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%.

High-throughput extraction of ß-carotene from Blakeslea trispora based on a newly developed setup.

Rapid and efficient extraction of bioactive compounds from biomass for sample analysis presents one of the major challenges in high-productive strain screening and bioprocess development. In this work, a high-throughput method based on a new setup was developed for the extraction of ß-carotene from Blakeslea trispora. After the cell was completely disrupted by high-throughput machinery grinding, ß-carotene was completely extracted by chloroform and dimethylsulfoxide (1:1, v/v). According to the experimental results, the newly developed high-throughput extraction (HTPE) method could simultaneously process 96 samples within 30 Min, and only 5-mL solvent was used for each sample. The sufficient extraction of ß-carotene by the HTPE method, which was confirmed by the conventional reference extraction method, further demonstrated that the newly developed HTPE method was more efficient and economical than the methods developed previously for the extraction of ß-carotene.