Classical cloning, expression, and purification

Unfortunately the enzyme titer in the fermentation broth, even after classical mutation and several years of process development, was too low for the biocatalyt-ic process. With respect to the economics, the gene encoding for glutaryl-7-ACA acylase has been cloned, sequenced and expressed in E. coli to produce sufficient amounts. The enzyme titer could be increased by a factor of more than 100. Even the purification of the enzyme became much easier and resulted in higher yields with less side-activities, e.g., esterases. Two chromatographic purification steps were substituted by crystallization of the enzyme. The enzyme crystals could be stored long term without deactivation. To allow for reuse, the glutaryl-7-ACA acylase was immobilized on a polymeric carrier. 

This classical cloning strategy has proven to be successful in many cases, but it is time-consuming and laborious. Purification of a native enzyme to homogeneity is difficult, and any undesired contaminant protein can be mistakenly subjected to sequencing. Cloning efforts from plant material expressing low levels of the desired enzyme are often unsuccessful. Plant tissues that are known to be enriched in the desired activity provide the best results. Furthermore, the enzymatic activity must be maintained in vitro as the desired enzyme is diluted aud purified. If accessory proteins or cofactors are required for stability or fimctioual activity, the purified protein will become inactive as it is isolated from the crude cell extract. A robust in vitro assay with correct substrates is also required, and this may not be possible if the substrates are unavailable or if the enzymatic products are unstable. 

In summary, the de novo isolation of the cDNAs encoding enzymes of alkaloid biosynthesis is still achieved by using a variety of classical techniques, such as protein purification followed by partial amino acid sequence determination, and by newer techniques such as proteomics coupled to functional heterologous expression. The current status of cloned cDNAs specifically related to isoquinoline alkaloid biosynthesis is schematically presented in Figure 10.8. New additions to this list will certainly be made in the future as a result of a combination of approaches both new and old. 

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