β-果糖基转移酶的性质研究

目的研究低聚果糖产生菌黑曲霉 (Aspergillusniger)SIPI 6 0 2来源的 β 果糖基转移酶 (β FT)的性质。 方法经典的酶学研究方法。结果 β FT催化蔗糖转化成蔗果三糖的Km和Vmax分别为 2 6 8.37g L和 2 .37g (min·L) ;将蔗果三糖转化成蔗果四糖的Km和Vmax分别为 173.0 8g L和 0 .10g (min·L)。葡萄糖是 β FT的竞争性抑制剂 ,其Ki=2 4 5 .2 3g L。β FT反应的最适pH为 4 .5～ 6 .0 ,最适温度为 5 5℃。β FT在中性条件、5 0℃以下稳定性良好。β FT转化蔗糖生成低聚果糖的反应进程实验表明 ,高浓度的底物有利于提高反应速度和转化率 ,并且产物中果糖浓度较低浓度底物中低。结论黑曲霉SIPI 6 0 2来源的 β FT适合用于工业化生产低聚果糖     

用固定化氧化葡萄糖酸菌转化6-(N-羟乙基)胺基-6-脱氧-L-山梨呋喃糖

目的探索用固定化氧化葡萄糖酸菌转化6-(N-羟乙基)胺基-6-脱氧-L-山梨呋喃糖的最优条件。方法以海藻酸钠为载体把氧化葡萄糖酸菌固定化,考察固定化细胞在不同转化条件时对底物的转化率。结果通过条件优化,确定固定化细胞转化时间为24 h,底物质量浓度为10 g.L-1,采用的海藻酸钠质量浓度为40g.L-1,菌体包埋量为每g凝胶包埋3.1 g菌体,最适pH为7.0,最适温度为28℃,摇床转速要求高于100r.min-1;用60mmol.L-1的MnCl2替代原转化液中20mmol.L-1的MgSO4;固定化细胞与原游离细胞工艺相比,24h内底物转化率由原来的47%上升到93%,连续转化10次后,转化率仍为38%。结论用固定化氧化葡萄糖酸菌转化6-(N-羟乙基)胺基-6-脱氧-L-山梨呋喃糖,转化率提高46%并能连续转化约10次。     

固定化细胞转化脱氧胸苷合成2'-脱氧腺苷

目的选择最优的载体包埋固定化AEM0957细胞酶促催化脱氧胸苷和腺嘌呤合成脱氧腺苷。方法分别用琼脂、聚丙烯酰胺、明胶、卡拉胶、海藻酸钠、聚乙烯醇等6种材料包埋固定细菌AEM0957细胞,以此为催化剂转化脱氧胸苷合成脱氧腺苷。结果通过优化反应条件,聚丙烯酰胺固定化细胞催化脱氧胸苷的转化率可达57%,高于游离细胞反应能力,并能多次重复使用,10次重复反应的转化率仍能够保持在32%左右。结论聚丙烯酰胺在稳定性、转化性能等方面优于其它材料,该研究为脱氧腺苷的工业化生产奠定了重要基础。     

开孔明胶固定酵母细胞生产三磷酸腺苷

用开孔明胶包埋啤酒酵母制成固定化细胞，在含15g／L单磷酸腺苷、150mmol／L葡萄糖、16mmol／L硫酸镁的250mmol／L磷酸二氢钠缓冲液（pH7．0）中35℃振荡反应2h，生产三磷酸腺苷（ATP）的转化率可达95％。在前一批底物溶液灭菌和添加烟酰胺腺嘌呤二核苷酸的条件下，固定化酵母细胞可反复使用10次，ATP转化率稳定维持在90％以上。     

两相体系中固定化细胞生物合成(S)-3,5-双三氟甲基苯乙醇

建立了在有机/水两相体系中固定化酵母细胞牛物合成(S)-3,5-双三氟甲基苯乙醇的方法.考察了有机溶剂、缓冲液pH、底物浓度、温度、转化时间、辅助底物对转化率和对映体过量值(ee)的影响.结果显示,采用辛烷-缓冲液两相体系,缓冲液pH 8,初始底物浓度5.55 mmol/L,30℃转化36 h,转化率和产物的ee值分别为93.2%和96.3%.添加与底物等当量的葡萄糖或蔗糖作为辅助底物,固定化细胞可以反复使用6次,转化率维持在50%以上.     

固定化β-葡萄糖苷酶制备人参皂苷F_1的研究

本文探讨了β-葡萄糖苷酶的固定化方法,及利用固定化β-葡萄糖苷酶转化人参皂苷Rg1为人参皂苷F1的转化工艺。确定交联-包埋法为最佳固定化方法,应用正交实验得出最佳制备条件为:交联时间3h,戊二醛浓度0.1%,海藻酸钠浓度1%,CaCl2浓度2%。固定化酶与游离酶在热稳定性和pH值稳定性方面显示出不同的性质,其中固定化β-葡萄糖苷酶的最适反应温度为70℃,最适反应pH值为5.5。固定化β-葡萄糖苷酶在15℃环境中保存30d后,酶活回收率为68.82%。固定化β-葡萄糖苷酶转化人参皂苷Rg1为人参皂苷F1的转化条件为:固定化酶承载量为3.76U/g固定化酶载体,底物浓度为0.2mg/mL,转化温度为40℃,转化周期为2d,转化次数为4次,平均转化率为80.49%。     

β-呋喃果糖苷酶的性质研究

研究了低聚果糖产生菌黑曲霉 (Aspergillus niger) SIPI-60 2的β-呋喃果糖苷酶 (1)的性质。 1水解蔗糖的 Km和 Vm ax分别为 0 .0 2 5 mol/ L和 1.3 9mmol· L-1 · min-1 。果糖是 1的竞争性抑制剂 ,Ki=10 .0 5 mmol/ L。通过对菌丝体、β-果糖基转移酶 (2 )与 1的混合物及纯的 2转化蔗糖的反应进程的比较 ,考察了 1对 2反应进程的影响。结果表明 ,1对低聚果糖生成速率和最终产率均有影响。结果还提示在用菌丝体转化蔗糖过程中低聚果糖可能会诱导出更多的 1     

利用κ-卡拉胶-魔芋多糖复配胶固定化酵母生产三磷酸腺苷

利用κ-卡拉胶-魔芋多糖复配胶包埋的固定化啤酒酵母细胞,由腺苷(AR)生产三磷酸腺苷(ATP).采用HPLC法分析固定化细胞反应过程核苷(酸)的变化以及用琼脂糖平板电泳法对其工艺条件进行了优化.固定化细胞反应1 h,ATP转化率接近100%.底物溶液灭菌和添加NAD+后,该固定化酵母细胞可反复使用12次,ATP的转化率稳定维持在95%以上.     

固定化嗜热脂肪芽孢杆菌生产1,6-二磷酸果糖的研究

采用卡拉胶包埋嗜热脂肪芽孢杆菌，制成块状固定化细胞，对固定化细胞包埋方法及其用于生产1，6－二磷酸果糖（FDP）的工艺条件作了探索，在固定化细胞包埋前，包埋后及用于生产FDP 10批次后采用活化培养工艺，均可提高固定化细胞生产FDP的能力，转化培养液中的FDP产量达到40－65mg/ml，固定化细胞用于生产FDP的批次可达到20次左右。     

固定化细胞转化阿糖尿苷合成阿糖腺苷

采用聚乙烯醇、卡拉胶和聚丙烯酰胺为混合载体固定化埃希菌AEM0812细胞.以8.0％聚乙烯醇、1.0％卡拉胶和10％聚丙烯酰胺作为载体,菌体浓度为30％,在pH 7.0、含4.0％氯化钾的饱和硼酸溶液(成型剂)中处理36h,得相对酶活力为98.17％且较稳定的颗粒型固定化细胞,用来催化转化阿糖尿苷合成阿糖腺苷,在54℃、pH 6.5和缓冲液离子浓度120 mmol/L的最适条件下,阿糖尿苷的转化率为68％.固定化细胞连续5次反应的平均转化率约54.3％.     

Comparison and Suitability of Gel Matrix for Entrapping Higher Content of Enzymes for Commercial Applications

To check the suitability of enzyme entrapped beads for use in pharmaceutical industry, amylase enzyme was entrapped in agar/agarose, polyacrylamide gels and calcium alginate beads. Sodium alginate of 1% concentration was found to be best with respect to immobilization efficiency and calcium alginate beads so obtained were not much susceptible to breakage. When sodium alginate- amylase mixture was added from a height of about 20-30 cm. into CaCl₂ solution, size of beads was large at higher alginate concentration due to the increase in the size of droplet formation before entering into CaCl₂ solution. Enzyme entrapped polyacrylamide and agar/agarose gels were fragile and could not withstand repeated use whereas enzyme entrapped in large calcium alginate beads was used successfully for 50 cycles for the conversion of starch into product without much damage to the beads under stirring conditions. Amylase preparation was also mixed with urease, lysozyme and coimmobilized in large sized calcium alginate beads. These beads were used for 10 repeated cycles to check the conversion of substrates into their products by their respective enzymes and we concluded that an enzyme or mixture of two or three enzymes can be immobilized in the same large sized calcium alginate beads. This will save the additional cost of bioreactor, manpower, maintenance conditions required for the conversion of one drug into another using enzyme/s entrapped in large sized beads.     

两相体系中固定化细胞生物合成L-苯基乙酰基甲醇

目的生物合成L-苯基乙酰基甲醇(L-PAC)。方法在10%石油醚/水两相体系中,利用固定化酵母细胞生物合成L-麻黄素的重要中间体L-PAC。结果确定在最佳温度28℃,pH 6.46,活化时间30 min,反应时间10 h,苯甲醛浓度为3 mL/L,添加少量β-环糊精等的条件下,以2%海藻酸钙和10%聚乙烯醇为复合载体,生物合成L-PAC,产量可达8.34 g/L。结论为生物合成L-PAC提供了一种很好的方法。     

色氨酸酶基因工程菌固定化及其培养条件的研究

对色氨酸酶基因工程菌 ＷＷ－ １１进行固定化及培养条件研究,为工业化生产 Ｌ－色氨酸奠定基础。方法;通过色氨酸酶活力测定,考察三种固定化材料及温度、 ｐＨ、单价阳离子和乙醇对固定化 ＷＷ－ １１色氨酸酶活力的影响。结果：以聚乙烯醇作为ＷＷ－１１的固定化载体,其活力回收为６０．９％。固定化ＷＷ－１１色氨酸酶降解反应最适ｐＨ为９．０、最适温度为５０℃;固定化ＷＷ－１１色氨酸酶合成反应最适ｐＨ为７．５、最适温度为４５℃。Ｋ＋、ＮＨ＋对固定化工程菌色氨酸酶有明显激活作用,而Ｎａ＋则有一定的抑制作用。结论：固定化工程菌色氨酸酶对温度、乙醇等的稳定性比游离菌有显著提高。     

Immobilization of Paecilomyces variotii tannase and properties of the immobilized enzyme

Tannase produced by Paecilomyces variotii was encapsulated in sodium alginate beads and used for the effective hydrolysis of tannic acid; the efficiency of hydrolysis was comparable to that of the free enzyme. The alginate beads retained 100% of their efficiency in the first three rounds of successive use and 60% in rounds 4 and 5. The response surface methodology showed that the best conditions to hydrolysis of tannic acid by immobilized tannase were: sodium alginate 5.2%, CaCl₂ 0.55?M and 9?h to curing time. The optimized process resulted in 2.4 times more hydrolysed tannic acid than that obtained before optimization. The optimum pH for the actions of both the encapsulated and the free enzymes was 5.5. The optimum temperature of the reaction was determined to be 40°C for the free enzyme and 60°C for the immobilized form. The immobilization process improved the stability at low pH.     

Immobilization of Paecilomyces variotii tannase and properties of the immobilized enzyme

Tannase produced by Paecilomyces variotii was encapsulated in sodium alginate beads and used for the effective hydrolysis of tannic acid; the efficiency of hydrolysis was comparable to that of the free enzyme. The alginate beads retained 100% of their efficiency in the first three rounds of successive use and 60% in rounds 4 and 5. The response surface methodology showed that the best conditions to hydrolysis of tannic acid by immobilized tannase were: sodium alginate 5.2%, CaCl? 0.55?M and 9?h to curing time. The optimized process resulted in 2.4 times more hydrolysed tannic acid than that obtained before optimization. The optimum pH for the actions of both the encapsulated and the free enzymes was 5.5. The optimum temperature of the reaction was determined to be 40 °C for the free enzyme and 60 °C for the immobilized form. The immobilization process improved the stability at low pH.     

雅致小克银汉霉对16α,17α-环氧黄体酮C_(11)α-羟基化的工艺研究

目的研究雅致小克银汉霉对16α,17α-环氧黄体酮的C11α-羟基化的工艺条件。方法通过单因素试验和正交设计,以C11α-羟基化转化率为指标,确定最佳发酵工艺条件。结果该菌株最佳发酵工艺条件为:葡萄糖3%,玉米浆2.8%,(NH4)2SO40.3%,MnSO40.01%,BaCl20.02%,ZnSO40.005%,LiCl 0.01%,初始pH4.5,接种量3%,装液量100 mL/500 mL,摇床选用往复式摇床。结论采用优化后的培养条件,C11α-羟基化转化率达65.16%。作为1株高羟化能力的新菌株,具有良好的应用前景。     

pH sensitive alginate-guar gum hydrogel for the controlled delivery of protein drugs

Design of a pH sensitive alginate-guar gum hydrogel crosslinked with glutaraldehyde was done for the controlled delivery of protein drugs. Alginate is a non-toxic polysaccharide with favorable pH sensitive properties for intestinal delivery of protein drugs. Drug leaching during hydrogel preparation and rapid dissolution of alginate at higher pH are major limitations, as it results in very low entrapment efficiency and burst release of entrapped protein drug, once it enters the intestine. To overcome these limitations, another natural polysaccharide, guargum was included in the alginate matrix along with a cross linking agent to ensure maximum encapsulation efficiency and controlled drug release. The crosslinked alginate-guar gum matrix is novel and the drug loading process used in the study was mild and performed in aqueous environment. The release profiles of a model protein drug (BSA) from test hydrogels were studied under simulated gastric and intestinal media. The beads having an alginate to guar gum percentage combination of 3:1 showed desirable characters like better encapsulation efficiency and bead forming properties in the preliminary studies. The glutaraldehyde concentration giving maximum (100%) encapsulation efficiency and the most appropriate swelling characteristics was found to be 0.5% (w/v). Freeze-dried samples showed swelling ratios most suitable for drug release in simulated intestinal media ( approximately 8.5). Protein release from test hydrogels was minimal at pH 1.2 ( approximately 20%), and it was found to be significantly higher ( approximately 90%) at pH 7.4. Presence of guar gum and glutaraldehyde crosslinking increases entrapment efficiency and prevents the rapid dissolution of alginate in higher pH of the intestine, ensuring a controlled release of the entrapped drug.