利用渗透交联固定化细胞促进生物转化

固定化技术已在生物工程中得到广泛的实际应用,特别是应用于生物转化以提高酶或细胞的稳定性,实现连续操作等。对于含胞内酶的细胞的生物转化．一般先破碎细胞,使酶释放出来,再进行酶固定化。由于酶的稳定性通常与细胞膜的结台有关^[1],细胞破碎中常导致酶的失活。如果不破碎细胞,对完整细胞固定化,又会有传质困难抑制酶活力的发挥。我们研究出渗透交联固定化细胞技术以解决这个矛盾。先采用某种试剂(多为表面活性剂)处理细胞,提高细胞的通透性,再进行交联固定化．可以保证酶的活力破坏较小,又减小了传质阻力。既提高了固定化细胞的稳定性,又提高了固定化细胞的表观酶活。称这种固定化技术为渗透交联固定化细胞技术。Prabhuaney等采用CTAB-戊二醛处理聚丙烯酰胺凝胶包理的含青霉素酰化酶E. coli细胞^[2]。Nmhida采用1,6-己二胺-戊二醛处理含天冬氨酸酶的E．Coli细胞^[3]。渗透交联固定化处理会损伤细胞和酶是这种技术的一个矛盾。本文采用多乙烯多胺-戊二处理方法．因多乙烯多胺既起到表面活性剂的作用．又是交联剂。而且渗透能力比CTAB和1,6-已二 胺为低,故对细胞和酶损伤较小。     

固定化Ralstonia metallidurans CH34降解苯酚的研究*

将既能耐抗重金属又能降解苯酚的细菌Ralstonia m etalliduransCH34固定化以提高其降酚效率。首先通过正交实验,得到了固定化该菌种的最优制备条件,然后对固定化细胞的降酚效果进行了研究。结果表明,固定化R.m etalliduransCH34的降酚效果明显优于游离细胞;抗重金属毒性方面也有较大提高;在加入额外碳源(甲苯,柠檬酸)情况下,固定化R.m etalliduransCH34进行苯酚降解时所受影响明显要小于游离态菌。     

肾胚细胞瘤原癌基因novC在tTA调控系统中的表达

nov基因与肾甲细胞瘤密切相关,在肾母细胞瘤细胞中大量表达,而在正常的成年肾中不表达。通过将Bujard′s tTA调控系统引入QT6细胞系中,同时将novC基因克隆到pUHD10-3质粒中,与pUHD-tTA质粒共转染细胞,诱导novC大量表达(其分子量约为48kD),建立了使nov基因大量表达的细胞系统,为研究novC基因对细胞的调控机制建立了体外细胞模型系统。     

温度对谷胱甘肽分批发酵的影响及动力学模型

研究了24～32℃范围内产朊假丝酵母生产谷胱甘肽的分批发酵过程,发现较高温度对细胞生长有促进作用,而较低温度则更有利于谷胱甘肽产量的提高。应用改进的Logistic和LuedekingPiret方程分别对细胞生长动力学和谷胱甘肽合成动力学进行了模拟,得到不同温度下各种动力学参数。在此基础上,进一步研究了温度同细胞生长动力学参数之间的内在联系,得到谷胱甘肽分批发酵过程中细胞浓度的变化同温度以及底物浓度之间的一般关系式：dX-dt＝［0.0224(T+1.7)］2X(1-X/X_max)1+S｛8.26×10.6×exp［-31477/R/(T+273)］｝。验证实验结果表明,该模型具有很好的适用性。     

固定化金霉素产生菌原生质体转化林可霉素反应模型的研究

建立了固定化金霉素产生菌金色链霉菌(Streptomyces aurofaciens)原生质体转化林可霉素反应动力学模型并模拟计算了反应过程,模型计算与实验结果有好的一致性。讨论了各反应参数对转化反应的影响,给出了转化率与反应时间的关系式和最大转化率计算式,还对转化反应动力学模型进一步应用进行了讨论。     

微囊化K562细胞生长周期及代谢特性的研究

以K562细胞为模型,分别进行微囊化和游离培养,运用流式细胞术考察两种培养体系下细胞周期和生长代谢变化;建立数学模型,模拟了两种培养体系下细胞的生长活性和代谢特性。实验发现：微囊化培养过程中的K562细胞处于DNA合成期（S期）的百分含量显著高于游离培养,并且细胞保持较高的增殖活性。模型计算表明,所建模型动力学参数能够很好地描述微囊化和游离两种培养体系下细胞的代谢情况;对细胞活性的理论计算表明,微囊化的细胞具有较高的增殖和代谢活性,同时细胞能够较长时间保持此活性;模型参数表明,两种培养体系下,葡萄糖对细胞生长的影响无显著差别 (k^Free_L≈k^APA_L),乳酸对游离培养细胞的生长具有明显抑制作用,但对微囊化培养细胞抑制作用较小(kFreeL>≈kAPAL)。     

锌酵母分批流加发酵动态优化

对锌酵母分批流加发酵过程的控制变量反应温度和pH的动态最优化进行研究。基于酵母流加发酵有抑制的状态模型．通过龙格一库塔法计算微分方程组、单纯形法优化对模型参数进行估计。采用不同的温度和pH控制策略进行研究,由此获得动力学模型参数与温度和pH关系的回归模型。在此基础上,以极大值原理、梯度法优化求解以获得最高锌酵母产量为目标的最优温度和pH分布T*（t）、pH*(t)。实验验证,在T*(t)和pH*(t)下操作,锌酵母产率可提高13.7％。     

PVA固定化的生黑醋菌生产L-山梨糖的研究

生黑醋菌可以将D-山梨醇转化为L-山梨塘,用微生物将D-山梨醇氧化为L-山梨糖是维生素C生产的一个重要部分,目前工业上用的都是游离菌批式生产工艺。由于固定化活细胞作为生物催化剂具有生产的连续性和稳定性．操作简便．产物易于分离纯化等优点^[1],已有不少实验室研究甩固定化微生物细胞将D-山梨醇转化为L-山梨糖^[1-6],国内也有用海藻酸固定化生黑醋菌Acetobacteriummelanogenum的报道^[2,3]。用海藻酸钙^[1-3]、聚丙烯酰胺^[4]、铝处理的海藻酸钙^[5]、水合聚丙烯酰胺与海藻酸钙混合固定化的微生物细胞^[6]转化D-山梨醇成为L-山梨糖,都有因机械强度差,而不适合在搅拌式发酵罐中生产的弱点。聚乙烯醇制备的固定化微生物细胞具有机械强度好、类似于橡皮的弹性、成低等特性^[7]。因此,我们选择聚乙烯醇作为固定化生黑醋菌的材料。     

固定化巨大芽孢杆菌青霉素G酰化酶酶促合成头孢氨苄的研究

β—内酰胺系列抗菌素抗菌谱广、疗效高、毒副作用小,国际上研究与应用日渐广泛深入。头孢氨苄(Cephalexin)是重要的半合成抗菌素之一,由头孢霉素母核7—氨基脱乙酰氧基头孢烷酸(简称7-ADCA)和侧链结构物苯甘氨酸或其甲酯(PGME)经酰化而生成。酰化有化学法和酶法两种。采用青霉素G酰化酶或a—氨基酸酯酶或,a—氨酰转移酶的酶法,具有工艺操作简单、无需基团保护、环境污染轻等优点。继日本人于70年代初试验成功酶法之后,80年代初我们开展了这方面研究。制备方面,胞外酶优于胞内酶;使用方面,固定化酶优于固定化细胞。在用具有青霉素G酰化酶活性的固定化大肠杆菌(Escherichia coli)细胞合成头孢氨苄的基础上,又研究了用固定化巨大芽孢杆菌(Bacillus megaterium)BP931胞外青霉素G酰化酶酰化合成头孢氨苄的条件。本文报道这一研究结果。     

絮凝颗粒酵母均匀悬浮体系生长动力学的研究

利用粟酒裂殖酵母(Schizosaccharomyces pombe)变异株自身絮凝形成的颗粒,作为细胞固定化方法。以双酶法制备的淀粉耱化液为底物,在有效容积2．35L的小型悬浮床生物反应器中连续生产酒精。研究了微量供氧条件下该絮凝颗粒酵母均匀悬浮体系的生长动力学,获得了描述其生长规律的模型方程。     

Production of Gluconobacter oxydans cells from low-cost culture medium for conversion of glycerol to dihydroxyacetone

Gluconobacter oxydans could be immobilized as a biocatalyst for the conversion of glycerol to dihydroxyacetone. To reduce the production cost, the cells were produced from agricultural byproducts. Corn meal hydrolysate and corn steep liquor were employed to replace of sorbitol and yeast extract as medium for G. oxydans cell production. The optimal medium contained 80 g/L reducing sugar, 25 g/L corn steep liquor, and 10 g/L glycerol. The cell mass was about 4.22 g/L and the glycerol dehydrogenase activity was about 5.23 U/mL. For comparison, the cell mass was about 4.0 g/L and the glycerol dehydrogenase activity was about 5.35 U/mL cultured in sorbitol and yeast extract medium. These studies shown the corn meal hydrolysate and corn steep liquor medium was similar in performance to a nutrient-rich medium, but the cost of production was only 15% of that cultured in sorbitol and yeast extract medium. It was an economical process for the production of G. oxydans cells as biocatalyst for the conversion of glycerol to dihydroxyacetone in industry.     

利用改进的卡拉胶载体固定化Gluconobacter oxydans和Bacilllus cereus活细胞的研究

本文利用氯化钙和脱乙酰几丁质改进卡拉胶载体用于固定化G.oxydans和B.cereus活细胞取得理想效果.脱乙酰几丁质与卡拉胶可以形成韧性和通透性良好的网络结构,改善了固定化细胞的各种性能.凝胶中的细胞密度可高达3.25×10~3个细胞/ml凝胶,固定化细胞可反复利用11批,每批发酵收率均可维持在65%左右,并且缩短了发酵周期     

Repeated use of immobilized Gluconobacter oxydans cells for conversion of glycerol to dihydroxyacetone

Dihydroxyacetone (DHA) is of great interest in the fine chemical and pharmaceutical industry; therefore, the discovery of suitable biocatalysts for the efficient production of it is very necessary. In the experiment, Gluconobacter oxydans was immobilized in polyvinyl alcohol (PVA). Various parameters of the immobilized cells were investigated. The results have shown that the optimal conversion conditions by the immobilized cells were at 30 degrees C and pH 6.0. The immobilized cells remained very active over the period of 14 days for storage and only lost 10% of its original activity. Repeated use of immobilized cells for conversion of glycerol to DHA was carried out in a 1.5 L stirred tank reactor, the average conversion rate was about 86%. Despite the high shear stress, bead shape was not affected, even after five consecutive conversion cycles. The regenerated biocatalyst could recover 90% of its initial activity.     

A model system for increasing the intensity of whole-cell biocatalysis: investigation of the rate of oxidation of D-sorbitol to L-sorbose by thin bi-layer latex coatings of non-growing Gluconobacter oxydans

We developed a novel <50-microm thick nano-porous bi-layer latex coating for preserving Gluconobacter oxydans, a strict aerobe, as a whole cell biocatalyst. G. oxydans was entrapped in an acrylate/vinyl acetate co-polymer matrix (T (g) approximately 10 degrees C) and cast into 12.7-mm diameter patch coatings (cellcoat) containing approximately 10(9) CFU covered by a nano-porous topcoat. The oxidation of D-sorbitol to L-sorbose was used to investigate the coating catalytic properties. Intrinsic kinetics was studied in microbioreactors using a pH 6.0 D-sorbitol, phosphate, pyruvate (SPP) non-growth medium at 30 degrees C, and the Michaelis-Menten constants determined. By using a diffusion cell, cellcoat and topcoat diffusivities, optimized by arresting polymer particle coalescence by glycerol and/or sucrose addition, were determined. Cryo-FESEM images revealed a two-layer structure with G. oxydans surrounded by <40-nm pores. Viable cell density, cell leakage, and oxidation kinetics in SPP medium for >150 h were investigated. Even though the coatings were optimized for permeability, approximately 50% of G. oxydans viability was lost during cellcoat drying and further reduction was observed as the topcoat was added. High reaction rates per unit volume of coating (80-100 g/L x h) were observed which agreed with predictions of a diffusion-reaction model using parameters estimated by independent experiments. Cellcoat effectiveness factors of 0.22-0.49 were observed which are 20-fold greater than any previously reported for this G. oxydans oxidation. These nano-structured coatings and the possibility of improving their ability to preserve G. oxydans viability may be useful for engineering highly reactive adhesive coatings for multi-phase micro-channel and membrane bioreactors to dramatically increase the intensity of whole-cell oxidations.     

Immobilization of mycobacterial cells onto silicone--assessing the feasibility of the immobilized biocatalyst in the production of androstenedione from sitosterol

Silicone rubbers are hydrophobic, a feature that may prove advantageous if this material is to be used as immobilization matrix in bioconversion systems where hydrophobic species are present, such as sterols and mycobacterial cells. Mycobacterium sp. cells with sitosterol side chain cleavage activity were accordingly effectively adsorbed onto silicone and the potential application of the concept was assessed by matching the behavior of the resulting immobilized biocatalyst with free cells and Celite immobilized cells. Mass transfer, kinetics, thermal and storage stability characterization of a biotransformation system based in the use of the silicone immobilized biocatalyst was performed. The feasibility of biocatalyst reutilization was tentatively explored.     

Kinetic aspects of glucose oxidation by Gluconobacter oxydans cells immobilized in calcium alginate

Summary Gluconobacter oxydans subspecies suboxydans (ATCC 621 H), when growing at high glucose concentrations, oxidizes this substrate incompletely and gluconic acid accumulates in the medium in almost stoichiometric amounts. Such cells were harvested and entrapped in various alginate gels. The preparation with the highest retention of glucose oxidizing activity was used in further studies with the aim of developing an efficient process for continuous gluconic acid production.The retention of activity increases (up to 95%) as the alginate concentration in the gel decreases or the cell/alginate weight ratio is enhanced. In the latter case, however, transport of oxygen to and inside the biocatalyst beads rapidly becomes rate-limiting and thus lowers the efficiency of the biocatalyst. Similarly, the efficiency decreases as the size of the biocatalyst beads increases. In no case rate-limitation by transport of glucose was found. Thus, biocatalyst activity per unit volume of support, diameter of the biocatalyst beads, and aeration efficiency are important parameters for reactor design.     

Design of a nonuniformly distributed biocatalyst

The properties of a nonuniformly distributed biocatalyst, where the active enzymes are immobilized on the exterior or the interior portions o a solid support, are compared with those of a conventional biocatalyst which is uniformly distributed in a spherical geometry. To investigate the performance of nonuniformly distributed biocatalysts their effectiveness factors are computed and compared for six different enzyme distribution configurations: one-half core, one-half shell, one-third center space, one-third middle annulus, one-third outer shell, and the uniformly distributed. According to the results of numerical analysis, the biocatalyst performance of the exterior "shell" configuration is always far more effective for the immobilized enzymes with positive order reaction kinetics such as Michaelis-Menten and competitive product inhibition. However, in the case of negative order enzymatic reaction kinetics such as substrate inhibition, the interior "core" configuration of the biocatalyst can render far greater enzyme utilization efficiency.     

A diffusion model and optimal cell loading for immobilized cell biocatalysts

A diffusion model based on the random pore model is derived for immobilized cell biocatalysts and verified with 19 sets of experimental diffusion data. The predicted effective diffusivity relative to that for the support matrix reflects a quadratic dependence on the cell loading and contains a single parameter that depends on the intracellular diffusivity and the chemical partitioning coefficient. The model is used to predict optimal cell loadings that maximize the total reaction rate in an immobilized cell biocatalyst. A rule of thumb based on the diffusion model is obtained to the effect that the cell loading should be at least (1/3) for single reactions regardless of the kinetics and diffusional resistances. A means of calculating improved lower bounds is provided for cases where the cellular diffusional resistance is known but the kinetics are not. The optimal cell loadings for reversible first-order and for Michaelis-Menten kinetics are presented and demonstrated to be within the range of conditions of practical interest.     

A Fortran Program for Evaluation of the Effectiveness Factor of Biocatalysts in the Presence of External and Internal Diffusional Limitations

A Fortran program called SPEFF for evaluation of the effectiveness factor of immobilized enzyme preparations of spherical form in the presence of external and internal mass transfer resistances is described, and a listing of the program is given. Enzyme distribution in the bioparticle may be uniform or nonuniform. In the latter case the enzyme distribution is approximated by fifth-order polynomial. In the program differential equations are replaced by the system of non-linear algebraic equations, and the latter are solved by Newton iteration technique. The program is developed for Michaelis-Menten kinetics with allowance for competitive product inhibition and substrate inhibition. After slight modifications the program can be used for computation of the effectiveness factor of a membrane with an immobilized enzyme, or in the case when the enzyme kinetics are more complex. A typical run on a PDP-11/45 computer took 10-20 seconds. A typical computation time in the case of IBM-compatible TURBO PC was 15-30 seconds.     

Comparative study of spent grains and delignified spent grains as yeast supports for alcohol production from molasses

Fresh, defrosted and delignified brewer's spent grains (BSG) were used as yeast supports for alcoholic fermentation of molasses. Glucose solution (12%) with and without nutrients was used for cell immobilization on fresh BSG, without nutrients for cell immobilization on defrosted and with nutrients for cell immobilization on delignified BSG. Repeated fermentation batches were performed by the immobilized biocatalysts in molasses of 7, 10 and 12 initial Baume density without additional nutrients at 30 and 20 degrees C. Defrosted BSG immobilized biocatalyst was used only for repeated fermentation batches of 7 initial Baume density of molasses without nutrients at 30 and 20 degrees C. After immobilization, the immobilized microorganism population was at 10(9) cells/g support for all immobilized biocatalysts. Fresh BSG immobilized biocatalyst without additional nutrients for yeast immobilization resulted in higher fermentation rates, lower final Baume densities and higher ethanol productivities in molasses fermentation at 7, 10 and 12 initial degrees Be densities than the other above biocatalysts. Adaptation of defrosted BSG immobilized biocatalyst in the molasses fermentation system was observed from batch to batch approaching kinetic parameters reported in fresh BSG immobilized biocatalyst. The results of this study concerning the use of fresh or defrosted BSG as yeast supports could be promising for scale-up operation.