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Combined strategies for improving the heterologous expression of an alkaline lipase from Acinetobacter radioresistens CMC-1 in Pichia pastoris

Paper ID Volume ID Publish Year Pages File Format Full-Text
34625 45037 2013 7 PDF Available
Title
Combined strategies for improving the heterologous expression of an alkaline lipase from Acinetobacter radioresistens CMC-1 in Pichia pastoris
Abstract

•Introduction of N-extension leader spacer doubled ARL activity in P. pastoris.•High-copy-number constructs and co-expression of HAC1 increased ARL productivity.•The maximum ARL lipase activity can be reached 1.06 × 104 U/mL in a 10-L fermenter.

In this study, a series of strategies was developed to enhance the expression of an alkaline lipase from Acinetobacter radioresistens (ARL) in Pichia pastoris. Activity of the lipase from recombinant strain carrying a single copy of codon-optimized ARL gene was 65 U/mL in shake flask culture with p-nitrophenyl caprylate as the substrate. The lipase yield was increased to 104 U/mL by introducing a short N-extension spacer peptide coding for the 10 amino acids (EEAEAEAEPK) between α-factor signal peptide and ARL. The N-terminal extension spacer did not affect the pH or temperature properties of the recombinant ARL. After the multi-copy constructs were identified by Q-PCR assay, a higher lipase activity of 180 U/mL was obtained. Further introduction of the spliced HAC1 gene into multi-copy integrants (>6 copies) extensively enhanced the ARL yield by 30–40%. As a result, the ARL yield reached 1.06 × 104 U/mL in a 10-L scaled-up fed-batch fermenter as well as the lipase showed some better properties compared to that wild one from A. radioresistens.

Keywords
Acinetobacter radioresistens CMC-1; ARL; Lipase; Secretion expression; Pichia pastoris
First Page Preview
Combined strategies for improving the heterologous expression of an alkaline lipase from Acinetobacter radioresistens CMC-1 in Pichia pastoris
Publisher
Database: Elsevier - ScienceDirect
Journal: Process Biochemistry - Volume 48, Issue 9, September 2013, Pages 1317–1323
Authors
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Subjects
Physical Sciences and Engineering Chemical Engineering Bioengineering