固定化黑曲霉β-葡萄糖苷酶制备染料木素活性苷元的研究

比较了以海藻酸钠为载体,用胶囊法、包埋-交联法、交联-包埋法三种不同方法固定化黑曲霉β-葡萄糖苷酶的效果,并研究了最佳固定化方法的固定化条件和固定化酶的部分性质。结果表明,交联-包埋法即β-葡萄糖苷酶与0.20%戊二醛交联后再用2.0%海藻酸钠包埋的固定化方法中酶结合效率和酶活力回收率最高。海藻酸钠浓度和戊二醛浓度对酶结合效率影响较大,戊二醛浓度和包埋颗粒直径大小对酶活力回收率影响显著。与游离酶相比,制备的固定化酶最适温度、最适pH值和Km值分别由50℃、4.5和2.57μg/mL下降到40℃、4.0和2.02μg/mL。固定化酶具有更强的耐酸性和稳定性。该固定化酶用于大豆异黄酮活性苷元染料木素的合成,重复使用6次后,固定化酶的活力仍保持84.94%,染料木苷转化率为56.04%。     

固定化黑曲霉β-葡萄糖苷酶制备染料木素活性苷元的研究

比较了以海藻酸钠为载体,用胶囊法、包埋-交联法、交联-包埋法三种不同方法固定化黑曲霉β-葡萄糖苷酶的效果,并研究了最佳固定化方法的固定化条件和固定化酶的部分性质。结果表明,交联-包埋法即β-葡萄糖苷酶与0.20%戊二醛交联后再用2.0%海藻酸钠包埋的固定化方法中酶结合效率和酶活力回收率最高。海藻酸钠浓度和戊二醛浓度对酶结合效率影响较大,戊二醛浓度和包埋颗粒直径大小对酶活力回收率影响显著。与游离酶相比,制备的固定化酶最适温度、最适pH值和K_m值分别由50℃、4.5和2.57 μg/mL下降到40℃、4.0和2.02 μg/mL。固定化酶具有更强的耐酸性和稳定性。该固定化酶用于大豆异黄酮活性苷元染料木素的合成,重复使用6次后,固定化酶的活力仍保持84.94%,染料木苷转化率为56.04%。     

乙二醇缩水甘油醚交联海藻酸钠-羧甲基纤维素钠固定化脂肪酶

以海藻酸钠、羧甲基纤维素钠(CMC)为载体,分别以乙二醇缩水甘油醚(EGDE)和戊二醛为交联剂,采用包埋交联法对脂肪酶进行固定化,结果显示EGDE的交联效果要优于戊二醛,添加EGDE的固定化酶酶活最好。得到制备固定化酶的最优方案为海藻酸钠2.5%,CMC浓度1.5%,给酶量800U/ml复配载体,氯化钙5%,以0.02%的EGDE交联固定30min,由此制备得到酶活约为380 U/g的固定化酶,酶活收率约为50.09%。固定化酶的最适反应p H为8.5,比游离酶增大0.5个单位;最适反应温度是45℃,比游离酶提高5℃;耐热性能变好,且重复使用7次后仍能保持60%左右的相对酶酶活。     

包埋法固定化真菌漆酶及其应用研究

采用海藻酸钠包埋法固定真菌漆酶,海藻酸钠和CaCl2的最佳浓度分别为3%和4%,最佳给酶量为30U,最大回收率为48.0%.与游离漆酶相比,固定化漆酶的热稳定性有明显改善,最适反应pH向酸性方向漂移0.5,最适反应温度提高了5℃.使用固定化酶处理低浓度造纸废水,运行8批次后残留酶活为64%.     

交联壳聚糖微球固定化β-葡萄糖苷酶工艺研究

目的:以戊二醛交联壳聚糖微球为载体,通过共价连接反应固定化β-葡萄糖苷酶.方法:以固定化酶比活和酶活回收率为目标,采用单因素方法优化固定化工艺、微球制备条件.结果:微球最佳制备条件:2.5%壳聚糖,2%乙酸,7.5%氢氧化钠,氢氧化钠:乙醇(v/v)=1:1.最佳固定化工艺为:0.1g壳聚糖微球在20mL 3%戊二醛溶液中50℃交联2h.加酶量为7 388mU/g干球,25℃吸附24h.固定化酶比活为6 188mU/g干球,酶活回收率为95.4%.结论:交联壳聚糖微球共价连接法可有效固定化β-葡萄糖苷酶.     

锰过氧化物酶的耦合固定化及其性质研究

分别采用海藻酸钠、明胶和壳聚糖为载体,并以戊二醛为交联剂,通过包埋-交联和吸附-交联两种耦合固定化方法制备固定化锰过氧化物酶。探讨了酶的不同固定化条件和固定化酶的部分性能。与游离酶相比,制备的3种固定化酶最适反应pH分别由7.0降低到5.0、5.0和3.0,最适反应温度分别由35℃升高到75℃、55℃和75℃。3种固定化酶的耐热性都显著提高,其中用壳聚糖制成的固定化酶在pH 2.2～11的宽范围内表现出很好的酸碱耐受性。30℃连续测定6～9次酶活力,重复使用的3种固定化酶显示出良好的稳定性。将固定化酶应用在偶氮染料的脱色中,用明胶制成的固定化酶在静置和摇床条件下,以及用海藻酸钠制成的固定化酶在摇床条件下,均表现出与游离酶相近的脱色能力,并且在重复进行的摇床实验中,脱色能力未降低,反应前后的酶活力均没有损失。     

石油生物脱硫菌Agrobacterium tumefaciens UP-3的固定化研究

对能降解二苯并噻吩(DBT)的根癌土壤杆菌AgrobacteriumtumefaciensUP3菌株进行了固定化研究,以聚乙烯醇(PVA)和海藻酸钠(SA)混合物为包埋法固定化载体,固定化最佳操作条件为4℃交联,PVA和SA混合物总浓度7%,两者最佳浓度比为6,细胞浓度为0.05g/mL。当DBT加入量为2.7mmol/L时,UP-3的静息细胞最高脱硫率为13%,而固定化细胞的脱硫效率超过了60%;固定化细胞的最佳使用条件为降解5d,温度28℃～32℃。     

锰过氧化物酶的耦合固定化及其性质研究

分别采用海藻酸钠、明胶和壳聚糖为载体,并以戊二醛为交联剂,通过包埋-交联和吸附-交联两种耦合固定化方法制备固定化锰过氧化物酶。探讨了酶的不同固定化条件和固定化酶的部分性能。与游离酶相比,制备的3种固定化酶最适反应pH分别由7·0降低到5·0、5·0和3·0,最适反应温度分别由35℃升高到75℃、55℃和75℃。3种固定化酶的耐热性都显著提高,其中用壳聚糖制成的固定化酶在pH2·2~11的宽范围内表现出很好的酸碱耐受性。30℃连续测定6~9次酶活力,重复使用的3种固定化酶显示出良好的稳定性。将固定化酶应用在偶氮染料的脱色中,用明胶制成的固定化酶在静置和摇床条件下,以及用海藻酸钠制成的固定化酶在摇床条件下,均表现出与游离酶相近的脱色能力,并且在重复进行的摇床实验中,脱色能力未降低,反应前后的酶活力均没有损失。     

壳聚糖固定化琼脂酶的研究

采用壳聚糖微球对琼脂酶进行固定化,在单因素实验的基础上用正交试验法确定最佳固定化工艺。结果表明:在戊二醛体积分数为2.5%,交联时间为6 h,加酶量为15 mL,固定时间为3 h时固定酶的活力最高;固定化酶的最适反应温度及最适pH分别为50℃和8.5,高于游离酶;同时其热稳定性及操作稳定性均高于游离酶。     

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。     

Immobilization of Laccase by Alginate–Chitosan Microcapsules and its Use in Dye Decolorization

Laccase is a ligninolytic enzyme that is widespread in white-rot fungi. Alginate–chitosan microcapsules prepared by an emulsification–internal gelation technique were used to immobilize laccase. Parameters of the immobilization process were optimized. Under the optimal immobilization conditions (2% sodium alginate, 2% CaCl₂, 0.3% chitosan and 1:8 ratio by volume of enzyme to alginate), the loading efficiency and immobilized yield of immobilized laccase were 88.12% and 46.93%, respectively. Laccase stability was increased after immobilization. Both the free and immobilized laccase alone showed a very low decolorization efficiency when Alizarin Red was selected for dye decolorization test. When 0.1 mM 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was added into the decolorization system, the decolorization efficiency increased significantly. Immobilized laccase retained 35.73% activity after three reaction cycles. The result demonstrated that immobilized laccase has potential application in dyestuff treatment.     

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。     

Study on Remediation-improvement of 2,4-Dichlorophenol Contaminated Soil by Organic Fertilizer Immobilized Laccase

ABSTRACT

In this paper, laccase is immobilized by the cross-linking method, using organic fertilizer as a carrier and glutaraldehyde as a cross-linking agent. Here, the optimal conditions of laccase immobilization were explored and the optimal operating conditions and stabilities of free laccase and immobilized laccase were also studied. Then, free laccase and immobilized laccase were applied to the soil remediation. Meanwhile, the effect of soil improvement treated with immobilized laccase was studied through ecological evaluation. The results showed that the optimal conditions for laccase immobilization were: the volume fraction of glutaraldehyde was 5%, the amount of enzyme added was 15 mL, and the immobilization time was 6 h. Under the same conditions, thermal stability and acid-base stability of immobilized laccase were better than free laccase. Under the optimal conditions, using laccase to treat 2,4-dichlorophenol in the soil, it was found that the free laccase group degraded 44.4% within 5 days, while the immobilized laccase group degraded 58.6%. Although both the degradation trends and route are the same, the degradation effect of the latter is obviously better. Ecological evaluation showed that organic fertilizer carrier had an impact on soil physical and chemical properties and soil enzymes, playing a positive role in soil ecological security and improving the soil.     

酶法提取玉米胚中总黄酮的研究

尝试使用α-淀粉酶进行糖苷转移提取玉米中总黄酮,同时对α-淀粉酶的固定化进行研究。对α-淀粉酶固定化的研究表明:以壳聚糖为载体,用体积分数4%戊二醛进行交联,加酶量为3 g.L-1条件下可以获得最佳的固定化效果;与游离酶相比,其最适作用温度范围、pH值范围均比游离酶范围宽;固定化酶的热稳定性优于游离酶,且具有良好的操作稳定性。本试验使用游离酶法、固定化酶法提取玉米中总黄酮,得率分别为2.82%、2.46%。     

Immobilization of a keratinolytic protease from Purpureocillium lilacinum on genipin activated-chitosan beads

Keratinase from Purpureocillium lilacinum LPSC # 876 was immobilized on chitosan beads using two different cross-linking agents: glutaraldehyde and genipin. For its immobilization certain parameters were optimized such as cross-linker concentration, activation time and activation temperature. Under optimum conditions, enzyme immobilization resulted to be 96 and 92.8% for glutaraldehyde and genipin, respectively, with an activity recovery reaching up to 81% when genipin was used. The immobilized keratinase showed better thermal and pH stabilities compared to the soluble form, retaining more than 85% of its activity at pH 11 and 74% at 50 °C after 1 h of incubation. The residual activity of immobilized keratinase remained more than 60% of its initial value after five hydrolytic cycles. The results in this study support that glutaraldehyde could be replaced by genipin as an alternative cross-linking eco-friendly agent for enzyme immobilization.     

Preparation of active corn peptides from zein through double enzymes immobilized with calcium alginate–chitosan beads

Double enzymes (alcalase and trypsin) were effectively immobilized in a composite carrier (calcium alginate–chitosan) to produce immobilized enzyme beads referred to as ATCC. The immobilization conditions for ATCC were optimized, and the immobilized enzyme beads were characterized. The optimal immobilization conditions were 2.5% of sodium alginate, 10:4 sodium alginate to the double enzymes, 3:7 chitosan solution to CaCl₂ and 2.5 h immobilization time. The ATCC beads had greatly enhanced stability and good usability compared with the free form. The ATCC residual activity was retained at 88.9% of DH (degree of hydrolysis) after 35 days of storage, and 36.0% of residual activity was retained after three cycles of use. The beads showed a higher zein DH (65.8%) compared with a single enzyme immobilized in the calcium alginate beads (45.5%) or free enzyme (49.3%). The ATCC kinetic parameters V_max and apparent K_m were 32.3 mL/min and 456.62 g⁻¹, respectively. Active corn peptides (CPs) with good antioxidant activity were obtained from zein in the ethanol phase. The ATCC might be valuable for preparing CPs and industrial applications.     

Immobilization of Candida rugosa lipase by cross-linking with glutaraldehyde followed by entrapment in alginate beads

Candida rugosa lipase was immobilized by first cross-linking with glutaraldehyde and then entrapping in calcium alginate beads. The presence of 2-propanol during cross-linking markedly improved the enzyme activity and activity recovery. Maximal enzyme activity (2.1?mmol?h^?1?g^?1 immobilized conjugate, wet weight) and activity recovery (117%) were observed at 30% (v/v) 2-propanol for hydrolysis of olive oil, which were 1.7 and 2.0 times higher than those of the immobilized enzyme prepared in the absence of 2-propanol. The half-life of the immobilized lipase prepared by entrapment after cross-linking in 30% 2-propanol was 1.6 times higher than that prepared by entrapment of the native lipase without cross-linking and 2-propanol pretreatment. The enantioselectivity of the former was 11 times higher than that of the latter for hydrolysis of racemic ketoprofen ethyl ester.