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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 Fe

L-asparaginases (L-ASNase, EC 3.5.1.1) are amidohydrolases catalysing the conversion of L-Asn to L-Asp and ammonia and to a lower extent L-Gln to L-Glu (L-glutaminase activity, L-GLNase). This enzyme is exploited as an anti-neoplastic agent to treat acute lymphoblastic leukaemia. Currently, Escherichia coli and Erwinia chrysanthemi L-ASNases are used as oncological drugs. However, harmful side-effects and hypersensitivity reactions are the main limitations of these therapeutics. A link has been proposed between L-GLNase activity and harmful side effects. Therefore, finding of new L-ASNases with low glutaminase activity, thus potentially with lower side effect risk, is important for medical applications. A detailed biochemical characterization of a wide range of L-ASNases linked with in silico approaches could contribute for discovering better oncologic ASNase drug candidates. In this study, ten bacterial and yeast L-ASNase genes belonging to type I and II class of L-ASNase families, were synthesized and cloned into T7 bacterial expression vector for production in E. coli BL21 cells. The optimum pH and temperature of the expressed L-ASNases were at pH 7.0-9.0 and 35°C-50°C range, respectively. While the optimum temperature and pH of the ten L-ASNases were similar as those previously reported for type I and II L-ASNases, none of them displayed detectable glutaminase activity. Structural comparisons of these ten ASNases with quite much differing kinetic properties showed that the residues with a catalytic role are conserved and some differences at position 59 close to the substrate may affect the kinetic parameters. The type I L-ASNase from Lachancea thermotolerans yeast ( LtASNase) exhibited the highest specific activity of 313.82 U/mg and catalytic efficiency for L-Asn. Therefore, LtASNase is a promising enzyme for further investigation in pharmaceutical applications.

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.

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.

The biochemical conversion route in the utilization of biomass has required intensive development of processing methods for reducing recalcitrance of cellulose to enzymatic hydrolysis. Since discov...

© 2020 Informa UK Limited, trading as Taylor & Francis Group.NADPH-dependent oxidoreductases are crucial biocatalysts for the industrial production of chiral compounds. For in situ recycling of required expensive cofactors in this biosynthetic process, NAD(P)+-dependent formate dehydrogenases (FDHs) are wanted to be employed as redox biocatalysts due to their greener and process friendly nature. However, their utilization is limited by their undesired cofactor preference that strongly prone to NAD+ more than NADP+ and catalytic efficiency. To mine NADP+-dependent FDHs in nature by the guidance of bioinformatic tools or re-engineering of their NAD+-dependent equivalents to get an applicable recycler are attractive topics in bioengineering. It can be said that, up to now, the attempts to switch the cofactor preference of the FDHs generally have resulted in NADP+-dependent enzymes that have not to catch the desired catalytic efficiencies or stability. In this review, all studies about the native NADP+-dependent FDHs and also engineered equivalents that reconstructed with different protein engineering approaches for altering the coenzyme specificity are outlined. To switch the coenzyme preference of FDHs or to find the native NADP+-dependent FDHs will be the hot topics in bioengineering until finding a feasible regenerator. Therefore, this study will be a useful guide to get a pathway for designing or discovering novel NADP+-dependent FDHs.

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.

NAD-dependent formate dehydrogenase (FDH) enzymes are frequently used in industrial and scientific applications. FDH is a reversible enzyme that reduces the NAD molecule to NADH and produces CO2 by oxidation of the formate ion, whereas it causes CO2 reduction in the reverse reaction. Some transition metal elements - Fe3+, Mo6+ and W6 + - can be found in the FDH structure of anaerobic and archaeal microorganisms, and these enzymes require cations and other redox-active cofactors for their FDH activity. While NAD-dependent FDHs do not necessarily require any metal cations, the presence of various metal cations can still affect FDH activities. To study the effect of 11 different metal ions, NAD-dependent FDH enzymes from ten different microorganisms were tested: Ancylobacter aquaticus (AaFDH), Candida boidinii (CboFDH), Candida methylica (CmFDH), Ceriporiopsis subvermispora (CsFDH), Chaetomium thermophilum (CtFDH), Moraxella sp. (MsFDH), Myceliophthora thermophila (MtFDH), Paracoccus sp. (PsFDH), Saccharomyces cerevisiae (ScFDH) and Thiobacillus sp. (TsFDH). It was found that metal ions (mainly Cu2+ and Zn2+) could have quite strong inhibition effects on several enzymes in the forward reaction, whereas several cations (Li+, Mg2+, Mn2+, Fe3+ and W6+) could increase the forward reaction of two FDHs. The highest activity increase (1.97 fold) was caused by Fe3+ in AaFDH. The effect on the reverse reaction was minimal. The modelled structures of ten FDHs showed that the active site is formed by 15 highly conserved amino acid residues spatially settling around the formate binding site in a conserved way. However, the residue differences at some of the sites close to the substrate do not explain the activity differences. The active site space is very tight, excluding water molecules, as observed in earlier studies. Structural examination indicated that smaller metal ions might be spaced close to the active site to affect the reaction. Metal ion size showed partial correlation to the effect on inhibition or activation. Affinity of the substrate may also affect the sensitivity to the metal's effect. In addition, amino acid differences on the protein surface may also be important for the metal ion effect. WOS:000576745100001 33043425 Q4

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. WOS:000572586600001 32974869 Q3

Formate dehydrogenases (FDHs) are NAD(P)H-dependent enzymes that catalyse the reversible oxidation of formate to CO2. The main goal was to use directed evolution to obtain variants of the FDH from Chaetomium thermophilum (CtFDH) with enhanced reduction activity in the conversion of CO2 into formic acid. Four libraries were constructed targeting five residues in the active site. We identified two variants (G93H/I94Y and R259C) with enhanced reduction activity which were characterised in the presence of both aqueous CO2(g) and HCO3−. The A1 variant (G93H/I94Y) showed a 5.4-fold increase in catalytic efficiency (kcat/KM) compared to that of the wild-type for HCO3− reduction. The improved biocatalysts were also applied as a coupled cofactor recycling system in the enantioselective oxidation of 4-phenyl-2-propanol catalysed by the alcohol dehydrogenase from Streptomyces coelicolor A3 (ScADH). Conversions in these reactions increased from 56 to 91% when the A1 variant was used instead of wild-type CtFDH. Two variants presenting up to five-fold increase in catalytic efficiency and kcat were obtained and characterised. They constitute a promising enzymatic alternative for CO2 utilization and will serve as scaffolds to be further developed in order to meet industrial requirements.

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.

In recent years, CO

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.

Immobilization of enzymes from different sources on various supports in designed systems increases enzymes’ stability by protecting the active site of it from undesired effect of reaction environme...

Staphylococcus aureus, among other staphylococcal species, developed multidrug resistance and causes serious health risks that require complex treatments. Therefore, the development of novel and effective strategies to combat these bacteria has been gaining importance. Since Staphylococcus simulans lysostaphin is a peptidoglycan hydrolase effective against staphylococcal species, the enzyme has a significant potential for biotechnological applications. Despite promising results of lysostaphin as a bacteriocin capable of killing staphylococcal pathogens, it is still not widely used in healthcare settings due to its high production cost. In this study, medium engineering techniques were applied to improve the expression yield of recombinant lysostaphin in E. coli. A new effective inducible araBAD promoter system and different mediums were used to enhance lysostaphin production. Our results showed that the composition of autoinduction media enhanced the amount of lysostaphin production 5-fold with the highest level of active lysostaphin at 30 degrees C. The production cost of 1000U of lysostaphin was determined as 4-fold lower than the previously proposed technologies. Therefore, the currently developed bench scale study has a great potential as a large-scale fermentation procedure to produce lysostaphin efficiently.

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 omega 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 omega 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 omega 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 omega 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.

Lysostaphin enzyme is an effective antibacterial agent proven to be promising for the prevention of healthcare-associated infections related to methicillin-resistant Staphylococcus aureus (MRSA). Here, safe, and non-toxic antibacterial hybrid coatings consisting of lysostaphin and waterborne polyurethane (PU) are presented. Lysostaphin was covalently immobilized onto polydopamine functionalized halloysite nanotubes (HNTs) to obtain stable HNT-lysostaphin nanohybrid structures with high effective lysostaphin concentrations, which were then incorporated into PU coatings by a facile spray coating process. Resulting PU/HNT-lysostaphin hybrid coatings presented strong antibacterial activity against S. aureus with a >99 % killing efficiency. The incorporation of lysostaphin into the polymer matrix via HNTs as a carrier enhanced the stability of the enzyme resulting in non-leaching coatings that presented high operational stability over multiple bacterial incubation cycles and high storage stability without any significant loss of enzymatic activity. Furthermore, PU/HNT-lysostaphin coatings demonstrated significant antibiofilm properties and reduced the formation of S. aureus biofilms by 70 % relative to neat PU coatings. The lysostaphin based antibacterial hybrid coatings developed in this study, which can be easily applied to surfaces in healthcare facilities and medical devices offer an effective approach for the prevention of S. aureus associated nosocomial infections.

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 (kLa) 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 kLa when the gas residence time and probe measuring the oxygen concentration of response time are in specific requirements.

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 KMand kcatvalues 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.

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 CO

In this study, a heterologously expressed exo-polygalacturonase from Sporothrix schenckii 1099-18 was purified and then its two magnetic immobilized counterparts were prepared in yolk–shell-structured mesoporous silica. The exo-polygalacturonase was initially precipitated on porous calcium carbonate in the presence of Fe3O4 magnetic nanoparticles. Then, a silica layer was created on the surface of the precipitated particles. The obtained immobilized exo-polygalacturonase (silica@Fe3O4/SsExo-PG) was further treated with glutaraldehyde (silica@Glu- Fe3O4/SsExo-PG). Fourier-transform infrared spectroscopy, X-ray diffraction and scanning electron microscopy techniques were used to confirm the immobilization of polygalacturonase. The optimum pH and temperature of all the exo-polygalacturonase preparations were determined to be 4.0 and 60 °C, respectively which are also optimum conditions for apple juice clarification. The silica@ Fe3O4/SsExo-PG and silica@Glu- Fe3O4/SsExo-PG showed 3- and 3.7-fold much stability, respectively than the free exo-polygalacturonase at 60 °C. The free and immobilized SsExo-PG preparations were used for the clarification of apple juice and the result showed that the turbidity of apple juice was reduced by 82% after 1 h treatment time. After ten consecutive uses, silica@Fe3O4/SsExo-PG and silica@Glu-Fe3O4/SsExo-PG retained 80% and 90% of their initial activities, respectively. Altogether, biochemical, thermal-stability, and apple juice clarification characteristics of the immobilized SsExo-PG suggest a high potential for biodegradation of pectin.

Nowadays, the use of formate dehydrogenase (FDH, EC 1.17.1.9) is well established as a means of NADH regeneration from NAD(+) via the coupled conversion of formate into carbon dioxide. Recent studies have been reported that specifically Chaetomium thermophilum FDH (CtFDH) is the most efficient FDH catalyzing this reaction in reverse (i.e. using CO2 as a substrate to produce formate, and thereby regenerating NAD(+)). However, to date the production of active CtFDH at high protein expression levels has received relatively little attention. In this study, we have tested the effect of batch and high cell density fermentation (HCDF) strategies in a small stirred fermenter, as well as the effect of supplementing the medium with casamino acids, on the expressed level of secreted CtFDH using P. pastoris. We have established that the amount of expressed CtFDH was indeed enhanced via a HCDF strategy and that extracellular protease activity was eliminated via the addition of casamino acids into the fermentation medium. On this basis, secreted CtFDH in an active form can be easily separated from the fermentation and can be used for subsequent biotechnological applications. Duzce University Scientific Research Project DepartmentDuzce University [2017.06.02.578] This work was supported by Duzce University Scientific Research Project Department with 2017.06.02.578 project number and special thanks to BERC Lab for providing pPICZa A vector with 6xHis-tagged CtFDH. The authors also gratefully acknowledge the help and support of Rowan M Lindeque (Department of Chemical and Biochemical Engineering, DTU, Denmark). WOS:000534056400009 2-s2.0-85081536738 PubMed: 32423672

Formate dehydrogenases (FDHs) are NAD(P)H-dependent enzymes that catalyse the reversible oxidation of formate to COFour libraries were constructed targeting five residues in the active site. We identified two variants (G93H/I94Y and R259C) with enhanced reduction activity which were characterised in the presence of both aqueous COTwo variants presenting up to five-fold increase in catalytic efficiency and k

Several protein expression systems can be used to get enzymes in required quantities and study their functions. Incorporating a polyhistidine tag is a beneficial way of getting various enzymes such as FDHs for industrial applications. The NAD(+) dependent formate dehydrogenase from Chaetomium thermophilum (CtFDH) can be utilized for interconversion of formate to carbon dioxide coupled with the conversion of NAD(+) to NADH. In this study, N-terminal His tagged CtFDH (N-CtFDH) and C-terminal His tagged CtFDH (C-CtFDH) was constructed to learn the effect of His tag location on the activity and kinetic parameters of the enzyme. The solubility of proteins was not affected by tag position, however, an interference on the N-terminal region caused a deterioration in specific activity and the kinetic ability of enzyme. The obtained results indicated that the C-terminus of the enzyme is an appropriate region for tag engineering. The C-CtFDH has an approximately three-fold larger specific activity and two-fold higher catalytic efficiency than N-CtFDH. The results suggest that insertion of a His-tag at the N-terminal or C-terminal end of CtFDH has different effects on the protein and the N-terminal fragment of the protein is crucial for the function of CtFDH.

Several protein expression systems can be used to get enzymes in required quantities and study their functions. Incorporating a polyhistidine tag is a beneficial way of getting various enzymes such as FDHs for industrial applications. The NAD

Yogurt can be considered one of the first products of biotechnology. It is the result of enzymatic and bioprocess innovations emerging around 5000 BC near the region of Mesopotamia. Later on, many ...

The gene of Thermotoga maritima GH10 xylanase (TmXYN10B) was synthesised to study the extreme limits of this hyperthermostable enzyme at high temperatures in the presence of biomass-dissolving hydrophilic ionic liquids (ILs). TmXYN10B expressed from Pichia pastoris showed maximal activity at 100 °C and retained 92 % of maximal activity at 105 °C in a 30-min assay. Although the temperature optimum of activity was lowered by 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc), TmXYN10B retained partial activity in 15–35 % hydrophilic ILs, even at 75–90 °C. TmXYN10B retained over 80 % of its activity at 90 °C in 15 % [EMIM]OAc and 15–25 % 1-ethyl-3-methylimidazolium dimethylphosphate ([EMIM]DMP) during 22-h reactions. [EMIM]OAc may rigidify the enzyme and lower Vmax. However, only minor changes in kinetic parameter Km showed that competitive inhibition by [EMIM]OAc of TmXYN10B is minimal. In conclusion, when extended enzymatic reactions under extreme conditions are required, TmXYN10B shows extraordinary potential. Electronic supplementary material The online version of this article (doi:10.1007/s00792-016-0841-y) contains supplementary material, which is available to authorized users.

Staphylococcus aureus (S. aureus), which has developed multidrug resistance, leads to many healthcare-associated infections resulting in significant medical and economic losses. Therefore, the development of new efficient strategies to deal with these bacteria has been gaining importance. Lysostaphin is a peptidoglycan hydrolase that has considerable potential as a bacteriocin. However, there have been few reported optimization and scale-up studies of the lysostaphin bioproduction process. Our preliminary results have revealed that the composition of auto-induction media at 30 °C increases the produced lysostaphin around 10-fold in shake flasks. In this study, achieving higher yields for recombinant lysostaphin in E. coli at a laboratory scale has been the aim, through the use of auto-induction media. Optimized medium composition and fermentation parameters were transferred to a laboratory-scale bioreactor. The tested conditions improved protein yields up to 184 mg/L in a 3 L stirred bioreactor and the productivity was improved 2-fold in comparison to previously published reports. Furthermore, this study also showed that lysostaphin is an effective bacteriocin on both commercially available and isolated S. aureus strains. These results will contribute to future larger-scale production of lysostaphin via the proposed fermentation conditions.

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. WOS:000600997800021 33148525 Q3

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.

WOS: 000434458100013, PubMed ID: 29777512, To identify a robust NADP(+) dependent formate dehydrogenase from Lactobacillus buchneri NRRL B-30929 (LbFDH) with unique biochemical properties. A new NADP(+) dependent formate dehydrogenase gene (fdh) was cloned from genomic DNA of L. buchneri NRRL B-30929. The recombinant construct was expressed in Escherichia coli BL21(DE3) with 6 x histidine at the C-terminus and the purified protein obtained as a single band of approx. 44 kDa on SDS-PAGE and 90 kDa on native-PAGE. The LbFDH was highly active at acidic conditions (pH 4.8-6.2). Its optimum temperature was 60 A degrees C and 50 A degrees C with NADP(+) and NAD(+), respectively and its T-m value was 78 A degrees C. Its activity did not decrease after incubation in a solution containing 20% of DMSO and acetonitrile for 6 h. The K-M constants were 49.8, 0.12 and 1.68 mM for formate (with NADP(+)), NADP(+) and NAD(+), respectively. An NADP(+) dependent FDH from L. buchneri NRRL B-30929 was cloned, expressed and identified with its unusual characteristics. The LbFDH can be a promising candidate for NADPH regeneration through biocatalysis requiring acidic conditions and high temperatures., Research Fund of the Yildiz Technical University [FDK-2018-3331], This work was supported by Research Fund of the Yildiz Technical University (Project Number: FDK-2018-3331) and special thanks to Dr. Siqing Liu from USDA ARS for providing chromosomal DNA of Lactobacillus buchneri NRRL B-30929.

To identify a robust NADPA new NADPAn NADP

Over the next decades, with the growing concern of rising atmospheric carbon dioxide (CO

Binay, Barış (Arel Author), This study aimed to prepare robust immobilized formate dehydrogenase (FDH) preparations which can be used as effective biocatalysts along with functional oxidoreductases, in which in situ regeneration of NADH is required. For this purpose, Candida methylica FDH was covalently immobilized onto Immobead 150 support (FDHI150), Immobead 150 support modified with ethylenediamine and then activated with glutaraldehyde (FDHIGLU), and Immobead 150 support functionalized with aldehyde groups (FDHIALD). The highest immobilization yield and activity yield were obtained as 90% and 132%, respectively when Immobead 150 functionalized with aldehyde groups was used as support. The half-life times (t1/2) of free FDH, FDHI150, FDHIGLU and FDHIALD were calculated as 10.6, 28.9, 22.4 and 38.5 h, respectively at 35 °C. FDHI150, FDHIGLU and FDHIALD retained 69, 38 and 51% of their initial activities, respectively after 10 reuses. The results show that the FDHI150, FDHIGLU and FDHIALD offer feasible potentials for in situ regeneration of NADH.

Over the next decades, with the growing concern of rising atmospheric carbon dioxide (CO2) levels, the importance of investigating new approaches for its reduction becomes crucial. Reclamation of CO2 for conversion into biofuels represents an alternative and attractive production method that has been studied in recent years, now with enzymatic methods gaining more attention. Formate dehydrogenases (FDHs) are NAD(P)H-dependent oxidoreductases that catalyze the conversion of formate into CO2 and have been extensively used for cofactor recycling in chemoenzymatic processes. A new FDH from Clostridium ljungdahlii (ClFDH) has been recently shown to possess activity in the reverse reaction: the mineralization of CO2 into formate. In this study, we show the successful homologous expression of ClFDH in Escherichia coli. Biochemical and kinetic characterization of the enzyme revealed that this homologue also demonstrates activity toward CO2 reduction. Structural analysis of the enzyme through homology modeling is also presented.

NAD + -dependent formate dehydrogenase(s) (EC 1.2.1.2, FDH) catalyzes the interconversion of formate anion to carbon dioxide coupled with the conversion of NAD + or NADH. FDHs attract significant attention in biotechnology due to their potential applications in NAD(H)-dependent industrial biocatalysis as well as in the production of renewable fuels and chemicals from carbon dioxide. In the present work, a new FDH from thermophilic fungus Myceliophthora thermophile ( Mt FDH) was characterized. The gene of the enzyme was synthesised, cloned, expressed in E. coli , as 6His-tagged protein, and purified to homogeneity by metal chelate affinity chromatography. Kinetic analysis suggested that Mt FDH exhibits higher catalytic efficiency on NaHCO 3 compared to formate. Notable, recombinant Mt FDH displays a pH optimum for the conversion of formate anion to carbon dioxide at extreme alkaline pH (pH 10.5). Thermal stability analysis showed that the enzyme displays good thermostability with T m 48 °C. Homology modelling and phylogenetic analysis suggested that the enzyme belongs to the D-specific 2-hydroxy acid dehydrogenases family. The active-site residues are well conserved compared to other homologous FDHs. The results of the present work provide new knowledge on the structure, function and diversity of FDHs and indicate that Mt FDH possess a huge potential for CO 2 reduction or NADH generation and under extreme alkaline conditions.

While formate dehydrogenases (FDHs) have been used for cofactor recycling in chemoenzymatic synthesis, the ability of FDH to reduce CO

Formate dehydrogenases are promising enzymes for in situ regeneration of reduced form of nicotinamide adenine dinucleotide. However, the low operational stability of free form of formate dehydrogenases restricts their industrial uses. In this study, three different aldehyde group containing supports, such as glyoxyl silica, glyoxyl agarose and aldehyde-functionalized Immobead 150 were evaluated for the covalent immobilization of formate dehydrogenase from Candida methylica. The immobilization - activity yields were determined as 75-68%, 60-84%, and 90-132%, respectively for glyoxyl silica, glyoxyl agarose and aldehyde-functionalized Immobead 150 supports for formate oxidation. The formate production activity of Candida methylica formate dehydrohenase was tested for different initial bicarbonate concentrations; however, no product was determined. The optimum pHs of free FDH and immobilized FDH on aldehyde-functionalized Immobead 150 were both 7.0, whereas the optimum pHs of the immobilized FDHs on glyoxyl silica and glyoxyl agarose were both 8.0. The optimum temperatures of all the FDH preparations were determined as 35 °C. KM values of free FDH and immobilized FDH on glyoxyl silica, glyoxyl agarose and aldehyde-functionalized Immobead 150 supports were determined as 4.18 ± 0.22, 3.06 ± 0.14, 3.22 ± 0.18, and 2.79 ± 0.15 mM, respectively for formate and 0.51 ± 0.05, 0.49 ± 0.02, 0.59 ± 0.05, and 0.15 ± 0.04 mM, respectively for NAD+. The half-life time values of immobilized FDH on glyoxyl silica, glyoxyl agarose and aldehyde-functionalized Immobead 150 supports were 2.8, 3.1 and 3.6 times higher than that of the free FDH, respectively at 35 °C. The immobilized FDH on glyoxyl silica, glyoxyl agarose and aldehyde-functionalized Immobead 150 supports were remained 60, 56 and 51% of their initial activities, respectively at the end of 10 reuses. These results show that the immobilized FDHs may be used for in situ regeneration of NADH along with oxidoreductases. © 2016 Elsevier B.V. All rights reserved.

Binay, Barış (Arel Author), Octyl-agarose is a support that permits the one step immobilization, stabilization and purification of lipases. However, the enzyme may be released from the support under drastic conditions. This paper describes a new heterofunctional support, octyl agarose beads activated with divinyl sulfone, that has proved to be useful to produce very stable and active biocatalysts of lipases from Candida rugosa (CRL), Rhizomucor miehei (RML) and Thermomyces lanuginosus (TLL), able to work under any reaction conditions without risking enzyme desorption. The three enzymes failed inimmobilizationonglyoxyl-octyl supports for different reasons. The immobilization at pH 5 permitted to keep the good properties of octyl agarose. Further incubation at pH 8 permitted to establish at least one covalent enzyme-support bond per enzyme molecule (preventing the risk of enzyme desorption), avoiding the inactivation produced at pH 10, and the final result is that all three new biocatalysts are more active than the octyl-glyoxyl counterparts and much more stable (e.g., 20 using CRL). The end of the enzyme-support reaction was achieved via blocking the vinylsulfone groups with different nucleophiles (cationic, anionic, hydrophobic, etc). This not only determined the final enzyme stability, but also the activity, selectivity and even specificity ofthe different immobilized preparations.