Chlorothalonil hydrolytic dehalogenase (Chd) is one of two reported hydrolytic dehalogenases for halogenated aromatics, and its catalysis is independent of coenzyme A and ATP. Earlier studies have established that the catalytic activity of Chd requires zinc ions. In this study, the metal center of Chd was systematically investigated. The metal content of Chd was determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES), and there were 2.14 equivalents of zinc/mol of protein, indicating that Chd contains a binuclear (Zn2+-Zn2+) center. It was found that other divalent cations, such as cobalt (Co2+) and cadmium (Cd2+), could substitute zinc (Zn2+) leading to relative activities of 91.6% and 120.0%, whereas manganese (Mn2+) and calcium (Ca2+) could substitute Zn2+ leading to relative activities of 29.1% and 57.0%, respectively. The enzymatic properties of these different metal ion-substituted Chd variants were also compared. Error-prone PCR and DNA shuffling methods were applied to directly evolve Chd to generate variants with higher catalytic efficiencies of chlorothalonil. Enhanced Chd variants were selected based on the formation of clear haloes on Luria-Bertani plates supplemented with chlorothalonil. One variant, Q146R/N168Y/S303G, exhibited a 4.43-fold increase in catalytic efficiency, showing the potential for application in the dehalogenation and detoxification of chlorothalonil contaminated-sites.

Abstract:A process, voltage, temperature (PVT) insensitive boosted charge transfer (BCT) circuit for charge-domain (CD) pipelined analog-to-digital converters (ADC) is presented. The output charge of existing BCT varies extensively with PVT variation, leading to large common-mode charge errors in each differential BCT stage when used in CD pipelined ADCs. Therefore, complicate commonmode control circuits must be adopted to stabilize the common-mode charge of each stage, which consumes large power and chip area. The proposed BCT circuit employs a differential difference amplifier and a differential voltage reference to reject the charge errors caused by PVT variations. A 125-MSPS, 10-bit CD pipelined ADC without commonmode control circuit is implemented based on the proposed BCT, consuming only 27 mW from a 1.8 V supply.

A low power 10-bit 125-MSPS charge-domain (CD) pipelined analog-to-digital converter (ADC) based on MOS bucket-brigade devices (BBDs) is presented. A PVT insensitive boosted charge transfer (BCT) that is able to reject the charge error induced by PVT variations is proposed. With the proposed BCT, the common mode charge control circuit can be eliminated in the CD pipelined ADC and the system complexity is reduced remarkably. The prototype ADC based on the proposed BCT is realized in a 0.18 m CMOS process, with power consumption of only 27 mW at 1.8-V supply and active die area of 1.04 mm 2 . The prototype ADC achieves a spurious free dynamic range (SFDR) of 67.7 dB, a signal-to-noise ratio (SNDR) of 57.3 dB, and an effective number of bits (ENOB) of 9.0 for a 3.79 MHz input at full sampling rate. The measured differential nonlinearity (DNL) and integral nonlinearity (INL) are C0:5/ 0:3 LSB and C0:7/ 0:55 LSB, respectively.

Identification of the metal center of chlorothalonil hydrolytic dehalogenase and enhancement of catalytic efficiency by directed evolution.