Downstream effects, solvents

The evolved enzymes were further characterized for their ability to carry out the desired pNB ester hydrolysis. Figure 4 shows the specific reaction rates for enzymes from the first four generations in 1% and 15% DMF. Each successive generation catalyst is more effective than its parent, and the best, pNB esterase 4-54B9, is 15 times more productive than wild type in 1% DMF. In 15% DMF, this enzyme makes product at 4 times the rate of the wild type enzyme in 1 % organic solvent. The impact of this improvement is not only the increased productivity of the evolved enzyme, but also in the 4-fold increase in solubility of the substrate in 15% DMF. The increased solubility reduces the size of the reactor and the downstream processes required to produce and purify a given amount of product. The 2-fold increase in enzyme expression level further reduces process costs. 

Membrane filtration is a widely used but narrowly understood technique for sample preparation in chemical analysis. This section has the goal of providing some basic information to aid in the use of filtration tools with drug impurities. Many of the common sample preparation approaches described elsewhere in this chapter, such as liquid extraction, supercritical fluid extraction, and accelerated solvent extraction are effective at removing the dissolved analytes of interest from the matrix while leaving behind many poorly soluble or insoluble matrix components. In contrast, filtration is designed to remove these suspended particles from the extract prior to subsequent analytical steps. Unfiltered samples can destroy the performance of a downstream analytical technique such as HPLC or optical spectroscopy.68,69... 

Another method for separating low-solubility samples, which is well known in the downstream processing of biopolymers, starts with a large volume of sample dissolved in a chromatographically weak solvent. This solution is fed to the column. As the solute has a relatively strong affinity to the stationary phase, e.g. silica, it is adsorbed and concentrated at the column inlet. This has the effect of introducing the sample as a concentrated plug. 

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