Lee TS, Vaghjiani JD, Lye GJ, Turner MK
The Advanced Centre for Biochemical Engineering, Department of Biochemical Engineering, University College London, Torrington Place, London, United Kingdom
Enzyme Microb Technol. 2000 May 1;26(8):582-592. doi: 10.1016/s0141-0229(99)00194-5.
Crystallization has recently emerged as a suitable process for the manufacture of biocatalysts in the form of cross-linked enzyme crystals (CLECs) or for the recovery of proteins from fermentation broths. In both instances it is essential to define conditions which control crystal size and habit, and that yield a reliable recovery of the active protein. Experiments to define the crystallization conditions usually depend on a factorial design (either incomplete or sparse matrix) or reverse screening techniques. In this work, we describe a simple procedure that allows the effect of three factors, for example protein concentration, precipitant concentration and pH, to be varied simultaneously and smoothly over a wide range. The results are mapped onto a simple triangular diagram where a 'window of crystallization' is immediately apparent, and that conveniently describes variations either in the crystal features, such as their yield, size, and habit, or in the recovery of biological activity. The approach is illustrated with two enzymes, yeast alcohol dehydrogenase (ADH I) and Candida rugosa lipase. For ADH the formation of two crystal habits (rod and hexagonal) could be controlled as a function of pH (6.5-10) and temperature (4-25 degrees C). At pH 7, in 10 to 16% w/v polyethylene glycol (PEG) 4000, only rod-shaped crystals formed whereas at pH 8, in 10 to 14% w/v PEG, only hexagonal crystals existed. For both enzymes, catalyst recovery was greatest at high crystallization agent concentrations and low protein concentration. For ADH, the greatest activity recovery was 87% whereas for the lipase crystals, by using 45% v/v 2-methyl-2,4-pentanediol (MPD) as the crystallization agent, a crystal recovery of 250 crystals per µl was obtained. For the lipase system, the use of crystal seeding was also shown to increase the crystal recovery by up to a factor of four. From the crystallization windows, the original conditions based on literature precedent (35% v/v MPD, 1 mM CaCl(2), 1.8 mg protein/ml) were altered (47.5% v/v MPD, 2 mM CaCl(2), 3 mg protein/ml). This led to an improved recovery of the lipase under conditions that scale reliably from 0.5 ml to 500 ml with no change in size, shape or recovery of the crystals themselves. Finally, these crystals were crosslinked with 5% v/v glutaraldehyde and mass and activity balances were calculated for the entire process of CLEC production. Up to 35% of the lipase activity present in the crude solid was finally recovered in the lipase CLECs after propan-2-ol fractionation, crystallization, and crosslinking.
结晶法最近已成为一种适用于制造交联酶晶体（CLEC）形式的生物催化剂或从发酵液中回收蛋白质的工艺。在这两种情况下，确定控制晶体尺寸和晶习并能可靠回收活性蛋白的条件至关重要。确定结晶条件的实验通常依赖于析因设计（不完全或稀疏矩阵）或反向筛选技术。在本工作中，我们描述了一种简单的程序，该程序能使蛋白质浓度、沉淀剂浓度和pH值这三个因素的影响在很宽的范围内同时且平稳地变化。结果被绘制到一个简单的三角图上，在该图上“结晶窗口”一目了然，它方便地描述了晶体特征（如产率、尺寸和晶习）或生物活性回收方面的变化。用两种酶，即酵母乙醇脱氢酶（ADH I）和皱褶假丝酵母脂肪酶对该方法进行了说明。对于ADH，两种晶习（棒状和六方晶）的形成可作为pH值（6.5 - 10）和温度（4 - 25℃）的函数进行控制。在pH 7时，在10%至16% w/v聚乙二醇（PEG）4000中，只形成棒状晶体，而在pH 8时，在10%至14% w/v PEG中，只存在六方晶体。对于这两种酶，在高结晶剂浓度和低蛋白质浓度下催化剂回收率最高。对于ADH，最大活性回收率为87%，而对于脂肪酶晶体，通过使用45% v/v 2 - 甲基 - 2,4 - 戊二醇（MPD）作为结晶剂，每微升可获得250个晶体的回收率。对于脂肪酶体系，还表明使用晶种可使晶体回收率提高至四倍。根据结晶窗口，基于文献先例的原始条件（35% v/v MPD，1 mM CaCl₂，1.8 mg蛋白质/ml）被改变（47.5% v/v MPD，2 mM CaCl₂，3 mg蛋白质/ml）。这使得在从0.5 ml可靠放大至500 ml且晶体本身的尺寸、形状或回收率不变的条件下，脂肪酶的回收率得到了提高。最后，这些晶体用5% v/v戊二醛交联，并计算了CLEC生产整个过程的质量和活性平衡。经过丙 - 2 - 醇分级分离、结晶和交联后，粗固体中存在的脂肪酶活性最终有高达35%在脂肪酶CLEC中得以回收。
