-4的微环境分析

以聚乙烯醇(polyvinyl alcohol,PVA)和海藻酸钠(sodium alginates,Na·Alg)作为包埋载体,以五硼酸铵和氯酸铁、氯酸铝溶液作为交联剂,固定微杆菌Microbacterium sp. S₂-4,制备得到固定化球形颗粒。利用扫描电子显微镜(SEM)分析了在去除污染地表水COD过程中固定化球形颗粒的微环境变化,指出物理阻隔、吸附和种群排斥联合作用是固定化微环境对不利外界环境的主要屏蔽机理;同时还指出了通过该方法制备得到的固定化颗粒存在的结构缺陷。     

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。     

MOFs固定5-羟甲基糠醛氧化酶及其催化活性的研究

固定化酶作为一种绿色高效的生物催化剂,其性能远超游离酶。目前酶的固定化技术适用范围仍然较小,酶的研究范围多停留在模型酶阶段,扩大固定化酶的研究范围具有十分重要的意义。金属有机骨架材料(MOFs)作为酶固定化的载体在近些年得到了广泛的探索,但是具有生物功能的酶-MOFs复合材料的许多特性仍有待挖掘。采用仿生矿化的合成方法将5-羟甲基糠醛氧化酶(HMFO)固定到以沸石咪唑酯(ZIF-8)为代表的MOFs材料中,制备得到一种新的生物催化剂HMFO@ZIF-8,扫描电子显微镜表征其形态区别于经典的菱形十二面体。采用考马斯亮蓝法测定蛋白质浓度,计算得到酶的固定化效率达到89. 0%。HMFO@ZIF-8催化5-羟甲基糠醛的转化率达到84. 3%,收率和选择性均高于游离酶。拓展了MOFs固定化酶的研究范围,为研究其他生物大分子复合材料的生物催化剂提供一定的借鉴意义。     

多孔醋酸纤维素球形载体固定化糖化酶的研究

报道了一种多孔醋酸纤维素球形固定化酶载体的制备技术,以NaIO₄氧化法活化,固定糖化酶,测定了固定化糖化酶的催化反应特性,并与游离酶作了比较.固定化酶在55℃下水解10批淀粉溶液,总反应时间超过24 h,活力无显著变化.     

γ-Fe_2O_3-凹土超顺磁性纳米复合材料固载猪胰脂肪酶

以凹凸棒石黏土为原料,制备γ-Fe2O3-凹土超顺磁性纳米复合材料(γ-Fe2O3-ATP)作为猪胰脂肪酶(PPL)固定化的载体,利用透射电子显微镜(TEM)、N2吸附脱附等温图(BET)、振动试样磁强计(VSM)等对材料进行表征,同时对固定化条件和固定化酶的相关性质进行了研究。结果表明:制备的γ-Fe2O3-ATP是介孔材料,比表面积为102.63 m2/g,平均孔径为10.862 nm,饱和磁化强度为8.915 emu/g,其作为载体能实现固定化酶与反应介质简单、快速分离回收和重复利用。在固定化时间为4 h及pH 6.0时制备的固定化酶效果最佳;经过6 h高温保存后固定化酶可保留初始酶活的52%,而游离酶仅保留初始酶活的19%,同时固定化酶在重复使用5次后酶活仍保留初始酶活的60%。     

海藻酸盐固定化北京丙酸杆菌丙酸发酵的研究

本文报道利用海藻酸钠固定化北京丙酸杆菌,及其丙酸发酵的最适条件。最适海藻酸钠和菌体的起始浓度分别为2％和I00％(w／v)。菌体和海藻酸盐混合滴入CaCl₂：溶液中得到直径为3—4 mm球形颗粒。将固定化细胞放入25ml厌氧管中5 g颗粒/15ml培养基。其成分为(％)：葡萄糖1,酵母膏0．5,CaCI₂0.1。30℃静置培养产生大量的挥发酸,丙酸对乙酸的比例接近10：1。固定化细胞重复利用发酵30次,保持稳定活性65天。     

磁性纳米颗粒固定化乙醇脱氢酶的研究

本研究采用3-丙氨基三乙氧基硅烷(APTES)和戊二醛修饰包裹有SiO2磁性Fe3O4纳米颗粒表面,将其作为固定化载体固定化乙醇脱氢酶,研究固定化条件对固定化效率的影响,并对固定化酶性质进行分析。研究发现,当Fe3O4@SiO2纳米颗粒修饰上氨基和醛基后依然具有良好的水分散性和胶体稳定性,适合作为固定化载体。通过单因素优化,发现当最适给酶量为11. 3U/100 mg,搅拌转速为150 r/min,固定化p H和固定化温度分别控制在6. 5和5℃～15℃,固定化时长为45 min时,具有较好的固定化效果,固定化率可达到60. 2%。在此条件下制备得到的固定化酶与游离酶相比,固定化酶具有良好的耐高温和耐碱性。所得固定化乙醇脱氢酶在连续使用8次后,固定化率仍保留在57%左右,表明该固定化酶具有较好的操作稳定性,可为连续生产NADH提供技术依据。     

氨基化二氧化硅颗粒固定木瓜蛋白酶研究

采用正硅酸乙酯与N-(β-氨乙基)氨丙基三乙氧基硅烷在油包水形成的微胶囊中同步水解的方法,一步法制备了氨基化的二氧化硅颗粒,得到的颗粒粒径在0.3～0.5μm之间,平均大小为0.37μm, 氨基含量和颗粒大小可控,氨基含量高达56mmol/g。此颗粒经戊二醛处理后,采用共价法固定木瓜蛋白酶,固定化最适pH6.5,最佳给酶量为15mg/g载体,固定化酶的最适反应温度为70℃,最适反应pH为6.5,固定化酶热稳定性,pH耐受性,贮存稳定性都明显高于游离酶,表明此颗粒可作为一种优良的酶固定化载体。     

陶瓷载体固定化酵母发酵动力学研究

固定化细胞和固定化酶一样,作为固液两相的非均相催化反应系统,其反应速度无疑受到内外扩散的影响。世界各国的化学工程学者在固定化酶反应动力学方面研究得较多,固定化细胞反应动力学研究虽有些报道,但主要集中在以海藻酸钙为载体的固定化系统,且载体形状为球形颗粒。对于其他材料的不同形状颗粒载体的固定化细胞动力学研究报道较少。陶瓷作为固定化细胞载体是我们研究的一种固定化细胞的新型载体,在机械强度,孔径大小,细胞与之结合牢固度及其稳定活性,再生性能方面有其特有的优越性^[10]。为了给在生产实践中使用这种新型载体提供理论依据,本文采用球型陶瓷和拉西环陶瓷为载体固定化酵母,对它们的发酵动力学进行了比较研究。     

固定化细胞有机相催化不对称还原β-羰基酯

将酵母细胞用海藻酸钙包埋后用于有机相催化不对称还原4-氯乙酰乙酸乙酯制备光学活性的4-氯-3-羟基丁酸乙酯,从中筛选得到具有较高立体选择性和还原能力的菌株假丝酵母SW0401,将此菌株的细胞固定化细胞作为研究对象,系统考察了固定化条件、固定化细胞大小、反应溶剂、初始底物浓度、辅助底物、固定化细胞热处理和抑制剂对还原反应的影响。结果表明,上述因素对反应的摩尔转化率和产物（S）-CHBE光学纯度有显著影响。固定化时所用缓冲液的pH值为7．0时和固定化细胞颗粒平均直径为2.5mm较合适,以正己烷为反应介质时反应的摩尔转化率和产物光学纯度最优,初始底物浓度以54.7mmol／L为宜,辅助底物以1-己醇为佳。对固定化细胞的热处理和添加抑制剂烯丙醇均能够明显改善产物的光学纯度,但对提高摩尔转化率有负面影响。     

固定化微生物处理模拟污染地表水

以聚乙烯醇和海藻酸钠为包埋剂、驯化后的活性污泥为包埋菌剂,制备固定化微生物颗粒,其中包埋剂与包埋菌剂的比例为2:1。将该固定化微生物颗粒按20%的填充率装填到自制反应器中,用于处理模拟污染地表水,研究该固定化微生物的性能特点及其对模拟污染地表水的净化效果。结果表明:固定化微生物反应器的最佳水力停留时间为10h,最佳进水COD负荷为1.15～1.85g·L-1·d-1。在水温为20～29℃、溶解氧为3～4mg·L-1、水力停留时间为10h的条件下,当进水COD浓度为70.58～91.76mg·L-1、铵氮浓度为13.68～17.82mg·L-1时,COD去除率>62.3%,铵氮去除率>90.6%,表明固定化微生物能够有效地去除污染地表水中的COD和铵氮。     

Immobilization of chloroperoxidase from Serratia marcescens

A bacterial non-heme chloroperoxidase from Serratia marcescens W 250 was immobilized in calcium-alginate gel. Methods for stabilization of the immobilized enzyme were developed, and some kinetic parameters of the immobilized preparations were determined. The enzyme encapsulated into the gel granules in the presence of potassium ferricyanide followed by treatment with glutaraldehyde demonstrated the highest stability under the reaction conditions.     

Conditions for immobilizing Streptomyces erythreus in a calcium alginate gel and the apparatus setup for the process

A laboratory unit for production of calcium alginate gel granules with immobilized microorganisms is described. It provides sterile production of particles from tens micrometers to 2 mm in diameter. Expediency of using biocatalysts in the form of fine granules is exemplified with a number of immobilized microorganisms. Conditions for immobilizing the erythromycin producing organism by its incorporation into the calcium alginate gel were studied. Viability of the actinomycete in the gel was shown by consumption of the nutrients and biosynthesis of the antibiotic.     

Immobilization of Chloroperoxidase from Serratia marcescens

A bacterial non-heme chloroperoxidase from Serratia marcescensW 250 was immobilized in calcium alginate gel. Methods for stabilization of the immobilized enzyme were developed, and some kinetic parameters of the immobilized preparations were determined. The enzyme encapsulated into the gel granules in the presence of potassium ferricyanide followed by treatment with glutaraldehyde demonstrated the highest stability under the reaction conditions.     

Immobilization of yeast cells by adhesion on tuff granules

Summary A method for immobilizing yeast cells (Saccharomyces cerevisiae) possessing invertase activity by direct adhesion on tuff granules coated with insolubilized gelatin is described. The immobilized cells, firmly fixed as a monolayer onto the surface of the support granules display catalytic properties (in terms of apparent K _m) close to free cells and are particularly suitable for continuous sucrose hydrolysis in a fixed-bed reactor. From an industrial point of view, the immobilization method described here has two advantages over other immobilization methods, i.e. the immobilized yeast cells have a fairly good operational stability and their proliferation on tuff granules can be controlled.     

Biodegradation of phenol by Trichosporon cutaneum cells covalently bound to polyamide granules

Polyamide granules with high specific area were used for covalent immobilization of Trichosporon cutaneum R57. In order to increase the concentration of active (amino) groups necessary for cell immobilization, the polyamide (PA) sorbent was chemically modified. The optimal conditions for covalent immobilization of the cells were determined. Phenol degradation was studied with chemically immobilized cells. For comparison, parallel experiments were carried out with physically immobilized and free cells. Both covalently-bound and free cells fully degraded phenol at concentrations up to 1·0 g/litre. The optimal pH of phenol degradation by covalently bound cells was 6·0. The number of cycles of effective phenol degradation by immobilized cells was studied. The results obtained for covalently bound Trichosporon cutaneum R57 cells on PA granules clearly show the possibility for their application for the purification of waste water containing phenol.     

White‐rot fungi combined with lignite granules and lignitic xylite to decolorize textile industry wastewater

The feasibility of using immobilized fungi to decolorize textile industry wastewater containing dyes was examined in experiments with: two species of white‐rot fungi (a Marasmius species from Indonesia, which produces copious biomass, and Trametes hirsuta, which produces high levels of laccase); two types of lignite products as adsorbents and solid substrates (lignitic xylite and lignite granules); and four simulated wastewaters, each containing a different kinds of reactive textile azo dye. The growth, extracellular enzyme production, dye degradation and dye absorption parameters afforded by each permutation of fungus, substrate and dye were then measured. Both fungal species grew poorly on xylite, but much better on lignite granules. Marasmius sp. produced up to 67 U/L laccase on lignite granules, but just 10 U/L on xylite, and no other detectable extracellular enzymes. T. hirsuta produced 1343 U/L laccase and up to 12 U/L unspecific peroxidase when immobilized on lignite granules, and 898 U/L laccase with 14 U/L unspecific peroxidase when immobilized on xylite. The amount of color lost from the dye solutions depended on both the type of dye and the enzyme levels in the fermenter.     

T cell antigen receptor triggered exocytosis in cytotoxic T lymphocytes is inhibited by soluble, but not immobilized monoclonal antibodies to Lyt-2 antigen

The effect of monoclonal antibodies (mAb) to surface antigens on the T cell antigen receptor (TcR)-triggered exocytosis of intracellular granules in cytotoxic T lymphocytes (CTL) was studied. Soluble anti-LFA-1, anti-TcR, and anti-Lyt-2 mAb inhibited both CTL-inflicted 51Cr-release from the target cell (TC) and TC-stimulated exocytosis of granules from cloned CTL. Soluble anti-TcR and anti-Lyt-2 mAb but not soluble anti-LFA-1 mAb inhibited exocytosis, which was triggered by solid-phase anti-TcR mAb. Immobilized anti-Lyt-2 did not inhibit secretion triggered by immobilized anti-TcR mAb; immobilized anti-LFA-1 mAb had an modest inhibiting effect. Inhibition of exocytosis by soluble anti-Lyt-2 mAb was greater when stimulating anti-TcR mAb were immobilized at a lower density on a plastic surface. When the requirement for TcR cross-linking was bypassed by synergistic action of phorbol ester and ionophore A23187, no inhibition of exocytosis by soluble anti-Lyt-2 mAb was detected. The obtained data point to steric hindrance as the most likely explanation of the inhibition of TcR-triggered CTL activation by anti-Lyt-2 mAb.     

Enhanced Toluene Degradation Under Chlorate-Reducing Conditions by Bioaugmentation of Sand Columns with Chlorate- and Toluene-Degrading Enrichments

Chlorate was examined as a potential electron acceptor for enhancing toluene degradation. Most chlorate respiring bacteria (CRB) use nitrate as an electron acceptor, and toluene is known to be degraded under denitrifying conditions. Therefore, it was hypothesized that there would be bacteria that could degrade toluene using chlorate as an electron acceptor, and that chlorate could be used to stimulate toluene degradation. Repeated tests and different approaches in batch tests failed to produce an enrichment capable of toluene degradation supported by chlorate reduction. However, the addition of chlorate increased the overall rate of toluene degradation in bioaugmented columns that were fed chlorate vs. a control column. Toluene removal at an influent toluene concentration of 11 mg/L was 93±5%, which was larger by a factor of 1.95 than toluene removal in a nonbioaugmented control column. Following the discontinued feed of chlorate, toluene removal decreased to 69±4%, demonstrating that chlorate could be used to produce a 1.36-fold increase in toluene removal.     

Chlorate Toxicity and Nitrate Reductase Activity in Tomato Plants

Chlorate damage was studied in tomato plants ( Lycopersicum esculentum cv. Moneymaker) that were supplied with a nitrogen-free nutrient solution or with a nutrient solution, containing either nitrate or ammonium as a nitrogen source. Damage was low in ammonium-fed plants and high in nitrate-fed plants and in nitrogen-less plants. Nitrate reductase activity could be detected in all treatments, although the activity was highest in the nitrate-fed plants.
The hypothesis that chlorate can be used as a substrate by the enzyme nitrate reductase in higher plants, was studied and proved to be true for the tomato plants, as was found earlier for Escherichia and Chlorella . The affinity of the enzyme for chlorate was lower than for nitrate, the K _m being 4 m M and 0.15 m M respectively. Induction of the enzyme by chlorate could not be detected. The enzyme activity was lowered in leaf discs after a 7 h treatment with chlorate and the inhibition was proportional to the chlorate concentration of the medium.
The results were discussed in terms of competition between nitrate and chlorate at the uptake and the enzyme site and with regard to a possible influence of chlorate on synthesis and breakdown of the enzyme.