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Andreas S Bommarius

Publications and source records attributed to Andreas S Bommarius.

14 recordsLinked to original sources
PubMed2007

Better library design: data-driven protein engineering.

Data-driven protein engineering is increasingly used as an alternative to rational design and combinatorial engineering because it uses available knowledge to limit library size, while still allowing for the identification of unpredictable substitutions that lead to large effects. Recent advances in computational modeling and bioinformatics, as well as an increasing databank of experiments on functional variants, have led to new strategies to choose particular amino acid residues to vary in order to increase the chances of obtaining a variant protein with the desired property. Strategies for limiting diversity at each position, design of small sub-libraries, and the performance of scouting experiments, have also been developed or even automated, further reducing the library size.

Computational Biology↗
PubMed2006

High-throughput screening for enhanced protein stability.

High thermostability of proteins is a prerequisite for their implementation in biocatalytic processes and in the evolution of new functions. Various protein engineering methods have been applied to the evolution of increased thermostability, including the use of combinatorial design where a diverse library of proteins is generated and screened for variants with increased stability. Current trends are toward the use of data-driven methods that reduce the library size by using available data to choose areas of the protein to target, without specifying the precise changes. For example, the half-lives of subtilisin and a Bacillus subtilis lipase were increased 1500-fold and 300-fold, respectively, using a crystal structure to guide mutagenesis choices. Sequence homology based methods have also produced libraries where 50% of the variants have improved thermostability. Moreover, advances in the high-throughput measurement of denaturation curves and the application of selection methods to thermostability evolution have enabled the screening of larger libraries. The combination of these methods will lead to the rapid improvement of protein stability for biotechnological purposes.

Computational Biology↗
PubMed2006

The crystal structure of NAD(P)H oxidase from Lactobacillus sanfranciscensis: insights into the conversion of O2 into two water molecules by the flavoenzyme.

The FAD-dependent NAD(P)H oxidase from Lactobacillus sanfrancisensis (L.san-Nox2) catalyzes the oxidation of 2 equivalents of either NADH or NADPH and reduces 1 equivalent of O(2) to yield 2 equivalents of water. During steady-state turnover only 0.5% of the reducing equivalents are detected in solution as hydrogen peroxide, suggesting that it is not released from the enzyme after the oxidation of the first equivalent of NAD(P)H and reaction with O(2). Here we report the crystal structure of L.san-Nox2 to 1.8 A resolution. The enzyme crystallizes as a dimer with each monomer consisting of a FAD binding domain (residues 1-120), a NAD(P)H binding domain (residues 150-250), and a dimerization domain (residues 325-451). The electron density for the redox-active Cys42 residue located adjacent to the si-face FAD is consistent with oxidation to the sulfenic acid (Cys-SOH) state. The side chain of Cys42 is also observed in two conformations; in one the sulfenic acid is hydrogen bonded to His10 and in the other it hydrogen bonds with the FAD O2' atom. Surprisingly, the NAD(P)H binding domains each contain an ADP ligand as established by electron density maps and MALDI-TOF analysis of the ligands released from heat-denatured enzyme. The ADP ligand copurifies with the enzyme, and its presence does not inhibit enzyme activity. Consequently, we hypothesize that either NADPH or NADH substrates bind via a long channel that extends from the enzyme exterior and terminates at the FAD re-face. A homology model of the NADH oxidase from Lactococcus lactis (L.lac-Nox2) was also generated using the crystal structure of L.san-Nox2, which reveals several important similarities and differences between the two enzymes. HPLC analysis of ligands released from denatured L.lac-Nox2 indicates that it does not bind ADP, which correlates with the specificity of the enzyme for oxidation of NADH.

Binding Sites↗
PubMed2006

Identifying the interacting positions of a protein using Boolean learning and support vector machines.

It is known that in the three-dimensional structure of a protein, certain amino acids can interact with each other in order to provide structural integrity or aid in its catalytic function. If these positions are mutated the loss of this interaction usually leads to a non-functional protein. Directed evolution experiments, which probe the sequence space of a protein through mutations in search for an improved variant, frequently result in such inactive sequences. In this work, we address the use of machine learning algorithms, Boolean learning and support vector machines (SVMs), to find such pairs of amino acid positions. The recombination method of imparting mutations was simulated to create in silico sequences that were used as training data for the algorithms. The two algorithms were combined together to develop an approach that weighs the structural risk as well as the empirical risk to solve the problem. This strategy was adapted to a multi-round framework of experiments where the data generated in the present round is used to design experiments for the next round to improve the generated library, as well as the estimation of the interacting positions. It is observed that this strategy can greatly improve the number of functional variants that are generated as well as the average number of mutations that can be made in the library.

Algorithms↗
PubMed2006

Structure-guided consensus approach to create a more thermostable penicillin G acylase.

The thermostabilization of penicillin G acylase (PGA) is a difficult problem due to the large size of the protein and its complex maturation process. We developed a data-driven protein design method that requires fewer homologous sequences than the traditional consensus approach and utilizes structural information to limit the number of variants created. Approximately 50% of our 21 single-point mutants were found experimentally to be more thermostable than the wild-type PGA, two had almost threefold longer half-life at 50 degrees C, with very little effect on activity. An analysis of four programs that predict the thermostability conferred by point mutations shows little agreement between the programs and with the experimental data, emphasizing that the chosen stabilizing mutations are very difficult to predict, but that our data-driven design method should prove useful.

Bacteria↗
PubMed2006

Search for extraterrestrial life using chiral molecules: mandelate racemase as a test case.

We have investigated an enzymatic racemization reaction as a marker for extraterrestrial life, which resulted in a change in optical rotation of a mandelic acid over time, as measured by polarimetry. Mandelate racemase was active in aqueous buffer in a temperature range between 0 degrees C and 70 degrees C and also in concentrated ammonium salt solutions and water-in-oil microemulsions in a temperature range between -30 degrees C and 60-70 degrees C; however, the enzyme was not active in several organic cryosolvents. Thus, we have demonstrated that concentrated ammonium salt solutions and water-in-oil microemulsions, both of which are able to form on extraterrestrial planets and moons in the presence of liquid water, are suitable media for enzyme reactions at subzero temperatures. Kinetic data for the mandelate racemase reaction obtained by polarimetry, while reproducible and internally consistent, differed significantly from several sets of data obtained previously by other methods such as chromatography and hydrogen-deuterium exchange. However, we conclude that reactions yielding a polarimetric signal, such as the racemizations employed in this work, are suitable mechanisms by which to utilize a change in chirality over time as a tool to detect signs of life.

Buffers↗
PubMed2005

Evaluation of Hofmeister effects on the kinetic stability of proteins.

Dissolved salts are known to affect properties of proteins in solution including solubility and melting temperature, and the effects of dissolved salts can be ranked qualitatively by the Hofmeister series. We seek a quantitative model to predict the effects of salts in the Hofmeister series on the deactivation kinetics of enzymes. Such a model would allow for a better prediction of useful biocatalyst lifetimes or an improved estimation of protein-based pharmaceutical shelf life. Here we consider a number of salt properties that are proposed indicators of Hofmeister effects in the literature as a means for predicting salt effects on the deactivation of horse liver alcohol dehydrogenase (HL-ADH), alpha-chymotrypsin, and monomeric red fluorescent protein (mRFP). We find that surface tension increments are not accurate predictors of salt effects but find a common trend between observed deactivation constants and B-viscosity coefficients of the Jones-Dole equation, which are indicative of ion hydration. This trend suggests that deactivation constants (log k(d,obs)) vary linearly with chaotropic B-viscosity coefficients but are relatively unchanged in kosmotropic solutions. The invariance with kosmotropic B-viscosity coefficients suggests the existence of a minimum deactivation constant for proteins. Differential scanning calorimetry is used to measure protein melting temperatures and thermodynamic parameters, which are used to calculate the intrinsic irreversible deactivation constant. We find that either the protein unfolding rate or the rate of intrinsic irreversible deactivation can control the observed deactivation rates.

Alcohol Dehydrogenase↗
PubMed2005

Simulation modeling of pooling for combinatorial protein engineering.

Pooling in directed-evolution experiments will greatly increase the throughput of screening systems, but important parameters such as the number of good mutants created and the activity level increase of the good mutants will depend highly on the protein being engineered. The authors developed and validated a Monte Carlo simulation model of pooling that allows the testing of various scenarios in silico before starting experimentation. Using a simplified test system of 2 enzymes, betagalactosidase (supermutant, or greatly improved enzyme) and beta-glucuronidase (dud, or enzyme with ancestral level of activity), the model accurately predicted the number of supermutants detected in experiments within a factor of 2. Additional simulations using more complex activity distributions show the versatility of the model. Pooling is most suited to cases such as the directed evolution of new function in a protein, where the background level of activity is minimized, making it easier to detect small increases in activity level. Pooling is most successful when a sensitive assay is employed. Using the model will increase the throughput of screening procedures for directed-evolution experiments and thus lead to speedier engineering of proteins.

Cells, Cultured↗
PubMed2005

Support vector machines for learning to identify the critical positions of a protein.

A method for identifying the positions in the amino acid sequence, which are critical for the catalytic activity of a protein using support vector machines (SVMs) is introduced and analysed. SVMs are supported by an efficient learning algorithm and can utilize some prior knowledge about the structure of the problem. The amino acid sequences of the variants of a protein, created by inducing mutations, along with their fitness are required as input data by the method to predict its critical positions. To investigate the performance of this algorithm, variants of the beta-lactamase enzyme were created in silico using simulations of both mutagenesis and recombination protocols. Results from literature on beta-lactamase were used to test the accuracy of this method. It was also compared with the results from a simple search algorithm. The algorithm was also shown to be able to predict critical positions that can tolerate two different amino acids and retain function.

Algorithms↗
PubMed2004

Crystallization and preliminary analysis of a water-forming NADH oxidase from Lactobacillus sanfranciscensis.

Single crystals have been obtained of NADH oxidase (Nox), a flavoenzyme cloned from Lactobacillus sanfranciscensis. The enzyme catalyzes the oxidation of two equivalents of NAD(P)H and reduces one equivalent of oxygen to yield two equivalents of water, without releasing hydrogen peroxide after the reduction of the first equivalent of NAD(P)H. The enzyme crystallizes in space group P2(1)2(1)2(1), with unit-cell parameters a = 59.6, b = 92.6, c = 163.5 A. The crystals diffract to 1.85 A resolution using synchrotron radiation. Matthews coefficient calculations suggest the presence of two molecules per asymmetric unit (V(M) = 2.3 A(3) Da(-1), 45.5% solvent content), which has been confirmed by the molecular-replacement solution using a search molecule derived from NADH peroxidase (PDB code 1f8w).

Crystallization↗
PubMedDate not supplied

Accelerated biocatalyst stability testing for process optimization.

The deactivation of protein biocatalysts even at relatively low temperatures is one of the principal drawbacks to their use. To aid in the development of novel biocatalysts, we have derived an equation for both time- and temperature-dependent activity of the biocatalyst based on known concepts such as transition state theory and the Lumry-Eyring model. We then derived an analytical solution for the total turnover number (ttn), under isothermal operation, as a function of the catalytic constant kcat, the unfolding equilibrium constant K, and the intrinsic first-order deactivation rate constant(s) k(d,i). Employing an immobilized glucose isomerase biocatalyst in a CSTR and utilizing a linear temperature ramp beyond the Tm of the enzyme, we demonstrate an accelerated method for extracting the thermodynamic and kinetic constants describing the biocatalyst system. In addition, we demonstrate that the predicted biocatalyst behavior at different temperatures and reaction times is consistent with the experimental observations.

Aldose-Ketose Isomerases↗
PubMedDate not supplied

Deactivation of formate dehydrogenase (FDH) in solution and at gas-liquid interfaces.

Enzymes, increasingly important in the synthesis of fine chemicals and pharmaceutical intermediates, are often insufficiently stable under reacting conditions. We have investigated the stability, in homogeneous aqueous solution and at gas-liquid interfaces, of formate dehydrogenase (FDH), important for cofactor regeneration, from Candida boidinii and overexpressed in E. coli. When exposed to mechanical stress, residual activity, [E](t)/[E](0), and residual protein were found to scale proportionally with gas-liquid surface area in the bubble column, verifying a surface-driven process, and with time and total throughput in a gear pump, but did not seem to be influenced much by shear in a Couette viscometer. All FDH variants are deactivated by chaotropes but not kosmotropes: the first-order deactivation constant k(d) correlates well with the Jones-Dole coefficient B but not well with the surface tension increment deltasigma of various concentrated ammonium salt solutions. This finding might provide guidance for focusing the search for quantitative theories of Hofmeister effects.

Bioreactors↗
PubMedDate not supplied

Pooling for improved screening of combinatorial libraries for directed evolution.

Following diversity generation in combinatorial protein engineering, a significant amount of effort is expended in screening the library for improved variants. Pooling, or combining multiple cells into the same assay well when screening, is a means to increase throughput and screen a larger portion of the library with less time and effort. We have developed and validated a Monte Carlo simulation model of pooling and used it to screen a library of beta-galactosidase mutants randomized in the active site to increase their activity toward fucosides. Here, we show that our model can successfully predict the number of highly improved mutants obtained via pooling and that pooling does increase the number of good mutants obtained. In unpooled conditions, we found a total of three mutants with higher activity toward p-nitrophenyl-beta-D-fucoside than that of the wild-type beta-galactosidase, whereas when pooling 10 cells per well we found a total of approximately 10 improved mutants. In addition, the number of "supermutants", those with the highest activity increase, was also higher when pooling was used. Pooling is a useful tool for increasing the efficiency of screening combinatorial protein engineering libraries.

Binding Sites↗