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Site-saturation Mutagenesis (site-saturation + mutagenesi)

Distribution by Scientific Domains

Life Sciences	50%
Chemistry	50%

Selected Abstracts

Engineering thermal stability of l- asparaginase by in vitro directed evolution

FEBS JOURNAL, Issue 6 2009
Georgia A. Kotzia
l- Asparaginase (EC 3.5.1.1, l- ASNase) catalyses the hydrolysis of l- Asn, producing l- Asp and ammonia. This enzyme is an anti-neoplastic agent; it is used extensively in the chemotherapy of acute lymphoblastic leukaemia. In this study, we describe the use of in vitro directed evolution to create a new enzyme variant with improved thermal stability. A library of enzyme variants was created by a staggered extension process using the genes that code for the l- ASNases from Erwinia chrysanthemi and Erwinia carotovora. The amino acid sequences of the parental l- ASNases show 77% identity, but their half-inactivation temperature (Tm) differs by 10 °C. A thermostable variant of the E. chrysamthemi enzyme was identified that contained a single point mutation (Asp133Val). The Tm of this variant was 55.8 °C, whereas the wild-type enzyme has a Tm of 46.4 °C. At 50 °C, the half-life values for the wild-type and mutant enzymes were 2.7 and 159.7 h, respectively. Analysis of the electrostatic potential of the wild-type enzyme showed that Asp133 is located at a neutral region on the enzyme surface and makes a significant and unfavourable electrostatic contribution to overall stability. Site-saturation mutagenesis at position 133 was used to further analyse the contribution of this position on thermostability. Screening of a library of random Asp133 mutants confirmed that this position is indeed involved in thermostability and showed that the Asp133Leu mutation confers optimal thermostability. [source]

The generation of nisin variants with enhanced activity against specific Gram-positive pathogens

MOLECULAR MICROBIOLOGY, Issue 1 2008
Des Field
Summary Nisin is the prototype of the lantibiotic group of antimicrobial peptides. It exhibits broad spectrum inhibition of Gram-positive bacteria including important food pathogens and clinically relevant antibiotic-resistant bacteria. Significantly, the gene-encoded nature of nisin means that it can be subjected to gene-based bioengineering to generate novel derivatives. Here, we take advantage of this to generate the largest bank of randomly mutated nisin derivatives reported to date, with the ultimate aim of identifying variants with enhanced bioactivity. This approach led to the identification of a nisin-producing strain with enhanced bioactivity against the mastitic pathogen Streptococcus agalactiae resulting from an amino acid change in the hinge region of the peptide (K22T). Prompted by this discovery, site-directed and site-saturation mutagenesis of the hinge region residues was employed, resulting in the identification of additional derivatives, most notably N20P, M21V and K22S, with enhanced bioactivity and specific activity against Gram-positive pathogens including Listeria monocytogenes and/or Staphylococcus aureus. The identification of these derivatives represents a major step forward in the bioengineering of nisin, and lantibiotics in general, and confirms that peptide engineering can deliver derivatives with enhanced antimicrobial activity against specific problematic spoilage and pathogenic microbes or against Gram-positive bacteria in general. [source]

Construction of cellobiose phosphorylase variants with broadened acceptor specificity towards anomerically substituted glucosides

BIOTECHNOLOGY & BIOENGINEERING, Issue 3 2010
Manu R.M. De Groeve
Abstract The general application of glycoside phosphorylases such as cellobiose phosphorylase (CP) for glycoside synthesis is hindered by their relatively narrow substrate specificity. We have previously reported on the creation of Cellulomonas uda CP enzyme variants with either modified donor or acceptor specificity. Remarkably, in this study it was found that the donor mutant also displays broadened acceptor specificity towards several ,-glucosides. Triple mutants containing donor (T508I/N667A) as well as acceptor mutations (E649C or E649G) also display a broader acceptor specificity than any of the parent enzymes. Moreover, further broadening of the acceptor specificity has been achieved by site-saturation mutagenesis of residues near the active site entrance. The best enzyme variant contains the additional N156D and N163D mutations and is active towards various alkyl ,-glucosides, methyl ,-glucoside and cellobiose. In comparison with the wild-type C. uda CP enzyme, which cannot accept anomerically substituted glucosides at all, the obtained increase in substrate specificity is significant. The described CP enzyme variants should be useful for the synthesis of cellobiosides and other glycosides with prebiotic and pharmaceutical properties. Biotechnol. Bioeng. 2010;107: 413,420. © 2010 Wiley Periodicals, Inc. [source]

Natural Diversity to Guide Focused Directed Evolution

CHEMBIOCHEM, Issue 13 2010
Helge Jochens Dr.
Abstract Simultaneous multiple site-saturation mutagenesis was performed at four active-site positions of an esterase from Pseudomonas fluorescens to improve its ability to convert 3-phenylbutyric acid esters (3-PBA) in an enantioselective manner. Based on an appropriate codon choice derived from a structural alignment of 1751 sequences of ,/,-hydrolase fold enzymes, only those amino acids were considered for library creation that appeared frequently in structurally equivalent positions. Thus, the number of mutants to be screened could be substantially reduced while the number of functionally intact variants was increased. Whereas the wild-type esterase showed only marginal activity and poor enantioselectivity (Etrue=3.2) towards 3-PBA-ethyl ester, a significant number of hits with improved rates (up to 240-fold) and enantioselectivities (up to Etrue=80) were identified in these "smart" libraries. [source]