Nitrogenase is the only enzyme capable of reducing N2 to NH3. This challenging reaction requires the coordinated transfer of multiple electrons from the reductase, Fe-protein, to the catalytic component, MoFe-protein, in an ATP-dependent fashion. In the last two decades, there have been significant advances in our understanding of how nitrogenase orchestrates electron transfer (ET) from the Fe-protein to the catalytic site of MoFe-protein and how energy from ATP hydrolysis transduces the ET processes. In this review, we summarize these advances, with focus on the structural and thermodynamic redox properties of nitrogenase component proteins and their complexes, as well as on new insights regarding the mechanism of ET reactions during catalysis and how they are coupled to ATP hydrolysis. We also discuss recently developed chemical, photochemical, and electrochemical methods for uncoupling substrate reduction from ATP hydrolysis, which may provide new avenues for studying the catalytic mechanism of nitrogenase.

Electron Transfer in Nitrogenase

Chemical and physical Chitosan modification for designing enzymatic industrial biocatalysts: How to choose the best strategy?

Hydrogenases catalyze the reversible conversion of molecular hydrogen to protons and electrons via a heterolytic splitting mechanism. The active sites of [NiFe] hydrogenases comprise a dinuclear Ni-Fe center carrying CO and CN- ligands. The catalytic activity of the standard (O2-sensitive) [NiFe] hydrogenases vanishes under aerobic conditions. The O2-tolerant [NiFe] hydrogenases can sustain H2 oxidation activity under atmospheric conditions. These hydrogenases have very similar active site structures that change the ligand sphere during the activation/catalytic process. An important structural difference between these hydrogenases has been found for the proximal iron-sulphur cluster located in the vicinity of the active site. This unprecedented [4Fe-3S]-6Cys cluster can supply two electrons, which lead to rapid recovery of the O2 inactivation, to the [NiFe] active site.

/pdf/structure-and-function-of-nife-hydrogenases-4z9rarl8ki.pdf

Structure and function of [NiFe] hydrogenases.

Polyacrylic resins were employed to study how immobilization resin particle size influences Candida antarctica Lipase B (CALB) loading, fraction of active sites, and catalytic properties for polyester synthesis. CALB adsorbed more rapidly on smaller beads. Saturation occurred in less than 30 seconds and 48 h for beads with diameters 35 and 560-710 μm, respectively. Infrared microspectroscopy showed that CALB forms protein loading fronts for resins with particle sizes 560-710 and 120 μm while CALB appears evenly distributed throughout 35 μm resins. The fraction of active CALB molecules adsorbed onto resins not influenced by particle size was less than 50 %. At about 5% w/w CALB loading, decrease in the immobilization support diameter from 560-710 to 120, 75 and 35 μm increased conversion of e-CL to polyester (20 to 36, 42 and 61%, respectively, at 80 min). Similar trends were observed for condensation polymerizations of 1,8-octanediol and adipic acid.

Effects of Porous Polyacrylic Resin Parameters on Candida antarctica Lipase B Adsorption, Distribution, and Polyester Synthesis Activity

The market for industrial enzymes has witnessed constant growth, which is currently around 7% a year, projected to reach $10.5 billion in 2024. Lipases are hydrolase enzymes naturally responsible for triglyceride hydrolysis. They are the most expansively used industrial biocatalysts, with wide application in a broad range of industries. However, these biocatalytic processes are usually limited by the low stability of the enzyme, the half-life time, and the processes required to solve these problems are complex and lack application feasibility at the industrial scale. Emerging technologies create new materials for enzyme carriers and sophisticate the well-known immobilization principles to produce more robust, eco-friendlier, and cheaper biocatalysts. Therefore, this review discusses the trending studies and industrial applications of the materials and protocols for lipase immobilization, analyzing their advantages and disadvantages. Finally, it summarizes the current challenges and potential alternatives for lipases at the industrial level.

Current Status and Future Perspectives of Supports and Protocols for Enzyme Immobilization

Four major enzymes commonly used in the market are lipases, proteases, amylases, and cellulases For instance, in both academic and industrial levels, microbial lipases have been well studied for industrial and biotechnological applications compared to others Immobilization is done to minimize the cost The improvement of enzyme properties enables the reusability of enzymes and facilitates enzymes used in a continuous process Immobilized enzymes are enzymes physically confined in a particularly defined region with retention to their catalytic activities Immobilized enzymes can be used repeatedly compared to free enzymes, which are unable to catalyze reactions continuously in the system Immobilization also provides a higher pH value and thermal stability for enzymes toward synthesis The main parameter influencing the immobilization is the support used to immobilize the enzyme The support should have a large surface area, high rigidity, suitable shape and particle size, reusability, and resistance to microbial attachment, which will enhance the stability of the enzyme The diffusion of the substrate in the carrier is more favorable on hydrophobic supports instead of hydrophilic supports The methods used for enzyme immobilization also play a crucial role in immobilization performance The combination of immobilization methods will increase the binding force between enzymes and the support, thus reducing the leakage of the enzymes from the support The adsorption of lipase on a hydrophobic support causes the interfacial activation of lipase during immobilization The adsorption method also causes less or no change in enzyme conformation, especially on the active site of the enzyme Thus, this method is the most used in the immobilization process for industrial applications

https://www.mdpi.com/2073-4344/10/7/744/pdf

The Immobilization of Lipases on Porous Support by Adsorption and Hydrophobic Interaction Method

Microbial lipases represent one of the most important groups of biotechnological biocatalysts. However, the high-level production of lipases requires an understanding of the molecular mechanisms of gene expression, folding, and secretion processes. Stable, selective, and productive lipase is essential for modern chemical industries, as most lipases cannot work in different process conditions. However, the screening and isolation of a new lipase with desired and specific properties would be time consuming, and costly, so researchers typically modify an available lipase with a certain potential for minimizing cost. Improving enzyme properties is associated with altering the enzymatic structure by changing one or several amino acids in the protein sequence. This review detailed the main sources, classification, structural properties, and mutagenic approaches, such as rational design (site direct mutagenesis, iterative saturation mutagenesis) and direct evolution (error prone PCR, DNA shuffling), for achieving modification goals. Here, both techniques were reviewed, with different results for lipase engineering, with a particular focus on improving or changing lipase specificity. Changing the amino acid sequences of the binding pocket or lid region of the lipase led to remarkable enzyme substrate specificity and enantioselectivity improvement. Site-directed mutagenesis is one of the appropriate methods to alter the enzyme sequence, as compared to random mutagenesis, such as error-prone PCR. This contribution has summarized and evaluated several experimental studies on modifying the substrate specificity of lipases.

Main structural targets for engineering lipase substrate specificity

Due to its thermostability and high pH compatibility, subtilisin is most known for its role as an additive for detergents in which it is categorized as a serine protease according to MEROPS database. Subtilisin is typically isolated from various bacterial species of the Bacillus genus such as Bacillus subtilis, B. amyloliquefaciens, B. licheniformis, and various other organisms. It is composed of 268–275 amino acid residues and is initially secreted in the precursor form, preprosubtilisin, which is composed of 29‐residues signal peptide, 77‐residues propeptide, and 275‐residues active subtilisin. Subtilisin is known for the presence of high and low affinity calcium binding sites in its structure. Native subtilisin has general properties of thermostability, tolerance to neutral to high pH, broad specificity, and calcium‐dependent stability, which contribute to the versatility of subtilisin applicability. Through protein engineering and immobilization technologies, many variants of subtilisin have been generated, which increase the applicability of subtilisin in various industries including detergent, food processing and packaging, synthesis of inhibitory peptides, therapeutic, and waste management applications.

Versatility of subtilisin: A review on structure, characteristics, and applications

https://pubs.rsc.org/en/content/articlepdf/2018/cc/c8cc06261g

Redox-dependent conformational changes of a proximal [4Fe-4S] cluster in Hyb-type [NiFe]-hydrogenase to protect the active site from O2.

The purification procedure of Hyd-2-type [NiFe]-hydrogenase from Citrobacter sp. S-77 was improved by applying treatment with trypsin before chromatography. Purified protein samples both with and without trypsin treatment were successfully crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol as a precipitant. Both crystals belonged to space group P21, with unit-cell parameters a = 63.90, b = 118.89, c = 96.70 A, β = 100.61° for the protein subjected to trypsin treatment and a = 65.38, b = 121.45, c = 98.63 A, β = 102.29° for the sample not treated with trypsin. The crystal obtained from the trypsin-treated protein diffracted to 1.60 A resolution, which is considerably better than the 2.00 A resolution obtained without trypsin treatment. The [NiFe]-hydrogenase from Citrobacter sp. S-77 retained catalytic activity with some amount of O2, indicating that it has clear O2 tolerance.

Noor Dina Muhd Noor

Papers

The Immobilization of Lipases on Porous Support by Adsorption and Hydrophobic Interaction Method

Main structural targets for engineering lipase substrate specificity

Versatility of subtilisin: A review on structure, characteristics, and applications

Redox-dependent conformational changes of a proximal [4Fe-4S] cluster in Hyb-type [NiFe]-hydrogenase to protect the active site from O2.

Improved purification, crystallization and crystallographic study of Hyd-2-type [NiFe]-hydrogenase from Citrobacter sp. S-77