ABSTRACT
BACKGROUND: Through modification of the flagellin type III secretion pathway of Bacillus halodurans heterologous peptides could be secreted into the medium as flagellin fusion monomers. The stability of the secreted monomers was significantly enhanced through gene-targeted inactivation of host cell extracellular proteases. In evaluating the biotechnological potential of this extracellular secretion system an anti-viral therapeutic peptide, Enfuvirtide, was chosen. Currently, Enfuvirtide is synthesised utilizing 106 chemical steps. We used Enfuvirtide as a model system in an effort to develop a more cost-effective biological process for therapeutic peptide production. RESULTS: An attempt was made to increase the levels of the fusion peptide by two strategies, namely strain improvement through gene-targeted knock-outs, as well as vector and cassette optimization. Both approaches proved to be successful. Through chromosomal inactivation of the spo0A, lytC and lytE genes, giving rise to strain B. halodurans BhFDL05S, the secretion of recombinant peptide fusions was increased 10-fold. Cassette optimization, incorporating an expression vector pNW33N and the N- and C-terminal regions of the flagellin monomer as an in-frame peptide fusion, resulted in a further 3.5-fold increase in the secretion of recombinant peptide fusions. CONCLUSIONS: The type III flagellar secretion system of B. halodurans has been shown to successfully secrete a therapeutic peptide as a heterologous flagellin fusion. Improvements to both the strain and expression cassette led to increased levels of recombinant peptide, showing promise for a biotechnological application.
Subject(s)
Bacillus/metabolism , Flagellin/metabolism , Recombinant Fusion Proteins/metabolism , Bacterial Proteins/antagonists & inhibitors , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Enfuvirtide , Flagellin/genetics , Gene Knockout Techniques , HIV Envelope Protein gp41/biosynthesis , HIV Envelope Protein gp41/genetics , N-Acetylmuramoyl-L-alanine Amidase/antagonists & inhibitors , N-Acetylmuramoyl-L-alanine Amidase/genetics , N-Acetylmuramoyl-L-alanine Amidase/metabolism , Peptide Fragments/biosynthesis , Peptide Fragments/genetics , Recombinant Fusion Proteins/genetics , Transcription Factors/antagonists & inhibitors , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
A purine nucleoside phosphorylase from the alkaliphile Bacillus halodurans Alk36 was cloned and overexpressed in Escherichia coli. The enzyme was purified fivefold by membrane filtration and ion exchange. The purified enzyme had a V (max) of 2.03 x 10(-9) s (-1) and a K (m) of 206 microM on guanosine. The optimal pH range was between 5.7 and 8.4 with a maximum at pH 7.0. The optimal temperature for activity was 70 degrees C and the enzyme had a half life at 60 degrees C of 20.8 h.
Subject(s)
Bacillus/enzymology , Bacillus/genetics , Purine-Nucleoside Phosphorylase/genetics , Purine-Nucleoside Phosphorylase/isolation & purification , Amino Acid Sequence , Base Sequence , Cloning, Molecular , DNA Primers/genetics , DNA, Bacterial/genetics , Escherichia coli/genetics , Genes, Bacterial , Hydrogen-Ion Concentration , Kinetics , Models, Molecular , Molecular Sequence Data , Protein Structure, Tertiary , Purine-Nucleoside Phosphorylase/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Structural Homology, Protein , ThermodynamicsABSTRACT
An esterase, designated EstTs1, was identified and characterized from a genomic library of Thermus scotoductus SA-01 (ATCC 700910). The library was screened in Escherichia coli for lipolytic activity on tributyrin agar plates. A 1.7-kb DNA fragment from a lipolytic positive clone was sequenced and two open reading frames (ORFs) were identified. A 774-bp ORF, designated EstTs1 with an estimated molecular mass of 28.6 kDa, and a 693-bp ORF, designated EstTs2 with an estimated molecular mass of 25.6 kDa, were identified. These two ORFs appear to form part of an operon. Sequence analysis showed that both proteins contained the G-X-S-X-G signature sequence motif present in most esterases and lipases. The deduced amino sequence of EstTs1 was found to display significant sequence identity with putative hydrolase proteins from both Thermus aquaticus Y51MC23 and Thermus thermophilus HB27. Similarly, EstTs2, also displayed significant homology to a second putative hydrolase protein present in the same two organisms. The cloning and characterization of these two ORFs from T. aquaticus Y51MC23 and T. thermophilus strain HB27 encoding putative hydrolase genes have not been reported. E. coli cells harbouring EstTs1 on a multicopy vector produced a clearing zone on tributyrin agar plates, whereas no enzymatic activity was observed for E. coli harbouring EstTs2 on a multicopy vector. EstTs1 displayed optimum activity at pH 7 and 80 degrees C with a half life of 48 h at 70 degrees C.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Esterases/genetics , Esterases/metabolism , Thermus/enzymology , Bacterial Proteins/chemistry , Cluster Analysis , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Enzyme Stability , Escherichia coli/genetics , Esterases/chemistry , Half-Life , Hot Temperature , Hydrogen-Ion Concentration , Lipolysis , Molecular Sequence Data , Molecular Weight , Phylogeny , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Thermus/geneticsABSTRACT
A genomic library of Bacillus coagulans strain 81-11 was screened in Escherichia coli JM83 for lipolytic activity by using tributyrin agar plates. A 2.4 kb DNA fragment was subcloned from a lipolytic-positive clone and completely sequenced. Nucleotide sequence analysis predicted a 723 bp open reading frame (ORF), designated estC1, encoding a protein of 240 amino acids with an estimated molecular mass of 27,528 Da and a pI of 9.15. The deduced amino acid sequence of the estC1 gene exhibited significant amino acid sequence identity with carboxylesterases from thermophilic Geobacillus spp. and sequence analysis showed that the protein contains the signature G-X-S-X-G included in most esterases and lipases. Enzyme assays using p-nitrophenyl (p-NP) esters with different acyl chain lengths as the substrate confirmed the esterase activity. EstC1 exhibited a marked preference for esters of short-chain fatty acids, yielding the highest activity with p-NP butyrate. Maximum activity was found at pH 8 and 50 degrees C, although the enzyme displayed stability at temperatures up to 60 degrees C.
