表面活化甲壳素固定化Gibberella intermedia生物转化制备异烟酸

甲壳素及其衍生物资源来源广泛、具有生物安全性,在材料、食品、化工等领域拥有良好的应用前景。本研究首次尝试采用经表面活化的甲壳素为包埋材料对产腈水解酶的G. intermedia游离细胞进行固定化,对固定化条件进行了初步优化,选取甲壳素质量分数3%,三聚磷酸钠质量分数7%,固定化时间5 h,固定菌体浓度为20g/L时催化活力达到最高。对固定化细胞的催化应用特性进行了表征,结果表明固定化细胞对4-氰基吡啶转化反应的最适反应温度为50℃,最适pH值为7.0;最适底物浓度为125mmol/L,最大产物耐受浓度为400mmol/L,均明显优于游离细胞。探索了将制备的固定化细胞直接应用于4-氰基吡啶生物转化合成异烟酸,固定化细胞可重复利用14批次,而相应游离细胞仅为3次。     

3-氰基吡啶转烟酸菌株的筛选及发酵条件研究

从土壤中筛选出高效催化3-氰基吡啶转化烟酸的菌株NA-6,对该菌株进行形态鉴定,初步鉴定为恶臭假单胞菌.同时对发酵条件进行研究,结果表明:该菌株在温度30℃,起始pH值为7.0,3-氰基吡啶浓度为0.3％时,腈水解酶活性最大,比原始提高了1.89倍.     

壳聚糖-聚乙烯醇固定化细胞在生物转化合成烟酸中的应用

传统的腈水解酶游离催化剂在腈类化合物生物转化过程中存在易失活、稳定性低、重复利用率不高等特点，本研究采用复合固定方式对基因工程腈水解酶进行固定化以期提升其应用性能。通过固定化壳聚糖和聚乙烯醇（PVA）作为包埋材料，获得了具有高机械强度和酶活性的固定化细胞。初步探究了固定化聚乙烯醇和壳聚糖的浓度及固定剂的优选条件，分别为80g/L的PVA、40g/L的壳聚糖及60g/L三聚磷酸钠的饱和硼酸溶液；与游离细胞相比，固定化细胞的热稳定性和贮藏稳定性均显著提高，可增加约2倍；在525min的转化时间内，采用底物流加模式可转化合成烟酸208g/L。本文为复合固定化催化剂在腈化合物生物转化及烟酸合成中的应用奠定了坚实基础。     

用固定化L-赖氨酸脱羧酶细胞制备1,5-戊二胺

研究了4种固定化蜂房哈夫尼菌(H.alveiAS1.1009)菌体细胞的材料和方法,包括海藻酸钙包埋法、半透膜透析袋法、海藻酸钙-明胶交联包埋法和明胶包埋法。其中海藻酸钙包埋法稳定性最好,该方法最优转化条件为:在ρ(海藻酸钠)=30g/L的水溶液中,最适菌体质量浓度为ρ(菌体)=30.8g/L,100mL转化液中海藻酸钙球体体积为30mL,转化最适温度为37℃,最适pH=5.0。用该方法转化测得比酶活可达1028.9U。重复性佳:第一批固定化细胞酶活可达游离菌体的98.62%,第四批可达第一批酶活的38.68%。     

固定化细胞催化合成6-氰基-(3R,5R)-二羟基己酸叔丁酯工艺研究

采用硅藻土作载体,聚乙烯亚胺和戊二醛作交联剂来固定化E.coli BL21(DE3)/pCDFDuet-gdh-cr共表达菌株,以此来催化6-氰基-(5R)-羟基-3-羰基己酸叔丁酯[(5R)-1]合成6-氰基-(3R,5R)-二羟基己酸叔丁酯[(3R,5R)-2],考察了硅藻土-聚乙烯亚胺-戊二醛固定化细胞的催化性能,进一步优化了固定化细胞催化合成(3R,5R)-2工艺。结果表明:在固定化细胞用量100g/L、葡萄糖与底物质量浓度比为1:1、转化温度30℃、pH7.0、流速12mL/min、100 g/L的(5R)-1转化完全的条件下,填充床式反应器可以连续反应五个批次,转化570 g/L的(5R)-1,较搅拌式反应器提高了107.3%,较游离细胞间歇催化操作提高了185%。     

精细化工中间体2-氯烟酸的合成

2-氯烟酸是一种重要的医药和农药中间体,用途广泛.采用3步合成法,即以3-氰基吡啶和双氧水为起始原料,经氧化生成3-氰基吡啶N氧化物,再氯化生成2-氯-3-氰基吡啶,再水解生成2-氯烟酸,产物纯度99.5%,总收率70.0%以上,确定了反应物配比、反应温度、反应时间等工艺参数.     

复合载体固定化大肠杆菌制备γ-氨基丁酸

用卡拉胶与明胶复合载体固定大肠杆菌,优化了固定化细胞的制备条件,并利用该复合载体固定大肠杆菌细胞进行酶法制备γ-氨基丁酸的研究。实验结果表明,最佳工艺条件为:m(卡拉胶):m(明胶)=3:2,ρ(混合胶)=12g/L,ρ(KCl)=60g/L,固定化时间为3h,ρ(菌体)=80g/L,反应温度为37℃,转化体系pH=4.8,ρ(底物)=40g/L。固定化细胞在最适条件下比酶活高达10740U。包埋1.0g湿菌体的固定化细胞重复使用7次,可把42gL-谷氨酸完全转化为γ-氨基丁酸。     

外加剂对提高固定化细胞制备L-苯丙氨酸能力的作用

在固定化细胞制备L -苯丙氨酸的研究体系中 ,以卡拉胶为固定化载体 ,所得的固定化细胞在使用过程中的催化稳定性较差 ,难以满足大规模生产的需要。故在卡拉胶包埋固定E .coli细胞过程中 ,对交联剂的处理方式和吸附剂的添加进行了研究。研究发现 ,复合交联剂对固定化细胞的处理能使固定化细胞的转化能力明显提高 :多乙烯多胺 (质量分数 0 .2 5 % )和已二胺 (质量分数 0 .2 5 % )对固定化细胞的组合处理 ,固定化细胞的转化能力提高了 3 1.6%。在细胞包埋过程中添加质量分数 4%的吸附剂E ,固定化细胞的转化能力提高了近 5 2 %。     

Acinetobacter johnsonii G2细胞的固定化及其非水相介质中催化合成葛根素糖苷

利用海藻酸钠包埋法固定化Acinetobacter johnsonii G2细胞,在非水相介质中生物催化葛根素合成葛根素糖苷,考察了细胞的固定化条件、转化条件以及固定化细胞的操作稳定性。结果表明,最佳固定化条件为:海藻酸钠质量分数为2%,氯化钙质量分数为2%,细胞包埋量的体积分数为50%;最佳转化条件为:温度为40℃,p H为6.47,二甲基亚砜体积分数为20%。在最佳条件下,葛根素在催化体系中质量浓度提高至31.92 g/L,产率达93%,且固定化细胞的重复使用稳定性好。与游离细胞相比,固定化细胞对p H、温度及有机溶剂表现出更强的稳定性,且在非水相介质中催化效率更高。     

氧化葡萄糖酸杆菌的固定化及在鼓泡式反应器中制备乙醇酸

考察了包埋法固定氧化葡萄糖酸杆菌的过程,并且研究了利用该固定化细胞在鼓泡塔生物反应器中转化乙二醇制备乙醇酸。固定化的最优条件为:聚乙烯醇的最佳质量分数为8%,海藻酸钠为0.8%,最适湿细胞包埋量为每毫升凝胶0.27 g。固定化细胞对酸碱的抗性和热稳定性均较游离细胞有所提高。利用固定化细胞在鼓泡式反应器中转化乙二醇制备乙醇酸,在乙二醇质量浓度70 g/L的条件下,首批转化率达到92.6%,并且可以实现连续转化6个批次,平均转化率在85%以上。     

Covalent immobilization of α‐amylase onto thermally crosslinked electrospun PVA/PAA nanofibrous hybrid membranes

Poly(vinyl alcohol)/poly(acrylic acid) (PVA/PAA) nanofibers with the fiber diameter of 100–150 nanometers were fabricated by electrospinning. PVA/PAA nanofibers were crosslinked by heat‐induced esterification and resulting nanofiber mats insoluble in water. α‐Amylase was covalently immobilized onto the PVA/PAA nanofiber surfaces via the activation of amine groups in the presence of 1,1′‐carbonyldiimidazole. The immobilized α‐amylase has more resistance to temperature inactivation than that of the free form and showed maximum activity at 50°C. pH‐dependent activities of the free and immobilized enzymes were also investigated, and it was found that the pH of maximum activity for the free enzyme was 6.5, while for the optimal pH of the immobilized enzyme was 6.0. Reuse studies demonstrated that the immobilized enzyme could reuse 15 times while retaining 81.7% of its activity. Free enzyme lost its activity completely within 15 days. Immobilized enzyme lost only 17.1% of its activity in 30 days. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

2-氯烟酸的合成

综述了2-氯烟酸的合成工艺,并提出了改进的工艺路线。3-氰基吡啶先经乙酰丙酮酸钼催化氧化得到烟酸N-氧化物,再经有机碱和苯磷酰二氯作用下,三氯氧磷氯化得到2-氯烟酸,总收率95.0%。     

Bioconversion of corn stover hydrolysate to ethanol by a recombinant yeast strain

Three corn stover hydrolysates, enzymatic hydrolysates prepared from acid and alkaline pretreatments separately and hemicellulosic hydrolysate prepared from acid pretreatment, were evaluated in composition and fermentability. For enzymatic hydrolysate from alkaline pretreatment, ethanol yield on fermentable sugars and fermentation efficiency reached highest among the three hydrolysates; meanwhile, ethanol yield on dry corn stover reached 0.175 g/g, higher than the sum of those of two hydrolysates from acid pretreatment. Fermentation process of the enzymatic hydrolysate from alkaline pretreatment was further investigated using free and immobilized cells of recombinant Saccharomyces cerevisiae ZU-10. Concentrated hydrolysate containing 66.9 g/L glucose and 32.1 g/L xylose was utilized. In the fermentation with free cells, 41.2 g/L ethanol was obtained within 72 h with an ethanol yield on fermentable sugars of 0.416 g/g. Immobilized cells greatly enhanced the ethanol productivity, while the ethanol yield on fermentable sugars of 0.411 g/g could still be reached. Repeated batch fermentation with immobilized cells was further attempted up to six batches. The ethanol yield on fermentable sugars maintained above 0.403 g/g with all glucose and more than 92.83% xylose utilized in each batch. These results demonstrate the feasibility and efficiency of ethanol production from corn stover hydrolysates.     

Properties of cathepsin B immobilized in calcium alginate beads

Cathepsin B (EC 3.4.22.1), purified from goat brain, was immobilized in calcium alginate beads in the presence of bovine serum albumin. The immobilized enzyme retained ∼63% of the original activity and could be used for seven successive batch reactions with retention of 22–30% of the initial activity. Immobilized cathepsin B hydrolysed α-N-benzoyl-D ,L -arginine-β-naphthylamide (BANA) maximally at pH 5·5, exhibiting a shift of 0·5 pH unit from that of the soluble enzyme (pH optima 6·0). It showed enhanced stability in acidic as well as alkaline environments in comparison to the free enzyme. The optimal temperature and thermal stability were not altered significantly after immobilization. The K_m value for the immobilized enzyme was two-fold higher than for the soluble enzyme.     

Immobilization of β-galactosidase onto polymeric supports

Poly(vinyl alcohol) cross-linked with para-formaldehyde (PVA–F) and natural polysaccharide–chitosan in bead form and salicylic acid–resorcinol–formaldehyde polymeric resin (SRF) in powder form were used for immobilization of β-galactosidase through covalent linkages. Various activation processes and conditions were optimized. Immobilized enzyme showed very good storage stability at room temperature. Durability of the enzyme was also improved on immobilization. On repeated use of enzyme immobilized on chitosan beads, no loss was observed in enzyme activity even after 10 batches. Michaelis constant K_m and maximum reaction velocity V_m were calculated for free and immobilized enzyme systems. Effect of pH and temperature on enzyme activity was estimated and energy of activation (E_a) and inactivation constant (K_i) for free and immobilized enzyme were calculated. © 1995 John Wiley & Sons, Inc.     

明胶膜固定化脲酶的制备及性质

以明胶为载体,戊二醛为交联剂,采用包埋-交联联用法制备了明胶膜固定化脲酶,其酶活力为6 07U/g载体,酶活力收率为66 1%。最优固定化条件是包酶量为10mg酶/g明胶,ρ(明胶)=100g/L,φ(戊二醛)=0 5%。研究了固定化酶的性质,并与游离酶作了比较,游离酶的最适pH=7 0,固定化酶的最适pH=6 5;游离酶的最适温度为60℃,固定化酶的最适温度升至70℃;固定化酶与游离酶的米氏常数Km分别为11 7mM和12 4mM;固定化酶在80℃下180min仍保留初始活力的10%,而游离酶几乎完全失活。固定化酶重复使用20次其活力仅下降15%,4℃下贮存35d后仍保持初始活力的55%。     

Lipase-catalyzed synthesis of isoamyl butyrate: Optimization by response surface methodology

Immobilized lipase from Mucor miehei (Lipozyme IM-20) was employed in the esterification of butyric acid and isoamyl alcohol to synthesize isoamyl butyrate in n-hexane. Response surface methodology based on five-level, five-variable central composite rotatable design was used to evaluate the effects of important variables—enzyme/substrate (E/S) ratio (5–25 g/mol), acid concentration (0.2–1.0 M), alcohol concentration (0.25–1.25 M), incubation period (12–60 h), and temperature (30–50°C)—on esterification yield of isoamyl butyrate. In the range of parameters studied, the extent of esterification decreased with temperature, lower E/S ratios, and incubation periods. Excess acid and alcohol concentrations (i.e., acid/alcohol >1.4 or alcohol/acid >1.4) were found to decrease yield probably owing to inhibition of the enzyme by acid or alcohol, the former being more severe. The optimal conditions achieved are as follows: E/S ratio, 17 g/mol; acid concentration, 1.0 M; incubation period, 60 h; alcohol concentration, 1.25 M; and temperature, 30°C. With these conditions, the predicted value was 1.0 M ester, and the actual experimental value was 0.98 M.     

泡沫分离法回收酶法生产生物柴油中的液体脂肪酶

采用泡沫分离法对酶法生产生物柴油过程中的水相脂肪酶进行回收并富集. 通过正交实验考察了液体脂肪酶溶液中pH值、通气速度、初始酶浓度对富集比、酶蛋白回收率和酶活回收率的影响. 结果表明,气速10 L/(L×h)、进料酶浓度0.2 g/L及pH 7.0时蛋白回收率和酶活回收率接近100%,相应的富集比为3.67;初始酶浓度对富集比和蛋白回收率有显著影响,回收过程中脂肪酶活性无损失. 甲醇的存在能显著提高回收速率,甘油的存在降低了富集比,而生物柴油的存在影响了泡沫的稳定性,要形成稳定的泡沫,生物柴油含量需小于0.01%.