Tuyển tập các báo cáo nghiên cứu về hóa học được đăng trên tạp chí hóa hoc quốc tế đề tài : Production optimization of cyanophycinase ChpEal from Pseudomonas alcaligenes DIP1 | Sallam et al. AMB Express 2011 1 38 http content 1 1 38 o AMB Express a SpringerOpen Journal ORIGINAL Open Access Production optimization of cyanophycinase ChpEal from Pseudomonas alcaligenes DIP1 Ahmed Sallam Dimitar Kalkandzhiev and Alexander Steinbuchel 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 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. Keywords .
