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A novel and simple method of using penicillin for screening of mutant strains with a high penicillin acylase (PAC) activity was developed. Random mutagenesis was conducted using a PAC-producing strain resistant to 6-aminopenicillanic acid (6-APA) as the parent strain and mutants were screened with penicillin at a high concentration. Results suggest that mutants with a high minimum inhibitory concentration for penicillin (MICpenG) usually overproduce PAC. Both volumetric and specific PAC activities of a mutant, MD7, were significantly higher than those of the parent strain, HBPAC101 harboring pCLL2902. The mutation(s) resulting in the enhanced expression was mapped on the host chromosome rather than the plasmid. In addition, the mutant strain of MDΔP7, derived by elimination of the harbored plasmid in MD7, was demonstrated to be efficient in production of PAC by using the expression plasmids for which expression of the pac gene is limited by translation. An extremely high specific PAC activity of more than 350 U/L/OD600 was reached upon cultivation of MDΔP7 harboring pTrcKnPAC2902 in a bioreactor. As such, the strategy is effective in terms of constructing PAC overproducers and improving the process yield for production of PAC. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 65: 219–226, 1999.

We have identified the bottleneck steps limiting expression of penicillin acylase (PAC) through comparison of the expression performance for various PAC-expression vectors constructed by genetically modulating the efficiencies of transcription and/or translation of the pac gene. To our knowledge, this is the first report demonstrating that expression of PAC could be limited by various steps, such as transcription, translation, and post-translational steps (i.e. translocation and periplasmic processing), depending on the host/vector systems. Results also indicate that the structure of the wild-type pac gene might not be optimal for direct use in production of PAC using recombinant DNA technology. To improve the gene expression, transcription was enhanced by manipulating certain DNA bases in the pac regulatory region, whereas translation was enhanced by enlarging the spacing between the ribosome binding site and the ATG initiation codon to increase the initiation efficiency. The information is useful in terms of developing genetic strategies for overproduction of recombinant PAC in Escherichia coli. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 63: 263–272, 1999.

In this paper, we propose a system to detect and track fingertips online and recognize Mandarin Phonetic Symbol (MPS) for user-friendly Chinese input purposes. Using fingertips and cameras to replace pens and touch panels as input devices could reduce the cost and improve the ease-of-use and comfort of computer-human interface. In the proposed framework, particle filters with enhanced appearance models are applied for robust fingertip tracking. Afterwards, MPS combination recognition is performed on the tracked fingertip trajectories using Hidden Markov Models. In the proposed system, the fingertips of the users could be robustly tracked. Also, the challenges of entering, leaving and virtual strokes caused by video-based fingertip input can be overcome. Experimental results have shown the feasibility and effectiveness of the proposed work.