酶制剂的生产及其应用课件

第十三讲 酶制剂的生产及其应用,1894年，日本科学家首次从米曲霉中提炼出淀粉酶，并将淀粉酶用作治疗消化不良的药物，从而开创了人类有目的地生产和应用酶制剂的先例。 1911年，美国科学家从木瓜中提取出木瓜蛋白酶，并将木瓜蛋白酶用于除去啤酒中的蛋白质浑浊物。此后，酶制剂 的生产和应用就逐步发展起 1949年，科学家成功地用液体深层发酵法 生产出了细菌淀粉酶，从此揭开了近代酶工业的序幕。 1971年，第一次国 际酶工程学术会议在美国召开，会议的主题就是固定化酶的研制和应用。 20世纪70年代后期，酶工程领域又出现了固定化细胞技术。 1986年，我国科学家利用固定化原生质体发酵生产碱性磷酸酶和葡萄糖氧化酶等相继获得成功，为酶工程的进一步发展开辟了新的途径。 近20年来，随着基因工程的渗入，使酶的定向改造成为可能，所以在固定化酶、固定化细胞和固定化原生质体发展的同时，酶分子修饰技术、酶的化学合成以及酶的人工合成等方面的研究，也在积极地开展中，从而使酶工程更加显示出广阔而诱人的前景。,酶制剂生产的历史,酶制剂的产品市场,全世界已发现的酶有3000多种 目前工业上生产的酶有60多种 真正达到工业规模的只有20多种 剂型和品种有60多个 2001年为16亿美元 每年约为78的增长率发展 预计到2008年销售额将达到30亿美元,酶制剂的产品结构,80的工业酶是水解酶，主要用于降解自然界中的高聚物，如淀粉、蛋白质、脂肪等物质。 蛋白酶、淀粉酶和脂肪酶是目前工业应用的3大主要酶制剂。 蛋白酶可用于去污剂、奶制品业、皮革业等； 淀粉酶用于烘焙、酿造、淀粉糖化和纺织业； 脂肪酶用于去污剂、食品和精细化工工业等。,生产厂家,世界上酶生产厂商有70多家，其中较大的有25家。最大的8家酶制剂生产厂商是： （1） Novozymes （诺维信公司，丹麦） （2） Gist Broccdes（荷兰） （3） Cultot （科特公司，芬兰） （4） Genencor International （杰能科公司，美国） （5） Solvay （苏尔威公司，比利时） （6） Clr Hansen （汉森公司，丹麦） （7） Rhone Ponlene （罗兰，普朗克公司，法国） （8） Quest （荷兰）,The best known examples to the public are use of enzymes in detergents. At present a detergent also contains lipases and amylases to dissolve fat and starch stains. In order to restore colour of cotton that has been washed several times, cellulases are added. Cellulases are also used to give jeans the so-called “stone-wash” look.,Enzymes are also extensively used in other industries such as pulp and paper industry, textile industry, leather industry and for baking. Enzymes are also used in dairy industry and for the production of wine, fruit juice, beer and alcohol.,In particular due to need of enantiopure pharmaceuticals and building blocks for organic synthesis, the use of enzyme catalysis in fine chemicals and pharmaceutical industry is increasing.,Why are enzymes of interest as catalysts in synthesis?,There are unique advantages that enzymes offer which are difficult to obtain by conventional catalysis.,First of all, they have great selectivity and specificity. No matter how simple the enzyme-catalysed chemical reaction is, this may be on three level: chemoselectivity, regioselectivity and stereo selectivity and stereospecificity.,Chemoselectivity is the ability of the enzyme to direct the catalytic action to a specific functional group in the molecule so as to distinguish between OH or NH. When the substrate contains several functional groups of the same kind, as seen in carbohydrates, the enzyme is able to catalyse a regioselective reaction of one particular OH-group.,The stereochemcial properties of enzymes are extremely attractive in organic synthesis. Enzyme catalysis may be used for production of enantiopure chiral molecules either by enantioselective asymmetric synthesis or to resolve racemic mixtures.,The stereochemical properties of enzymes are important but so is the fact that enzymes work under mild conditions. The latter is becoming more and more important as greater demands are made on chemical process industry concerning environmental aspects.,Importance of enantiopure compounds,No matter if a pair of enantiomers have exactly the same chemical and physical properties, such as melting point, boiling point and spectra and even show the same reactivity in and achiral environment, they are, in principle, totally different compounds when they interact with chiral molecules.,It is well known that some enantiomers may have different odours and tastes. For example, (S)-carvone tastes of caraway while the the (R)-enantiomer tastes of spearmint.,The effect of different enantiomers may be particularly significant for drugs. Hence drugs that are chiral must be administered as single enantiomers.,Biotransformation deals with the use of biological catalysts to convert a substrate into a product in a limited number of enzymatic steps.,Biological catalysts when compared with chemical catalysts have the advantages of their regioselectivity and stereospecificity which lead to single enantiomeric products with regulatory requisites for pharmaceutical, food and agricultural use. They are also energy effective catalysts working at moderate temperatures, pressures and pH values.,Biotransformations have been performed by a variety of biological catalysts, such as isolated enzymes, cells, immobilized enzymes and cells the developments of recombinant DNA technology have led to improvements in the enzyme production in different host organisms giving the bioprocess engineer a greater choice of biocatalyst option.,诱导酶与组成型酶,需要加入诱导物才可以产生的酶叫诱导酶 不需加入诱导物就可以产生的酶叫组成型酶 不需加入诱导物就可以产生诱导酶的突变株，叫做组成型突变株（调节性突变）,酶的生产,酶的生产技术,培养基 pH 温度 DO,培养基,碳源 氮源 碳氮比 无机盐 生长因子 产酶促进剂,碳源,许多酶类生产以玉米粉、甘薯粉、淀粉等为碳源，价格便宜，而且对产酶有诱导作用 有些碳源对酶的合成有分解代谢产物阻遏作用，要注意控制其浓度,氮源,氮源对微生物产酶有诱导和抑制作用 多数情况下将有机氮源和无机氮源配合使用，在高浓度的有机氮源外添加1-3%的无机氮源,碳氮比,碳氮比低有利于菌体生长的需要，种子与发酵前期采用低碳氮比 碳氮比高有利于菌体产酶的需要，发酵中后期采用高碳氮比,无机盐,同其它微生物产品生产一样,氨基酸、维生素、嘌呤碱和嘧啶碱，通过加入玉米浆、酵母膏、麸皮、米糠以及豆饼和玉米来提供,生长因子,产酶促进剂,诱导物或诱导物前体 表面活性剂，胞外酶，一般使用非离子表面活性剂，而不用阳离子或阴离子试剂,pH,黑曲霉生产糖化酶同时产生-淀粉酶和葡萄糖苷转移酶 pH倾向中性时，糖化酶活性低，另两种酶活性高 pH倾向酸性时，糖化酶活性高，另两种酶活性低,温度控制,根据菌体生长和酶合成的需要，进行变温生产，以枯草杆菌As1.398进行中性蛋白酶生产，培养温度必须从31C逐渐升温至40C，然后再降温至31C，蛋白酶产量比不升温者高66,DO,一般，通气量少对霉菌的孢子萌发和菌丝生长有利，对酶生产不利；通气量大则相反 但也有相反的情况，如黑曲霉的-淀粉酶的生产，酶生产时菌的需氧量为生长旺盛时的36-40%,-淀粉酶生产工艺,生产菌种：芽孢杆菌，BF-7658,生产工艺流程,斜面 孢子悬浮液 种子罐 发酵罐 提取,斜面,马铃薯培养基或淀粉蛋白胨培养基 茄子瓶50ml左右，37 72h,孢子悬浮液,用50 ml无菌生理盐水洗下,种子及发酵培养工艺配方,不同阶段通风比为： 种子罐 0-10小时，1: 1.3； 10-14小时 1:1.45 发酵罐 0-12小时，1: 0.74；12小时以后1:1.46-1.1,中间补料:12小时后，每隔1小时补料一次直至多40小时左右。 pH高于6.5，菌体空泡多，意味着出现衰老，多补一些。补料结束后6-8小时，升至7.5，营养细胞80%不空泡，酶活不增加，放罐。 补料优点：（低浓度发酵和高浓度补料） 1、有利于菌体生长产酶 2、发酵后残糖、残氮低，便于提取 3、高浓度补料可以保持环境稳定，延长产酶期，增强菌体产酶的诱导作用，增加产酶量,Production of amino acids by biotransformations,Production of L-phenylalanine,苯丙酮酸天冬氨酸,苯丙氨酸,天冬氨酸转氨酶,酶转化过程,天冬氨酸转氨酶的制备,菌种及种子培养： 菌种：大肠杆菌H201 实验室筛选保藏 种子培养基：葡萄糖1.5、蛋白胨0.6、玉米浆1.0、MgSO40.05、牛肉膏0.15、NaCl 0.05、pH7.0-7.5； 配好的培养基分装后，0.1Mpa，121灭菌20min，冷却备用，接种后37振荡培养12-13hr。,发酵实验,发酵培养基:同上 发酵罐装料系数为0.7，0.1Mpa，121灭菌20min，冷却备用，接种后37 搅拌通气培养1524hr。,微生物细胞的生长曲线和酶活曲线,细胞生长曲线,细胞生长过程中的酶活曲线,产转氨酶的大肠杆菌在该培养体系中适应期不明显，且对数生长期较长。 从酶活曲线可以看出，酶活的增加在培养10hr左右已达到较高的水平，到15hr基本已平衡，结合上述的细胞生长曲线，可以发现酶活在细胞的对数生长末期达到最高。所以，在实际培养体系中最佳产酶时间为1516hr。,酶法转化,苯丙酮酸30g/L 天冬氨酸：苯丙酮酸（mol） 1.1:1 发酵液：转化液1：2，V/V pH8.5-9.0 36下转化16-20h,苯丙酮酸为一不稳定的有机物，在空气中能缓慢氧化成苯甲醛，在转氨反应的游离细胞体系中，仍有可能被其他酶所作用，产生副反应，影响了苯丙氨酸的转氨得率。,酶量的选择,酶作为反应的催化剂，其用量的增大有利于反应速度的提高,表面活性剂对加快反应速度的影响,金属离子对转氨反应的影响,金属离子对酶的活性表达一般均有一定的作用（激活或抑制作用），为提高转氨酶的活性，选择了以下几种金属离子考察对酶的激活或抑制作用，发现金属离子对产物之一L苯丙氨酸的得率的影响较小。,Production of D-p-hydroxyphenylglycine from DL-5-p-hydroxyphenylhydantoin,酶法转化过程,Microorganism and culture media,Burkholderia cepecia JS-02 isolated from a soil sample was used in this study. The culture media per liter contains: 20 g sucrose, 25 mL corn steep liquid, 2 g KH2PO4, 3 g NaCl, 0.025 g MgSO47H2O and 8 mM inducers.,Preparation of resting cells,Seed culture was prepared in 500 mL flasks each containing 50 mL culture medium by incubating at 30 for 16 h. Flask culture was operated in 500 mL flasks each containing 50 mL culture medium with 5 % inoculants for 24 h.,Preparation of resting cells,Aerobic fermentation was done in a 5-L fermentor (Marubishi Japan) containing 3 L culture medium for 1618h. Different aeration rate (0.20.6 vvm), temperature (2535 ), and agitation rate (250600 rpm) were employed for the process optimization.,Preparation of resting cells,At the end of the exponential phase the cells were harvested by centrifugation and washed twice using cold water.,Bioconversion with resting cells,5-L DL-HPH solution (25 g/L) was mixed with the harvested cells from 3 L culture broth (about 125 g wet cells) the initial pH was adjusted to 9.0 with NaOH After striped off the oxygen by gassing nitrogen for stabilization of hydantoin and enzymes, the reaction mixture was maintained at 2540 for 3040 h with a moderate agitation to perform bioconversion.,Enzyme assay,A predetermined amount of resting cells were incubated with 1% DL-5-HPH or N-C-D-HPG in 100 mL Na2HPO4- NaH2PO4 buffer solution (pH 8.0) for 30 min, with gentle shaking at 35 . Aliquots of samples were withdrawn for determining the concentrations of N-C-D-HPG and D-HPG. Specific enzymes activities, defined as mole of product made per minute by resting cells harvested from per milliliter culture broth.,Analytical methods,Cells concentration in culture broth was monitored by measuring the absorbance at 640 nm (A640). In bioconversion process, the concentrations of D-HPG, N-C-D-HPG and DL-5-HPH were determined at 210nm with a high performance liquid chromatography equipped with a Kromasil C18 column (4.6250 mm). The mobile phase consisted of H2O/CH3CN/H3PO4 (80/20/0.02 by volume), and was inje