Heterologous Expression and Partial Characterization of a Putative Opine Dehydrogenase from a Metagenomic Sequence of Desulfohalobium retbaense.

The aim of this research was to prove the function of the putative opine dehydrogenase from Desulfohalobium retbaense and to characterize the enzyme in terms of functional and kinetic parameters. A putative opine dehydrogenase was identified from a metagenomic library by a sequence-based technique search of the metagenomic library, and afterward was successfully heterologously produced in Escherichia coli. In order to examine its potential for applications in the synthesis of secondary amines, first the substrate specificity of the enzyme towards different amino donors and amino acceptors was determined. The highest affinity was observed towards small amino acids, preferentially L-alanine, and when it comes to α-keto acids, pyruvate proved to be a preferential amino acceptor. The highest activity was observed at pH 6.5 in the absence of salts. The enzyme showed remarkable stability in a wide range of experimental conditions, such as broad pH stability (from 6.0-11.0 after 30 min incubation in buffers at a certain pH), stability in the presence of NaCl up to 3.0 M for 24 h, it retained 80 % of the initial activity after 1 h incubation at 45 °C, and 65 % of the initial activity after 24 h incubation in 30 % dimethyl sulfoxide.

The Influence of Isoenzyme Composition and Chemical Modification on Horseradish Peroxidase@ZIF-8 Biocomposite Performance.

Many articles in the literature deal with horseradish peroxidase (HRP) biomineralization, but none pay attention to the isoenzyme composition of commercial HRP or the influence of the carbohydrate component of the protein molecule on the biomineralization process. To study the impact of these factors, we performed periodate oxidation of commercial HRP and a purified HRP-C isoform for biomineralization within ZIF-8. With purified HRP, enzyme@ZIF-8 biocomposites with higher activity were obtained, while periodate oxidation of the carbohydrate component of both commercial HRP and purified HRP-C yields biocomposites with very high activity in acetate buffer that does not degrade the ZIF-8 structure. Using acetate instead of phosphate buffer can prevent the false high activity of HRP@ZIF-8 biocomposites caused by the degradation of ZIF-8 coating. At the same time, purification and especially oxidation of the carbohydrate component of enzymes prior to biomineralization lead to significantly improved activity of the biocomposites.

Chemical Modification of Glycoproteins' Carbohydrate Moiety as a General Strategy for the Synthesis of Efficient Biocatalysts by Biomimetic Mineralization: The Case of Glucose Oxidase.

Zeolitic imidazolate framework-8 (ZIF-8) is widely used as a protective coating to encapsulate proteins via biomimetic mineralization. The formation of nucleation centers and further biocomposite crystal growth is entirely governed by the pure electrostatic interactions between the protein's surface and the positively charged Zn(II) metal ions. It was previously shown that enhancing these electrostatic interactions by a chemical modification of surface amino acid residues can lead to a rapid biocomposite crystal formation. However, a chemical modification of carbohydrate components by periodate oxidation for glycoproteins can serve as an alternative strategy. In the present study, an industrially important enzyme glucose oxidase (GOx) was selected as a model system. Periodate oxidation of GOx by 2.5 mM sodium periodate increased negative charge on the enzyme molecule, from -10.2 to -36.9 mV, as shown by zeta potential measurements and native PAGE electrophoresis. Biomineralization experiments with oxidized GOx resulted in higher specific activity, effectiveness factor, and higher thermostability of the ZIF-8 biocomposites. Periodate oxidation of carbohydrate components for glycoproteins can serve as a facile and general method for facilitating the biomimetic mineralization of other industrially relevant glycoproteins.

Flow cytometry-based system for screening of lignin peroxidase mutants with higher oxidative stability.

Lignin peroxidase (LiP) is a heme-containing oxidoreductase that oxidizes structurally diverse substrates in an H2O2-dependent manner. Its ability to oxidize many pollutants makes it suitable for bioremediation applications and an ideal candidate for optimization by mutagenesis and selection. In order to increase oxidative stability of LiP we generated a random mutagenesis library comprising 106 mutated LiP genes and screened for expressed enzymes with higher than wild-type activity after incubation in 30 mM H2O2 by flow cytometry with fluorescein-tyramide as a substrate. To preserve the genotype-phenotype connection, the LiP mutants were displayed on the yeast cell surface. Two rounds of sorting were performed, recovered colonies were then screened in microtiter plates, and activity analysis revealed a significant increase in the percentage of cells expressing LiP variants with higher oxidative stability than wtLiP. Two rounds of sorting increased the proportion of more-stable variants from 1.4% in the original library to 52.3%. The most stable variants after two rounds of sorting featured between two and four mutations and retained up to 80% of initial activity after 1 h incubation in 30 mM H2O2. We for the first-time applied flow cytometry for screening of any ligninolytic peroxidase library. Obtained results suggest that developed system may be applied for improvement of industrially important characteristics of lignin peroxidase.

Semi-rational design of cellobiose dehydrogenase for increased stability in the presence of peroxide.

Cellobiose dehydrogenase (CDH, EC 1.1.99.18) from white rot fungi Phanerochaete chrysosporium can be used for constructing biosensors and biofuel cells, for bleaching cotton in textile industry, and recently, the enzyme has found an important application in biomedicine as an antimicrobial and antibiofilm agent. Stability and activity of the wild-type (wt) CDH and mutants at methionine residues in the presence of hydrogen peroxide were investigated. Saturation mutagenesis libraries were made at the only methionine in heme domain M65 and two methionines M685 and M738 in the flavin domain that were closest to the active site. After screening the libraries, three mutants with increased activity and stability in the presence of peroxide were found, M65F with 70% of residual activity after 6 h of incubation in 0.3 M hydrogen peroxide, M738S with 80% of residual activity and M685Y with over 90% of residual activity compared to wild-type CDH that retained 40% of original activity. Combined mutants showed no activity. The most stable mutant M685Y with 5.8 times increased half-life in the presence of peroxide showed also 2.5 times increased kcat for lactose compared to wtCDH and could be good candidate for applications in biofuel cells and biocatalysis for lactobionic acid production.

Development of GFP-based high-throughput screening system for directed evolution of glucose oxidase.

Glucose oxidase (GOx) mutants with higher activity or stability have important role in industry and in the development of biosensors and biofuel cells. Discovering these mutants can be time-consuming if appropriate high-throughput screening (HTS) systems are not available. GOx gene libraries were successfully screened and sorted using a HTS system based on GOx activity dependent fluorescent labeling of yeast cells with tyramids and quantification of the amount of expressed enzyme by yeast enhanced green fluorescent protein (yGFP) tagging and flow cytometry. For this purpose, we expressed wild type and a mutant GOx as a chimera with the yGFP to confirm differences in catalytic activity between wild-type and mutant GOx. Fluorescence of yGFP is preserved during expression of chimera, and also after the oxidative enzymatic reaction. We have obtained a 2.5-fold enrichment in population of cells expressing active enzyme, and percentage of enzyme variants with enzymatic mean activity higher than wild type activity was increased to 44% after a single round of GOx gene library sorting. We have found two mutants with 1.3 and 2.3-fold increase in Vmax values compared to the wtGOx. By simultaneous detection of protein expression level and enzyme activity we have increased the likelihood of finding GOx variants with increased activity in a single round of flow cytometry sorting.