Deacetylcephalosporin C synthase (DACS), a 2-oxoglutarate-dependent oxygenase synthesized by Streptomyces clavuligerus, transforms an inert methyl group of deacetoxycephalosporin C (DAOC) into an active hydroxyl group of deacetylcephalosporin C (DAC) during the biosynthesis of cephalosporin. It is a step which is chemically difficult to accomplish, but its development by use of an enzymatic method with DACS can facilitate a cost-effective technology for the manufacture of semisynthetic cephalosporin intermediates such as 7-amino-cephalosporanic acid (7ACA) and hydroxymethyl-7-amino-cephalosporanic acid (HACA) from cephalosporin G. As the native enzyme showed negligible activity toward cephalosporin G, an unnatural and less expensive substrate analogue, directed-evolution strategies such as random, semirational, rational, and computational methods were used for systematic engineering of DACS for improved activity. In comparison to the native enzyme, several variants with improved catalytic efficiency were found. The enzyme was stable for several days and is expressed in soluble form at high levels with significantly higher k cat /K m values. The efficacy and industrial scalability of one of the selected variants, CefF GOS , were demonstrated in a process showing complete bioconversion of 18 g/liter of cephalosporin G into deacetylcephalosporin G (DAG) in about 80 min and showed reproducible results at higher substrate concentrations as well. DAG could be converted completely into HACA in about 30 min by a subsequent reaction, thus facilitating scalability toward commercialization. The experimental findings with several mutants were also used to rationalize the functional conformation deduced from homology modeling, and this led to the disclosure of critical regions involved in the catalysis of DACS. IMPORTANCE7ACA and HACA serve as core intermediates for the manufacture of several semisynthetic cephalosporins. As they are expensive, a cost-effective enzyme technology for the manufacture of these intermediates is required. Deacetylcephalosporin C synthase (DACS) was identified as a candidate enzyme for the development of technology from cephalosporin G in this study. Directedevolution strategies were employed to enhance the catalytic efficiency of deacetylcephalosporin C synthase. One of the selected mutants of deacetylcephalosporin C synthase could convert high concentrations of cephalosporin G into DAG, which subsequently could be converted into HACA completely. As cephalosporin G is inexpensive and readily available, the technology would lead to a substantial reduction in the cost for these intermediates upon commercialization. S emisynthetic cephalosporins, a class of ␤-lactam antibiotics, have shown remarkable effectiveness in the treatment of infectious diseases. Together with penicillins, they comprise nearly 65% of anti-infectives used worldwide. Their high specificity and low toxicity, coupled with the evolvability of newer generations of antibiotics, have led to ␤-lactams being by far the most fr...

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The aim of the present study was to develop a linear regression model aiding to a quick scan of the most important sites for mutation of an anticancer biologic trastuzumab. The important sites identified on trastuzumab can be used to carry out site-directed mutagenesis to improve the binding affinity of the drug towards its antigen, human epidermal growth factor receptor 2 (HER2). This will lead to low dosage requirement of the drug for treating cancer patients, which in turn help to cut the cost and combat development of resistance. A quantitative structure-activity relationship (QSAR) model was built by multiple linear regressions using genetic algorithm-based feature selection (GA-MLR) method using 48 dependent variables (dissociation constant K d ) and 226 independent variables (theoretical descriptors generated using a proteometrics approach). The final QSAR model selected in the study was more on the basis of ability to predict accurately independent test data and generalization ability of the model rather than mere statistical significance of the model. With combined analysis of descriptors presented in final QSAR model and most frequent descriptors pooled from all solution models, it was demonstrated that the modeling procedure was able to bring on the factors important for antigen-antibody interactions with an example of HER2-trastuzumab interaction reported in previous experimental studies. This paper will allow the prediction of the most preferable site to mutate for improving the binding affinity of trastuzumab with HER2 and also will be helpful in selecting most preferable amino acids to substitute in the selected site for mutations. This is the novel report on proteometrics approach with autocorrelation formalism for antibody engineering, which can be extended to other antibodyantigen pairs.