprylate synthesis with Novozym 435. When molar ratios of caprylic acid to n-octanol (1:1 and 1:3) were evaluated with enzyme loadings of 1% to 4% (wt/wt acid), an interdependence between the masses of reagents and the enzymes was observed, that was expressed as a mathematical relation. The study of this relation, named as SER, indicated a specific range of reaction conditions that resulted in conversions above 90%. The most suitable condition corresponded to an acid:alcohol molar ratio of 1:1.3 and an enzyme loading of 1.5%, resulting in 94.5% of conversion at 65?°C in 3?hours of reaction. A different reaction system (bottle reactor) was used to evaluate the influence of reagents mixture and heat distribution. The use of a bottle reactor allowed yield improvement that reached 99.3%. At this condition, Novozym 435 was reused, without washing steps, in three subsequent batches keeping high conversion. A possible balance between the shift of chemical equilibrium by stoichiometric excess of reagents and enzymatic inhibition effects by substrates can be expressed mathematically in a convenient way, helping to predict the behaviour of synthesis in different conditions. The mathematical relation proposed, SER, allowed the achievement of 99% of conversion on enzymatic synthesis of octyl caprylate.     

β-galactosidase-catalyzed synthesis of 3-O-β-D-galactopyranosyl-sn-glycerol: Optimization by response surface methodology

In this study, the synthesis of 3-O-β-D-galactopyranosyl-sn-glycerol (GG) was performed by the reverse hydrolysis of D-galactose and glycerol using β-galactosidase from Kluyveromyces lactis. Four process variables, reaction temperature (30.0–45.0?°C), reaction time (24–48?h), enzyme concentration (150.00–350.00?U/mL), and substrate molar ratio (glycerol:D-galactose, 7.5:12.5?mmol/mmol) were investigated and optimized via response surface methodology (RSM) for optimal GG synthesis. Both quadratic equations and the optimal reaction conditions were established. Results showed that the four variables, i.e., reaction temperature, reaction time, enzyme concentration, and substrate molar ratio had significant (p?β-galactosidase concentration and 8.65:1.00 of substrate molar concentration ratio (glycerol: D-galactose) at 39.8?°C and 48?h of reaction. Under these conditions, the GG concentration was 140.03?g/L and GG yield was 55.71%, which both were close to the predicted values (143.26?g/L and 56.73%). This finding proves the RSM to be a useful tool in optimizing process conditions for GG synthesis.     

Bioreaction Engineering Leading to Efficient Synthesis of L‐Glyceraldehyd‐3‐Phosphate

Enantiopure L‐glyceraldehyde‐3‐phosphate (L‐GAP) is a useful building block in natural biological and synthetic processes. A biocatalytic process using glycerol kinase from Cellulomonas sp. (EC 2.7.1.30) catalyzed phosphorylation of L‐glyceraldehyde (L‐GA) by ATP is used for the synthesis of L‐GAP. L‐GAP has a half‐life of 6.86 h under reaction conditions. The activity of this enzyme depends on the Mg²⁺ to ATP molar ratio showing maximum activity at the optimum molar ratio of 0.7. A kinetic model is developed and validated showing a 2D correlation of 99.9% between experimental and numerical data matrices. The enzyme exhibits inhibition by ADP, AMP, methylglyoxal and Ca²⁺, but not by L‐GAP and inorganic orthophosphate. Moreover, equal amount of Ca²⁺ exerts a different degree of inhibition relative to the activity without the addition of Ca²⁺ depending on the Mg²⁺ to ATP molar ratio. If the Mg²⁺ to ATP molar ratio is set to be at the optimum value or less, inorganic hexametaphosphate (PPi6) suppresses the enzyme activity; otherwise PPi6 enhances the enzyme activity. Based on reaction engineering parameters such as conversion, selectivity and specific productivity, evaluation of different reactor types reveals that batchwise operation via stirred‐tank reactor is the most efficient process for the synthesis of L‐GAP.