糖对溶胶-凝胶法固定化脂肪酶CALB的影响

选择糖类物质作为sol-gel法固定脂肪酶Candida antarctic lipase B,CALB）过程的保护剂,研究了蔗糖、葡萄糖、海藻糖、木糖、麦芽糖、乳糖等糖分子对固定化酶活的影响,结果显示：添加木糖、海藻糖、葡萄糖进行固定化后CALB的活性较高,其中加入木糖的作用最为显著,CALB的活性最高。并借助红外分析、热分析研究了木糖对CALB脂肪酶的作用机理。认为木糖对CALB的保护作用为：木糖分子的羟基同酶的酰胺基形成了氢键,而且木糖分子与酶分子之间还发生了其它相互作用,糖分子能够紧密地包裹在酶分子周围, 使酶分子得到很好的保护,脂肪酶CALB的活性和热稳定性均有所提高。     

水解条件对纤维素酶解速度的影响

纤维素酶水解速度与水解温度、pH值、水解时间等因素有关。超声波可加速纤维素的酶水解速度，用N2保护能延缓纤维素酶的失活。     

工业纤维素酶水解效率的影响因素分析

研究了影响工业纤维素酶Accellerase 1000水解效率的因素,及Accellerase 1000、Spezyme CP和Novozyme 188在不同水解状态下失活情况和葡萄糖对纤维素酶的抑制情况。结果表明反应器类型对水解影响不大,而固含量、酶量、搅拌速率和温度对Accellerase 1000水解效率影响很大。在醋酸缓冲液中,Accellerase 1000的滤纸酶活和纤维二糖酶活失活速率最快;在有木质素存在的醋酸缓冲液中相比各纤维素酶的滤纸酶活失活变慢,但纤维二糖酶失活加快;在滤纸水解过程中,Spezyme CP和Novozyme 188相对较为稳定。葡萄糖对纤维素酶有明显的抑制作用。     

纤维素酶固定化载体的研究进展

重点介绍了可溶性载体、不溶性载体以及可溶-不可溶性载体固定化纤维素酶的研究进展,3种载体都能不同程度地提高纤维素酶的稳定性与重复使用性。可溶性载体能提高纤维素酶的操作稳定性,有利于水解不溶性的纤维素,但回收不方便。不溶性载体固定化纤维素酶,回收方便,操作稳定性提高,但即使是提高了比表面积和减少了酶与底物的传质阻力的不溶性磁性纳米材料与膜材料固定化纤维素酶,也大多停留在水解可溶的羧甲基纤维素(CMC)阶段,不能高效率地水解不溶的纤维素底物。可溶-不可溶性载体固定纤维素酶,既能方便回收,又能水解不溶性的纤维素底物,但存在难固定,沉淀-溶解过程酶活损失大的缺点,期待开发新的固定方法与新的可溶-不可溶性载体。     

竹浆分批加酶工艺对长时间纤维素酶解性能的影响

优化纤维素酶水解工艺对于纤维素的高效利用具有重要意义。实验在保证纤维素酶相同的添加量时,采用多种方式分批添加纤维素酶水解竹浆,在长时间(7 d)的酶解过程中测定每天还原性糖产量,最后对长时间酶解过程中的影响因素进行了探究。实验结果表明:采用(0.35 mL+0.15 mL+0.1mL)添加工艺提高了酶解效率,酶解7d后糖产量提高了4.36%;纤维素酶随着培育时间的进行活性不断减小,培育1 d后的酶活性仅为原酶活性的53.87%;酶解糖产量随葡萄糖浓度的增加而降低,当葡萄糖浓度为30 mg/mL时,糖产量较无葡萄糖实验组降低了30.7%。酶解过程中酶活的丧失以及底物的抑制可能是影响纤维素酶长时间水解过程中的重要因素。     

共固定化细胞协同糖化发酵纤维素原料产乳酸

为提高纤维素原料对乳酸的转化率,将富含纤维二糖酶的黑曲霉（Aspergillus niger ZU-07）孢子和德氏乳酸杆菌（Lactobacillus delbrium）细胞共固定在海藻酸钙凝胶珠中,将共固定化细胞体系与纤维素原料的酶水解相耦联,组建成新型串联式生物反应器。研究表明,共固定化细胞中的纤维二糖酶可将纤维素水解液中的纤维二糖迅速转化成葡萄糖,而葡萄糖又能被乳酸杆菌迅速转化成乳酸,从而解除了纤维二糖及葡萄糖对纤维素酶的反馈抑制作用。当酶解罐和共固定化细胞反应柱的温度分别控制在50 ℃和48 ℃,共固定化细胞反应柱的装填量为40％时,串联式生物反应器中生成的乳酸浓度和纤维素对乳酸的转化率分别达到55.7 g·L^-1和91.5%。采用分批添料工艺,乳酸终浓度和反应器生产效率分别提高到106.7 g·L^-1和1.270 g·L^-1·h^-1,而单位底物的纤维素酶用量降低了25%。     

酶法合成海藻糖的研究

麦芽糖苷基海藻糖合成酶(MTSase)和麦芽糖苷基海藻糖水解酶(MTHase)可将淀粉转化为海藻糖,以改性壳聚糖为载体固定化海藻糖合成酶,通过比较固定化过程中各个因素的影响,得出结论如下:海藻糖酶液与5%戊二醛交联,交联温度为35℃、交联时间为16h条件下,固定化酶活性最高,再与淀粉溶液反应12h,海藻糖转化率达45.47%,与未固定化酶法制备相比有明显提高,符合工业化生产的需要并为进一步纯化海藻糖打下良好基础。     

以凹土颗粒稳定的乳液为模板制备复合微球固定化酶

以凹土颗粒稳定的Pickering乳液为模板聚合有机/无机复合微球,并以此为载体固定化脂肪酶,当脂肪酶浓度为0.020wt％,固定化温度为45 ℃及pH=7.4的条件下,固定化效果较好,酶活达到最大.脂肪酶固定化后显示出较好的热稳定性、储存稳定性,重复使用三次后酶活仍与游离酶的初始酶活相近.从而为酶的固定化的提供了一条新的途径.     

环氧树脂固定化拟南芥腈水解酶NIT的研究

游离酶不易回收,很难重复利用,而固定化酶重复利用度高。利用LX-1000EP(C)环氧树脂作为载体对腈水解酶进行固定化,研究了其最优固定化条件及其稳定性。最佳固定化条件为:固定化温度为20℃,固定化过程中缓冲液为磷酸钾缓冲液,pH8.0,浓度为0.1mol/L,加量为每克载体加10mL的粗酶液。固定化腈水解酶的最高酶活回收率达到98.1%,固定化酶在重复使用6次后,酶活仍能保持在初始酶活的30%以上。     

压热声处理对酶活性影响的评述

介绍了食品加工中压热声处理(MTS)对酶失活的影响，阐述了温度、压力、振幅和介质等因素对MTS失活酶的影响。MTS处理过程中多数酶的失活速率随温度升高而增大，且失活效率比热处理高；压力对MTS失活酶的影响不大；超声振幅增大，MTS失活酶的速率增大；处理介质对MTS失活酶的影响是复杂的。在不同的介质中酶失活效果不同。同时介绍了MTS失活酶的自由基机理和剪应力机理，为MTS的进一步研究和开拓应用提供了理论基础。     

Preparation of clay–poly(glycidyl methacrylate) composite support for immobilization of cellulase

A composite material was synthesized by grafting of glycidyl methacrylate onto clay using surface initiation atom transfer radical polymerization (SI-ATRP) technique. Epoxy group of the grafted p(GMA) chains was reacted with hexamethylenediamine (HMDA). The composite material was characterized using scanning electron microscopy (SEM) and FTIR. Cellulase from Trichoderma reesei was immobilized on the aminated composite particles via adsorption and covalent binding methods. The amounts of adsorbed enzyme on the aminated composite particles were 43.4 mg/g. The recovered activities of the adsorbed and covalently immobilized cellulase were found to be 87.7% and 73.2% for the substrate, carboxymethyl cellulose (CMC, 1.0 g/L). The pH stabilities and thermo-stabilities, repeated use and storage stabilities of both immobilized cellulase preparations were evaluated. The immobilized cellulase preparations have better stabilities and higher retained activities with respect to pH, temperature and storage than those of the free enzyme. Operational stability of the covalently immobilized cellulase was tested in a continuous flow system, and the activity loss was about 4% at the end of 48 h operation period.     

Spouted Bed Drying as a Method for Enzyme Immobilization

Usually immobilization is a requirement for the use of enzymes as an industrial biocatalyst. In this work, endophytic fungus Cercospora kikuchii lipase was immobilized by covalent binding on agricultural by-products and microcrystalline cellulose. The enzyme support system was submitted to spouted bed drying. Lipase immobilized on microcrystalline cellulose with 1.5% of glutaraldehyde showed the best results, presenting 179.1% of the original activity after drying, followed by rice husk (173.9%), corn stover (169.8%), sugarcane bagasse (157.3%), green coconut fiber (102.3%), and corncob (99.8%). The immobilized derivatives obtained showed a decreased enzyme activity with an average of only 17.31%, whereas the enzyme in its free form lost 85.8% of its initial activity after storage for 6 months. The operational stability showed that the biocatalysts prepared retained an average of 67.2% of the initial activity after five reuse cycles. The results showed that the use of agricultural by-products as low-cost support material associated with the spouted bed drying is promising and can contribute to industrial application of biocatalysts.     

DRYING TECHNOLOGY IN AGRICULTURE AND FOOD SCIENCE

ABSTRACT

The retentions of alcohol dehydrogenase (ADH) activity during drying and storage were investigated at various temperatures and relative humidities for various carbohydrate solutions. The highest retention of ADH activity after drying was obtained for the mixture of trehalose and methyl-β-cyclodextrin. For a single dried droplet, the effects of humidity on the storage stability of ADH were reversed between the trehalose solution and the blend of trehalose and methyl-β-cyclodextrin. However, the addition of methyl-β-cyclodextrin decreased the residual activity of ADH during storage. The carbohydrate matrix structure after drying might influence mainly the storage stability of ADH encapsulated in the mixed carbohydrate of trehalose and methyl-β-cyclodextrin.     

Enzyme immobilization on polyglycidylmethacrylate graft-copolymer of different polysaccharides

Comparative results obtained in preparing and characterizing samples of enzymes immobilized by reaction with polyglycidylmethacrylate (PGMA) copolymers with different polysaccharide matrices are reported. Sepharose copolymers having between 25 and 50% synthetic polymer were used to find the best immobilization conditions of horseradish peroxidase (HRP) and glucose-oxidase (GOD) (pH, time, temperature, enzyme cncentration). Activity, enzyme loading and coupling efficiency of immobilized HRP and GOD are greatly dependent on the type of matrix while the polymer content is less important. Coupling efficiencies between 0.8 and 1.5% have been obtained for HRP samples, whereas for GOD samples coupling efficiencies three times greater were obtained. HRP and GOD immobilized samples show K_m′ values greater than those of corresponding free enzymes and this indicates diffusion limitation phenomena. Storage, thermal and operational stability were also studied. In general the storage stability could be considered satisfactory (50% residual activity after 360 days). Sepharose and starch HRP-copolymers had an improved thermal stability compared with that of free enzyme. Residual activity found in continuous operation tests carried out on HRP-immobilized samples turned out to be dependent on support. HRP-PGMA-Cellulose sample gave the best results (50% residual activity after 16 days). PGMA-graft-copolymers have also been used to immobilize other enzymes such as α-amylase, α-chymotrypsin and cellulase.     

Immobilization of cellulase using porous polymer matrix

A new method is discussed for the immobilization of cellulase using porous polymer matrices, which were obtained by radiation polymerization of hydrophilic monomers. In this method, the immobilized enzyme matrix was prepared by enzyme absorbtion in the porous polymer matrix and drying treatment. The enzyme activity of the immobilized enzyme matrix varied with monomer concentration, cooling rate of the monomer solution, and hydrophilicity of the polymer matrix, taking the change of the nature of the porous structure in the polymer matrix. The leakage of the enzymes from the polymer matrix was not observed in the repeated batch enzyme reactions.     

A novel method to prepare chitosan powder and its application in cellulase immobilization

In this study, chitosan microspheres and sponges with uniform spherical and porous morphologies were prepared by coiling the stretched chains of chitosan with addition of salt and choosing different kinds of organic solvents as evaporation solvents. Cellulase was immobilized to the support by a covalent method. The enzyme exhibited a considerable affinity to the support, and the protein loading of 145.5 mg g^?1 support was fairly high. The immobilized cellulase had a higher K_m than free cellulase and had better stability with respect to pH, thermal stability, reuses and storage stability than free cellulase. Copyright © 2005 Society of Chemical Industry     

Storage stability of vacuum-dried probiotic bacterium Lactobacillus paracasei F19

There is still lack of the insight into the storage stability of dry probiotics produced by vacuum drying. Therefore, in this study we assessed the stability of a vacuum-dried Lactobacillus paracasei F19 under varying storage conditions. L. paracasei F19 was vacuum-dried with and without sorbitol and trehalose. The dried cells were stored at 4, 20 and 37 °C, and at a_w = 0.07, 0.22 and 0.33. The survival was determined by viable counts on MRS agar plates. The inactivation rate constants were determined for each storage condition. The survival after drying of cells dried without and with trehalose and sorbitol was 29, 70 and 54%, respectively. All vacuum-dried cells were very stable at 4 °C. However, high stability at non-refrigerated temperatures was obtained only in the presence of sorbitol. In contrast to sorbitol, the supplementation of trehalose did not stabilize cells during storage. This is supposedly due to the rapid crystallization of trehalose during storage. While glass transition temperatures of dry cell-sorbitol increased from ?32 °C to 12 °C during storage at 37 °C and a_w = 0.07, T_g of dry cell-trehalose (?15 °C after drying) could not be determined after storage for only 24 h. In conclusion, we showed that high stability of probiotic cells at non-refrigerated temperatures could be obtained by vacuum drying process with appropriate protectant.     

Characterizing the properties and evaluating the efficiency of biocatalysts based on immobilized fungal amylase

Preparations based on native enzymes have limited industrial use because of their instability and sensitivity to the changes in pH, temperature, and other external factors. It is therefore essential to create biocatalysts based on immobilized enzymes that are more stable and thus more efficient in practical application. To stabilize fungal amylase (EC 3.2.1.1.), the enzyme is covalently immobilized on a chitosan-containing cellulose. It is shown that the thermal stability of the immobilized amylase is increased by 350%, compared to the native enzyme, and its resistance to pH-inactivation is also improved. The reduction of the inactivation rate constant and the increase of the Gibbs free energy for the immobilized enzyme, relative to the native enzyme, testify to its increased stability resulting from steric factors associated with the formation of azomethine bonds with cellulose and chitosan. It is shown that using the immobilized enzyme preparation instead of the native amylase increases the product yield in barley malt hydrolysis by a factor of 1.5, allowing us to use this preparation in the food industry.     

喷雾干燥法制备1型糖尿病反义肽噬菌体壳聚糖微球疫苗

目的制备1型糖尿病反义肽噬菌体壳聚糖微球疫苗,并观察其热稳定性。方法采用水溶性壳聚糖、海藻糖、甘氨酸作为保护剂,通过喷雾干燥技术制备1型糖尿病反义肽噬菌体壳聚糖微球疫苗,扫描电镜下观察其形态及粒径大小,并检测入口温度及海藻糖浓度对壳聚糖微球疫苗活性的影响。将喷雾干燥样品置37℃放置6d,检测其热稳定性。结果通过喷雾干燥法制备的1型糖尿病反义肽噬菌体壳聚糖微球疫苗电镜下呈不规则的球形,粒径大小为20μm左右。喷雾干燥后样品的滴度与喷雾干燥前的疫苗原液相比均降低,且随着入口温度的升高而先升高后降低;喷雾干燥后的样品随着海藻糖浓度的增加,滴度也相应升高。与喷雾干燥前的疫苗原液相比,壳聚糖微球疫苗具有更好的热稳定性。结论已成功制备了1型糖尿病反义肽噬菌体壳聚糖微球疫苗,其具有良好的热稳定性。     

Covalent immobilization of cytochrome P450 BM3 (R966D/W1046S) on glutaraldehyde activated SPIONs

Selective hydroxylation of the C‐H bond of saturated hydrocarbon chains at room temperature is the signature of an invaluable biocatalyst, cytochrome P450 BM3 from Bacillus megaterium. Despite this remarkable ability, because of the enzyme's inherent low stability and dependence on electron supply by expensive NADPH, developing stable and economic BM3 systems is a challenging subject. To improve BM3 stability, facilitate its reuse, and reduce the process cost, this study suggests covalent immobilization of R966D/W1046S P450 BM3 on glutaraldehyde pre‐activated super paramagnetic iron oxide nanoparticles (SPIONs). This double mutant consumes less expensive cofactors like NADH and BNAH and its immobilization on magnetic support facilitates its separation and reuse. Free and immobilized enzyme performances were evaluated by 10‐pNCA hydroxylation and BM3 selectivity (hydroxylation at ω (1–3) positions of a fatty acid) was confirmed in a reaction involving myristic acid. The enzyme activity recovery was up to 60 % with 100 % enzyme binding efficiency. BM3‐SPIONs were easily separated from the reaction medium by applying a magnet, and recycled for 5 times, after which they could still present half of their initial activity. The enzyme storage stability was significantly improved: after one month of storage at 4 °C, the immobilized enzyme showed 80 % residual activity toward NADH while the soluble enzyme was inactive after a week. Binding an enzyme to fabricated SPIONs is a promising technique to increase enzyme stability and prevent downstream contamination in biocatalytic processes. In this context, BM3‐SPIONs can be a practical model system in cost‐effective large‐scale applications of such enzymes.