菠萝果蛋白酶生产工艺的改进

本试验研究了菠萝果蛋白酶生产工艺中的各个重要环节。结果表明:用0.08～0.1％的丹宁作沉淀剂较适宜,既可保持产品质量,又可兼顾产品产量;酶膏在-12℃以下低温冻结和真空干燥两个因素能显著提高产品酶活性;1000 ug/g硫代硫酸钠加62.5 ug/g半胱氨酸是菠萝果蛋白酶活性的有效保护剂,可使酶活性比对照提高32.57％;提取过程中用抗坏血酸、乙二胺四乙酸二钠、氯化钠和醋酸锌溶液对酶复合物进行洗涤可有效地提高产品的活性和质量。     

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%.     

中性蛋白酶AS 1.398固定化酶的研究

枯草芽孢杆菌AS1.398中性蛋白酶经海藻胶包埋制成固定化酶,用396的胶包埋率达98%,表现活力为7%左右。其最适反应pH为7.0,最适温度为55℃,与自然酶相一致。固定化酶的耐热性比自然酶高,最稳定的pH为7.0左右,pH8.0以上不稳定。海藻胶容易解体,球形破裂,因此只适合在中性条件下使用。     

聚乙二醇对菠萝蛋白酶的化学修饰

方法:用琥珀酸酐法活化的聚乙二醇对菠萝蛋白酶进行化学修饰,得到菠萝蛋白酶的修饰酶,对比研究三种菠萝蛋白酶:修饰酶、混合酶、天然酶的热稳定性及酸碱稳定性,考察金属离子对三种菠萝蛋白酶的影响。结果:当在55℃水浴保温100min后天然酶活力只保留20%,混合酶活力保留37%,修饰酶活力保留58%;在pH3.0-4.5及pH6.0-7.0的条件下,修饰酶活力高于天然酶活力。当Ca2 的浓度达到0.05mg/mL时,修饰酶的活力高达257.66%;当Mg2 的浓度达到0.035mg/mL时,修饰酶的活力高达147.25%。一价离子Na 对三种菠萝蛋白酶无明显影响。结论:修饰的菠萝蛋白酶对温度和pH值的稳定性均比天然酶有很大程度的提高。混合酶的活力介于天然酶和修饰酶之间说明聚乙二醇对菠萝蛋白酶有一定的保护作用。二价离子Ca2 、Mg2 对三种菠萝蛋白酶活力均有不同程度的激活作用。     

中性蛋白酶AS 1.398固定化酶的研究

枯草芽孢杆菌AS1.398中性蛋白酶经海藻胶包埋制成固定化酶,用396的胶包埋率达98%,表现活力为7%左右。其最适反应pH为7.0,最适温度为55℃,与自然酶相一致。固定化酶的耐热性比自然酶高,最稳定的pH为7.0左右,pH8.0以上不稳定。海藻胶容易解体,球形破裂,因此只适合在中性条件下使用。     

固定化芽孢菌产碱性蛋白酶的研究

本文报导了用海藻酸钙固定地衣芽孢杆菌(Bacilluslicheniformis),发酵生产碱性蛋白酶的研究。固定化细胞颗粒在低浓度的玉米粉、黄豆饼粉为原料的培养基中发酵24h,酶活高达1724u/ml,充分利用并节省了原材料,缩短了发酵周期。发酵液菌浓度的测定结果表明,固定化细胞凝胶粒细菌的渗漏程度较低,有利于提取工艺的简化。     

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。     

假菠萝麻蛋白酶分离纯化及特性研究

从假菠萝麻叶汁中用乙醇分部沉淀法得到一种蛋白酶,对酪蛋白有强烈的水解活性,经Ｓｅｐｈａｄｅｘ Ｇ－１００柱层析和ＳＰ－Ｓｅｐｈａｄｅｘ Ｃ－５０离子交换柱层析等步骤纯化,可得到六角形结晶。结晶酶液经ＰＡＧＥ圆盘电泳,每条胶柱上只显示一条蛋白带,其活性在ｐＨ４。５－１０、５５℃范围内较稳定,最适ｐＨ８．５最适温度５０℃,Ｋｍ（酪蛋白）值为０．１８５％（Ｗ／Ｖ）。用ＳＤＳ－ＰＡＧＥ和Ｓｅｐｈａｄｅｘ     

Immobilization of the proteins in the natural rubber with dialdehyde sodium alginate

The biodegradable dialdehyde sodium alginate (DASA) was exploited to immobilize the proteins in the natural rubber latex (NRL) and the variations of the properties for the NRL films were estimated in detail. As demonstrated, the proteins were distributed more uniformly in the NRL films with DASA and the extractable protein (EP) content was effectively decreased. Particularly, the EP content was lowered to a value about 46 μg/g with 0.40% DASA, which could meet with the demands of the allergy protein threshold limit of 50 μg/g as described in ASTM D 5712 standard. Furthermore, there was some improve on the burial degradability of the NRL films modified with DASA. The mechanical properties, however, had no evident variation in the presence of DASA. In conclusion, the immobilization of the proteins with DASA should be a potential alternative to tackle the protein allergy problem for the NRL and its products.     

固定化酶法手性转化右旋磷霉素的初步研究

研究确定沙门柏干酪青霉菌（Penicillium camemberti）2221能够产生手性转化右旋磷霉素的酶,以及以海藻酸钠为载体固定化酶转化的最适条件。以海藻酸钠为载体,分别考察了海藻酸钠浓度、氯化钙浓度、海藻酸钠和酶用量的体积比、固定化时间等条件,以及固定化酶的最适反应pH、最适反应温度和反复利用的稳定性等。结果表明,最优固定化条件是3.0%（质量与体积比）海藻酸钠、3.0%（质量与体积比）氯化钙、酶液（浓缩20倍）和3.0%（质量与体积比）海藻酸钠溶液的体积比为1 2、固定化时间为6 h。固定化酶的最适温度为37℃,最适pH为6.2,转化率为14.3%,连续反应4次后转化率为最初的57.57%。利用海藻酸钠包埋固定化沙门柏干酪青霉菌产生的手性转化右旋磷霉素的酶,能够连续转化生成左旋磷霉素,提高酶的利用效率,延长使用时间,具有进一步研究开发的价值。     

生物脱硫菌根癌土壤杆菌UP-3的固定化研究

生物脱硫催化剂固定化研究对生物脱硫技术的推广应用具有重要的意义。该文以筛选出的具有脱硫能力的根癌土壤杆菌UP-3为固定化研究对象,二苯并噻吩(DBT)为生物催化脱硫的模型化合物,主要考察了菌株UP-3的培养条件、固定化方法和载体、固定化操作条件和固定化细胞的使用条件。结果表明：以桑特斯培养基在30℃下培养28h的根癌土壤杆菌UP-3具有最佳活性。采用3wt％海藻酸钠水溶液为包埋载体,液菌比为20：1,在4℃下1wt％CaCl2水溶液中固定化24h,得到的固定化细胞脱硫性能最好。在30℃下,反应6d可将浓度为625mg／L的DBT降解60％以上。     

Immobilization of catalase on cotton fabric oxidized by sodium periodate

Cotton fabric was first oxidized with sodium periodate, and then employed to immobilize catalase. Optimization studies for oxidation of the fabric and immobilization of the enzyme were performed. The properties of the immobilized catalase were examined and compared with those of the free enzyme. A high activity of the immobilized enzyme was obtained when the fabric was oxidized at 40°C and pH 6.0 for 8h in a bath containing 0.20 mol L^?1 sodium periodate and the enzyme was immobilized at 4°C for 24h with a catalase dosage of 120.0 U mL^?1. The immobilized enzyme exhibited optimum activity at 40°C, while the free enzyme had optimal temperature of 30°C, suggesting that the immobilized catalase could be used in a broader temperature range. Both the immobilized and free enzyme had pH optima of 7.0. The staining test and reusability showed that the catalase was fixed covalently on the oxidized cotton fabric.     

Additional stabilization of stem bromelain coupled to a thermosensitive polymer by uniform orientation and using polyclonal antibodies

Stem bromelain was covalently coupled to a thermosensitive polymer of N-isopropylacrylamide (p(NIPAm)) either through the amino groups of the enzyme (randomly coupled) or via the lone oligosaccharide chain (uniformly coupled). The enzyme coupled via the oligosaccharide chain exhibited better access to the substrate casein as compared to the preparation in which the amino groups formed the point of contact between the enzyme and the polymer. Native bromelain exhibited a pH optimum of 8.0 and a broad pH-activity profile. The polymer-coupled preparations exhibited broader pH-activity profiles and shifting of pH optimum to 10.0 at 35°C. At 25°C, the shifting of pH optimum was observed for the randomly coupled enzyme only. The temperature-activity profiles of bromelain coupled to p(NIPAm) also showed appreciable broadening and the preparations retained greater fraction of maximum activity above the temperature optimum. The optimum temperature of the uniformly oriented preparation also rose to 70°C. Inactivation rates of the polymer-coupled bromelain were remarkably low at 60°C as compared to the native protease, and binding of antibromelain antibodies improved the resistance to inactivation of the polymer-coupled preparations. The cleavage patterns of hemoglobin and IgG by the native bromelain and the polymer-coupled preparations were comparable. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 3, pp. 375–382.     

Immobilization and characterization of cellulase on concanavalin A (Con A)-layered calcium alginate beads

Cellulase has been immobilized on hybrid concanavalin A (Con A)-layered calcium alginate–starch beads. Immobilized cellulase retained about 82% of its activity. Con A was extracted from jack bean and the obtained crude protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The immobilized beads showed high mechanical and storage stability; immobilized cellulase retained 100% and 85% activity at 4°C and 30°C, respectively, over one month. The immobilized cellulase retained about 70% of its activity after five cycles of use. The immobilized cellulase retained 70% activity after 120-min exposure to 60°C, whereas the soluble form only retained about 20%, showing that immobilization improved thermal stability. Surface morphology and elemental analysis of immobilized cellulase were examined using scanning electron microscope equipped with energy-dispersive X-ray. Based on the enzyme stability and reuse, this method of immobilization is both convenient and cheap.     

高效苯酚降解菌细胞固定化方法与条件的研究

含酚废水是一种难降解有机废水,对环境污染非常严重。目前常利用细菌处理含酚废水。但利用细菌处理含酚废水存在一些缺点,为此将1株高效苯酚降解菌进行细胞固定化。采用正交实验设计方法确定了该菌株固定化的最佳条件,并且考察了该固定化细胞降解苯酚的最佳条件。实验表明:该菌株的固定化细胞降解苯酚能力和耐受苯酚能力均大于游离细胞,经36 h可将1 800 mg/L苯酚降解完全。其降解苯酚的最适温度为30℃,最佳pH值为5~9。     

Oriented immobilization of stem bromelain via the lone histidine on a metal affinity support

Bromelain is a basic, 23.8 kDa thiol proteinase obtained from the stem of the pineapple plant (Ananas comosus) and is unique for it contains a single histidine residue (His-158) in the polypeptide. Based on the technology of protein separation with immobilized metal ion affinity chromatography (IMAC), a method for oriented immobilization of bromelain was selected. Bromelain was successfully immobilized on iminodiacetic acid carrier Sepharose 6B. Cu²⁺ complexed with iminodiacetate (IDA) was used as the chelating ligand to bind the lone histidine on bromelain. Simultaneously, preparation of a high affinity immobilized preparation was attempted using a soluble cross-linked preparation of bromelain on Cu-IDA-Sepharose. However this second method proved unsuccessful, possibly due to poor histidine accessibility in the cross-linked preparation. The immobilized preparation obtained using uncrosslinked bromelain was more resistant to thermal inactivation, as evidenced by retention of over enzyme 50% activity after incubation at 60 °C, as compared to 20% retained by the native enzyme. The immobilized preparation also exhibited a broader pH-activity profile in acidic range. The native, immobilized and soluble cross-linked bromelain showed apparent Michaelis constant (K_m) values of 1.08, 0.42, 1.56 mg/ml, respectively, using casein as the substrate. While the maximum velocity (V_max) values of the soluble and immobilized preparations were comparable, cross-linked preparation showed a 20% decrease, suggesting inactivation. The mild conditions used for predominantly oriented immobilization exploiting the unique property of single histidine, the high recovery of immobilized preparations, the stability, reusability and the regenerability of the matrix are the main features of the method reported here.     

CO2转化酶的固定化研究

CO2是导致温室效应的主要气体,固定和转化CO2的研究对于温室效应的减缓和环境保护方面具有重要意义。近年来CO2转化的研究取得了迅猛发展,其中生物法固定CO2由于其反应条件温和且绿色无污染的优点而备受关注。本文对转化CO2有关的乳酸脱氢酶（LDH）、苹果酸脱氢酶（MDH）和草酰乙酸脱羧酶（OAADC）进行了初步的固定化分析。首先以碳纳米管、壳聚糖和海藻酸钠为原料,制备了包埋上述CO2转化酶的微胶囊固定化体系,然后分别比较了游离酶和固定化酶的操作稳定性和储存稳定性。研究结果表明,固定化的CO2转化酶的操作稳定性和储存稳定得到明显的提高。本研究对CO2的转化和应用方面具有重要参考价值。