Production optimization of cyanophycinase ChpEal from Pseudomonas alcaligenes DIP1

Ahmed Sallam,Dimitar Kalkandzhiev,Alexander Steinbüchel

doi:10.1186/2191-0855-1-38

Abstract

Pseudomonas alcaligenes DIP1 produces an extracellular cyanophycinase (CphEal). The corresponding gene (cphEal) was identified from subclones of a genomic DNA gene library by heterologously expressing the functionally active enzyme in Escherichia coli. The nucleotide sequence of the gene (1260 base pairs) was determined indicating a theoretical mass of 43.6 kDa (mature CphEal) plus a leader peptide of 2,6 kDa which corresponds well to the apparent molecular mass of 45 kDa as revealed by SDS-PAGE. The enzyme exhibited a high sequence identity of 91% with the extracellular cyanophycinase from P. anguilliseptica strain BI and carried an N-terminal Sec secretion signal peptide. Analysis of the amino acid sequence of cphE revealed a putative catalytic triad consisting of the serine motif GXSXG plus a histidine and a glutamate residue, suggesting a catalytic mechanism similar to serine-type proteases. The cyanophycinase (CphEal) was heterologously produced in two different E. coli strains (Top10 and BL21(DE3)) from two plasmid vectors (pBBR1MCS-4 and pET-23a(+)). The signal peptide of CphEal was cleaved in E. coli, suggesting active export of the protein at least to the periplasm. Substantial enzyme activity was also present in the culture supernatants. The extracellular cyanophycinase activities in E. coli were higher than activities in the wild type P. alcaligenes DIP1 in complex LB medium. Highest extracellular enzyme production was achieved with E. coli BL21(DE3) expressing CphEal from pBBR1MCS-4. Using M9 minimal medium was less effective, but the relatively low cost of mineral salt media makes these results important for the industrial-scale production of dipeptides from cyanophycin.

Highlights

Cyanophycin is a naturally occurring poly(amino acid) that was first observed in cyanobacteria (Borzi 1887)
Cloning and analysis of cphEal from P. alcaligenes DIP1 Partial digestion of genomic DNA isolated from P. alcaligenes DIP1 with the restriction endonuclease PstI yielded fragments with a broad size range that were subsequently ligated into the plasmid vector pBluescriptSK
Molecular characterization of the CGPase gene from P. alcaligenes DIP1 The N-terminus of CphEal was identified in antilinear orientation to the lacZ promoter of the vector by DNA sequence analysis of the cloned fragment; it was concluded that the gene was expressed under the control of its own promoter

Summary

Introduction

Cyanophycin (cyanophycin granule polypeptide, CGP) is a naturally occurring poly(amino acid) that was first observed in cyanobacteria (Borzi 1887). It is accumulated in the early stationary growth phase (Mackerras et al 1990; Liotenberg et al 1996) and functions as storage compound for nitrogen, carbon, and energy (Elbahloul et al 2005; Füser and Steinbüchel 2007). Most genera of cyanobacteria (Simon 1987; Allen 1988; Mackerras et al 1990; Liotenberg et al 1996; Wingard et al 2002) and some heterotrophic bacteria (Krehenbrink et al 2002; Ziegler et al 2002; Füser and Steinbüchel 2007) harbor a cyanophycin synthetase gene (cphA) and are able to synthesize CGP. CGP from cyanobacteria exhibits a molecular mass of 25-100 kDa (Simon 1976), while CGP from heterotrophic bacteria and recombinant strains is smaller and much less polydisperse (25-30 kDa) (Ziegler et al 1998; Aboulmagd et al 2001; Krehenbrink et al 2002)

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