Cloning and expression heterologous alanine dehydrogenase genes: Investigation of reductive amination potential of L-alanine dehydrogenases for green synthesis of alanine derivatives.

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 α-ketobutyrate (34.0 s-1mM-1) and α-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.

A newly synthesized magnetic nanoparticle coated with glycidyl methacrylate monomer and 1,2,4-Triazole: Immobilization of α-Amylase from Bacillus licheniformis for more reuse, stability, and activity in the presence of H2O2.

α-Amylase is a secretory enzyme commonly found in nature. The α-Amylase enzyme catalyzes the hydrolysis of α-D-(1,4)-glucosidic bonds in starch, glycogen, and polysaccharides. The chemical characterization of the composite carrier and the immobilized enzyme was performed, and the accuracy of the immobilization was confirmed by FTIR, SEM, and EDS analyses. The X-ray diffraction (XRD) analysis indicates that the magnetic nanoparticle retained its magnetic properties following the modification process. Based on the Thermogravimetric Analysis (TGA) outcomes, it was evident that the structural integrity of the FPT nanocomposite remained unchanged at 200°C. The optimal pH was determined to be 5.5, and no alteration was observed following the immobilization process. Purified α-amylases usually lose their activity rapidly above 50°C. In this study, Bacillus licheniformis α-Amylase enzyme was covalently immobilized on the newly synthesized magnetic composite carrier having more azole functional group. For novelty-designed immobilized enzymes, while there is no change in the pH and optimum operating temperature of the enzyme with immobilization, two essential advantages are provided to reduce enzyme costs: the storage stability and reusability are increased. Furthermore, our immobilization technique enhanced enzyme stability when comparing our immobilized enzyme with the reference enzyme in industrial applications. The activity of the immobilized enzyme was higher in presence of 1-3% H2O2.

Application of reductive amination by heterologously expressed Thermomicrobium roseumL-alanine dehydrogenase to synthesize L-alanine derivatives.

Unnatural amino acids are unique building blocks in modern medicinal chemistry as they contain an amino and a carboxylic acid functional group, and a variable side chain. Synthesis of pure unnatural amino acids can be made through chemical modification of natural amino acids or by employing enzymes that can lead to novel molecules used in the manufacture of various pharmaceuticals. The NAD+ -dependent alanine dehydrogenase (AlaDH) enzyme catalyzes the conversion of pyruvate to L-alanine by transferring ammonium in a reversible reductive amination activity. Although AlaDH enzymes have been widely studied in terms of oxidative deamination activity, reductive amination activity studies have been limited to the use of pyruvate as a substrate. The reductive amination potential of heterologously expressed, highly pure Thermomicrobium roseum alanine dehydrogenase (TrAlaDH) was examined with regard to pyruvate, α-ketobutyrate, α-ketovalerate and α-ketocaproate. The biochemical properties were studied, which included the effects of 11 metal ions on enzymatic activity for both reactions. The enzyme accepted both derivatives of L-alanine (in oxidative deamination) and pyruvate (in reductive amination) as substrates. While the kinetic KM values associated with the pyruvate derivatives were similar to pyruvate values, the kinetic kcat values were significantly affected by the side chain increase. In contrast, KM values associated with the derivatives of L-alanine (L-α-aminobutyrate, L-norvaline, and L-norleucine) were approximately two orders of magnitude greater, which would indicate that they bind very poorly in a reactive way to the active site. The modeled enzyme structure revealed differences in the molecular orientation between L-alanine/pyruvate and L-norleucine/α-ketocaproate. The reductive activity observed would indicate that TrAlaDH has potential for the synthesis of pharmaceutically relevant amino acids.

Second generation Pichia pastoris strain and bioprocess designs.

Yeast was the first microorganism used by mankind for biotransformation processes that laid the foundations of industrial biotechnology. In the last decade, Pichia pastoris has become the leading eukaryotic host organism for bioproduct generation. Most of the P. pastoris bioprocess operations has been relying on toxic methanol and glucose feed. In the actual bioeconomy era, for sustainable value-added bioproduct generation, non-conventional yeast P. pastoris bioprocess operations should be extended to low-cost and renewable substrates for large volume bio-based commodity productions. In this review, we evaluated the potential of P. pastoris for the establishment of circular bioeconomy due to its potential to generate industrially relevant bioproducts from renewable sources and waste streams in a cost-effective and environmentally friendly manner. Furthermore, we discussed challenges with the second generation P. pastoris platforms and propose novel insights for future perspectives. In this regard, potential of low cost substrate candidates, i.e., lignocellulosic biomass components, cereal by-products, sugar industry by-products molasses and sugarcane bagasse, high fructose syrup by-products, biodiesel industry by-product crude glycerol, kitchen waste and other agri-food industry by products were evaluated for P. pastoris cell growth promoting effects and recombinant protein production. Further metabolic pathway engineering of P. pastoris to construct renewable and low cost substrate utilization pathways was discussed. Although, second generation P. pastoris bioprocess operations for valorisation of wastes and by-products still in its infancy, rapidly emerging synthetic biology tools and metabolic engineering of P. pastoris will pave the way for more sustainable environment and bioeconomy. From environmental point of view, second generation bioprocess development is also important for waste recycling otherwise disposal of carbon-rich effluents creates environmental concerns. P. pastoris high tolerance to toxic contaminants found in lignocellulosic biomass hydrolysate and industrial waste effluent crude glycerol provides the yeast with advantages to extend its applications toward second generation P. pastoris strain design and bioprocess engineering, in the years to come.

Immobilization of the Bacillus licheniformis α-Amylase on Azole Functionalized Nanoparticle: More Active, Stable, and Usability.

Enzymes are a powerful tool employed in industrial applications due to their high specificity and efficiency. Amylase enzymes play an important role in detergent, textile, analytical chemistry, and paper industries. Here we present the design, synthesis, and characterization of azole functionalized nanoparticles for the immobilization of α-amylase from Bacillus licheniformis (BlA). A modest binding efficiency (47%) was determined by the BCA assay. Enzymatic activity was measured using DNS method and illustrated the immobilization of amylase with the designed nanoparticles enhanced the thermal stability and long-term storage of amylases at a wide range of temperatures and pHs. With the required scale-up study, these implications amplify novel ways to implement this Fe3O4-PGMA-5A immobilized BlA enzyme in particular industrial applications.

Volumetric Mass Transfer Coefficient Measurement in a Stirred Tank Reactor.

A bioreactor is a controlled vessel which provides biological conversions into bioactive components using cells or enzymes. In the aerobic processes, it is important to know oxygen requirements of the cells which may vary during fermentation as a result of microbial activity, aging, substrate depletion and product formation, etc. Here we describe the measurement of volumetric mass transfer coefficient (k L a) in a stirred tank reactor using dynamic method based on unsteady state which is also one of the significant parameter especially in scaling-up. The equipment in the measurement according to dynamic method has low cost compared to steady-state methodology. This method is reliable in the determination of k L a when the gas residence time and probe measuring the oxygen concentration of response time are in specific requirements.

The challenges of using NAD+-dependent formate dehydrogenases for CO2 conversion.

In recent years, CO2 reduction and utilization have been proposed as an innovative solution for global warming and the ever-growing energy and raw material demands. In contrast to various classical methods, including chemical, electrochemical, and photochemical methods, enzymatic methods offer a green and sustainable option for CO2 conversion. In addition, enzymatic hydrogenation of CO2 into platform chemicals could be used to produce economically useful hydrogen storage materials, making it a win-win strategy. The thermodynamic and kinetic stability of the CO2 molecule makes its utilization a challenging task. However, Nicotine adenine dinucleotide (NAD+)-dependent formate dehydrogenases (FDHs), which have high selectivity and specificity, are attractive catalysts to overcome this issue and convert CO2 into fuels and renewable chemicals. It is necessary to improve the stability, cofactor necessity, and CO2 conversion efficiency of these enzymes, such as by combining them with appropriate hybrid systems. However, metal-independent, NAD+-dependent FDHs, and their CO2 reduction activity have received limited attention to date. This review outlines the CO2 reduction ability of these enzymes as well as their properties, reaction mechanisms, immobilization strategies, and integration with electrochemical and photochemical systems for the production of formic acid or formate. The biotechnological applications of FDH, future perspectives, barriers to CO2 reduction with FDH, and aspects that must be further developed are briefly summarized. We propose that constructing hybrid systems that include NAD+-dependent FDHs is a promising approach to convert CO2 and strengthen the sustainable carbon bio-economy.

Recombinant protein production in Pichia pastoris: from transcriptionally redesigned strains to bioprocess optimization and metabolic modelling.

Pichia pastoris is one of the most widely used host for the production of recombinant proteins. Expression systems that rely mostly on promoters from genes encoding alcohol oxidase 1 or glyceraldehyde-3-phosphate dehydrogenase have been developed together with related bioreactor operation strategies based on carbon sources such as methanol, glycerol, or glucose. Although, these processes are relatively efficient and easy to use, there have been notable improvements over the last twenty years to better control gene expression from these promoters and their engineered variants. Methanol-free and more efficient protein production platforms have been developed by engineering promoters and transcription factors. The production window of P. pastoris has been also extended by using alternative feedstocks including ethanol, lactic acid, mannitol, sorbitol, sucrose, xylose, gluconate, formate or rhamnose. Herein, the specific aspects that are emerging as key parameters for recombinant protein synthesis are discussed. For this purpose, a holistic approach has been considered to scrutinize protein production processes from strain design to bioprocess optimization, particularly focusing on promoter engineering, transcriptional circuitry redesign. This review also considers the optimization of bioprocess based on alternative carbon sources and derived co-feeding strategies. Optimization strategies for recombinant protein synthesis through metabolic modelling are also discussed.

Enzymes for Efficient CO2 Conversion.

The accumulation of carbon dioxide in the atmosphere as a result of human activities has caused a number of adverse circumstances in the world. For this reason, the proposed solutions lie within the aim of reducing carbon dioxide emissions have been quite valuable. However, as the human activity continues to increase on this planet, the possibility of reducing carbon dioxide emissions decreases with the use of conventional methods. The emergence of compounds than can be used in different fields by converting the released carbon dioxide into different chemicals will construct a fundamental solution to the problem. Although electro-catalysis or photolithography methods have emerged for this purpose, they have not been able to achieve successful results. Alternatively, another proposed solution are enzyme based systems. Among the enzyme-based systems, pyruvate decarboxylase, carbonic anhydrase and dehydrogenases have been the most studied enzymes. Pyruvate dehydrogenase and carbonic anhydrase have either been an expensive method or were incapable of producing the desired result due to the reaction cascade they catalyze. However, the studies reporting the production of industrial chemicals from carbon dioxide using dehydrogenases and in particular, the formate dehydrogenase enzyme, have been remarkable. Moreover, reported studies have shown the existence of more active and stable enzymes, especially the dehydrogenase family that can be identified from the biome. In addition to this, their redesign through protein engineering can have an immense contribution to the increased use of enzyme-based methods in CO2 reduction, resulting in an enormous expansion of the industrial capacity.

Enhancing recombinant Chaetomium thermophilium Formate Dehydrogenase Expression with CRISPR Technology.

Genetic manipulation of Escherichia coli influences the regulation of bacterial metabolism, which could be useful for the production of different targeted products. The RpoZ gene encodes for the ω subunit of the RNA polymerase (RNAP) and is involved in the regulation of the relA gene pathway. RelA is responsible for the production of guanosine pentaphosphate (ppGpp), which is a major alarmone in the stringent response. Expression of relA is reduced in the early hours of growth of RpoZ mutant E. coli. In the absence of the ω subunit, ppGpp affinity to RNAP is decreased; thus, rpoZ gene deleted E. coli strains show a modified stringent response. We used the E. coli K-12 MG1655 strain that lacks rpoZ (JEN202) to investigate the effect of the modified stringent response on recombinant protein production. However, the absence of the ω subunit results in diminished stability of the RNA polymerase at the promoter site. To avoid this, we used a deactivated CRISPR system that targets the ω subunit to upstream of the promoter site in the expression plasmid. The expression plasmid encodes for Chaetomium thermophilum formate dehydrogenase (CtFDH), a valuable enzyme for cofactor regeneration and CO2 reduction. A higher amount of CtFDH from the soluble fraction was purified from the JEN202 strain compared to the traditional BL21(DE3) method, thus offering a new strategy for batch-based recombinant enzyme production.

Highly-stable Madurella mycetomatis laccase immobilized in silica-coated ZIF-8 nanocomposites for environmentally friendly cotton bleaching process.

In this study, a laccase from Madurella mycetomatis (MmLac) was produced heterologously in Pichia pastoris; the initial immobilization in a metal-organic framework (MOF) (MmLac/ZIF-8) was achieved using zinc nitrate and 2-methylimidazole. Due to the instability of MmLac/ZIF-8 in an acidic medium, a silica layer was created on the surface of MmLac/MOF-8. The immobilized laccase composite (silica@MmLac/ZIF-8) obtained was further treated with glutaraldehyde (silica@Glu-MmLac/ZIF-8) to increase stability of composite. Fourier-transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy techniques were used to confirm the immobilization of MmLac and to investigate the morphology of the immobilized laccase samples. The MmLac samples were also characterised in terms of optimum pH, temperature and thermal stability. The optimum pH of all the MmLac samples was determined to be 4.0. The free MmLac showed maximum activity at 55 °C, whereas both silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 were maximumly active at 65 °C. The silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 were 9.3- and 11.8-fold higher in stability, respectively, than the free MmLac at 65 °C. Furthermore, both silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 showed a higher bleaching performance than free MmLac on cotton woven fabric. According to these results, silica@MmLac/ZIF-8 and silica@Glu-MmLac/ZIF-8 may be promising candidates for biocatalysts in laccase-based biotechnological applications.

Production of Industrial Enzymes via Pichia pastoris as a Cell Factory in Bioreactor: Current Status and Future Aspects.

Industrial enzymes have been widely preferred in various industries such as chemical production, food & beverage, pharmaceutical, textile, cosmetics, etc. due to the advancements in recent years. They are considered more economic than using whole cells and more environmental-friendly than chemical alternatives. Since the demand for industrial enzymes has been rising, the development of production strategies has been gathered speed. In this respect, the efficiency of Pichia pastoris (P. pastoris) as a host for heterologous protein expression has proved and gained attention due to its great potential for large-scale studies. Especially high-cell density fermentation of P. pastoris is a well-studied and efficient method. Moreover, the improvements in the state of art gene-editing tools have broadened the possibilities of strain improvement for P. pastoris. This review summarized the role of P. pastoris as a cell factory by accentuating the accomplishments in biocatalyst production. Moreover, the benefits and challenges of the most relevant expression systems named Escherichia coli (E. coli), Saccharomyces cerevisiae (S. cerevisiae), P. pastoris and recent evolvements and future directions were revealed in detail. Subsequently, offers for prospects and the latest evolvements to enhance the recombinant protein production were discussed.

From Secretion in Pichia pastoris to Application in Apple Juice Processing: Exo-Polygalacturonase from Sporothrix schenckii 1099-18.

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 SsExo- PG are somewhat similar to other Exo-PGs. The KM and kcat values for the degradation of polygalacturonic acid (PGA) by the purified enzyme were found to be 0.5868 µM 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.

Optimisation of the Production and Bleaching Process for a New Laccase from Madurella mycetomatis, Expressed in Pichia pastoris: from Secretion to Yielding Prominent.

Laccases are polyphenol oxidoreductases used in a number of industrial applications. Due to the increasing demand for these "green catalysis" enzymes, the identification and biochemical characterisation of their novel properties is essential. In our study, cloned Madurella mycetomatis laccase (mmlac) genes were heterologously expressed in the methylotrophic yeast host Pichia pastoris. The high yield of the active recombinant protein in P. pastoris demonstrates the efficiency of a reliably constructed plasmid to express the laccase gene. The optimal biochemical conditions for the successfully expressed MmLac enzyme were identified. Detailed structural properties of the recombinant laccase were determined, and its utility in decolourisation and textile bleaching applications was examined. MmLac demonstrates good activity in an acidic pH range (4.0-6.0); is stable in the presence of cationic metals, organic solvents and under high temperatures (50-60 °C); and is stable for long-term storage at - 20 °C and - 80 °C for up to eight weeks. The structural analysis revealed that the catalytic residues are partially similar to other laccases. MmLac resulted in an increase in whiteness, whilst demonstrating high efficiency and stability and requiring the input of fewer chemicals. The performance of this enzyme makes it worthy of investigation for use in textile biotechnology applications, as well as within environmental and food technologies.

Conserved Amino Acid Residues that Affect Structural Stability of Candida boidinii Formate Dehydrogenase.

The NAD+-dependent formate dehydrogenase (FDH; EC 1.2.1.2) from Candida boidinii (CboFDH) has been extensively used in NAD(H)-dependent industrial biocatalysis as well as in the production of renewable fuels and chemicals from carbon dioxide. In the present work, the effect of amino acid residues Phe285, Gln287, and His311 on structural stability was investigated by site-directed mutagenesis. The wild-type and mutant enzymes (Gln287Glu, His311Gln, and Phe285Thr/His311Gln) were cloned and expressed in Escherichia coli. Circular dichroism (CD) spectroscopy was used to determine the effect of each mutation on thermostability. The results showed the decisive roles of Phe285, Gln287, and His311 on enhancing the enzyme's thermostability. The melting temperatures for the wild-type and the mutant enzymes Gln287Glu, His311Gln, and Phe285Thr/His311Gln were 64, 70, 77, and 73 °C, respectively. The effects of pH and temperature on catalytic activity of the wild-type and mutant enzymes were also investigated. Interestingly, the mutant enzyme His311Gln exhibits a large shift of pH optimum at the basic pH range (1 pH unit) and substantial increase of the optimum temperature (25 °C). The present work supports the multifunctional role of the conserved residues Phe285, Gln287, and His311 and further underlines their pivotal roles as targets in protein engineering studies.

Structural insights into the NAD+-dependent formate dehydrogenase mechanism revealed from the NADH complex and the formate NAD+ ternary complex of the Chaetomium thermophilum enzyme.

The removal of carbon dioxide from the waste streams of industrial processes is a major challenge for creation of a sustainable circular economy. This makes the synthesis of formate from CO2 by NAD+ dependent formate dehydrogenases (FDHs) an attractive process for this purpose. The efficiency of this reaction is however low and to achieve a viable industrial process an optimised engineered enzyme needs to be developed. In order to understand the detailed enzymatic mechanism of catalysis structures of different cofactor and substrate complexes of the FDH from the thermophilic filamentous fungus, Chaetomium thermophilum have been determined to 1.2-1.3 Å resolution. The substrate formate is shown to be held by four hydrogen bonds in the FDH catalytic site within the ternary complex with substrate and NAD+and a secondary formate binding site is observed in crystals soaked with substrate. Water molecules are excluded from the FDH catalytic site when the substrate is bound. The angle between the plane of the NAD+ cofactor pyridine ring and the plane of the formate molecule is around 27°. Additionally, structures of a FDH mutant enzyme, N120C, in complex with the reduced form of the cofactor have also been determined both in the presence and absence of formate bound at the secondary site. These structures provide further understanding of the catalytic mechanism of this fungal enzyme.