谷胱甘肽硫转移酶(GST)的固定化及酶学特性研究

对谷胱甘肽硫转移酶的固定化、游离酶和固定化酶的酶学特性进行了研究,通过试验,确定谷胱甘肽硫转移酶的最佳固定化条件为先用2％壳聚糖吸附酶,然后再加戊二醛交联,交联用戊二醛浓度为1．2％,交联时间6h;游离酶的最适温度为45—55℃,最适pH值为6．5-7．0：固定化酶的最适温度为45-50℃,最适pH值为7．0;游离酶和固定化酶的最适酶促反应时间为30min。     

聚乙二醇二缩水甘油醚交联氨基载体LX-1000EA固定化脂肪酶 *

聚乙二醇二缩水甘油醚(PEGDGE)作为双功能环氧试剂,在实验中被用于交联氨基载体LX-1000EA共价固定化海洋脂肪酶,经过处理后的载体共价固定化脂肪酶具有良好的效果。实验经过单因素初筛和正交试验,得到最佳的交联及固定化条件为0.75%交联剂浓度、交联温度35℃、交联时间3h、载体量1.25g、pH9.0、固定化温度55℃、固定化时间1h。对LX-1000EA-PEGDGE固定化酶与游离酶、戊二醛(GA)交联LX-1000HA-GA的固定化酶进行酶学性质的比较,发现LX-1000EA- PEGDGE固定化酶较游离酶最适反应温度未改变,与LX-1000HA-GA相同的是最适反应pH都由7.0提高为8.0。在最适条件中所测LX-1000EA-PEGDGE酶活达到78.84U/g,固定化改变了游离酶的酸碱耐受性,热稳定性和操作稳定性较游离酶和LX-1000HA-GA固定化酶均有提高。LX-1000EA-PEGDGE的热稳定表现优异,在60℃孵育3h后保留90%酶活;使用5次后仍能残余50%酶活;保存30天酶活仍保留60%。首次使用新型双环氧交联剂PEGDGE交联有机氨基载体共价结合固定化脂肪酶,为更有效的固定化方法提供了技术支持,同时也发现交联剂对固定化酶的性质存在较大影响。     

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%。     

介孔分子筛MCM-41固定中性脂肪酶条件的研究

以介孔分子筛MCM-41材料为载体,采用物理吸附法对中性脂肪酶进行了固定化处理,并研究不同条件对固定化脂肪酶催化活性的影响,从而得到该种材料对脂肪酶的最佳固定化条件。给酶量为45960 U/g,固定化温度为45℃,pH值为7.5,时间为3 h,此时固定化酶的活力约为4666 U/g。固定化酶和游离酶的最适反应温度都为40℃,最适pH值为7.5,比游离酶低。固定化酶温度稳定性和pH稳定性较游离酶有所提高。     

聚乙二醇二缩水甘油醚交联氨基载体LX-1000EA固定化脂肪酶

聚乙二醇二缩水甘油醚(PEGDGE)作为双功能环氧试剂,在实验中被用于交联氨基载体LX-1000EA共价固定化海洋脂肪酶,经过处理后的载体共价固定化脂肪酶具有良好的效果。实验经过单因素初筛和正交试验,得到最佳的交联及固定化条件为0. 75%交联剂浓度、交联温度35℃、交联时间3h、载体量1. 25g、pH9. 0、固定化温度55℃、固定化时间1h。对LX-1000EAPEGDGE固定化酶与游离酶、戊二醛(GA)交联LX-1000HA-GA的固定化酶进行酶学性质的比较,发现LX-1000EA-PEGDGE固定化酶较游离酶最适反应温度未改变,与LX-1000HA-GA相同的是最适反应pH都由7. 0提高为8. 0。在最适条件中所测LX-1000EA-PEGDGE酶活达到78. 84U/g,固定化改变了游离酶的酸碱耐受性,热稳定性和操作稳定性较游离酶和LX-1000HA-GA固定化酶均有提高。LX-1000EA-PEGDGE的热稳定表现优异,在60℃孵育3h后保留90%酶活;使用5次后仍能残余50%酶活;保存30天酶活仍保留60%。首次使用新型双环氧交联剂PEGDGE交联有机氨基载体共价结合固定化脂肪酶,为更有效的固定化方法提供了技术支持,同时也发现交联剂对固定化酶的性质存在较大影响。     

壳聚糖固定化真菌漆酶及其用于处理酚类污染物的研究

Trametessp. AH282在液体培养条件下经邻甲苯胺诱导能有效合成漆酶同工酶A。以壳聚糖为载体,戊二醛为交联剂进行了漆酶A的固定化研究,确定酶固定化适宜条件为：0.1g壳聚糖与15 mL 5%戊二醛交联8 h后,加入30.0U酶固定12h。在此条件下获得的固定化漆酶催化能力为176.4U/g载体,酶活回收率58.5%。与游离酶相比,固定化漆酶与作用底物愈创木酚的亲和力降低,但固定化酶的稳定性有明显改善。固定化漆酶的最适温度为55℃,比游离酶提高5℃;70℃条件下保温8 h,固定化酶保留酶活56.5%,而在相同条件下游离酶酶活明显下降。使用固定化漆酶反应装置进行酚类化合物转化实验,连续进行12批次操作,固定化酶酶活仍保持60%以上,漆酶使用效率明显提高。     

金属框架结构材料MOF-199对漆酶的固定化及其性质

以金属框架结构材料MOF-199为载体对漆酶进行固定化,并对固定化酶的性质进行初步研究。首先,以3-氨基丙基三乙氧基硅烷对载体MOF-199进行表面氨基化修饰,再用戊二醛对载体进行活化,最后对漆酶进行固定化。固定化条件优化结果表明:在漆酶质量浓度0.3 g/L,戊二醛用量1%(体积分数),pH 4.8下固定7 h,制得固定化酶活性最高。对固定化酶的研究发现:最适反应温度为40℃,最适pH为5.2,在连续操作7次后,固定化酶的活力仍能保持在51%。固定化漆酶热稳定性,pH耐受性,贮存稳定性均明显高于游离漆酶。     

固定化解淀粉芽孢杆菌a—淀粉酶的研究

本文对固定化ａ－淀粉酶进行了初步的研究,固定化酶采用海藻酸钙凝胶球来包埋一定纯度的解淀粉芽孢杆菌ａ－淀粉酶,并且把固定化酶的特点和性质同游离酶进行了比较。同游离酶相比固定比酶明显提高了酶的耐热性和贮藏稳定性,游离酶的最适作用温度为６０℃,而固定化酶最适温度为７０℃。     

大孔吸附树脂固定猪胰脂酶的初步研究

目的：为促进脂肪酶在工业上的应用,对以大孔吸附树脂为载体的猪胰脂酶固定进行了研究。方法：以吸附法固定,用单因素法考察了吸附条件对固定的影响。按Eadie—HOfstee法测定了固定化酶及游离酶的表观米氏常数,并具体研究了固定化酶的操作稳定性、热稳定性等性质。结果：固定化酶的比酯交换能力比游离酶上升6倍;最优的吸附条件为：pH7．0,酶加量300mg/g,水分含量20％;吸附时间4h;固定化酶在含水量10％时可稳定操作5批次;同时固定化酶有比游离酶有更好的热稳定性。结论：以大孔吸附树脂为载体对猪胰脂酶进行固定是可行的;与游离酶相比,所得固定化酶有着更高的比酯交换能力和更好的稳定性,更为适用于工业化生产。     

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。     

Repeated batch production of agar-oligosaccharides from agarose by an amberlite IRA-900 immobilized agarase system

The production of agar-oligosaccharides from agarose by free and immobilized agarase, obtained from a Pseudomonas aeruginosa AG LSL-11 was investigated and the activity, longevity and the operational stability of immobilized enzyme was compared with that of the free enzyme. The agar hydrolyzed products of free enzyme and immobilized enzyme were neoagarobiose, neoagarotetraose and neoagarohexaose as evidenced by LC-MS analysis. The immobilization of agarase was confirmed by SEM and also by the enzymatic transformation of agarose into agaroligosaccharides. The free agarase showed maximum activity at 40°C, whereas it’s immobilized counterpart showed maximum activity at 45oC, however, the optimum pH for both systems remained unchanged (pH 8.0). The relative activities of free agarase at pH 9.0 and 10.0 were 90 and 74%, respectively, whereas, the corresponding activities of the immobilized system were determined to be 97 and 90%. The stabilities of free agarase at pH 9.0 and 10.0 were 80 and 60% respectively, but for the immobilized system the respective residual activities were estimated to be 97 and 85%. Immobilized agarase appears to be more tolerant to high temperatures in terms of its activity and stability as it is compared to that of the free enzyme which retained 74 and 50.84% of relative activity at 55 and 60°C while, free agarase retained only 40 and 16.79% of its original activity. Furthermore, the immobilized agarase could be reused in batches efficiently for eight cycles, and could be stored for 3 months at 4°C as wet beads and for more than 6 months as dry beads.     

漆酶在磁性壳聚糖微球上的固定及其酶学性质研究

以磁性壳聚糖微球为载体,戊二醛为交联剂,共价结合制备固定化漆酶。探讨了漆酶固定化的影响因素,并对固定化漆酶的性质进行了研究。确定漆酶固定化适宜条件为：50 mg磁性壳聚糖微球,加入10mL 0.8mg/mL 漆酶磷酸盐缓冲液（0.1mol/L,pH 7.0）,在4℃固定2h。固定化酶最适pH为3.0, 最适温度分别为10℃和55℃,均比游离酶降低5℃。在pH 3.0,温度37℃时,固定化酶对ABTS的表观米氏常数为171.1μmol/L。与游离酶相比,该固定化漆酶热稳定性明显提高,并具有良好的操作和存储稳定性。     

桦褶孔菌漆酶固定化及其对染料的降解

采用壳聚糖交联法和海藻酸钠-壳聚糖包埋交联法固定化桦褶孔菌产生的漆酶,探讨最佳固定化条件,固定化漆酶的温度,pH稳定性及操作稳定性,并以两种固定化酶分别对4种染料进行了降解.结果表明:(1)壳聚糖交联法固定化漆酶的最佳条件为:壳聚糖2.5%,戊二醛7%,交联时间2h,固定化时间5h,给酶量1g壳聚糖小球:1mL酶液(1U/mL),固定化效率56%;(2)海藻酸钠-壳聚糖包埋交联法固定化漆酶的最佳条件为:海藻酸钠浓度4%,壳聚糖浓度0.7%,氯化钙浓度5%,戊二醛浓度0.6%,给酶量4mL 4%海藻酸钠:1mL酶液(1U/mL),固定化效率高达86%;(3)固定化的漆酶相比游离漆酶有更好的温度和pH稳定性;(4)比较两种固定化漆酶,海藻酸钠-壳聚糖包埋交联法固定化酶的温度及酸度稳定性要优于壳聚糖固定化酶,但可重复操作性要弱于后者,两者重复使用8次后的剩余酶活比率分别为71%及64%;(5)两种固定化酶对所选的4种不同结构的合成染料均有较好的降解效果,其中壳聚糖固定化酶对茜素红的降解效果及重复使用性极佳,重复降解40mg/L的茜素红10次,降解率仍保持在100%.     

Application and characterization of magnetic chitosan microspheres for enhanced immobilization of cellulase

In this study, a unique carrier magnetic chitosan microspheres (MCTS) was simply synthesized by anchoring Fe₃O₄ onto chitosan for direct immobilization of cellulases cross-linked by gluteraldehye. The structure and morphology were characterized using FT-IR, TGA, VSM and SEM. The optimum immobilization conditions were investigated: immobilized pH 7.0, amount of enzyme 15?mL (0.1?mg/mL), immobilization temperature 30?°C, immobilization time 5?h. At optimum conditions, MCTS achieved maximum enzyme solid loading rate of 73.5?mg/g, while recovery of enzyme activity approached to 71.6%. In the recycle test, immobilized cellulases operated without significant loss in its initial performances after 3 cycles, which indicated that immobilized cellulases can be regenerated and reused. The immobilized enzyme has better values of thermal and storage stability than that of free enzyme. Therefore, MCTS may be considered as a candidate with potential value of application in large-scale operations for cellulases immobilization.     

纳米磁性壳聚糖微球固定化酵母醇脱氢酶的研究

建立了以纳米级磁性壳聚糖微球(magnetic chitosan microspheres , M-CS)为载体固定化酵母醇脱氢酶(yeast alcohol dehydrogenase,YADH)的方法,优化了YADH的固定化条件,考察了固定化酶的性质。结果表明,M-CS 呈规则的圆球形,粒径在30nm 左右,具有较好的磁响应性。酵母醇脱氢酶固定化适宜条件为:50 mg 磁性壳聚糖微球,加入20mL 0.25 mg/mL 酵母醇脱氢酶(蛋白质含量)磷酸盐缓冲液(0.05 mol/L ,pH 7.0) ,在4 ℃固定2h。M-CS 容易吸附酵母醇脱氢酶,但吸附的酶量受载体与酶的比例、溶液的离子浓度、溶液pH的影响明显,而温度对吸附的酶量的影响则相对较弱。相对于游离的酵母醇脱氢酶,固定化酶的最适温度略有升高,可明显改善其热稳定性、酸碱稳定性、操作稳定性和贮存稳定性。     

Covalent immobilization of ω-transaminase from Vibrio fluvialis JS17 on chitosan beads

Chitosan beads were prepared by emulsion method and used for the immobilization of ω-transaminase of Vibrio fluvialis. The yield of enzyme immobilization (54.3%) and its residual activity (17.8%) were higher than those obtained with other commercial beads. ω-Transaminase was effectively immobilized on the chitosan beads at pH 6.0. The optimal pH of the immobilized enzyme was pH 9.0, which is the same as that of the free enzyme. The immobilized enzyme on chitosan beads retained ca. 77% of its conversion after five consecutive reactions with the 25 mM substrate, while the immobilized enzyme on Eupergit^® C retained 12%. Also, the immobilized ω-transaminase on chitosan bead retained 70% of initial activity when it's stored at 4 °C for 3.5 weeks. Addition of the co-factor, pyridoxal 5-phosphate (PLP), was needed to maintain the stability of the immobilized ω-transaminase.     

Immobilization of alpha-glucosidase in chitosan coated polygalacturonic acid

Crude alpha-glucosidase from Baker's yeast was immobilized in polygalacturonic acid beads and coated with chitosan. Chemical and physical characterization were performed by using p-nitrophenyl-alpha-D-glucopyranoside (pNPG) as an artificial substrate. Operation, thermal, pH, and strorage stabilities of the free and immobilized enzyme were also examined. The stabilities of immobilized enzyme were found to be better than that of the free enzyme. Furthermore, the hydrolysis rate of the chitosan coated alpha-glucosidase polygalacturonic acid beads were studied. In conclusion, the enzyme beads appear to have good characteristics and offer the prospect that this system may find application in enzyme immobilization, in addition to controlled drug release studies.     

吸附-交联法固定D-泛解酸内酯水解酶的研究

选择6种吸附树脂和离子交换树脂对D-泛解酸内酯水解酶进行固定化,筛选出了固定化效果较好的大孔弱碱性丙烯酸系阴离子交换树脂D-380为载体,用先吸附后交联的方法固定化。通过实验对固定化条件进行了优化,得出最佳的固定化条件为：加酶量6U／g树脂、吸附pH7．5、吸附时间4h、吸附温度30℃、交联剂戊二醛终浓度0．1％、交联时间2h。实验表明在此条件下制得的固定化酶有很好的稳定性：固定化酶在连续20次的底物水解反应后,剩余酶活达到71％。当温度达到80℃时游离酶几乎失去酶活,而固定化酶剩余酶活为60％以上。游离酶的pH稳定性范围为pH7～8,而固定化酶为pH6．5～8．5。     

Enhanced stabilization of mungbean thiol protease immobilized on glutaraldehyde-activated chitosan beads

A thiol protease purified from mungbean seedlings was immobilized on chitosan beads cross-linked with glutaraldehyde. The yield of the immobilized enzyme was maximum (～99%) at 1% concentration each of chitosan and glutaraldehyde. The immobilized enzyme showed reusability for 15 batch reactions. Immobilization shifted the optimum pH of the enzyme to a more acidic range and enhanced its stability both at acidic as well as alkaline pH values compared to the free enzyme. The stability of the enzyme to temperature and in aqueous non-conventional medium (ethanol and DMSO) was significantly improved by the immobilization process. The immobilized enzyme exhibited mass transfer limitation reflected by a higher apparent K_m value. This study produced an immobilized biocatalyst having improved characteristics and better operational stability than the soluble enzyme. The increase in stability in the presence of high concentrations of ethanol and DMSO may make it useful for catalyzing organic reactions such as trans-esterification and trans-amidation similar to other cysteine proteinases.     

Immobilization and characterization of horseradish peroxidase into chitosan and chitosan/PEG nanoparticles: A comparative study

Chitosan (CS) is considered a suitable biomaterial for enzyme immobilization. CS combination with polyethylene glycol (PEG) can improve the biocompatibility and the properties of the immobilized system. Thus, the present work investigated the effect of the PEG in the horseradish peroxidase (HRP) immobilization into chitosan nanoparticles from the morphological, physicochemical, and biochemical perspectives. CS and CS/PEG nanoparticles were obtained by ionotropic gelation and provided immobilization efficiencies (IE) of 65.8 % and 51.7 % and activity recovery (AR) of 76.4 % and 60.4 %, respectively. The particles were characterized by DLS, ZP, SEM, FTIR, TGA and DSC analysis. Chitosan nanoparticles showed size around 135 nm and increased to 229 nm after PEG addition and HRP immobilization. All particles showed positive surface charges (20−28 mV). Characterizations suggest nanoparticles formation and effective immobilization process. Similar values for optimum temperature and pH for immobilized HRP into both nanoparticles were found (45 °C, 7.0). V_max value decreased by 5.07 to 3.82 and 4.11 mM/min and K_M increased by 17.78 to 18.28 and 19.92 mM for free and immobilized HRP into chitosan and chitosan/PEG nanoparticles, respectively. Another biochemical parameters (K_cat, K_e, and K_α) evaluated showed a slight reduction for the immobilized enzyme in both nanoparticles compared to the free enzyme.