柠檬酸发酵过程的动力学

发酵动力学是微生物培养过程研究中的一个重要部分,它让人们从理论和定量的角度了解和分析微生物的培养过程,是过程设计和控制的基础．柠檬酸发酵过程的动力学．Chemiel^[1]、Kristiansen^〔2〕、Khan^〔3〕、Rohr^〔4〕、Vaija^〔5〕等曾作过研究。本文在考察前人工作的基础上．结合实验研究．进行如下探索。     

人促红细胞生成素基因的合成及在大肠杆菌中的表达

人促红细胞生成素(简称hEPO)是一种能够促进祖红细胞分化发育并进而促进红细胞生成的细胞生长因子,它在临床上可用以治疗某些贫血病人特别是由于慢性肾衰所引起的肾性贫血^〔1－3〕．hEPO是一种由166个氨基酸组成的糖基化蛋白质．它的前体还带有27个氨基酸的信号肽^〔4－6〕。有两对由Cys7-Cys161和Cys29－Cys33组成的二硫键,其糖基化位点为Ash--24。Ash一38．Asn一83和Ser一126^〔7－8〕。由于天然来源hEPO的量极少．因此通过基因工程的手段是当前获得大量hEPO的较好的途径^〔9－10〕。我们在前文中曾经报道用单链方法合成天花粉胰蛋白酶抑制荆基因〔11〕以及用单链和PCR相结合来合成绿豆胰蛋白酶抑制荆基因的方法〔12〕．我们在合成hEPO基固(包括带有信号肽的hEPO基因)时也采用了这两种方法：合成的hEPO基因在大脑杆菌、CHO细胞和昆虫细胞中均得到表达。本文报道hEPO基因的合成及在大肠杆菌中的表达结果。     

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。     

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。     

利用固定化酵母细胞生产1，6-二磷酸果糖的研究

目前对1,6-二磷酸果糖(简称FDP)生产基本采用Leisola^[1]等人在1974年提出的以酵母蕾为转化 媒体。以蔗糖、磷酸盐为底物的酶促磷酸化法来进行制备,但在工艺条件上已经有了很大的改进。利用固定化细胞生产FDP是现在人们进行研究的主要方向^[2]。在对常用的几种固定化方法进行比较后．本试验采用聚乙烯醇(PVA)和海藻酸钠为复合载体．对酵母细胞进行包埋,然后在饱和硼酸和CaCl₂溶藏中制成固定化小球。用其制备FDP,转化能力增强,转化周期明显缩短。如果进行工业化生产,可大大高设备利用率．简化工艺条件。     

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。     

固定化原生质体产谷氨酸脱氢酶的研究

本文报道了用海藻酸钙凝胶包埋法制备固定化谷氨酸捧杆菌T6—13原生质体及其用于生产谷氨酸脱氢酶(GDH,E．C．1．4．1．4)的研究。在一定条件下游离细胞和固定化细胞胞内可积累谷氨酸脱氢酶,但并不分泌到胞外。对数生长前期的细胞经蛋清溶菌酶处理14h后分离得到原生质体,游离原生质体和固定化原生质体可产胞外GDH。用3％海藻酸钙凝胶包埋10%的原生质体制备的固定化原生质体具有较高的产酶性,分批培养72h后发酵液中GDH活力可达到1．64×10^-2u／ml,为游离细胞胞内产酶的205％。固定化原生质体可用溶菌酶处理固定化细胞而制得,与直接固定化原生质体制备的固定化原生质体具有同样的产酶能力,且制备方便。固定化原生质体可重复使用6批次(约18天),且具有良好的贮藏稳定性。     

L－精氨酸组织传感器动力学响应机理的研究

生物组织中有丰富的酶源。由于酶的底物专一性,固此利用生物组织可研制对生物物质选择响应的组织传感器^〔1－4〕。为了制备性能良好的组织传感器及扩大它们的应用,必须对生物组织传感器的动力学响应机理进行研究。目前有关这方面的报道较步。本工作利用L－精氨酸厚叶景天叶组织传感器〔6〕研究了L－精氨酸组织传感器的动力学响应机理。     

L－亮氨酸发酵过程氮源反馈与非反馈控制的初步研究

氮源可直接影响氨基酸发酵过程中菌体生长与氨基酸生成．但是关于氨基酸发酵过程中氮源控制的研究报道并不多。本文作者在L－亮氨酸发酵过程碳源流加研究及动力学特征分析基础上^〔1〕,比较了几种非反馈型、反馈型补加硫酸铵的发酵结果．提出了较佳的控制方法。     

固定化技术研究的新进展

固定化生物催化剂的研究近一、二十年来发展非常迅速。它已由原来的单一固定化酶、固定化微生物细咆发展到动植物细胞、组织器官、微生物孢子^[1]、细胞与酶^[2]、好氧微生物与厌氧微生物^[3]的混合固定化等,其应用研究巳涉及发酵、食品、化工、分析、医疗、生化、环境净化等各个领域^[4],展示了广阔的发展前景。     

Citric acid production using immobilized conidia of Aspergillus niger TMB 2022

Conidia of Aspergillus niger TMB 2022 were immobilized in calcium alginate for the production of citric acid. A 1-mL conidia suspension containing ca. 2.32 x 10(8) conidia were entrapped into sodium alginate solution in order to prepare 3% Ca-alginate (w/v) gel bead. Immobilized conidia were inoculated into productive medium containing 14% sucrose, 0.25% (NH(4))(2)CO(3), 0.25% KH(2)PO(4), and 0.025% MgSO(4).7H(2)O with addition of 0.06 mg/L CuSO(4).5H(2)O, 0.25 mg/L ZnCl(2), 1.3 mg/L FeCl(3).6H(2)O, pH 3.8, and incubated at 35 degrees C for 13 days by surface culture to produce 61.53 g/L anhydrous citric acid. Under the same conditions with a batchwise culture, it was found that immobilized conidia could maintain a longer period for citric acid production (31 days): over 70 g/L anhydrous citric acid from runs No. 2-4, with the maximum yield for anhydrous citric acid reaching 77.02 g/L for run No. 2. In contrast, free conidia maintained a shorter acid-producing phase, ca. 17 days; the maximum yield for anhydrous citric acid was 71.07 g/L for run No. 2 but dropped quickly as the run number increased.     

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.     

黑曲霉菌株柠檬酸合成酶基因的敲除及功能分析

【背景】柠檬酸合成酶是碳代谢途径的中心酶,其在三羧酸循环(tricarboxylic acid cycle,TCA)、氨基酸合成和乙醛酸循环中发挥着重要作用,是柠檬酸合成的关键酶。本论文所选用的是一株高产柠檬酸的黑曲霉菌株CGMCC10142。【目的】克隆柠檬酸合成酶关键基因,构建柠檬酸合成酶的敲除菌株并鉴定其在黑曲霉菌株高产柠檬酸过程中的功能及影响。【方法】采用根癌农杆菌转化方法并利用同源重组原理,采用抗性筛选和致死型反向筛选的双重筛选方法获得正确敲除株。对转化子在不同碳源下的生长情况进行观察并对柠檬酸发酵过程中菌丝球变化和产酸量进行分析,最后通过荧光定量PCR分析柠檬酸合成酶基因对黑曲霉积累柠檬酸的影响,及其对主要代谢途径中重要酶相关基因和其他的表达量的影响。【结果】以柠檬酸高产菌株黑曲霉CGMCC10142为出发菌,构建一株遗传稳定的柠檬酸合成酶敲除的菌株T1-2。结果发现该菌株在以葡萄糖为碳源的培养基上生长缓慢并且产生孢子量减少。通过摇瓶发酵产酸实验,结果表明敲除菌在84 h产酸量为64.3 g/L,相对于出发菌的98.7g/L降低了34.85%。通过荧光定量PCR发现柠檬酸合成酶的表达量是下降的,同时重要酶的表达量都下降。【结论】该菌株的柠檬酸合成酶基因对柠檬酸积累具有重要作用,但存在其他同工酶基因,该基因敲除仅使产酸合成降低34.85%,同时发现该柠檬酸合成酶的顺畅表达有助于主代谢途径中各关键酶的高效表达,本研究可为研究黑曲霉高产柠檬酸机理奠定基础。     

A novel recycle batch immobilized cell bioreactor for propionate production from whey lactose

Recycle batch fermentations using immobilized cells of Propionibacterium acidipropionici were studied for propionate production from whey permeate, de-lactose whey permeate, and acid whey. Cells were immobilized in a spirally wound fibrous sheet packed in a 0.5-L column reactor, which was connected to a 5-L stirred tank batch fermentor with recirculation. The immobilized cells bioreactor served as a breeder for these recycle batch fermentations. High fermentation rates and conversions were obtained with these whey media without nutrient supplementation. It took approximately 55 h to ferment whey permeate containing approximately 45 g/L lactose to approximately 20 g/L propionic acid. Higher propionate concentrations can be produced with various concentrated whey media containing more lactose. The highest propionic acid concentration obtained with the recycle batch reactor was 65 g/L, which is much higher than the normal maximum concentration of 35 to 45 g/L reported in the literature. The volumetric productivity ranged from 0.22 g/L . h to 0.47 g/L . h, depending on the propionate concentration and whey medium used. The corresponding specific cell productivity was 0.033 to 0.07 g/L . g cell. The productivity increased to 0.68 g/L . h when whey permeate was supplemented with 1% (w/v) yeast extract. Compared with conventional batch fermentation, the recycle batch fermentation with the immobilized cell bioreactor allows faster fermentation, produces a higher concentration of product, and can be run continually without significant downtime. The process also produced similar fermentation results with nonsterile whey media. (c) 1995 John Wiley & Sons, Inc.     

碳源和氮源对5-酮基-葡萄糖酸生成的影响

氧化葡萄糖杆菌Gluconobacter oxydans可以将葡萄糖氧化成葡萄糖酸,并进一步氧化成2-酮基-葡萄糖酸(2KGA)和5-酮基-葡萄糖酸(5KGA),其中5KGA在催化剂的作用下能够转化为L(+)-酒石酸。为了提高5-酮基-葡萄糖酸产量,以仅生成5KGA的氧化葡萄糖杆菌Gluconobacter oxydans HGI-1为出发菌株,研究不同碳源(蔗糖、乳糖、麦芽糖、淀粉、葡萄糖)和有机氮源(酵母浸粉、鱼粉、玉米浆、黄豆饼粉、棉籽饼粉)对5KGA产量的影响。500 mL摇瓶试验结果表明,当葡萄糖浓度为100 g/L时,5KGA产量最高为98.20 g/L;当有机氮源为酵母浸粉、鱼粉和玉米浆,其添加量的蛋白含量为1.60%时,5KGA产量分别为100.20 g/L、109.10 g/L和99.83 g/L,其中,使用鱼粉的5KGA产量最高,使用玉米浆的5KGA产量比酵母浸粉略低。出于经济考虑,文中选择玉米浆作有机氮源,并在5 L发酵罐中进行分批发酵放大试验,5KGA的产量为93.80 g/L,最大生成速率为3.48 g/(L·h),平均生成速率为1.56 g/(L·h)。结果表明,葡萄糖和玉米浆分别为Gluconobacter oxydans HGI-1规模化生产5KGA的最适碳源和氮源,可利用葡萄糖几乎全部(85.93%)转化为5KGA。     

Proteomic analysis of calcium alginate-immobilized Saccharomyces cerevisiae under high-gravity fermentation conditions

Saccharomyces cerevisiae KAY446 cells immobilized in calcium alginate gel, and supplemented with additional amino acids, were successfully used in enhancing ethanol production. This combination succeeded in improving the ethanol yield and reducing the fermentation time. The ethanol yield under these conditions was 0.40 g of ethanol/g of glucose, with a final ethanol concentration of 118 g/L after 72 h. This is compared to yields with immobilized cells alone of 0.35 g of ethanol/g of glucose and freely suspended cells with no amino acid supplementation of 0.30 g of ethanol/g of glucose, under the same VHG conditions. The maximum specific ethanol production rates were 0.98, 0.73, and 0.61 g (g dry weight) (-1) h (-1) for immobilized cells under VHG conditions with and without amino acid supplementation and free cells, respectively. A proteomic analysis showed significant stimulation of many pathways during fermentation under these conditions, including the Ras/cAMP, glycolysis, starch, and sucrose pathways, amino acids biosynthesis, and aminoacyl-tRNA synthetases. The upregulation of ribosomal, heat-shock proteins and proteins involved in cell viability confirmed that protein biosynthesis was accelerated and revealed likely mechanisms for improving cellular viability.     

Citric acid production from sucrose by recombinant Yarrowia lipolytica using semicontinuous fermentation

The genetically modified yeast strain Yarrowia lipolytica H222‐S4(p67ICL1)T5 is able to utilize sucrose as a carbon source and to produce citric and isocitric acids in a more advantageous ratio as compared to its wild‐type equivalent. In this study, the effect of pH of the fermentation broth (pH 6.0 and 7.0) and proteose‐peptone addition on citric acid production by the recombinant yeast strain were investigated. It was found that the highest citric acid production occurred at pH 7.0 without any addition of proteose‐peptone. Furthermore, two process strategies (fed‐batch and repeated fed‐batch) were tested for their applicability for use in citric acid production from sucrose by Y. lipolytica. Repeated fed‐batch cultivation was found to be the most effective process strategy: in 3 days of cycle duration, approximately 80 g/L citric acid was produced, the yield was at least 0.57 g/g and the productivity was as much as 1.1 g/Lh. The selectivity of the bioprocess for citric acid was always higher than 90% from the very beginning of the fermentation due to the genetic modification, reaching values of up to 96.4% after 5 days of cycle duration.     

粘质沙雷氏菌产2,3-丁二醇培养基的优化

研究了各种碳源、氮源、柠檬酸及无机盐对细胞生长与产物形成的影响,通过单因子、正交及中心组合设计响应面分析优化发酵培养基。结果表明在培养基中添加柠檬酸不但可以促进细胞生长与糖耗速度,还可以缩短发酵周期,提高2,3-丁二醇的产量。采用优化后的培养基,2,3-丁二醇的产量由14.03g/L增加到39.27g/L,提高了近3倍。     

Ethanol production by Kluyveromyces lactis immobilized cells in copolymer carriers produced by radiation polymerization

The conditions for batch and continuous production of ethanol, using immobilized growing yeast cells of Kluyveromyces lactis, have been optimized. Yeast cells have been immobilized in hydrogel copolymer carriers composed of polyvinyl alcohol (PVA) with various hydrophilic monomers, using radiation copolymerization technique. Yeast cells were immobilized through adhesion and multiplication of yeast cells themselves. The ethanol production of immobilized growing yeast cells with these hydrogel carriers was related to the monomer composition of the copolymers and the optimum monomer composition was hydroxyethyl methacrylate (HEMA). In this case by using batch fermentation, the superior ethanol production was 32.9 g L(-1) which was about 4 times higher than that of cells in free system. The relation between the activity of immobilized yeast cells and the water content of the copolymer carriers was also discussed. Immobilized growing yeast cells in PVA: HEMA (7%: 10%, w/w) hydrogel copolymer carrier, were used in a packed-bed column reactor for the continuous production of ethanol from lactose at different levels of concentrations (50, 100 and 150) g L(-1). For all lactose feed concentrations, an increase in dilution rates from 0.1 h(-1) to 0.3 h(-1) lowered ethanol concentration in fermented broth, but the volumetric ethanol productivity and volumetric lactose uptake rate were improved. The fermentation efficiency was lowered with the increase in dilution rate and also at higher lactose concentration in feed medium and a maximum of 70.2% was obtained at the lowest lactose concentration 50 g L(-1).     

Production of ethanol by immobilized Saccharomyces bayanus in an extractive fermentation system

An extractive fermentation system using immobilized yeast cells was developed to study the ethanol production at high sugar concentrations. Organic acids were used as extracting solvents of ethanol and their toxicity was tested in free and k-carrageenan entrapped cell preparations. Immobilization seems to protect cells against solvent toxicity, when long-chain organic acids, e.g., oleic acid, were used, probably due to steric and diffusional limitations, the free cells not being viable at high oleic acid concentrations. The entrapped cells also present a higher metabolic activity than their free counterparts at high glucose concentrations. A solution of 300 g/L of glucose was totally fermented by the immobilized yeast cells, which when free cannot normally convert more than 200 g/L. In situ recovery of ethanol by oleic acid in a batch immobilized cell system led to higher ethanol productivities and to the fermentation of 400 g/L, when an oleic acid/medium ratio of 5 was used.