固定化解淀粉芽孢杆菌α－淀粉酶的研究

本文对固定化α－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α－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。同游离酶相比固定化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℃。     

玉米须总黄酮抗糖基化、乙酰胆碱酯酶和α-葡萄糖甘酶抑制活性分析

研究玉米须总黄酮抗糖基化、乙酰胆碱酯酶和α-葡萄糖甘酶抑制活性。采用内部沸腾法提取玉米须总黄酮,优化其工艺条件,并对其进行分析。在单因素实验基础上,采用Plackett-Burman法对7个相关因素进行评价,筛选出具有显著性差异的4个因素,并对其进行响应面优化。结果表明,内部沸腾法提取玉米黄酮最佳条件为解析时间16min,解析剂浓度73%,提取时间20min,提取温度为61℃,总黄酮提取得率为4.75±0.69%;玉米须黄酮对晚期糖基化终产物、乙酰胆碱酯酶和α-葡萄糖苷酶具有一定抑制效果,呈现浓度依赖性,IC50分别为467.90、73.07、151.04μg/mL。抗糖基化、乙酰胆碱酯酶和α-葡萄糖甘酶抑制活性与黄酮含量呈正相关(相关系数分别为0.83、0.90和0.87;P<0.05),该研究结果可为潜在的天然产物抑制剂开发提供理论依据。     

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℃。     

N-乙酰神经氨酸醛缩酶的固定化及固定化酶性质研究

使用LX-1000HFA氨基树脂对N-乙酰神经氨酸醛缩酶(NAL)进行固定化,并对游离酶与固定化酶的酶学性质及稳定性进行了对比研究。结果显示,最佳固定化条件为载体投放量5.0 g,固定化时间12 h,缓冲液浓度1.0 mol/L,pH7.5,温度25℃。在此条件下制备的固定化NAL活力最高,比酶活可达200 U/g湿载体。与游离酶相比,最适反应温度提高了5℃,最适反应pH没有变化,温度和pH耐受性明显提升。同时固定化酶储存稳定性和操作稳定性也显著增强,在4℃条件下储存10 d后其酶活仅损失6%,重复使用10次后仍保持初始酶活的80%。因此,该固定化酶具有良好的温度稳定性、pH稳定性、储存稳定性和操作稳定性,为酶法工业化生产N-乙酰神经氨酸研究提供了理论依据。     

磁性聚乙二醇载体固定化α淀粉酶的研究

α淀粉酶广泛应用于粮食加工、食品、酿造、发酵、纺织品和医药工业［1］．由于固定化酶的优点,国内外研究人员对固定化糖化酶［2,3］和固定化α淀粉酶[4]的制备及在淀粉酶法生产葡萄糖方面的应用作了大量的研究,显示了工业应用前景．然而,迄今为止,用磁性载体固定化α淀粉酶尚未见报道．我们用磁性聚乙二醇胶体粒子作载体,制备出具有磁响应性强、稳定性强、活力高的固定化α淀粉酶．由于具有磁响应性,可借助外部磁场方便简单地回收酶,为该酶工业化生产葡萄糖提供了一种新的途径．而且,由于磁性的优点,也为该酶在食品、医药、纺织…     

游离及固定化果糖基转移酶部分酶学性质的比较研究

 从诱变、筛选的米曲霉GX0 0 10菌株所产生的果糖基转移酶 ,经过纯化和固定化操作分别制备游离酶和固定化酶 ,对两者的酶学性质进行了比较研究 .结果表明 ,两者在蔗糖转化为蔗果低聚糖的酶促反应中 ,最适pH为 5 5,在pH5 0～ 7 5之间酶活性相对稳定 .游离酶和固定化酶的适宜温度范围分别是 4 5～ 52℃和 4 0～ 55℃ .在 55℃保温 60min ,酶活性保存率分别是 61 6%和 87 5% .固定化酶的热稳定性提高 .0 1mmol LHg2 +和 1mmol LAg+能完全抑制游离酶的活性 ,但只能部分抑制固定化酶的活性 ,1mmol L的Ti2 +能完全抑制两者的活性 .以蔗糖为底物时 ,游离酶的米氏常数Km=2 15mmol L ,而固定化酶Km =386mmol L .游离酶只能使用一次 ,固定化酶反复使用 54次后 ,剩余活力为 55 2 % .用 55% (W V)蔗糖溶液与固定化酶在pH5 0 ,4 6℃下作用 12h ,可获得61 5% (总低聚糖 总糖 )产物 ,其中蔗果五糖含量达到 7 2 % .     

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。     

丝素蛋白膜固定β-葡萄糖苷酶及其改良食品风味的研究

从黑曲霉发酵液中提取β-葡萄糖苷酶酶液,用丝素蛋白将其固定,探讨酶固定化的影响因素及固定化酶的性质。β-葡萄糖苷酶的固定化条件为:取0.8 Uβ-葡萄糖苷酶与4.0%戊二醛和10%牛血清白蛋白混合(体积比为5:3:2),涂布于1cm2丝素蛋白膜上交联作用8h。在此条件下获得的固定化酶性质为:最适温度为60℃,比游离酶提高10℃;最适pH为5.0;t1/2为75℃,热稳定性比游离酶有明显改善;最佳反应时间为15 min;与游离酶相比,与底物亲和力降低。将固定化酶膜应用于果汁、果酒、茶汁等食品的增香,经感官鉴评,样品间存在显著差异,进一步经色谱一质谱联用仪分析,发现酶解后的样品,原有香气物质有不同程度的增加,4-萜品醇增加了107%、紫苏醇增加了42%,还有三种未知的香气组分分别增加了251%、79%和33%;并有新风味物质——芳樟醇、香叶醇和2-羟基-5-甲基苯乙酮产生,显示了较好增香效果。     

氨基树脂固定化L-苏氨酸醛缩酶及其应用*

L-苏氨酸醛缩酶(L-Threonine aldolase,L-TA)可以催化甘氨酸和醛合成β-羟基-α-氨基酸。β-羟基-α-氨基酸具有两个手性中心,是多种手性药物的中间体。但是,游离的L-TA难以重复利用,分离纯化困难,严重阻碍了工业化应用。固定化技术可以有效解决这些问题。利用氨基树脂NAA固定化来源于Bacillus nealsonii的L-苏氨酸醛缩酶,采用戊二醛作为交联剂,经过条件优化确定最佳固定化条件为:加酶量13 U、载体量0.6 g、0.4%(V/V)戊二醛、活化时间2 h、pH 8.5、35℃、固定化5 h。在此条件下,固定化酶酶活回收率为85.7%。在30℃下半衰期可达59天,为游离酶的6.5倍。将其应用于合成L-syn-对甲砜基苯丝氨酸,使用460 h后,残余酶活为79.4%。进一步开发了载体再利用策略,将失活固定化酶表面的氨基用戊二醛活化后,再与新的游离酶进行固定化,实现载体的再利用。利用该方法载体可重复利用两次,制备的固定化酶仍能使用460 h。该方法大大降低了固定化成本,为固定化L-TA的工业化应用打下坚实的基础。     

产α-淀粉酶菌株的分离、鉴定及酶学性质研究

目的:筛选高产α-淀粉酶菌株,为工业生产α-淀粉酶提供储备菌株。方法:利用碘液显色法和摇瓶发酵法,从土壤中筛选产α-淀粉酶菌株;通过菌落形态、菌体特征观察和16S rDNA序列比对对菌种进行鉴定;发酵粗酶液经硫酸铵沉淀、透析脱盐后,对其酶学性质进行初步研究。结果:从土壤中筛选到一株高产α-淀粉酶菌株,枯草芽孢杆菌Bacillus subtilis XL-15。该菌株所产α-淀粉酶的最适反应温度为50℃,最适作用pH为6.5;Ca2 和Mn2 对酶有激活作用,而Cu2 、Zn2 和EDTA对酶有抑制作用;酶的动力学研究测出米氏常数Km值为1.726mg/mL。结论:该菌株是产α-淀粉酶的较好材料,且具有一定的应用前景。     

Immobilization of α-amylase on gum acacia stabilized magnetite nanoparticles,an easily recoverable and reusable support

In this work, α-amylase is immobilized, using glutaraldehyde, onto magnetite nanoparticles prepared using gum acacia as the steric stabilizer (GA-MN), for the first time. The immobilization of amylase to GA-MN is very fast and the synthesis of GA-MN is very simple. The use of GA enables higher immobilization of α-amylase (60%), in contrast to the unmodified magnetite nanoparticles (∼20%). The optimum pH and temperature for maximum enzyme activity for the immobilized amylase are identified to be 7.0 and 40 °C, respectively, for the hydrolysis of starch. The kinetic studies confirm the Michaelis–Menten behavior and suggests overall enhancement in the performance of the immobilized enzyme with reference to the free enzyme. Similarly the thermal stability of the enzyme is found to increase after the immobilization. The GA-MN bound amylase has also been demonstrated to be capable of being reused for at least six cycles while retaining ∼70% of the initial activity. By using a magnetically active support, quick separation of amylase from reaction mixture is enabled. The catalytic rate of amylase is actually found to enhance by twofold after the immobilization, which is extremely advantageous in industry. At higher temperature, the immobilized enzyme exhibits higher enzyme activity than that of the free enzyme.     

α-Amylase immobilization on gelatin: Optimization of process variables

α-Amylase was extracted and purified from soybean seeds to apparent homogeneity by affinity precipitation. The homogeneous enzyme preparation was immobilized on gelatin matrix using glutaraldehyde as an organic hardener. Response surface methodology (RSM) and 3-level-3-factor Box–Behnken design was employed to evaluate the effects of immobilization parameters, such as gelatin concentration, glutaraldehyde concentration and hardening time on the activity of immobilized α-amylase. The results showed that 20% gelatin (w/v), 10% glutaraldehyde (v/v) and 1 h hardening time yielded an optimum immobilization of 82.5%.     

Immobilization of Bacillus subtilis α-amylase on zirconia-coated alkylamine glass with glutaraldehyde

Bacillus subtilis α-amylase (EC 3.2.1.1) has been immobilized on zirconia-coated alkylamine glass by using the process of glutaraldehyde coupling. The immobilized enzyme preparation exhibited 52% of the initial enzyme activity and a conjugation yield of 28 mg/g support. The K_m value of the immobilized α-amylase was decreased by immobilization while V_max was unaltered. E_a of the enzyme was decreased upon conjugation. The soluble enzyme was optimally active at pH 5.6 while the immobilized enzyme exhibited optimal activity in the pH range 5.4–6.2. The alkylamine-immobilized enzyme has also been characterized through its isoelectric point. The industrial importance of this work is discussed.     

INFLUENCE OF pH AND TEMPERATURE ON THE ACTIVITY OF SnO2-BOUND α-AMYLASE: A GENOTOXICITY ASSESSMENT OF SnO2 NANOPARTICLES

Immobilization of biologically important molecules on a myriad of nanosized materials has attracted great attention due to their small size, biocompatibility, higher surface-to-volume ratio, and lower toxicity. These properties make nanoparticles (NPs) a superior matrix over bulk material for the immobilization of enzymes and proteins. In the present study, Bacillus amyloliquefaciens α-amylase was immobilized on SnO₂ nanoparticles by a simple adsorption mechanism. Nanoparticle-adsorbed enzyme retained 90% of the original enzyme activity. Thermal stability of nanosupport was investigated by thermogravimetric and differential thermal analysis. Scanning electron microscopic studies showed that NPs have porous structure for the high-yield immobilization of α-amylase. The genotoxicity of SnO₂-NPs was analyzed by pUC¹⁹ plasmid nicking and comet assay and revealed that no remarkable DNA damage occurred in lymphocytes. The pH-optima was found to be the same for both free and SnO₂-NPs bound enzyme, while the temperature-optimum for NPs-adsorbed α-amylase was 5°C higher than its free counterpart. Immobilized enzyme retained more than 70% enzyme activity even after its eight repeated uses.     

Immobilization of the α-amylase of Bacillus amyloliquifaciens TSWK1-1 for the improved biocatalytic properties and solvent tolerance

The α-amylase of Bacillus amyloliquifaciens TSWK1-1 (GenBank Number, GQ121033) was immobilized by various methods, including ionic binding with DEAE cellulose, covalent coupling with gelatin and entrapment in polyacrylamide and agar. The immobilization of the purified enzyme was most effective with the DEAE cellulose followed by gelatin, agar and polyacrylamide. The K _m increased, while V _max decreased upon immobilization on various supports. The temperature and pH profiles broadened, while thermostability and pH stability enhanced after immobilization. The immobilized enzyme exhibited greater activity in various non-ionic surfactants, such as Tween-20, Tween-80 and Triton X-100 and ionic surfactant, SDS. Similarly, the enhanced stability of the immobilized α-amylase in various organic solvents was among the attractive features of the study. The reusability of the immobilized enzyme in terms of operational stability was assessed. The DEAE cellulose immobilized α-amylase retained its initial activity even after 20 consequent cycles. The DEAE cellulose immobilized enzyme hydrolyzed starch with 27 % of efficiency. In summary, the immobilization of B. amyloliquifaciens TSWK1-1 α-amylase with DEAE cellulose appeared most suitable for the improved biocatalytic properties and stability.     

Immobilization and stabilization of α-galactosidase on Sepabeads EC-EA and EC-HA

α-Galactosidase from tomato has been immobilized on Sepabead EC-EA and Sepabead EC-HA, which were activated with ethylendiamino and hexamethylenediamino groups, respectively. Two strategy was used for the covalent immobilization of α-galactosidase on the aminated Sepabeads: covalent immobilization of enzyme on glutaraldehyde activated support and cross-linking of the adsorbed enzymes on to the support with glutaraldehyde. By using these two methods, all the immobilized enzymes retained very high activity and the stability of the enzyme was also improved. The obtained results showed that, the most stable immobilized α-galactosidase was obtained with the second strategy. The immobilized enzymes were characterized with respect to free counterpart. Some parameters effecting to the enzyme activity and stability were also analyzed. The optimum temperature and pH were found as 60 °C and pH 5.5 for all immobilized enzymes, respectively. All the immobilized α-galactosidases were more thermostable than the free enzyme at 50 °C. The stabilities of the Sepabead EC-EA and EC-HA adsorbed enzymes treated with glutaraldehyde compared to the stability of the free enzyme were a factor of 6 for Sepabead EC-EA and 5.3 for Sepabead EC-HA. Both the free and immobilized enzymes were very stable between pH 3.0 and 6.0 and more than 85% of the initial activities were recovered. Under the identical storage conditions the free enzyme lost its initial activity more quickly than the immobilized enzymes at the same period of time. The immobilized α-galactosidase seems to fulfill the requirements for different industrial applications.     

α淀粉酶与糖化酶的共固定化研究

以纤维素为载体用重氮化法同时固定了糖化酶和α淀粉酶,确定了共固定化的最适条件,研究了共固定化酶的性质,发现共固定化酶较固定化单酶能更好地发挥协同效应,能在较低温度下将淀粉一步水解为葡萄糖。共固定化酶在３０℃下的操作半寿期可达９２０小时。     

Immobilization of a thermostable α-amylase, Termamyl, onto nitrocellulose membrane by Cibacron Blue F3GA dye binding

Highly porous nitrocellulose membranes were prepared by a solvent casting technique for the first time to immobilize α-amylase. An affinity dye, namely Cibacron Blue F3GA (CB), was incorporated covalently within the structure. The nitrocellulose–CB derivatized membranes were used for the immobilization of a starch degrading enzyme, α-amylase. Optimum conditions of immobilization for highest apparent activity were determined as pH 6.0, temperature 50°C and initial enzyme concentration 0.317 KNU/l. Under these optimum conditions, maximum enzyme immobilization yield was around 21% of the initial amount of the enzyme in the solution. Performance of free and immobilized enzymes at the same amount was compared for repeated runs. Up to the third use, immobilized enzyme showed higher activity than that of free enzyme mainly due to higher enzyme concentration in the membrane structure, then the apparent activity decreased gradually. However, when regenerated by switching pH to cause contraction/expansion of the structure, the membrane showed the highest activity, almost 2.5 times than that of the free enzyme. This unusual feature along with inexpensive cost may well make the nitrocellulose membrane an economical material for industrial application in glucose syrup production.     

Starch conversion by soluble and immobilized α-amylase

B. subtilis α-amylase was immobilized on cyanogen bromide activated carboxymethyl cellulose. The conversion of wheat starchwas carried out at 72°C in a stirred tank by soluble and immobilized α-amylase. The initial reaction rate with immobilized α-amylase was lower than with the soluble enzyme, but after 1 hr immobilized α-amylase produced a higher quantity of reducing sugars than the soluble enzyme. The action pattern of immobilized α-amylase was different from that of the soluble enzyme: immobilized α-amylase produced relatively more glucose and maltose, except at the beginning of conversion. Immobilized α- readily hydrolyze G₆. The starch conversion by immobilized α-amylase was not diffusion controlled at a stirring rate of 100-300 rpm.