ORCID Profile
0000-0002-0529-404X
Current Organisation
Queensland University of Technology
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Publisher: Informa UK Limited
Date: 2010
Publisher: Elsevier BV
Date: 04-2022
Publisher: Royal Society of Chemistry (RSC)
Date: 2010
DOI: 10.1039/C0CC02657C
Abstract: Mutants of the lipase from Bacillus subtilis, previously engineered for enhanced thermostability using directed evolution based on the B-FIT method, show significantly increased tolerance to hostile organic solvents.
Publisher: Wiley
Date: 20-04-2012
Abstract: Directed evolution is a method to tune the properties of enzymes for use in organic chemistry and biotechnology, to study enzyme mechanisms, and to shed light on darwinian evolution in nature. In order to enhance its efficacy, iterative saturation mutagenesis (ISM) was implemented. This involves: 1) randomized mutation of appropriate sites of one or more residues 2) screening of the initial mutant libraries for properties such as enzymatic rate, stereoselectivity, or thermal robustness 3) use of the best hit in a given library as a template for saturation mutagenesis at the other sites and 4) continuation of the process until the desired degree of enzyme improvement has been reached. Despite the success of a number of ISM-based studies, the question of the optimal choice of the many different possible pathways remains unanswered. Here we considered a complete 4-site ISM scheme. All 24 pathways were systematically explored, with the epoxide hydrolase from Aspergillus niger as the catalyst in the stereoselective hydrolytic kinetic resolution of a chiral epoxide. All 24 pathways were found to provide improved mutants with notably enhanced stereoselectivity. When a library failed to contain any hits, non-improved or even inferior mutants were used as templates in the continuation of the evolutionary pathway, thereby escaping from the local minimum. These observations have ramifications for directed evolution in general and for evolutionary biological studies in which protein engineering techniques are applied.
Publisher: American Chemical Society (ACS)
Date: 21-10-2008
DOI: 10.1021/MA801763T
Publisher: Springer New York
Date: 2014
DOI: 10.1007/978-1-4939-1053-3_21
Abstract: Directed evolution methods have proved to be highly effective in the design of novel proteins and in the generation of large libraries of erse sequences. However, searching through the vast number of mutants produced during such experiments in order to find the best represents a daunting and difficult task. In recent years, a number of computational tools have been developed to provide guidance during this exploratory process. It can, however, be unclear as to which tool or tools best complement the chosen library design strategy. In this review, we describe and critically evaluate some of the more notable tools in this area, discussing the rationale behind each, the requirements for their implementation, and potential issues faced when using them. Some ex les of their application in an experimental setting are also provided. The tools have been classified based on contrasting strategies as to how they function: prospective tools SCHEMA and OPTCOMB use extant sequence and structural data to predict optimal locations for crossover sites, whereas retrospective tools ProSAR and ASRA use property data from the mutant library to predict beneficial mutations and features. From our evaluation, we suggest that each tool can play a role in the design process however this is largely dictated by the data available and the desired experimental strategy for the project.
Publisher: American Chemical Society (ACS)
Date: 10-06-2010
DOI: 10.1021/JA1030479
Abstract: Efficacy in laboratory evolution of enzymes is currently a pressing issue, making comparative studies of different methods and strategies mandatory. Recent reports indicate that iterative saturation mutagenesis (ISM) provides a means to accelerate directed evolution of stereoselectivity and thermostability, but statistically meaningful comparisons with other methods have not been documented to date. In the present study, the efficacy of ISM has been rigorously tested by applying it to the previously most systematically studied enzyme in directed evolution, the lipase from Pseudomonas aeruginosa as a catalyst in the stereoselective hydrolytic kinetic resolution of a chiral ester. Upon screening only 10,000 transformants, unprecedented enantioselectivity was achieved (E = 594). ISM proves to be considerably more efficient than all previous systematic efforts utilizing error-prone polymerase chain reaction at different mutation rates, saturation mutagenesis at hot spots, and/or DNA shuffling, pronounced positive epistatic effects being the underlying reason.
Publisher: Wiley
Date: 25-06-2022
DOI: 10.1111/NPH.18285
Abstract: The strigolactone (SL) class of phytohormones shows broad chemical ersity, the functional importance of which remains to be fully elucidated, along with the enzymes responsible for the ersification of the SL structure. Here we explore the functional evolution of the highly conserved CYP711A P450 family, members of which catalyze several key monooxygenation reactions in the strigolactone pathway. Ancestral sequence reconstruction was utilized to infer ancestral CYP711A sequences based on a comprehensive set of extant CYP711 sequences. Eleven ancestral enzymes, corresponding to key points in the CYP711A phylogenetic tree, were resurrected and their activity was characterized towards the native substrate carlactone and the pure enantiomers of the synthetic strigolactone analogue, GR24. The ancestral and extant CYP711As tested accepted GR24 as a substrate and catalyzed several ersifying oxidation reactions on the structure. Evidence was obtained for functional ergence in the CYP711A family. The monocot group 3 ancestor, arising from gene duplication events within monocot grasses, showed both increased catalytic activity towards GR24 and high stereoselectivity towards the GR24 isomer resembling strigol-type SLs. These results are consistent with a role for CYP711As in strigolactone ersification in early land plants, which may have extended to the ersification of strigol-type SLs.
Publisher: Wiley
Date: 13-09-2011
Abstract: In a previous directed evolution study, the B-FIT approach to increasing the thermal robustness of proteins was introduced and applied to the lipase from Bacillus subtilis. It is based on the general concept of iterative saturation mutagenesis (ISM), according to which sites in an enzyme are subjected to saturation mutagenesis, the best hit of a given library is then used as a template for randomization at other sites, and the process is continued until the desired catalyst improvement has been achieved. The appropriate choice of the ISM sites is crucial in the B-FIT method the criterion is residues characterized by highest B factors available from X-ray crystallography data. In the present study, B-FIT was employed in order to increase the thermal robustness of the epoxide hydrolase from Aspergillus niger. Several rounds of ISM resulted in the best variant showing a 21 °C increase in the T(60)(50) value, an 80-fold improvement in half-life at 60 °C, and a 44 kcal mol(-1) improvement in inactivation energy. Seven other variants were also evolved with moderate yet significant improvements these were characterized by 10-14 °C increases in T(60)(50), 20-30-fold improvement in half-lives at 60 °C and 15-20 kcal mol(-1) elevations in activation energy. Unexpectedly, in the ISM process the best variants were obtained from essentially neutral or even inferior mutant parents, that is, when a given library contains no improved mutants. This constitutes a practical way to escape from what appear to be local minima ("dead ends") in the fitness landscape-a finding of notable significance in directed evolution.
Publisher: American Chemical Society (ACS)
Date: 09-02-2018
Abstract: Molecular dynamics simulations and free energy calculations have been used to investigate the effect of ligand binding on the enantioselectivity of an epoxide hydrolase (EH) from Aspergillus niger. Despite sharing a common mechanism, a wide range of alternative mechanisms have been proposed to explain the origin of enantiomeric selectivity in EHs. By comparing the interactions of ( R)- and ( S)-glycidyl phenyl ether (GPE) with both the wild type (WT, E = 3) and a mutant showing enhanced enantioselectivity to GPE (LW202, E = 193), we have examined whether enantioselectivity is due to differences in the binding pose, the affinity for the ( R)- or ( S)- enantiomers, or a kinetic effect. The two enantiomers were easily accommodated within the binding pockets of the WT enzyme and LW202. Free energy calculations suggested that neither enzyme had a preference for a given enantiomer. The two substrates s led a wide variety of conformations in the simulations with the sterically hindered and unhindered carbon atoms of the GPE epoxide ring both coming in close proximity to the nucleophilic aspartic acid residue. This suggests that alternative pathways could lead to the formation of a ( S)- and ( R)-diol product. Together, the calculations suggest that the enantioselectivity is due to kinetic rather than thermodynamic effects and that the assumption that one substrate results in one product when interpreting the available experimental data and deriving E-values may be inappropriate in the case of EHs.
Publisher: Springer Science and Business Media LLC
Date: 11-2008
Publisher: Springer Science and Business Media LLC
Date: 12-2017
DOI: 10.1007/S10822-017-0090-X
Abstract: Enzymes with a high selectivity are desirable for improving economics of chemical synthesis of enantiopure compounds. To improve enzyme selectivity mutations are often introduced near the catalytic active site. In this compact environment epistatic interactions between residues, where contributions to selectivity are non-additive, play a significant role in determining the degree of selectivity. Using support vector machine regression models we map mutations to the experimentally characterised enantioselectivities for a set of 136 variants of the epoxide hydrolase from the fungus Aspergillus niger (AnEH). We investigate whether the influence a mutation has on enzyme selectivity can be accurately predicted through linear models, and whether prediction accuracy can be improved using higher-order counterparts. Comparing linear and polynomial degree = 2 models, mean Pearson coefficients (r) from [Formula: see text]-fold cross-validation increase from 0.84 to 0.91 respectively. Equivalent models tested on interaction-minimised sequences achieve values of [Formula: see text] and [Formula: see text]. As expected, testing on a simulated control data set with no interactions results in no significant improvements from higher-order models. Additional experimentally derived AnEH mutants are tested with linear and polynomial degree = 2 models, with values increasing from [Formula: see text] to [Formula: see text] respectively. The study demonstrates that linear models perform well, however the representation of epistatic interactions in predictive models improves identification of selectivity-enhancing mutations. The improvement is attributed to higher-order kernel functions that represent epistatic interactions between residues.
Location: Australia
No related grants have been discovered for Yosephine Gumulya.