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    Cloning and expression heterologous alanine dehydrogenase genes: Investigation of reductive amination potential of L-alanine dehydrogenases for green synthesis of alanine derivatives
    (Cell Press, 2024) Demir, Garip; Valjakka, Jarkko; Turunen, Ossi; Aktas, Fatih; Binay, Baris
    Unnatural amino acids (UAAs) offer significant promise in a wide range of applications, including drug discovery, the custom design of peptides and proteins, and their utility and use as markers for monitoring molecular interactions in biological research. The synthesis of UAAs presents a formidable challenge and can be classified into two primary categories: enzymatic and chemical synthesis. Notably, the enzymatic route, specifically asymmetric synthesis, emerges as a an attractive method for procuring enantiopure UAAs with high efficiency, owing to its streamlined and concise reaction mechanism. The current study investigated the reductive amination activity mechanisms of alanine dehydrogenase (L-AlaDH), sourced from a combination of newly and previously characterized microorganisms. Our principal aim was to evaluate the catalytic efficiency of these L-AlaDH enzymes concerning a range of specific ketoacids and pyruvate to ascertain their capability for facilitating the production of both natural and unnatural amino acids. After the characterization processes, mutation points for TtAlaDH were determined and as a result of the mutations, mutants that could use ketocaproate and ketovalerate more effectively than the wild type were obtained. Among the enzymes studied, MetAlaDH exhibited the highest specific activity against pyruvate, 173 U/mg, and a KM value of 1.3 mM. VlAlaDH displayed the most favourable catalytic efficiency with a rate constant of 170 s- 1mM- 1. On the other hand, AfAlaDH demonstrated the highest catalytic efficiency against alpha-ketobutyrate (34.0 s- 1mM-1) and alpha-ketovalerate (2.7 s- 1mM-1). Of the enzymes investigated in the study, TtAlaDH exhibited the highest effectiveness among bacterial enzymes in catalyzing ketocaproate with a measured catalytic efficiency of about 0.6 s- 1mM-1 and a KM value of approximately 0.3 mM. These findings provide valuable insights into the substrate specificity and catalytic performance of L-AlaDHs, enhancing our understanding of their potential applications in various biocatalytic processes.
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    From Secretion in Pichia pastoris to Application in Apple Juice Processing: Exo-Polygalacturonase from Sporothrix schenckii 1099-18
    (Bentham Science Publ Ltd, 2021) Karatas, Ersin; Tulek, Ahmet; Cakar, Mehmet Mervan; Tamturk, Faruk; Aktas, Fatih; Binay, Baris
    Background: Polygalacturonases are a group of enzymes under pectinolytic enzymes related to enzymes that hydrolyse pectic substances. Polygalacturonases have been used in various industrial applications such as fruit juice clarification, retting of plant fibers, wastewater treatment drinks fermentation, and oil extraction. Objectives: The study was evaluated at the heterologous expression, purification, biochemical characterization, computational modeling, and performance in apple juice clarification of a new exo-polygalacturonase from Sporothrix schenckii 1099-18 (SsExo-PG) in Pichia pastoris. Methods: Recombinant DNA technology was used in this study. Two different pPIC9K plasmids were constructed with native signal sequence-ssexo-pg and alpha signal sequence-ssexo-pg separately. Protein expression and purification performed after plasmids transformed into the Pichia pastoris. Biochemical and structural analyses were performed by using pure SsExo-PG. Results: The purification of SsExo-PG was achieved using a Ni-NTA chromatography system. The enzyme was found to have a molecular mass of approximately 52 kDa. SsExo-PG presented as stable at a wide range of temperature and pH values, and to be more storage stable than other commercial pectinolytic enzyme mixtures. Structural analysis revealed that the catalytic residues of SsEx-o-PG are somewhat similar to other Exo-PGs. The K-M and k(cat) values for the degradation of polygalacturonic acid (PGA) by the purified enzyme were found to be 0.5868 mu M and 179 s(-1), respectively. Cu2+ was found to enhance SsExo-PG activity while Ag2+ and Fe2+ almost completely inhibited enzyme activity. The enzyme reduced turbidity up to 80% thus enhanced the clarification of apple juice. SsExo-PG showed promising performance when compared with other commercial pectinolytic enzyme mixtures. Conclusion: The clarification potential of SsExo-PG was revealed by comparing it with commercial pectinolytic enzymes. The following parameters of the process of apple juice clarification processes showed that SsExo-PG is highly stable and has a novel performance.
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    Heterologous Expression and Partial Characterization of a New Alanine Dehydrogenase from Amycolatopsis sulphurea
    (Springer, 2021) Aktas, Fatih
    A novel alanine dehydrogenase (AlaDH; EC.1.4.1.1) was isolated from Amycolatopsis sulphurea and the AlaDH gene was cloned into a pET28a(+) plasmid and expressed in E. coli BL21 (DE3). The molecular mass of this enzyme was calculated as 41.09 kDa and the amino acid residues of the pure protein indicated the presence of N terminus polyhistidine tags. Its enzyme kinetic values were K-m 2.03 mM, k(cat) 13.24 (s(-1)), and k(cat)/K-m 6.53 (s(-1) mM(-1)). AlaDH catalyzes the reversible conversion of l-alanine and pyruvate, which has an important role in the TCA energy cycle. Maximum AlaDH activity occurred at about pH 10.5 and 25 degrees C for the oxidative deamination of l-alanine. AlaDH retained about 10% of its relative activity at 55 degrees C and it remained about 90% active at 50 degrees C. These findings show that the AsAlaDH from A. sulphurea has the ability to produce valuable molecules for various industrial purposes and could represent a new potential biocatalyst for biotechnological applications after further characterization and improvement of its catalytic properties.
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    Screening, partial purification and characterization of the hyper-thermophilic lipase produced by a new isolate ofBacillus subtilisLP2
    (Taylor & Francis Ltd, 2020) Yasar, Gulhan; Gulhan, Unzile Guven; Guduk, Elif; Aktas, Fatih
    Lipases are one of the most important catalysts for several industries such as detergent, dairy, and textile industry due to their bio-catalytic ability in aqueous and non-aqueous media. Stability to extreme conditions is an important property since it makes enzymes suitable to several industrial processes. In this study, lipase producing soil bacteria were screened and identified with 16S rDNA sequencing. A new hyper-thermophilic lipase named asBacillus subtilis LP2isolate was partially purified by ammonium sulphate precipitation with 17.8-fold purification and 583 U/mg specific activity. Maximum activity was exhibited at pH 7 and 80 degrees C with the substrate tween 80 K(M)and V(max)values were calculated as 18.3 mM and 680 U/mg with a catalytic efficiency (k(cat)/K-M) of 307 s(-1)M(-1). These results indicate that lipase fromBacillus subtilis LP2can be a valuable candidate for industrial applications such as organic synthesis and fats and oils industry due to their efficient catalysis in higher temperatures.