假丝酵母产脂肪酶在壳聚糖和硅胶上的固定化研究

本实验分别壳聚糖和氨基化后的硅胶作为固定化载体,用戊二醛作为交联剂,将脂舫酶共价固定化于载体上,壳聚糖的最佳固定化条件是戊二醛浓度2．5％,给酶量为4125U／g,氨基化硅胶的最佳固定化条件为戊二醛浓度2％,最佳给酶量在3456U／g,固定化酶活为2952U／g,最佳酶活表现率为85％。两种方法所得的固定化酶的耐温性和储藏稳定性都有了显著提高。氨基化硅胶固定化酶在50℃下保温3小时后仍保留有14％的酶活。结果显示假丝酵母产脂肪酶固定于弱亲水性载体上要比固定于强亲水性载体上效果更好。     

交联烯丙基葡聚糖凝胶共价偶联脂肪酶

研究了以交联烯丙然葡聚糖凝胶（ＣＡＤＢ）为载体,对－β硫酸酯乙砜基苯胺（ＳＥＳＡ）为偶联剂固定化脂肪酶的工艺条件,并考察了固定化脂肪酶的稳定性。试验结果表明：ＣＡＤＢ与ＳＥＳＡ反应的最适条件是ＰＨ－１２．０,ＳＥＳＡ与ＣＡＤＢ质量比为６：１,反应生成的对基苯磺酰乙基交联烯丙基葡聚糖凝胶（ＡＢＳＥ－ＣＡＤＢ）经重氮化后与脂肪酶偶联的最适条件是ＰＨ７．５,偶联时间≥１２ｈ,加酶量为ｕ／ｇ～１２００ｕ／     

磁性微球的制备及其固定化脂肪酶的研究

将Lipozyme CALB脂肪酶成功地固定于磁性纳米颗粒Fe3O4@SiO_2-p(NIPAM-co-GMA)的表面,研究了固定化过程中给酶量、温度、时间对固载量的影响。此外,稳定性试验表明:固定化脂肪酶的操作稳定性以及热稳定性都有提高。     

三维有序大孔硅材料载体固定化脂肪酶研究

以聚苯乙烯胶体晶体为模板制备三维有序大孔硅材料(3DOM-SiO_2),以其作为载体来固定脂肪酶。分别考察了脂肪酶加入量、反应体系pH、固定反应时间对固定化效果的影响。结果表明,3DOM-SiO_2材料固定脂肪酶的最佳酶液加入量为200 mL/g,固定化最适宜pH为7.0,最佳反应时间为5 h。固定化的脂肪酶在催化性能上与游离脂肪酶相比优势明显,最适宜反应温度提高到40℃左右,并且酶活随温度变化率低,热稳定性明显提高;脂肪酶固定化后对pH的敏感度降低,适应范围更宽,催化反应的最适pH为8.0;固定化脂肪酶重复使用8次后,相对酶活保持在62%。由此可见,3DOMSiO_2材料是固定脂肪酶的优良载体,在酶固定化领域应用前景广阔。     

磁性纳米粒子的制备及脂肪酶的固定化

建立了以纳米级磁性粒子为载体固定化脂肪酶的方法,优化了脂肪酶的固定化条件,考察了固定化酶的性质. 制备的磁性载体平均粒径20 nm,具有超顺磁性,分散和再分散效果好. 固定化酶的最适吸附时间为60 min,酶用量:载体量为1:1,固定化酶的酶活达到718 U/g. 结果表明,经纳米磁性粒子固定化后,脂肪酶得到活化,固定化酶比活为游离酶的1.8倍. 同时,固定化脂肪酶的pH稳定性显著提高.     

磁性琼脂糖复合微球固定化纤维素酶的研究

以磁性琼脂糖复合微球为载体,采用物理吸附法,制备出磁性固定化纤维素酶。确定了固定化工艺条件：ｐＨ２－２,吸附时间８ ｈ,酶用量为１５０ ｍｇ／ｇ 微球,在最佳固定化条件下,磁性固定化酶的活力为１９１－７ Ｕ／ｇ 微球,蛋白载量为１００ ｍｇ／ｇ 微球,比活为１－９ Ｕ／ｍｇ 蛋白,活性回收率为７３－１ ％ 。并对磁性固定化酶的理化性质进行了研究：磁性固定化酶的最适温度（５５ ℃） 与天然酶相同,最适ｐＨ（５－０）较天然酶提高１－０ 个单位,磁性固定化酶Ｋｍ 值（４－１×１０ －３ｇ／Ｌ）较天然酶Ｋｍ值（７－８×１０－３ ｇ／Ｌ） 小,热稳定性较天然酶有所提高,磁性固定化酶重复使用１０ 次,其相对活性保持在６０ ％ 。     

改性硅藻土固定化脂肪酶催化蓖麻油制备生物柴油

采用改性硅藻土做为载体,用吸附法对脂肪酶进行固定化,对固定条件进行优化,利用固定化脂肪酶催化蓖麻油制备生物柴油,考察反应时间、温度、醇油比及酶用量对转化率的影响和固定化脂肪酶催化合成生物柴油的稳定性。研究表明,硅烷化试剂添加量0.4%、温度30℃和时间4~6 h时固定化脂肪酶的活性最高,在醇油比为9:1,固定化酶用量为蓖麻油质量的4%,温度为60℃,反应时间为10 h的条件下,生物柴油的产率最高,经过3个批次的反应后,其产率都在40%以上,该固定化酶催化合成生物柴油有良好的工业化前景。     

载脂肪酶壳聚糖/海藻酸钙微胶囊的制备

针对固定化脂肪酶的研究背景,以壳聚糖、海藻酸钠为微载体制备材料,采用脉冲电场液滴工艺制备壳聚糖/海藻酸钙微胶囊。以脂肪酶为生物模型,系统考察了制备条件对载脂肪酶壳聚糖/海藻酸钙微胶囊酶活力的影响。结果表明:海藻酸钠质量浓度和酶与海藻酸钠载体配比是影响固定化酶活力的主要因素,载酶量为15mg/mL,海藻酸钠质量浓度为10mg/mL时载酶微胶囊酶活力最高,球形度好。通过改变壳聚糖质量浓度和相对分子质量,可以调控微胶囊膜的厚密程度进而影响固定化酶活力。成膜液pH值依次影响壳聚糖与海藻酸盐分子中官能团的电离状态、成膜反应静电络合程度、酶蛋白包封率,最终影响固定化酶活力。在载酶量为15mg/mL,海藻酸钠质量浓度为10mg/mL,壳聚糖相对分子质量、质量浓度和pH值依次为50kDa、1mg/mL和3.0的条件下,固定化酶活力为187IU/g。     

Cu(Ⅱ)螯合壳聚糖磁性微球固定化胃蛋白酶的制备及性能研究

将Cu(Ⅱ)螯合壳聚糖磁性微球用作固定化胃蛋白酶的载体,讨论了固定化时间、pH值和给酶量对酶固定化的影响.确定最适固定化条件为:固定化时间1.0 h、pH值4～5、给酶量150 mg·(g载体)-1.与自由酶比较,固定化胃蛋白酶的催化特性和稳定性均令人满意.     

海藻酸钠明胶协同固定化黑曲霉脂肪酶

以海藻酸钠明胶为复合载体,采用包埋法制备固定化黑曲霉脂肪酶,考察了海藻酸钠、明胶浓度等因子对固定化效果的影响,比较固定化酶和游离酶对温度、pH等条件的稳定性。结果表明,制备固定化黑曲霉脂肪酶的最优条件为:海藻酸钠、明胶浓度分别为1.25%和0.5%,CaC l2浓度为10%,给酶量为450 IU/g;固定化酶最适温度为35℃,最适pH为9.0,常见有机溶剂和金属离子对固定化酶的活力影响较小。     

融合蛋白CR2-GDH固定化及不对称还原制备(S)-4-氯-3-羟基丁酸乙酯

比较介孔分子筛材料SBA-15、MCM-41、海藻酸钙、改性二氧化硅4种载体固定化融合蛋白CR2-GDH其酶固载量和酶活回收率,选择SBA-15为固定化载体。研究固定化条件对固定化融合酶量的影响以及固定化酶的稳定性,固定化酶在双相体系催化不对称还原反应。结果表明,在pH值为5.5、酶浓度为1.4mg/mL、反应1h条件下,固定化酶量为27.7mg/g。加入25mmol/L的Ca²⁺,固定化酶的酶活回收率由58.6%提高到78.1%。与游离酶相比,固定化酶的热稳定性显著提高,40℃条件下酶活回收率提高19.1%。固定化酶水相中反复使用7批次后,剩余活性仍超过30%,具有较好的操作稳定性。与游离酶相比,固定化酶更耐受烷烃类有机溶剂。在水/有机溶剂双相反应体系中,Ca²⁺/SBA-15固定化酶和游离酶催化相比,产物得率提高23.8%。     

The effect of immobilization on the hydrolytic/interesterification activity of lipase from Rhizopus niveus

Lipase from Rhizopus niveus was immobilized by physical adsorption on Celite 545 and glass beads. The results showed that the highest immobilization efficiency and specific hydrolytic activity of 96% and 9.2 meq/mg protein/min, respectively, were obtained with Celite as the carrier. However, the specific hydrolytic activity of lipase adsorbed on glass beads by acetone precipitation was similar to that obtained by the Celite carrier, although the protein loading capacity was relatively low. The results showed that lipase immobilized on glass beads exhibited similar activity profiles with respect to reaction time, different enzyme concentrations, and water content, using trimyristin and tripalmitin as substrates, to those obtained with the free enzyme. In contrast, the immobilized lipase on Celite exhibited a considerably lower hydrolytic activity. However, the results also showed that the lipase activities of the free enzyme and the immobilized Celite enzyme were similar when the more hydrophilic triolein was used as the substrate. The interesterification of a mixture of tripalmitin and trimyristin or triolein was carried out using both the free and immobilized enzymes. The results indicated that the hydrolytic activity of lipase was similar in both cases for the first 24 h, after which it decreased dramatically. These findings suggest that at this late stage an equilibrium between the hydrolytic and interesterification reactions was reached.     

使用生物反应器酶促合成酸酐（英）

用装有固定化脂肪酶的生物反应器系统对有效地合成蓖麻油酸酐进行了尝试。合成酸酐试图证明：通过控制反应混合物的最适含水量，反应是否能达到目标的缩合度。结果表明，使用生物反应器，反应能迅速地进行，在生物反应器内进行重复间歇反应是可行的，这样与不同脂肪酶相比较，每次脂肪酶的用量均能大大减少。     

Immobilization of alcohol dehydrogenase on the surface of polyethylene membrane

Using polyacrylic acid (PAA) modified polyethylene (PE) membrane as a carrier, two immobilization routes of alcohol dehydrogenase (ADH) were studied, and the catalytic performance of immobilized enzyme was investigated using formaldehyde as a substrate. In the first route, PAA-PE membrane was further modified by polyethyleneimine (PEI) and then ADH was covalently linked by glutaraldehyde (GA) to the surface of PEI/PAA-PE. The results show that the optimal immobilization pH was 6.0, immobilization temperature was 5—15℃, ADH and GA concentrations were 1.0mg/ml and 0.01%(mass). For immobilized enzyme, the optimal reaction pH was 6.5, temperature was 15—30℃, and the highest reaction rate was 9.6 μmol/(L·min), the remaining activity was 47.3% after 10 use cycles. In the second route, ADH was immobilized on PAA-PE membrane with 1-(3-dimethylaminopropyl)-2-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as activators. The results show that the optimal molar ratio of EDC and NHS was 1∶0.5, and the immobilization time was 24 h. For immobilized enzyme, the optimal reaction pH was 6.5, temperature was 20—37℃, and the highest reaction rate was 15.58 μmol/(L·min), 53.8% activity was remained after 10 cycles.     

醇脱氢酶在聚乙烯膜表面的固定化研究

以聚丙烯酸(PAA)改性的聚乙烯(PE)膜为载体,研究了醇脱氢酶(ADH)的两种固定化路线,并以甲醛为底物考察了固定化酶的催化性能。路线1用聚乙烯亚胺(PEI)进一步改性,使用戊二醛(GA)固定化ADH。最优固定化pH为6.0,温度为5～15℃,酶浓度为1.0 mg/ml,GA浓度为0.01%(质量);固定化酶的最适反应pH为6.5,温度为15～30℃,反应速率最高为9.6μmol/(L·min);重复利用10次后可保持47.3%的活性。路线2以PAA-PE为载体,用1-(3-二甲氨基丙基)-2-乙基碳二亚胺盐酸盐(EDC)和N-羟基琥珀酰亚胺(NHS)为活化剂,固定化ADH。EDC和NHS最优摩尔比为1∶0.5,固定化时间为24 h;固定化酶的最适反应pH为6.5,温度为20～37℃,反应速率为15.58μmol/(L·min);重复利用10次后可保持53.8%的活性。     

Continuous production of fatty acids from palm olein by immobilized lipase in a two-phase system

Commercial lipases were tested for the ability to hydrolyze palm olein in isooctane in a two-phase system. Lipase OF (from Candida rugosa) showed the highest specific activity of 209 U/mg protein where 1 U is the amount of lipase enzyme required to produce 1 μmol of fatty acid (as palmitic acid) per minute. The enzyme was adsorbed completely on Accurel EP 100 (particle size <200 μm) with 20.5% activity retained. The soluble and the immobilized lipase OF showed optimal activity at the same pH and temperature (pH 6.5–7.5 and 35°C). However, the immobilized lipase had a wider range of pH and higher temperature stability. Continuous hydrolysis of palm olein was performed in a packed-bed reactor with 656 U of immobilized enzyme. The substrate (20% palm olein in isooctane) and Tris/maleate buffer were fed concurrently at the flow rates of 0.08 and 0.04 mL/min, respectively. The system gave a degree of hydrolysis (DH) of 90–100% for up to 250 h. A more stable system allowing for more than 300 h operation at DH>95% was achieved by mixing the immobilized enzyme with 1000–1500 μm Accurel EP100 to increase the system porosity and continuous feeding of the aqueous phase recycling from the product mixture. A similar result was also obtained using 1007 U of the immobilized enzyme and 60% palm olein in isooctane fed at 0.06 mL/min.     

Immobilization of α-amylase on polymeric carriers having different structures

α-Amylase was immobilized onto several polymeric carriers having carboxyl groups by the Woodward's reagent K better than by other methods. The amount of immobilized α-amylase mainly depended on the surface area of carriers, while the enzymic activity depended on the texture of carrier surface. Flat surface was favorable for making the enzyme exhibit high activity and for keeping it active in wide pH range. The durability of immobilized enzyme was excellent at 20°C and pH 5.5.     

DEAE-E/H固定化氨基酰化酶

对树脂DEAE-E/H(功能基化甲基丙烯酸环氧丙酯共聚物)固定化氨基酰化酶进行了研究。考察了树脂功能基含量、比表面和孔径对固定化酶活的影响。筛选出树脂Ⅱ进行了固定化条件的研究,具体分析了酶液溶度、吸附方式和吸附时间等影响因素。结果表明当酶液质量浓度为2.0mg/mL),循环吸附8 h时,树脂Ⅱ固定化酶的酶活为980 U/g。并通过电子扫描电镜(SEM)观察分析了DEAE-E/H的结构。     

树脂吸附法固定Candida rugosa脂肪酶

Candida rugosa脂肪酶具有优良的催化性能,对其进行固定化可以很方便地实现酶的回收和再利用。采用南开大学化工厂生产的4种阴离子交换树脂和4种大孔吸附树脂为载体,对来源于Candida rugosa的脂肪酶进行了吸附固定化,结果表明,以大孔吸附树脂AB-8为载体的固定酶比活性最高。固定化酶制备过程中缓冲液的最适宜pH值为7.2,最佳固定化时间为1 h,载体和酶的最佳质量配比为10∶1。与游离酶相比,固定化后酶活损失大约30%,但稳定性平均约提高60%。