Unusually broad substrate profile of self-sufficient cytochrome P450 monooxygenase CYP116B4 from Labrenzia aggregata.

A new member of the CYP116B subfamily-P450LaMO-was discovered in Labrenzia aggregata by genomic data mining. It was successfully overexpressed in Escherichia coli, purified, and subsequently characterized spectroscopically, and its catalytic properties were assessed. Substrate profiling of the P450LaMO revealed that it was a versatile catalyst, exhibiting hydroxylation and epoxidation activities as well as O-dealkylation and asymmetric sulfoxidation activities. Diverse compounds, including alkylbenzenes, aromatic bicyclic molecules, and terpenoids, were shown to be hydroxylated by P450LaMO. Such diverse catalytic activities are uncommon for the bacterial P450s, and the P450LaMO-mediated stereoselective hydroxylation of inactivated C-H bonds-ubiquitous and relatively unreactive in organic molecules-is particularly unusual. The self-sufficient nature of P450LaMO, coupled with its broad substrate range, highlights it as an ideal template for directed evolution towards various applications.

Refolding with Simultaneous Purification of Recombinant Serratia marcescens Lipase by One-Step Ultrasonication Process.

A new lipase from Serratia marcescens SRICI-01 (Trx-SmL) was successfully overexpressed in Escherichia coli with thioredoxin (Trx) fusion tag. Intriguingly, the concentration of potassium phosphate buffer (KPB) showed significant impact on the aggregation state of Trx-SmL during ultrasonic disruption. The proportion of inclusion bodies increased dramatically with the increase of KPB concentration from almost completely soluble in 10 mM KPB to insoluble in 200 mM KPB. Based on this new finding, a novel method for refolding and purification of recombinant Trx-SmL was developed by one-step ultrasonication. The Trx-SmL was firstly precipitated in 200 mM KPB, washed for three times, and subsequently subjected to ultrasonic process in 10 mM KPB where refolding and purification occurred simultaneously. This established method was proved to be a straightforward, economical, and efficient purification approach to facilely obtain recombinant Trx-SmL protein with high purity (> 90%) and activity recovery yield (> 80%) from cell lysates. The application potential of the purified fusion Trx-SmL was further demonstrated by kinetic bioresolution of (±)-trans-3-(4-methoxyphenyl)glycidic acid methyl ester [(±)-MPGM] producing optically pure (-)-MPGM, a key intermediate for diltiazem, with an overall yield of 41.5% and ee of 99%.