生物法合成维生素C棕榈酸酯

研究了不同的脂肪酶在有机溶剂体系中催化合成L-维生素C棕榈酸酯的反应。针对维生素C在有机溶剂中溶解度较低这一问题,对催化合成维生素C棕榈酸酯反应的脂肪酶和反应介质进行比较,同时对影响合成维生素C棕榈酸酯反应的因素（温度、底物浓度、底物摩尔比、反应时间和酶量等）进行探讨,优化了反应条件：在10mL的丙酮中,1.094g棕榈酸与0.107g维生素C在酶量为20％（W/W, 固定化酶/维生素C）的固定化脂肪酶催化下,初始含0.4nm分子筛20%,温度为60℃,转速为200r/min,反应48h转化率可以达到80%,产物维生素C棕榈酸酯的浓度可达20g/L。     

日本根霉IFO5318胞外β-葡萄糖苷酶的纯化及部分特性

采用硫酸铵沉淀及柱层析等步骤纯化了日本根霉IFO5318的β—葡萄糖苷酶,回收率为22％。该酶分子量约为4.0×10~5,由四个相同大小的亚基组成;最适反应温度55℃,最适反应pH5.5;对热较敏感,但能在较大的pH范围内保持稳定。用对硝基苯基—β-D-吡喃葡糖苷为底物,测得的K_m和V_(max)值分别为0.825mg·ml~(-1)和135.4μmol·min~(-1)·mg~(-1)。该酶对纤维二糖的水解能力最强,SDS、Fe³⁺、Hg²⁺等对酶活力有抑制作用。     

固定化诺卡氏菌细胞生产L(+)酒石酸的研究

用明胶包埋酒石酸诺卡氏菌(Nocardia tartaricans SWl3—57)得到顺式环氧琥珀酸开环水解酶活力较高的固定化细胞。固定化细胞的最适温度为30～45℃,而游离细胞的最适温度为35～45℃。两者的最适pH均为8．0～9．0,固定化细胞的表观米氏常数Km为0．256mol／L,而游离细胞有底物抑制作用,在底物浓度小于0．45mol／L时Km为0．246mol／L。用固定化细胞装柱(y=100ml),pH8．S,温度37℃,稀释速率D=0．25h^-1,以0．5mol／L浓度顺式环氧琥珀酸钠溶液为底物,连续运转53d,平均产L(+)酒石酸66．95g／L,克分子转化率为92．09％,反应器生产能力达到16．58g／L·h。     

微生物脂肪酶在正庚烷中合成短链芳香酯的研究

用微生物脂肪酶在溶剂相中合成短链芳香酯的研究表明：脂肪酶在正庚烷中催化合成芳香酯转化率比水相中有明显的优势。在10个不同来源的商品脂肪酶中,选择了其中对己酸乙酯转化率在85％以上来自Mucor miehei,Candida rugosa和Porcine pancreas的脂肪酶对7个短链脂肪酸酯进行合成,其中Mucor miehei脂肪酶对这几个芳香酯48 h合成转化率均在90％以上,乙酸乙酯21．28g^-1．丙酸乙酯19．6gL^-1,丁酸乙酯26．61gL^-1,戊酸乙酯24．95gL^-1,己酸乙酯产量34.60gL^-1,丁酸异戊酯30.76gL^-1,乙酸异戊酯12．76gL^-1。同时．Mucor miehei脂肪酶在正庚烷批式反应中稳定性也较好,己酸乙酯半衰期为120h,最少的乙酸乙酯也在140h。并且还发现脂肪酶在正庚烷中分解酶活降低很多,但对其酯合成转化率没有直接的影响。     

蜜环菌胞外漆酶的合成、纯化及性质研究

研究了蜜环菌胞外漆酶合成条件和酶学性质。实验表明,培养基初始pH5.5、培养温度25℃有利于菌株产酶;与麦芽糖、山梨糖和半乳糖相比,纤维二糖和棉子糖作为碳源时漆酶产量更高;有机氮源比无机氮源有利于漆酶合成。泥炭提取液可显著诱导漆酶生成,当其含量为50%时,菌株漆酶最高产量是对照组的7倍。在蜜环菌发酵上清液中检测到3个漆酶同功酶组分,其主要活性（约占75%）组份漆酶A经 (NH₄)₂SO₄沉淀、制备级PAGE电泳和阴离子交换柱层析被分离纯化至电泳均一,SDSPAGE法测得酶亚基分子量59kD,凝胶过滤色谱法测定活性酶分子量58kD。纯化的漆酶A等电点pI为4.0,氧化愈创木酚的最适反应pH为5.6,最适温度为60℃,在60℃和65℃时半衰期分别为45min和36.8min,在pH5.2～7.2范围内稳定性较好。100mmol/L Cl^-对该酶有显著抑制作用,1mmol/L SO^2-₄ 对漆酶有激活作用,1mmol/L NaN₃可完全抑制酶活性,10 mmol/L EDTA对漆酶活没有明显影响,1mmol/L Cu²⁺对漆酶有激活作用。以愈创木酚为底物时,测得酶的K_m=1.026mmol/L,V_max=5μmol/(min·mg);以ABTS为底物时,测得其K_m=0.22mmol/L,V_max=69μmol/(min·mg)。     

固定化纤维二糖酶的研究

黑曲霉（Aspergillus niger LORRE 012）的孢子中富含纤维二糖酶,将这些孢子用海藻酸钙凝胶包埋后,可以方便有效地固定纤维二糖酶。固定化后的纤维二糖酶性能稳定,半衰期为38 d,耐热性和适宜的pH范围均比固定化前有所增加,其K_m和V_max值分别为6.01 mmol/L和7.06 mmol/(min·L)。利用固定化纤维二糖酶重复分批酶解10 g/L的纤维二糖,连续10批的酶解得率均可保持在97%以上;采用连续酶解工艺,当稀释率为0.4 h^-1,酶解得率可达98.5%。玉米芯经稀酸预处理后,其纤维残渣用里氏木霉（Trichoderma reesei）纤维素酶降解,酶解得率为69.5%;通过固定化纤维二糖酶的进一步作用,上述水解液中因纤维二糖积累所造成的反馈抑制作用得以消除,酶解得率提高到84.2%,还原糖中葡萄糖的比例由53.6%升至89.5%,该研究结果在纤维原料酶水解工艺中具有良好的应用前景。     

灵芝UDP-葡萄糖4-差向异构酶酶学性质研究及其应用

【背景】灵芝多糖是灵芝的重要活性物质之一。UDP-葡萄糖4-差向异构酶（UDP-glucose 4-epimerase,UGE,EC 5.1.3.2）是灵芝多糖合成途径中糖供体生成的重要酶,其参与了UDP-葡萄糖与UDP-半乳糖的相互转化,与多糖中半乳糖残基含量密切相关。【目的】通过对来源于灵芝的UGE基因进行异源表达,丰富灵芝多糖糖供体合成途径重要酶的酶学特性信息,深入了解灵芝多糖代谢合成途径。【方法】以灵芝菌株（Ganoderma lingzhi） CGMCC 5.26的cDNA为模板,克隆得到UGE基因GL30389,并在Escherichia coli BL21（DE3）中诱导表达,产物纯化后进行酶学性质、酶动力学、底物专一性及转化率的研究。【结果】灵芝UGE的分子量为45 kDa。最适反应pH值为6.0,在pH 7.0—9.0范围内有较好的稳定性;最适反应温度为30℃,温度在40℃时稳定性最好。Fe²⁺和Mg²⁺对UGE有激活作用。以UDP-葡萄糖为底物时,K_m为0.824 mmol/L,V_max为769.230 μmol/（L·min）,k_cat为1.333 s^—1,k_cat/K_m为1.618 L/（mmol·s）。灵芝UGE对D-葡萄糖、半乳糖醛酸及N-乙酰葡萄糖胺有催化活性。通过优化pH、温度、底物与酶的配比、添加金属离子将转化率从16.0%提升至39.4%。【结论】灵芝UGE与植物来源的UGE酶学性质较为相似,其催化效率优于大部分细菌来源的UGE。本研究丰富了灵芝多糖糖供体合成途径重要酶的酶学特性信息,有利于深入了解灵芝多糖代谢合成途径。     

微水体系中荧光假单胞菌脂肪酶催化合成单甘酯

研究了无溶剂微水体系中荧光假单胞菌脂肪酶 (PFL)催化油脂甘油解合成单甘酯的反应因素以及多温程非均相固液反应对单甘酯产率的影响。以初始体系最低共熔点 (PFL)取代临界温度学说中的油脂初熔点 ,通过考察不同IEP体系的甘油解 ,发现PFL酶促油脂甘油解时存在碳链基质特异性的函数关系 ,即反应物油脂中饱和碳残基的质量百分含量 (C₁₆+C₁₈)与单甘酯产率间符合以下多项式:Y =- 0.0006X³ +0.0592X²-0.8909X+26.753(13%<X<76.5%),式中X为C₁₆+C₁₈,Y为40℃时等温反应条件下的单甘酯产率。IEP为40℃时,最适等温反应条件如下：加水量3%～4.5%,加酶量为500μ/g油酯摩尔比1：2.5-5.0(油酯：甘油)反应温度40℃.实验条件下多步等程序降温反应48h后单甘酯最高产率为81.4%.     

甲基对硫磷水解酶参与催化相关结构的研究

甲基对硫磷水解酶（MPH）是一种新的有机磷水解酶。将完整的甲基对硫磷水解酶基因（mpd）构建入pUC19载体,使得mpd基因以自身的启动子在Escherichia coli DH5α中表达并得到了纯化。金属螯合实验发现MPH的活性不受金属螯合剂1, 10菲NFDA1啉的影响;但用电感耦合等离子发射光谱测定其金属含量显示MPH是金属酶,1mol酶中结合了2mol的Zn²⁺。为确定参与MPH催化活性的必需氨基酸,用化学修饰剂碳化二亚胺、二乙基焦磷酸酯、磷酸吡哆醛和丁二酮处理MPH,然后检测其残余酶活力,结果表明天冬氨酸、谷氨酸、赖氨酸和精氨酸残基与酶的催化活性无关;而二乙基焦磷酸酯对组氨酸侧链的化学修饰引起酶活性的大幅度的下降,其对酶活性的抑制率达到9.6h^-1,说明组氨酸是酶活力所必需的基团。这些结果为进一步研究酶的结构及对酶进行分子改造提供了必要的基础数据。     

金属螯合载体定向固定化木瓜蛋白酶的研究

以磁性金属螯合琼脂糖微球为载体,利用金属螯合配体（IDACu²⁺）与蛋白质表面供电子氨基酸相互作用的原理,定向固定了木瓜蛋白酶。固定化最适条件为Cu²⁺1.5×10^-2mol/g载体、固定化时间4h、固定化pH7.0、给酶量30mg/g载体。固定化酶的最适反应温度70℃、最适反应pH8.0,固定化酶的热稳定性明显高于溶液酶,固定化酶活力回收为68.4％,且有较好的操作稳定性,载体重复使用5次后固定化酶酶活为首次固定化酶79.71%。     

L-抗坏血酸洛芬酯非水相酶促合成的动力学与热力学

对酶法合成L-抗坏血酸洛芬酯(芬维C酯)的反应动力学与热力学进行研究,确定了最有效的酶促反应环境。合成布洛芬维C酯的最优条件:转速200r/min,温度65℃,加酶量5%(以底物的质量分数计),底物浓度1mol/L,平衡所需时间66h,平衡时产物质量分数为19.07%;合成酮洛芬维C酯的最优条件:200r/min,60℃,加酶量7.5%,底物浓度600mmol/L,平衡时间132h,产物质量分数为10.63%;合成氟比洛芬维C酯的最优条件:200r/min,65℃,加酶量5%,底物浓度400mmol/L,平衡时间144h,产物质量分数为6.76%。对底物进行了比较,得到了各自的动力学与热力学参数。布洛芬米氏常数为0.101μmol/L,vmax=32.68μmol/(min.g),热力学平衡常数为0.166;酮洛芬的分别为0.144μmol/L,12.97μmol/(min.g),0.091;氟比洛芬的分别为0.185μmol/L,9.35μmol/(min.g),0.055。     

酶法合成头孢环己二烯

以环己二烯甘氨酸甲酯盐酸盐为酰基供体,7-氨基脱乙酰氧基头孢烷酸为酰基受体,γ－氧化铝为载体的固定化巨大芽孢杆菌胞外青霉素G酰化酶为酰化剂,合成了头孢环己二烯。5％酰基供体,2％酰基受体,每毫升反应物加44IU固定化酶,pH7．5,25℃振荡反应5h,头孢环己二烯产率为81％。苯乙酸、苯氧乙酸和头孢霉素G对酶法合成有不同程度的抑制作用。     

Enzymatic synthesis of ethyl glucoside lactate in non-aqueous system

Ethyl glucoside lactate, a novel α-hydroxy acid derivative, was synthesized by transesterification in non-aqueous phase using immobilized lipase as biocatalyst. Parameters such as solvent type, substrate concentration, reaction temperature, and enzyme concentration were investigated to optimize the lipase-catalyzed transesterification. In solvent-free system with butyl lactate as both acyl donor and solvent, a 71% conversion was achieved. In order to investigate the effect of initial water content, the reactions were carried out in the mediums treated with molecular sieves. The results showed that conversion and initial rate decreased with the increase of water content. The conversion and initial rate reached to 95% and 67.4 mM/h, respectively, by carrying out the reaction under reduced pressure, which was employed to eliminate butanol and the initial water.     

Enzymic synthesis of fructose monooleate in a reduced pressure pilot scale reactor using various acyl donors

Enzymic synthesis of fructose esters was studied under reduced pressure. Different acyl donors were tested, and immobilized Candida antarctica lipase was used as biocatalyst. Influences of pressure, nature of the acyl donor, molar ratio sugar/acyl donor were investigated. Pressure had the greatest influence. At 200 mbar, more than 90% of fructose was acylated compared to 50% under atmospheric pressure. This is explained by the evaporation of reaction by-product (methanol or water) that shifted the equilibrium. C. antarctica lipase catalyzed sugar ester synthesis very efficiently using rapeseed oil as acyl donor. Moreover, synthesis performed with an equimolar mixture of both substrates gave promising results. Although the reaction rate was slower than synthesis performed with an excess of fatty acid, fructose monooleate concentration was still high (44 g l⁻¹ instead of 56 g l⁻¹) and the residual acyl donor concentration was very low. Downstream processes for the recovery of pure fructose monooleate were simplified in this case.     

Immobilized Pseudomonas cepacia lipase for biodiesel fuel production from soybean oil

Enzymatic transesterification of soybean oil with methanol and ethanol was studied. Of the nine lipases that were tested in the initial screening, lipase PS from Pseudomonas cepacia resulted in the highest yield of alkyl esters. Lipase from Pseudomonas cepacia was further investigated in immobilized form within a chemically inert, hydrophobic sol-gel support. The gel-entrapped lipase was prepared by polycondensation of hydrolyzed tetramethoxysilane and iso-butyltrimethoxysilane. Using the immobilized lipase PS, the effects of water and alcohol concentration, enzyme loading, enzyme thermal stability, and temperature in the transesterification reaction were investigated. The optimal conditions for processing 10 g of soybean oil were: 35 degrees C, 1:7.5 oil/methanol molar ratio, 0.5 g water and 475 mg lipase for the reactions with methanol, and 35 degrees C, 1:15.2 oil/ethanol molar ratio, 0.3 g water, 475 mg lipase for the reactions with ethanol. Subject to the optimal conditions, methyl and ethyl esters formation of 67 and 65 mol% in 1h of reaction were obtained for the immobilized enzyme reactions. Upon the reaction with the immobilized lipase, the triglycerides reached negligible levels after the first 30 min of the reaction and the immobilized lipase was consistently more active than the free enzyme. The immobilized lipase also proved to be stable and lost little activity when was subjected to repeated uses.     

Effect of acyl donor chain length and sugar/acyl donor molar ratio on enzymatic synthesis of fatty acid fructose esters

Lipase-catalyzed synthesis of fatty acid sugar esters through direct esterification was performed in 2-methyl 2-butanol as solvent. Fructose and saturated fatty acids were used as substrates and the reaction was catalyzed by immobilized Candida antarctica lipase. The effect of the initial fructose/acyl donor molar ratio and the carbon-chain length of the acyl donor as well as their reciprocal interactions on the reaction performance were investigated. For this purpose, an experimental design taking into account variations of the molar ratio (from 1:1 to 1:5) and the carbon-chain length of the fatty acid (from C8 to C18) was employed. Statistical analysis of the data indicated that the two factors as well as their interactions had significant effects on the sugar esters synthesis. The obtained results showed that whatever the molar ratio used, the highest concentration (73 g l⁻¹), fructose and fatty acid conversion yields (100% and 80%, respectively) and initial reaction rate (40 g l⁻¹ h⁻¹) were reached when using the C18 fatty acid as acyl donor. Low molar ratios gave the best fatty acid conversion yields and initial reaction rates, whereas the best total sugar ester concentrations and fructose conversion yields were obtained for high molar ratios.     

A feasible enzymatic process for D-tagatose production by an immobilized thermostable L-arabinose isomerase in a packed-bed bioreactor

To develop a feasible enzymatic process for d-tagatose production, a thermostable l-arabinose isomerase, Gali152, was immobilized in alginate, and the galactose isomerization reaction conditions were optimized. The pH and temperature for the maximal galactose isomerization reaction were pH 8.0 and 65 degrees C in the immobilized enzyme system and pH 7.5 and 60 degrees C in the free enzyme system. The presence of manganese ion enhanced galactose isomerization to tagatose in both the free and immobilized enzyme systems. The immobilized enzyme was more stable than the free enzyme at the same pH and temperature. Under stable conditions of pH 8.0 and 60 degrees C, the immobilized enzyme produced 58 g/L of tagatose from 100 g/L galactose in 90 h by batch reaction, whereas the free enzyme produced 37 g/L tagatose due to its lower stability. A packed-bed bioreactor with immobilized Gali152 in alginate beads produced 50 g/L tagatose from 100 g/L galactose in 168 h, with a productivity of 13.3 (g of tagatose)/(L-reactor.h) in continuous mode. The bioreactor produced 230 g/L tagatose from 500 g/L galactose in continuous recycling mode, with a productivity of 9.6 g/(L.h) and a conversion yield of 46%.     

大肠杆菌EP8—10转化苯丙酮酸生成L—苯丙氨酸的研究

E. coli EP8-10 was selected from the soil. It was able to produce the transaminase with high activity when it was cultivated on the medium containing peptone and beef extract. Optimum conditions of enzyme reaction was: phenylpyruvic acid's concentration of 0.3-0.5 mol/L, L-Asptaric acid used as amino donor, pH 8.5 37 degrees C. When phenylpyruvic acid was 0.3 mol/L, 48 g/L L-phenylalanine was produced after 6 h with 97% conversion rate.     

Fatty acid esterification using nylon-immobilized lipase

The esterification of a long-chain fatty acid was conducted using a nylon-immobilized lipase from Candida cylindracea in a nearly anhydrous, nonpolar organic medium, hexane. Butyl laurate was produced from lauric acid and n-butanol at a maximum initial reaction rate of 37 mmol/h. g immobilized enzyme when the substrates were present in equimolar amounts at an initial concentration of 0.5 mol/L. Lower rates were obtained using nonstoichiometric amounts of the substrates. The rate of reaction increased with temperature, reaching a maximum between 35 and 45 degrees C and decreasing sharply at higher temperatures. (c) 1995 John Wiley & Sons, Inc.