产糖化酶黑曲霉固定化方法比较的研究

采用海藻酸钙凝胶电埋法、以沸石、多孔聚酯等材料为固定化载体的吸附法固定黑曲霉（Aspergillus niger AS3.4309)菌丝细胞,以游离菌丝体作为对照,进行发酵产糖化酶的比较,结果表明：以聚酯泡沫作为固定化载体吸附固定化菌丝细胞产糖化酶活力最高。在产糖化酶的发酵过程中,与游离菌丝体细胞相比,固定化黑曲霉持续产酶时间有一定程度的延长。     

壳聚糖酶产生菌的筛选及固定化细胞产酶

旨在筛选得到一株壳聚糖酶产生菌,并研究固定化细胞产酶的条件。在培养基中以壳聚糖为唯一碳源,对土壤样品进行筛选,获得一株无花果沙雷氏菌(Serratia ficaria CH-0203),该菌可被壳聚糖诱导产生壳聚糖酶。固定化细胞产酶的研究结果表明,多孔玻璃可以有效吸附CH—0203菌细胞。在最适发酵条件下(pH6．5,培养基与载体的总体积48ml,载体与培养基的比例为1．5g/4．0ml,吸附时间是20h-26h),发酵液酶活达到4．5U／ml,比游离细胞发酵提高了16％。采用半连续发酵的方式,固定化的细胞可以稳定发酵产酶120h左右。固定化细胞产酶的效率大大高于游离细胞。     

多孔聚氨酯固定化黑曲霉产β-葡萄糖苷酶条件的研究

以多孔聚氨酯材料为固定化载体 ,就固定化培养条件中的孢子浓度、pH值、温度、固液比等进行考察 ,经正交试验优化后的最佳固定化条件为固液比为 1 / 90、孢子浓度为 1 0 5mL-1 、温度为 2 8℃ ,pH值 4 .5 ,固定化条件中影响黑曲霉产 β 葡萄糖苷酶的因素显著性顺序为固液比 >孢子浓度 >pH值 >温度。与游离态菌丝体相比较 ,固定化细胞发酵产酶的持续稳定性和活力都有所提高 ,固定化细胞酶产量达 1 30 .0 0u/mL。     

固定化桔青霉产生核酸酶P_1的研究

利用吸附固定在多孔聚酯载体上的桔青霉(Penicillium citrinum)菌丝细胞,在摇瓶中以分批发酵方式合成核酸酶P_1.试验结果表明:在固定化细胞产酶的条件下,培养液中葡萄糖和蛋白胨的最适浓度分别为10g/L和1g/L,摇瓶转速以180~200r/min为宜.固定化细胞经过48h的产酶周期,培养液中的核酸酶P_1活力可高达513.3U/ml,其产酶效率是游离菌丝的3.6倍,而葡萄糖和蛋白胨的用量仅为游离菌丝产酶的五分之一.在重复分批发酵试验中,固定化菌丝细胞形状稳定,连续28批(共56d)的产酶结果基本一致,每批的平均酶活力为507.4U/ml,在经济上和工艺上均显示了明显的优越性.     

固定化桔青霉产生核酸酶P_1的研究

利用吸附固定在多孔聚酯载体上的桔青霉(Penicillium citrinum)菌丝细胞,在摇瓶中以分批发酵方式合成核酸酶P_1.试验结果表明:在固定化细胞产酶的条件下,培养液中葡萄糖和蛋白胨的最适浓度分别为10g/L和1g/L,摇瓶转速以180~200r/min为宜.固定化细胞经过48h的产酶周期,培养液中的核酸酶P_1活力可高达513.3U/ml,其产酶效率是游离菌丝的3.6倍,而葡萄糖和蛋白胨的用量仅为游离菌丝产酶的五分之一.在重复分批发酵试验中,固定化菌丝细胞形状稳定,连续28批(共56d)的产酶结果基本一致,每批的平均酶活力为507.4U/ml,在经济上和工艺上均显示了明显的优越性.     

铜金属螯合载体固定糖化酶

[目的]制备出含Cu2+的琼脂糖-IDA螯合载体及对其固定糖化酶工艺条件进行优化.[方法]利用金属螯合配体(IDA-Cu2+)与蛋白质表面供电子氨基酸相互作用的原理制备载体,采用紫外分光光度法测定不同影响因素下固定化糖化酶的酶活.[结果]Cu2+的加入量和固定化过程的酸度比给酶量对固定化糖化酶的活性影响还要大,在给酶量80 mg/g载体、1.0× 10-2 mol Cu2+/g载体、pH 4.6和固定化4h的固定化条件下,固定化酶活为252.1 U/g,重复使用5次后酶活为首次固定化酶活的65.1％.[结论]该Cu2+-IDA-金属螯合琼脂糖可用于淀粉水解糖化酶的优良固定化载体材料.     

光交联聚氨酯作载体的固定化细胞产生a-淀粉酶的研究

将可光交联的聚氨酯预聚物与枯草杆菌细胞悬浮液混匀后经紫外光照射交联,制得固定化细胞,用于产生a-淀粉酶。探讨了交联和固定化条件对裁体结构和固定化细胞产酶性能的影响,研究了这种固定化细胞的使用特性。结果表明:光交联聚氨酯能有效地固定枯草杆菌细胞,进行正常增殖和产酶,改变交联和固定化条件能调节载体的孔容、孔径和比表面,从而调节固定化细胞的产酶性能和其它使用特性;枯草杆菌经光交联聚氮酯载体固定化后对温度和pH的适应性提高,在同样条件下,这种固定化细胞的产酶能力比游离细胞提高约30％,亦高于用k-角叉菜胶作载体的固定化细胞．     

Immobilization of D-lactonohydrolase by adsorption-crosslinking method

选择6种吸附树脂和离子交换树脂对D-泛解酸内酯水解酶进行固定化,筛选出了固定化效果较好的大孔弱碱性丙烯酸系阴离子交换树脂D-380为载体,用先吸附后交联的方法固定化.通过实验对固定化条件进行了优化,得出最佳的固定化条件为:加酶量6 U/g树脂、吸附pH7.5、吸附时间4h、吸附温度30℃、交联剂戊二醛终浓度0.1％、交联时间2h.实验表明在此条件下制得的固定化酶有很好的稳定性:固定化酶在连续20次的底物水解反应后,剩余酶活达到71％.当温度达到80℃时游离酶几乎失去酶活,而固定化酶剩余酶活为60％以上.游离酶的pH稳定性范围为pH7～8,而固定化酶为pH 6.5～8.5.     

固定化米根霉生产L－乳酸的研究

开发了利用聚氨酯泡沫为载体固定化米根霉生产Ｌ－乳酸的新工艺。确定了固定化细胞发酵条件：葡萄糖浓度为５０ｇ／ｌ,载体立方体边长为４～８ｍｍ,载体量为２０ｃｍ３／７０ｍｌ培养基,固定化细胞制备培养时间为２４ｈ．利用固定化细胞发酵产酸速率是游离菌的３倍以上,对糖转化率达７７．７０％,与理论转化率相近。该固定化细胞应用在反复间歇发酵中可稳定１０批次以上。     

D-阿洛酮糖3-差向异构酶在枯草芽孢杆菌中的高效表达及固定化细胞研究

将来源于Clostridium cellulolyticum H10的DPEase基因在食品级表达系统Bacillus subtilis中进行产酶研究,在3L发酵罐中高密度发酵最终酶活可达495U/ml,得到高表达量的DPEase酶液。通过硅藻土-海藻酸钠(吸附包埋法)对重组细胞进行固定化研究,结果表明,当海藻酸钠浓度为2%、细胞包埋量为50g/L、CaCl_2浓度为2%、硅藻土浓度为1%时,固定化细胞酶活回收率可达64%,固定化细胞与游离细胞相比最适pH不变,最适温度提高5℃,热稳定性明显提高,连续反应7个批次后转化率仍然为28%,仍保持81%的残余酶活,具有很高的工业应用价值。     

Production of cellulolytic enzymes by immobilized Sporotrichum thermophile.

Spores of Sporotrichum thermophile were immobilized in agar, polyacrylamide, and sodium alginate to generate in situ mycelium for production of cellulolytic enzymes. Immobilized mycelium was considerably less effective than free cells for cellulase productivity. Of the three gel types, agar beads proved to be the best carrier for the immobilized spores and subsequently generated mycelium. Results of repeated batch experiments suggested that the immobilized mycelia could be reused but at much reduced efficiency.     

硅藻土固定中性脂肪酶的制备及其性质

以硅藻土为载体,采用吸附法,对脂肪酶进行固定化,研究了固定化条件对固定化脂肪酶的催化活性的影响,得到最佳的固定化条件：给酶量为33374U/g,固定化温度为35℃,pH值为7.5,时间为4h,此时固定化酶的活力约为5833U/g载体。固定化酶的热稳定性较游离酶有了很大的提高,其在80℃以下能保持80%以上的酶活,而游离酶60℃残余酶活仅为5%。最适反应温度和最适pH值也分别由游离酶的40℃上升至50℃和由7上升到7.5。对固定化中的中性脂肪酶在生物柴油合成中的应用也进行了初步研究。     

Glucoamylase and α-amylase production by immobilized Aspergillus niger

Summary Aspergillus niger mycelia or spores were immobilized in calcium alginate gel beads and employed for production of glucoamylase and -amylase by repeated batch process. The immobilized mycelium produced lower enzyme activities than immobilized spores germinated in a growth medium and subsequently cultured in an enzyme production medium. In repeated batch experiments, free cells could be used for only 4 4-day batches, whereas with immobilized spores at least 11 4-day batches with a gradual increase in enzyme activities in each successive batch were possible. The activity ratio of glucoamylase and -amylase produced was altered by immobilization.     

环氧交联剂和氨基载体固定化海洋假丝酵母脂肪酶*

游离酶经过固定化后,稳定性和环境耐受性得到提高,在食品、医药、化工、环境和皮革等领域可以很好的提高酶的利用率并降低生产成本,具有极大的应用潜力。新型交联剂在固定化酶工艺的应用极大推进了固定化酶研究的深入。借助新型交联剂聚乙二醇二缩水甘油醚(PEGDGE),利用氨基载体LX-1000HA固定化海洋假丝酵母脂肪酶,结合单因素和正交试验优化得到交联及固定化条件为:交联温度30℃,交联2h,交联剂浓度0.75%,pH7.0,加酶量800U,载体量0.5g,固定化2h,固定化温度45℃。根据上述最佳固定化工艺,制备得到固定化酶LX-1000HA-PEGDGE-CRL在最适条件下测得酶活达到160.81U/g,约为此前制备的固定化酶LX-1000HA-GA-CRL(由LX-1000HA和戊二醛交联脂肪酶得到)和LX-1000EA-PEGDGE-CRL(由短链氨基载体LX-1000EA和PEGDGE交联脂肪酶得到)酶活的2倍,发现固定化酶LX-1000HA-PEGDGE-CRL的最适反应温度相比于游离酶提高15℃;在70℃的环境中3h后酶活仍存留70%;循环使用6次后残留65%左右的酶活;酸碱耐受性和储存稳定性也表现良好,4℃保存30天后剩余约70%的初始酶活。同时,将制备的固定化酶LX-1000HA-PEGDGE-CRL与游离酶、固定化酶LX-1000HA-GA-CRL、固定化酶LX-1000EA-PEGDGE-CRL进行了比较,发现固定化酶LX-1000HA-PEGDGE-CRL在温度耐受性和重复使用性等方面具有更好的使用效果。     

根瘤菌BK-20固定化细胞生产L(+)-酒石酸

顺式环氧琥珀酸水解酶(CESH)是根瘤菌BK-20生产L(+)-酒石酸的关键酶。为提高其生产效率和生产稳定性,首先优化根瘤菌BK-20的产酶条件,然后利用固定化细胞连续生产L(+)-酒石酸。结果显示,优化后游离细胞酶活达(3 498.0±142.6)U/g,较优化前提高643%。固定化细胞酶活达(2 817.2±226.7)U/g,其最适包埋剂、菌体浓度和凝胶浓度分别为海藻酸钠,10%(W/V)和1.5%(W/V)。固定化细胞连续反应10批后,其形状和酶活均无明显改变,单批次转化率达98%以上,具有良好的生产稳定性。     

Semicontinuous cultivation of immobilized Claviceps purpurea

Summary Mycelia of Claviceps purpurea CBS 164.59 were immobilized in 2%, 4%, and 8% calcium alginate. Alkaloid production by free cells declined after 60 days, while immobilized cells retained their activity for 200 days. The cumulative alkaloid production for all fermentation cycles using 8% calcium alginate immobilized mycelia was 25 times higher than that from free cells. The best yields of the ergopeptide ergometrine were reached with 4% gel immobilized mycelia, while higher gel concentrations caused a shift in the alkaloid biosynthesis towards high clavine alkaloid production.Beginning with the third cycle of reincubation the immobilized mycelia showed a marked tendency to fragmentize into vacuolated arthrosporoid-like structures and produced violet-black pigments so that the beads recalled sclerotial structures of parasitically living Claviceps.Dedicated to Prof. Dr. K. Esser to his 60th birthday     

Catalytic properties of the immobilized Aspergillus tamarii xylanase

Xylanase from Aspergillus tamarii was covalently immobilized on Duolite A147 pretreated with the bifunctional agent glutaraldehyde. The bound enzyme retained 54.2% of the original specific activity exhibited by the free enzyme (120 U/mg protein). Compared to the free enzyme, the immobilized enzyme exhibited lower optimum pH, higher optimum reaction temperature, lower energy of activation, higher K_m (Michaelis constant), lower V_max (maximal reaction rate). The half-life for the free enzyme was 186.0, 93.0, and 50.0 min for 40, 50, and 60°C, respectively, whereas the immobilized form at the same temperatures had half-life of 320, 136, and 65 min. The deactivation rate constant at 60°C for the immobilized enzyme is about 6.0 × 10⁻³, which is lower than that of the free enzyme (7.77 × 10⁻³ min). The energy of thermal deactivation was 15.22 and 20.72 kcal/mol, respectively for the free and immobilized enzyme, confirming stabilization by immobilization. An external mass transfer resistance was identified with the immobilization carrier (Duolite A147). The effect of some metal ions on the activity of the free and immobilized xylanase has been investigated. The immobilized enzyme retained about 73.0% of the initial catalytic activity even after being used 8 cycles.     

Decolorization of alcohol distillery wastewater by thermotolerant white-rot fungi

To detect thermotolerant fungus strain for decolorization of alcohol distillery wastewater (WAD), 38 fungus strains were studied. Ability of ligninolytic enzyme production was examined at 35 and 43 degrees C on agar media containing 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) and MnCl2. At 43 degrees C, four of Pycnoporus coccineus strains showed their higher potential for WAD decolorization both on agar media and in liquid media. Immobilized mycelia on polyurethane foam removed total phenol about threefold higher than free mycelia did in shaking condition at 43 degrees C. Moreover, color removed by immobilized mycelia nearly 50% higher than free mycelia did.     

Physiological aspects of free and immobilized Aspergillus niger cultures producing citric acid under various glucose concentrations

Similarities and differences between cultures of free and immobilized Aspergillus niger were identified under various glucose concentrations. Growth and citric acid production rates were compared, and the macro-morphology and fine structure of the mycelia examined to determine which parameters were significant in the production of citric acid. With free cultures the diameter of pellets was inversely related to glucose concentration, while biomass levels were lower for immobilized cultures than the equivalent free cultures. Rates of citric acid production were higher with immobilized mycelium, especially at higher glucose levels. The morphology that characterized high citric acid productivity was that of swollen hyphal tips which were seen at concentrations over 100 kg/m³ glucose in both free and immobilized mycelium. Although there is a characteristic morphology associated with high productivity it does not account for the difference observed between free and immobilized mycelia. The increased glucose uptake and productivity was not due to an increased surface area either, since the immobilized system was slightly lower in total surface area than the equivalent free cultures. The major difference was in the mean diffusion path in the two systems.