交联海藻酸钠-明胶固定化L-阿拉伯糖异构酶的研究

以海藻酸钠和明胶为载体,对L-阿拉伯糖异构酶进行固定化。为增强固定化酶的稳定性,又用戊二醛对其进一步交联。研究了海藻酸钠及明胶浓度、CaCl2浓度、硬化时间以及戊二醛浓度等因素对固定化效果的影响,并对固定化酶的酶学性质进行了研究。结果表明最佳固定化条件为:海藻酸钠浓度2.0%、明胶浓度2.0%、硬化时间6h、CaCl2浓度4.0%、戊二醛浓度0.02%,该条件下所得酶活回收率最高为82%,且具有较好的操作稳定性,重复操作7次后酶活损失不到50%。与游离酶相比,固定化酶的最适反应pH及反应温度没有变化,但pH稳定性和耐热性都有所提高。      

交联海藻酸钠-明胶固定化L-阿拉伯糖异构酶的研究

以海藻酸钠和明胶为载体,对L-阿拉伯糖异构酶进行固定化。为增强固定化酶的稳定性,又用戊二醛对其进一步交联。研究了海藻酸钠及明胶浓度、CaCl2浓度、硬化时间以及戊二醛浓度等因素对固定化效果的影响,并对固定化酶的酶学性质进行了研究。结果表明最佳固定化条件为：海藻酸钠浓度2.0%、明胶浓度2.0%、硬化时间6h、CaCl2浓度4.0%、戊二醛浓度0.02%,该条件下所得酶活回收率最高为82%,且具有较好的操作稳定性,重复操作7次后酶活损失不到50%。与游离酶相比,固定化酶的最适反应pH及反应温度没有变化,但pH稳定性和耐热性都有所提高。     

树脂固定化酶的制备及其在低pH值葡萄糖酸生产工艺中的应用

分别利用强碱性大孔树脂、氨基树脂、环氧树脂作为葡萄糖氧化酶（GOD）和过氧化氢酶（CAT）固定化载体制备固定化酶,并研究其在低pH值葡萄糖酸生产工艺中的应用效果。耐酸性试验表明固定化酶较游离酶具有更强的耐酸性能。分别利用游离酶和3种固定化酶在低pH值（pH值3.5）条件下制备葡萄糖酸,强碱性大孔树脂、氨基树脂和环氧树脂固定化酶所需反应时间分别为40 h、24 h和27 h,酶活损失率分别为46.44%、3.42%和21.84%,而游离酶在反应过程中完全失活。3种固定化酶反应液的澄清度及色度均显著优于游离酶反应液。正交试验进一步优化后氨基树脂固定化酶的制备工艺为混合酶液浓度10%、GOD:CAT=1.5:1、固定温度25 ℃。在该条件下,氨基树脂固定化酶的酶活回收率可达93.15%,转化100 g/L葡萄糖溶液所需时间为23 h,反应结束时葡萄糖酸:葡萄糖酸钠可达0.899:1（m/m）。并且氨基树脂固定化酶稳定性良好。因此,利用氨基树脂固定化酶可以建立低pH值固定化酶葡萄糖酸生产工艺,实现高品质葡萄糖酸产物的生产。     

纳米花固定化葡萄糖异构酶的制备及性能分析

该研究采用共沉淀法制备了葡萄糖异构酶（Glucose Isomerase,GI）纳米花,对固定化条件进行了优化,同时对纳米花固定化酶的形态特征以及酶学性质进行了探究。结果表明,40 μL酶液中加入9 mL、pH值7.4的PBS缓冲液后与30 μL CuSO4混合,在35 ℃条件下静置反应18 h,制得的纳米花固定化葡萄糖异构酶（Glucose Isomerase @ Nano flowers,GI@NFs）的酶活回收率高达183.06%。SEM表征结果显示GI@NFs有完整的纳米花结构,傅里叶红外光谱显示GI@NFs具有酶和PO43-的特征吸收,X-射线衍射结果进一步证明其载体为Cu3(PO4)2。酶学性质研究发现,GI@NFs的最适反应温度为60 ℃,比自由酶的提高了10 ℃;最适反应pH值为8,比游离酶的最适pH更高;GI@NFs的温度稳定性和pH稳定性均比自由酶的明显提高;固定化酶被循环使用8次,其酶活力仍保持最初活力的60.32%。实验结果表明,纳米花结构提高了葡萄糖异构酶的酶活,表现出较好的循环性能和稳定性,具有一定的应用价值。     

固定化葡萄糖异构酶的微波活化及其性质研究

为寻求微波处理对酶进行活化改性的可行性,以固定化葡萄糖异构酶为对象,研究微波处理对其活性、酶学性质及动力学参数等方面的影响。研究结果表明:微波处理对固定化葡萄糖异构酶具有活化作用,在微波功率400 W,温度70℃时,固定化葡萄糖异构酶活性增强最明显,此时固定化酶的酶活力为156.13 U/g,比水浴对照高24.50%;同时微波处理降低固定化葡萄糖异构酶Km,增大Vmax,但对其最适p H和最适金属离子浓度几乎没有影响;此外,微波处理在一定程度上降低固定化葡萄糖异构酶的最适反应温度及高温下的热稳定性、操作稳定性,但这些参数依旧较为理想,能够满足实际生产需求。     

共固定化乳糖酶和葡萄糖异构酶的研究

研究了明胶-戊二醛法在共固定化乳糖酶和葡萄糖异构酶中的应用,并与开孔明胶法、卡拉胶包埋法进行了比较.进一步研究pH、明胶质量浓度、前交联中戊二醛的体积分数和二次交联的时间对明胶-戊二醛法共固定乳糖酶和葡萄糖异构酶的影响.结果表明,共固定化的最佳条件为:pH8.6,明胶浓度27%(w/w),前交联戊二醛体积分数0.15%和二次交联时间10min.在此条件下共固定化,乳糖酶的活力回收率为30.85%,葡萄糖异构酶的活力回收率为83.48%.共固定化乳糖酶和葡萄糖异构酶用于制备乳果糖,间歇操作6批次后酶活力仍然保持在初始活力的75%以上.     

共固定化乳糖酶和葡萄糖异构酶的研究

研究了明胶-戊二醛法在共固定化乳糖酶和葡萄糖异构酶中的应用,并与开孔明胶法、卡拉胶包埋法进行了比较。进一步研究pH、明胶质量浓度、前交联中戊二醛的体积分数和二次交联的时间对明胶-戊二醛法共固定乳糖酶和葡萄糖异构酶的影响。结果表明,共固定化的最佳条件为:pH8.6,明胶浓度27%（w/w）,前交联戊二醛体积分数0.15%和二次交联时间10min。在此条件下共固定化,乳糖酶的活力回收率为30.85%,葡萄糖异构酶的活力回收率为83.48%。共固定化乳糖酶和葡萄糖异构酶用于制备乳果糖,间歇操作6批次后酶活力仍然保持在初始活力的75%以上。      

磁性壳聚糖复合微球固定化葡萄糖异构酶制备及性能研究

通过化学共沉淀法合成纳米级Fe3O4粒子,并将其作为磁核,采用乳化交联法制备磁性壳聚糖复合微球,用SEM、FT-IR及激光粒度仪对微球结构进行表征。以磁性复合微球为载体,对葡萄糖异构酶进行固定化,并对固定化酶的参数、性质以及动力学参数进行研究。试验结果表明:磁性复合微球呈圆球形,具有较好的磁性。在加酶量12 mg/mL、戊二醛体积分数2%、交联时间2 h、振荡时间6 h条件下可以得到较佳的固定化效果,其酶活回收率达84.7%。对固定化酶性质的测定结果显示,最适Mg2+浓度0.01 mol/L,最适Co2+浓度0.003 mol/L,最适pH 7.2,最适温度75℃。通过计算其半衰期为40 d。对动力学参数的测定结果是:固定化酶的米氏常数为9.720,游离酶的米氏常数为8.190。     

聚丙烯腈膜固定化海藻糖合成酶的研究

以聚丙烯腈膜(PAN)为载体,采用吸附法固定化海藻糖合成酶粗酶液。通过单因素法探讨最佳固定化条件,并分析固定化酶酶学性质。结果表明,最佳固定化条件为:pH 7.6、30℃、加酶量0.1 mg/cm~2条件下震荡吸附3 h;该条件下制备的固定化酶最适反应条件为:温度40℃、pH 7.4、初始麦芽糖底物浓度200 g/L。与游离酶相比,固定化酶热稳定性提高10℃、酸碱稳定性由pH 6.6～7.4扩展至pH 5.4～8.0;反应达到平衡时,反应液中海藻糖含量为50.9%,副产物葡萄糖含量为9.1%,较游离酶降低6个百分点,在目前已报道的固定化海藻糖合成酶催化反应中副产物含量最低;在40℃、pH 7.4、麦芽糖底物浓度200 g/L条件下,重复使用累计72 h后,固定化酶活仍保留在初始酶活的64.4%。     

海藻酸钠固定化亚油酸异构酶及其酶学性质初步研究

以海藻酸钠为载体固定化亚油酸异构酶。研究了海藻酸钠浓度、酶用量、CaC12浓度、固定化时间对固定化过程的影响。结果表明,最佳固定化工艺是：以4%的海藻酸钠为载体、应用海藻酸钠溶液用量与酶液量的体积比3：1、3%的CaC12固定化5h。固定化酶的最适pH值为7.0,与游离酶相比,提高了0.5个pH单位;固定化酶和游离酶最适温度分别为35℃和30℃;固定化酶比游离酶具有更好的温度和pH值适应性。     

A New Immobilized Glucose Isomerase with High Productivity Produced by a Strain of Streptomyces murinus

The properties of a new immobilized glucose isomerase, produced by a selected strain of Streptomyces murinus, are described. The major advantages of the new immobilized enzyme are a productivity of more than 10,000 kg syrup dry substance per kg enzyme under optimal industrial conditions, increased activity and a very low syrup by-product formation. The influence of process parameters (temperature, pH and feed syrup additives) on activity and stability is discussed based on laboratory and industrial plant data.     

Production of High Fructose Syrup by Glucose Isomerase Immobilized on Phenol-Formaldehyde Resin

An immobilized glucose isomerase which is active and stable in concentrated glucose solution has been developed. The enzyme has been immobilized to Duolite A7 resin simply by adsorption. Industrial application of this immobilized enzyme for the continuous production of high fructose syrup is quite promising because the immobilized enzyme having enough mechanical strength against the pressure of column flow, is active over broad ranges of both temperature and pH and is less dependent on Mg and Coions. Long-run continuous experiments in column system have been carried out, isomerizing 45% of glucose with flow rate of SV 1 at 60°C. A gradual decrease of activity was observed after 25 d operation and the half-life was calculated to be 36 d. One liter of the immobilized enzyme in column isomerized 288 kg of glucose in a month. The values of kinetic parameters of the immobilized enzyme in column operation varied with flow rates and approached to those of the native enzyme when they were assayed at the flow rate approaching to SV 0.     

Studies on the Combined Action of Amylases and Glucose Isomerase on Starch and its Hydrolysates. Part III. Conversion of Starch to Glucose-Fructose Syrup

Glucose syrup was produced by liquefaction of starch using acid, acid-enzyme, one-step enzyme and two-steps enzyme process followed by saccharification by free and immobilized glucoamylase. The effect of D-glucose concentration and temperature on conversion of glucose syrup by glucose isomerase was studied. 50% glucose concentration and temperature range of 65 – 70°C gave the maximum conversion value to fructose. The isomerization was applied as a pilot plant process. The relative sweetness of glucose-fructose syrup was calculated and found that the syrup with fructose content of 26% has the same sweetness as sucrose.     

Immobilization of Glucose Isomerase in Microbial Cells. Part 2. Properties of Immobilized Glucose Isomerase in Cell Granule Preparations

Some properties of immobilized glucose isomerase in whole cell preparations were investigated. Immobilized glucose isomerase showed a similar velocity pattern to the native free enzyme. But the reaction system of immobilized enzyme was thought to be influenced by diffusion. The immobilized enzyme was much superior to the free enzyme in heat and pH stability and resistance to the inhibitory effect of several metal ions. In continuous process immobilized glucose isomerase showed a considerable durability. Decrease of activity of the immobilized enzyme column was approximately expressed by an exponential function of time.     

A New Regenerable Immobilized Glucose Isomerase

A new, immobilized glucose isomerase high productivity has been developed for isomerization of glucose into high fructose syrups. Soluble glucose isomerase from Streptomyces rubiginosus is highly purified and electrostatically adsorbed onto a granular DEAE-cellulose comprised of fibrous DEAE-cellulose, food grade polystyrene and titania. The same enzyme on fibrous DEAE-cellulose has been in commercial use in the United States since 1968. Because the soluble enzyme is electrostatically adsorbed to the carrier, the immobilized enzyme can be regenerated after use and the carrier reloaded with fresh soluble enzyme. Regeneration and reimmobilization can be repeated several times thus providing a minimum cost enzyme-carrier system. The enzyme is tightly bound to the carrier during isomerization. The immobilized isomerase is compatible with bisulfite in the substrate sufficient to stabilize the enzyme and provide a degree of protection against infection at low isomerization temperature. High purity and high specific activity of the soluble enzyme make possible high activity levels for the immobilized enzyme with potencies of up to 1500 IGIU/g. There occurs essentially no elution of color from the immobilized enzyme during start up of isomerization. Due to the combination of low compressibility of the particles and high immobilized activity, productivities greater than 9 metric tons of 42% fructose syrup solids Perkg of immobilized enzyme are being achieved on a commercial scale.     

一株嗜热菌木糖异构酶的纯化与性质研究

研究了1株嗜热菌(Anoxybacillus flavithermus)所产木糖异构酶的分离纯化以及酶学性质。结果表明,经硫酸铵沉淀、Sephadex G-75凝胶过滤、纤维素DE-52弱阴离子交换柱和Q Sepharose Fast Flow强阴离子交换层析得到的木糖异构酶,分子量约为181 ku,由4个相同分子量的亚基组成。酶反应的最适温度为80℃,最适为pH为7.0且最适pH范围宽泛,pH6.0~11.0酶反应活性能保持80%左右。该酶热稳定性及耐碱性能良好,70℃保温1 h后酶活仍能保持近80%左右;pH5.0~8.0保温1 h后酶活仍能保持近80%以上,甚至pH12.0保温1 h后酶活性仍能保持40%左右。Mn2+和Co2+对酶活性有明显促进作用,Zn2+、Cu2+以及Al3+对酶活性有一定程度的抑制。     

Some Properties of Whole-Cell Glucose Isomerase Immobilized in Polyacrylamide Gel by Radiation

The whole-cell glucose isomerase was immobilized by the method of block polymerization of acrylamide initiated by ⁶⁰Co irradiation. Some properties of immobilized glucose isomerase were studied under various reaction conditions, in respect of its use as industrial catalyst. It was established that Co²⁺ ions did not increase stability of the immobilized enzyme at pH above 8.0. The half-life of the immobilized glucose isomerase was found to be 24 days at 60°C.     

一株高寒地区产纤维酶细菌的筛选、鉴定及酶学性质研究

从青海茶卡盐湖地区土壤中分离出一株产纤维素酶的细菌菌株,根据菌落形态特征以及16S rDNA序列分析,初步鉴定为琼斯氏菌属（Jonesia quinghaiensis sp.）。在25 ℃、初始pH值为7.0的发酵条件下,该菌株产酶量培养45 h达到最大值,发酵液酶活力为0.3 U/mL。酶学性质研究表明,该纤维素酶最适反应pH值为8.0,最适反应温度为35 ℃,在30～40 ℃的范围内保持60%以上的活性。金属离子Mn2+、Co2+和Cu2+对酶活力有较强的抑制作用,Ca2+具有显著的促进催化作用。     

黑曲霉5143脂肪酶离子注入诱变及酶学性质的研究

以产脂肪酶菌株黑曲霉Aspergillus niger51-43为出发菌株,经氮离子注入技术对其进行诱变,筛选获得酶活力有较大提高且传代稳定的正突变菌株L7,其脂肪酶活力达29.17U/mL,较原初酶活力19.28U/mL提高了151.3%。在此基础上,对该酶的酶学性质进行了研究。结果表明,高产突变株L7所产脂肪酶的最适作用温度为30℃,最适作用pH8.0,在50℃和pH6.0～9.0之间有很好的稳定性。Ca2+、Na+、Mg2+对该酶有一定的激活作用,而Cu2+、Mn2+、Fe2+、Co2+、Zn2+则抑制该酶的活性。      

固定化亚油酸异构酶制备及其性质

以海藻酸钠、壳聚糖为载体,分别采用直接包埋、交联-包埋法制备固定化亚油酸异构酶;研究酶的固定化条件和固定化酶的部分性质。结果表明：以海藻酸钠为载体,采用交联-包埋法以戊二醛为交联剂时固定化效果较好;最佳固定化条件为：海藻酸钠质量浓度为3g/100mL,戊二醛质量浓度为0.3g/100mL,CaCl2质量浓度为2g/100mL;固定化酶的最适反应温度为50℃,最适反应pH值为5.0;与游离酶相比,固定化酶的热稳定性显著提高,温度在20～60℃之间较稳定,pH值在2～8之间表现出较好的酸碱耐受性;固定化亚油酸异构酶的Km为0.36mg/mL。连续操作6次固定化相对酶活力仍保持70.6%,与游离酶相比,固定化亚油酸异构酶催化效率约提高了50%。