第十三講 酶制劑的生產及其應用,1894年，日本科學家首次從米曲霉中提煉出淀粉酶，并將淀粉酶用作治療消化不良的藥物，從而開創(chuàng)了人類有目的地生產和應用酶制劑的先例。 1911年，美國科學家從木瓜中提取出木瓜蛋白酶，并將木瓜蛋白酶用于除去啤酒中的蛋白質渾濁物。此后，酶制劑 的生產和應用就逐步發(fā)展起 1949年，科學家成功地用液體深層發(fā)酵法 生產出了細菌淀粉酶，從此揭開了近代酶工業(yè)的序幕。 1971年，第一次國 際酶工程學術會議在美國召開，會議的主題就是固定化酶的研制和應用。 20世紀70年代后期，酶工程領域又出現(xiàn)了固定化細胞技術。 1986年，我國科學家利用固定化原生質體發(fā)酵生產堿性磷酸酶和葡萄糖氧化酶等相繼獲得成功，為酶工程的進一步發(fā)展開辟了新的途徑。 近20年來，隨著基因工程的滲入，使酶的定向改造成為可能，所以在固定化酶、固定化細胞和固定化原生質體發(fā)展的同時，酶分子修飾技術、酶的化學合成以及酶的人工合成等方面的研究，也在積極地開展中，從而使酶工程更加顯示出廣闊而誘人的前景。,酶制劑生產的歷史,酶制劑的產品市場,全世界已發(fā)現(xiàn)的酶有3000多種 目前工業(yè)上生產的酶有60多種 真正達到工業(yè)規(guī)模的只有20多種 劑型和品種有60多個 2001年為16億美元 每年約為78的增長率發(fā)展 預計到2008年銷售額將達到30億美元,酶制劑的產品結構,80的工業(yè)酶是水解酶，主要用于降解自然界中的高聚物，如淀粉、蛋白質、脂肪等物質。 蛋白酶、淀粉酶和脂肪酶是目前工業(yè)應用的3大主要酶制劑。 蛋白酶可用于去污劑、奶制品業(yè)、皮革業(yè)等； 淀粉酶用于烘焙、釀造、淀粉糖化和紡織業(yè)； 脂肪酶用于去污劑、食品和精細化工工業(yè)等。,生產廠家,世界上酶生產廠商有70多家，其中較大的有25家。最大的8家酶制劑生產廠商是： （1） Novozymes （諾維信公司，丹麥） （2） Gist Broccdes（荷蘭） （3） Cultot （科特公司，芬蘭） （4） Genencor International （杰能科公司，美國） （5） Solvay （蘇爾威公司，比利時） （6） Clr Hansen （漢森公司，丹麥） （7） Rhone Ponlene （羅蘭，普朗克公司，法國） （8） Quest （荷蘭）,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.,誘導酶與組成型酶,需要加入誘導物才可以產生的酶叫誘導酶 不需加入誘導物就可以產生的酶叫組成型酶 不需加入誘導物就可以產生誘導酶的突變株，叫做組成型突變株（調節(jié)性突變）,酶的生產,酶的生產技術,培養(yǎng)基 pH 溫度 DO,培養(yǎng)基,碳源 氮源 碳氮比 無機鹽 生長因子 產酶促進劑,碳源,許多酶類生產以玉米粉、甘薯粉、淀粉等為碳源，價格便宜，而且對產酶有誘導作用 有些碳源對酶的合成有分解代謝產物阻遏作用，要注意控制其濃度,氮源,氮源對微生物產酶有誘導和抑制作用 多數(shù)情況下將有機氮源和無機氮源配合使用，在高濃度的有機氮源外添加1-3%的無機氮源,碳氮比,碳氮比低有利于菌體生長的需要，種子與發(fā)酵前期采用低碳氮比 碳氮比高有利于菌體產酶的需要，發(fā)酵中后期采用高碳氮比,無機鹽,同其它微生物產品生產一樣,氨基酸、維生素、嘌呤堿和嘧啶堿，通過加入玉米漿、酵母膏、麩皮、米糠以及豆餅和玉米來提供,生長因子,產酶促進劑,誘導物或誘導物前體 表面活性劑，胞外酶，一般使用非離子表面活性劑，而不用陽離子或陰離子試劑,pH,黑曲霉生產糖化酶同時產生-淀粉酶和葡萄糖苷轉移酶 pH傾向中性時，糖化酶活性低，另兩種酶活性高 pH傾向酸性時，糖化酶活性高，另兩種酶活性低,溫度控制,根據(jù)菌體生長和酶合成的需要，進行變溫生產，以枯草桿菌As1.398進行中性蛋白酶生產，培養(yǎng)溫度必須從31C逐漸升溫至40C，然后再降溫至31C，蛋白酶產量比不升溫者高66,DO,一般，通氣量少對霉菌的孢子萌發(fā)和菌絲生長有利，對酶生產不利；通氣量大則相反 但也有相反的情況，如黑曲霉的-淀粉酶的生產，酶生產時菌的需氧量為生長旺盛時的36-40%,-淀粉酶生產工藝,生產菌種：芽孢桿菌，BF-7658,生產工藝流程,斜面 孢子懸浮液 種子罐 發(fā)酵罐 提取,斜面,馬鈴薯培養(yǎng)基或淀粉蛋白胨培養(yǎng)基 茄子瓶50ml左右，37 72h,孢子懸浮液,用50 ml無菌生理鹽水洗下,種子及發(fā)酵培養(yǎng)工藝配方,不同階段通風比為： 種子罐 0-10小時，1: 1.3； 10-14小時 1:1.45 發(fā)酵罐 0-12小時，1: 0.74；12小時以后1:1.46-1.1,中間補料:12小時后，每隔1小時補料一次直至多40小時左右。 pH高于6.5，菌體空泡多，意味著出現(xiàn)衰老，多補一些。補料結束后6-8小時，升至7.5，營養(yǎng)細胞80%不空泡，酶活不增加，放罐。 補料優(yōu)點：（低濃度發(fā)酵和高濃度補料） 1、有利于菌體生長產酶 2、發(fā)酵后殘?zhí)?、殘氮低，便于提?3、高濃度補料可以保持環(huán)境穩(wěn)定，延長產酶期，增強菌體產酶的誘導作用，增加產酶量,Production of amino acids by biotransformations,Production of L-phenylalanine,苯丙酮酸天冬氨酸,苯丙氨酸,天冬氨酸轉氨酶,酶轉化過程,天冬氨酸轉氨酶的制備,菌種及種子培養(yǎng)： 菌種：大腸桿菌H201 實驗室篩選保藏 種子培養(yǎng)基：葡萄糖1.5、蛋白胨0.6、玉米漿1.0、MgSO40.05、牛肉膏0.15、NaCl 0.05、pH7.0-7.5； 配好的培養(yǎng)基分裝后，0.1Mpa，121滅菌20min，冷卻備用，接種后37振蕩培養(yǎng)12-13hr。,發(fā)酵實驗,發(fā)酵培養(yǎng)基:同上 發(fā)酵罐裝料系數(shù)為0.7，0.1Mpa，121滅菌20min，冷卻備用，接種后37 攪拌通氣培養(yǎng)1524hr。,微生物細胞的生長曲線和酶活曲線,細胞生長曲線,細胞生長過程中的酶活曲線,產轉氨酶的大腸桿菌在該培養(yǎng)體系中適應期不明顯，且對數(shù)生長期較長。 從酶活曲線可以看出，酶活的增加在培養(yǎng)10hr左右已達到較高的水平，到15hr基本已平衡，結合上述的細胞生長曲線，可以發(fā)現(xiàn)酶活在細胞的對數(shù)生長末期達到最高。所以，在實際培養(yǎng)體系中最佳產酶時間為1516hr。,酶法轉化,苯丙酮酸30g/L 天冬氨酸：苯丙酮酸（mol） 1.1:1 發(fā)酵液：轉化液1：2，V/V pH8.5-9.0 36下轉化16-20h,苯丙酮酸為一不穩(wěn)定的有機物，在空氣中能緩慢氧化成苯甲醛，在轉氨反應的游離細胞體系中，仍有可能被其他酶所作用，產生副反應，影響了苯丙氨酸的轉氨得率。,酶量的選擇,酶作為反應的催化劑，其用量的增大有利于反應速度的提高,表面活性劑對加快反應速度的影響,金屬離子對轉氨反應的影響,金屬離子對酶的活性表達一般均有一定的作用（激活或抑制作用），為提高轉氨酶的活性，選擇了以下幾種金屬離子考察對酶的激活或抑制作用，發(fā)現(xiàn)金屬離子對產物之一L苯丙氨酸的得率的影響較小。,Production of D-p-hydroxyphenylglycine from DL-5-p-hydroxyphenylhydantoin,酶法轉化過程,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