[Show abstract][Hide abstract] ABSTRACT: To improve enzymatic activity of Bacillus pumilus lipases, DNA shuffling was applied to two lipase genes from local B. pumilus isolates. Using a high-throughput activity assay, the mutant with highest activity was selected. This chimeric mutant (L3-3), carrying two crossover positions and three point mutations, has a specific activity 6.4 and 8.2 times higher than the two parent enzymes. The mutant also is more tolerant to various detergents and organic solvents, and has a 9 times longer half-life at 50°C. Homology modeling of mutant L3-3, based on the highly homologous B. subtilis Lipase A, shows that the increased thermostability is likely due to structural rigidification and reduced surface hydrophobicity. Increased specific activity may result from the location of mutations close to the active site. Together, our results show that it is possible to evolve, by DNA shuffling, B. pumilus lipase variants with improved applicability as biocatalysts, even if the two parent enzymes are highly similar.
Full-text · Article · Jan 2013 · Journal of Biotechnology
[Show abstract][Hide abstract] ABSTRACT: Penicillin G acylase (PA) is an important enzyme used in the industrial production of b-lactam antibiotics. In this study, the effects of mutations in the translation initiation region of the Escherichia coli pac gene, encoding periplasmic PA, were examined. Several mutations led to increased amounts of PA activity, including those that lengthened the spacer region between the ribosome binding site and the ATG start codon, and those with altered codons on positions +2 and +4 relative to the start codon. These results indicated that the wild-type sequence of the pac gene does not provide maximum expression levels and that the strategies applied in this study can be used to improve production of PA in E. coli. Unexpectedly, our study also suggested that translocation of PA was, in contrast to earlier reports, shown not to require the Twin-arginine translocation pathway for transport into the periplasm.
Full-text · Article · May 2012 · World Journal of Microbiology and Biotechnology (Formerly MIRCEN Journal of Applied Microbiology and Biotechnology)
[Show abstract][Hide abstract] ABSTRACT: 1The penicillin acylase gene (pac) from E. coli ATCC 11105 genomic DNA was cloned into pUC19 vector using conventional techniques. The E. coli strain JM109 transformed by this construct was shown to possess high plasmid stability. PAA upon initial addition to the cultivation medium inhibited cell growth as well as PA production in the recombinant strain. A repressor effect of glucose on PA production was also observed, although the construct did not contain the whole regulatory region of the pac gene. Regulation mechanisms of the pac gene are discussed in the light of previous data and the observed effects of PAA and glucose on penicillin acylase production by the recombinant strain. The penicillin acylase which was constitutively expressed by the recombinant strain was purified and optimal temperature and pH values were found to be 60°C and 8.5, respectively.
No preview · Article · Nov 2001 · Enzyme and Microbial Technology
[Show abstract][Hide abstract] ABSTRACT: NAD+ glycohydrolase activities in serum samples from cancer patients were two- to three-fold higher than the activities in samples from healthy controls. SDS-PAGE analysis of serum samples followed by Western blotting revealed the presence of two proteins of ~45 and ~21 kDa that were immunoreactive with human CD38-specific monoclonal antibodies T16, HIT2 and OKT10. These proteins appeared to be more abundant in serum from cancer patients. NAD+ glycohydrolase activity in serum could be enriched by immunoaffinity chromatography by using T16-Sepharose 4B. The results suggest that the relative abundance of proteins immunologically related to CD38 may account for the elevated levels of glycohydrolase activities in serum of tumour patients.
[Show abstract][Hide abstract] ABSTRACT: The penicillin acylase gene (pac) amplified by polymerase chain reaction (PCR) from Escherichia coli (E.coli) ATCC11105 genomic DNA was cloned into pUC9, pEMBL9+ and pCP40 vectors. A penicillin acylase precursor was overexpressed at 26 degrees C from pac-pEMBL9+ and pac-pCP40 constructs transformed into E.coli strains JM109 and pCI857 containing HB101, respectively. With the thermo-inducible pac-pCP40 construct some level of mature alpha and beta subunits and varying degrees of enzyme activity were also detected.
No preview · Article · Mar 1996 · Biochemistry and molecular biology international
[Show abstract][Hide abstract] ABSTRACT: We have modified elongation factor Tu (EF-Tu) from Escherichia coli via mutagenesis of its encoding tufA gene to study its function-structure relationships. The isolation of the N-terminal half molecule of EF-Tu (G domain) has facilitated the analysis of the basic EF-Tu activities, since the G domain binds the substrate GTP/GDP, catalyzes the GTP hydrolysis and is not exposed to the allosteric constraints of the intact molecule. So far, the best studied region has been the guanine nucleotide-binding pocket defined by the consensus elements typical for the GTP-binding proteins. In this area most substitutions were carried out in the G domain and were found to influence GTP hydrolysis. In particular, the mutation VG20 (in both G domain and EF-Tu) decreases this activity and enhances the GDP to GTP exchange; PT82 induces autophosphorylation of Thr82 and HG84 strongly affects the GTPase without altering the interaction with the substrate. SD173, a residue interacting with (O)6 of the guanine, abolishes the GTP and GDP binding activity. Substitution of residues Gln114 and Glu117, located in the proximity of the GTP binding pocket, influences respectively the GTPase and the stability of the G domain, whereas the double replacement VD88/LK121, located on alpha-helices bordering the GTP-binding pocket, moderately reduces the stability of the G domain without greatly affecting GTPase and interaction with GTP(GDP). Concerning the effect of ligands, EF-TuVG20 supports a lower poly(Phe) synthesis but is more accurate than wild-type EF-Tu, probably due to a longer pausing on the ribosome.(ABSTRACT TRUNCATED AT 250 WORDS)
[Show abstract][Hide abstract] ABSTRACT: Mutagenesis was carried out in the N-terminal domain of elongation factor Tu (EF-Tu) to characterize the structure-function relationships of this model GTP binding protein with respect to stability, the interaction with GTP and GDP, and the catalytic activity. The substitutions were introduced in elements around the guanine nucleotide binding site or in the loops defining this site, in the intact molecule or in the isolated N-terminal domain (G domain). The double substitution Val88----Asp and Leu121----Lys, two residues situated on two vicinal alpha-helices, influences the basic activities of the truncated factor to a limited extent, probably via long-range interactions, and induces a destabilisation of the G domain structure. The functional alterations brought about by substitutions on the consensus sequences 18-24 and 80-83 highlight the importance of these residues for the interaction with GTP/GDP and the GTPase activity. Mutations concerning residues interacting with the guanine base lead to proteins in large part insoluble and inactive. In one case, the mutated protein (EF-TuAsn135----Asp) inhibited the growth of the host cell. This demonstrates the crucial role of the base specificity for the active conformation of EF-Tu. The obtained results are discussed in the light of the three-dimensional structure of EF-Tu.
No preview · Article · Sep 1990 · Biochimica et Biophysica Acta