Industrial Biocatalysts Research Articles

Snapshots of the Catalytic Cycle of the Industrial Enzyme α-Amino-ε-Caprolactam Racemase (ACLR) Observed Using X-ray Crystallography

α-Amino-ε-caprolactam racemase (ACLR) is a PLP-dependent enzyme exploited in industry for the racemization of amino acid amides in dynamic kinetic resolutions that produce homochiral amino acids. We report high-resolution structures of wild-type and variant ACLRs from Rhizobium freirei, generated using the chiral substrate ACL and representative of covalent intermediates in the catalytic cycle. These complexes substantiate suggestions of a two-base mechanism, in which the PLP-binding K267 and D210 serve as proton donors in the racemization, and they constitute a robust basis on which to engineer ACLRs for improved activity as industrial biocatalysts.

Identification of BSA B1 Bacteria and Its Potency of Purified Cellulase to Hydrolyze Chlorella zofingiensis

Cellulase has been widely used as biocatalyst in industries. Production of cellulase from microorganismshas many advantages such as short production time and less expense. Our previous study indicated that oneof cellulolytic bacteria from digestive tract of milkfish (Chanos chanos), namely BSA B1, showed the highestcellulase activity. The objective of this study was to determine the phylogenetic of BSA B1 strain using 16SrRNA gene sequence. Furthermore, this study also determine the specific activity of purified cellulase from BSAB1 strain and its potency to hydrolyze Chlorella zofingiensis cellulose. Cellulase was purified using ammoniumsulphate precipitation, dialysis, and ion exchange chromatography. The purified cellulase was used to hydrolyzecellulose of C. zofingiensis. The result demonstrated that BSA B1 strain was closely related with Bacillus aeriusand Bacillus licheniformis. The specific activity of the crude enzyme was 1.543 U mL-1; after dialysis was 4.384 UmL-1; and after chromatography was 7.543 U mL-1. Purified cellulase exhibited activity in hydrolyzed both CMCand C. zofingiensis. Compared to commercial cellulase, purified cellulase had lower activity in hydrolyzed CMCbut higher activity in hydrolyzed C. zofingiensis. Ethanol dehydration could potentially increase the reducingsugar yield in cellulose hydrolysis when used appropriately. Morphology of C. zofingiensis cell has changedafter incubation with cellulases and ethanol dehydration indicated degradation of cell wall.

Understanding the activation mechanism of Thermomyces lanuginosus lipase using rational design and tryptophan‐induced fluorescence quenching

The attractiveness of lipases as industrial biocatalysts underlines the importance of understanding their molecular action in detail, helping future efforts to improve performance and applicability. Lipases represent a special class of carboxyl ester hydrolases, which act on long‐chained water insoluble acyl‐glycerols at the water‐lipid interface. This heterogeneous catalysis makes it very difficult to monitor the structural changes taking place in the enzyme during activation in its preferred milieu. In this review, we cover recent developments toward a deeper understanding of the interfacial activation phenomenon of lipases and discuss a selection of biophysical methods suitable for studying lipase activation. For many lipases, such as that from Thermomyces lanuginosus, a structural domain called the “lid” is directly involved in the activation process. This has been shown through alteration of the lid‐residue composition using rational design. The resulting lid‐mutants have highlighted the lid's role in governing enzymatic activity and interfacial activation. Moreover, a recently developed fluorescence‐based technique called the Tryptophan Induced Quenching method has proved to enable the direct measurement of lipase activation in water‐ethanol mixtures, and thus represents an intriguing method to gain further insight into the structural movements taking place at the water‐lipid interface.Practical applications: Lipases are important industrial biocatalysts widely used in industrial applications including detergent, leather, paper, fuel, fine chemicals, baking, cosmetics, and food. Knowledge of structural changes of lipase upon activation and identification of the amino acids involved in lipase activation is crucial for design of new variants with improved or new properties. Rational design of the lid‐region in lipases has an interesting potential for optimization of lipase function and adapting them to different environments. In order to leverage on the design of new lipase variants and their altered function, methods that enable direct measurement of lipase action directly in solution, in real time, are desired. In this context, fluorescence based methods have shown great potential and provides interesting perspectives for probing the delicate movements occurring in lipases upon activation.Interfacial activation of lipases and the lid movements studied by rational design, fluorescence quenching methods, and solvent effect – polarity driven activation.

Enhanced bio-catalytic performance and dye degradation potential of chitosan-encapsulated horseradish peroxidase in a packed bed reactor system

In this study, horseradish peroxidase (HRP) was immobilized onto chitosan beads by entrapment method and employed for the degradation of textile dyes. Stable and firm quality chitosan beads developed with 2.5% chitosan concentration exhibited maximum immobilization yield (~92.54±2.53%). The pH optimum of chitosan-immobilized HRP (CTS-HRP) was marginally displaced towards alkaline region (pH7.5) than that of F-HRP which displayed its optimum activity at pH7.0. The free HRP (F-HRP) and CTS-HRP enzyme presented their maximum catalytic activities at 30°C and 70°C, respectively. Relative activities of F-HRP and CTS-HRP were decreased following pre-incubation above 30°C and 50°C, respectively and after 120min at 70°C, the F-HRP, and CTS-HRP retained 19.3±1.3 and 48.3±2.4% activities, accordingly. The CTS-HRP exhibited remarkably better resistance towards heavy metal induced activity inhibition. The effect of potential inhibitors on the activity of F-HRP and CTS-HRP was investigated and found that CTS-HRP was significantly less vulnerable to the denaturation caused by urea, ethylenediaminetetraacetic acid (EDTA), cysteine, 1, 4-dithiothreitol and Triton X-100. Moreover, the CTS-assisted entrapped-HRP was also employed for the decolorization of four different textile dyes i.e. Remazol Brilliant Blue R (RBBR), Reactive Black 5 (RB5), Congo Red (CR) and Crystal Violet (CV). The CTS-HRP showed considerable decolorization efficacy in six consecutive batch operations. Results suggest that CTS-HRP is an attractive choice for use as industrial biocatalyst in larger scale bioremediation of textile dyes and effluents.

Site‐directed mutagenesis studies of hydrophobic residues in the lid region of T1 lipase

T1 lipase is a potential biocatalyst for industrial application since it is highly thermostable and displays optimal activity at 60–75°C. Structural analysis of T1 lipase shows that its lid region undergoes a spatial displacement along with a distinct secondary structure reorganization upon activation. To study structure/function of this atypical lid, we performed site‐directed mutagenesis on the hydrophobic residues in the lid region. These residues were mutated to hydrophilic ones and biochemical properties of mutants were investigated. Results showed that F181 might be an important residue for enzyme‐substrate binding. Mutants A186S and A190S had 35–50% increase in catalytic efficiencies compared to wild‐type T1, without compromising their functions at high temperatures. In general, mutagenesis did not cause large changes to chain‐length preference in T1 lipase. Mutants A186S and V187N were inactive towards long‐chain pNP esters (p‐Nitrophenyl stearate) and V187N showed lower activity towards long‐chain triacylglycerols than wt T1, which makes it a potential catalyst in dairy industry. Thermostability of mutants were affected at different extent due to the influence of hydrophobic contact between the lid and the protein core. These findings not only shed light on the lipase structure/function relationship but also lay the framework for further engineering to gain more potent, stable, and selective lipases.Practical applications: A thermostable T1 lipase owns an atypical lid and hydrophobic residues in the lid influence its catalytic properties. Here, we screened mutants A186S and A190S with 35–50% increase in catalytic efficiency, which have industrial application in the modification of fats and oils. Moreover, mutant V187N showed lower activity towards long‐chain TAGs than wt T1. This mutant can be a potential biocatalyst in dairy industry which promote generation of short‐chain fatty acids from milk fats to increase flavors in dairy products.The lid region of thermostable T1 lipase undergoes a spatial displacement along with a distinct secondary structure reorganization upon activation (PDB ID: 2DSN, 2W22 [16,17]). Mutagenesis on hydrophobic residues in the lid affect catalytic properties of T1 lipase.

QUANTITATIVE PROTEASE ASSAY BY SUBSTRATE-AGAROSE PLATE METHOD

Proteases are unique class of industrial biocatalyst; occupy a key chair with respect to wide range of utility in both physiological and commercial sector. Repertoire of various enzyme assays were suggested for determining enzyme units of protease. Majority of currently reported assays are expensive, time consuming and cumbersome. In this direction, a development of speedy and sensitive quantitative enzyme assay for the characterization of proteases is highly desirable. The present investigation reported a quick method for characterization of proteases using single substrate-agarose plate method. A trypsin (obtained from bovine pancreas) and bacterial protease (serine protease of Bacillus circulans MTCC 7942) on subsequent interaction with proteinaceous substrate gives proteolytic zone on substrate-agarose plate. The clear zone diameter of proteolysis was directly proportional to enzyme units of protease. Furthermore, enzyme activities of protease treated with various chemical activator/inhibitor gives vis-à-vis response and yielded significant data. The biochemical characteristics of proteases are documented using rapid single substrate-agarose plate method. A rapid, sensitive and reliable improved quantitative protease assay using single substrate-agarose plate method could be used at academic, research and commercial level.

Relevance of substrates and products on the desorption of lipases physically adsorbed on hydrophobic supports

Lipase B from Candida antarctica (CALB) has been physically immobilized on octyl-agarose via interfacial activation. The incubation of the enzyme in 80% ethanol at pH 5 and 25°C has not significant effect on enzyme activity. Moreover, the hydrolysis of 100mM tributyrin catalyzed by this biocatalyst exhibited a quite linear reaction course. However, a new cycle of tributyrin hydrolysis showed a drastic drop in the activity. SDS-PAGE gels of the supernatant and the biocatalyst showed a significant enzyme desorption after the reaction. Similar results could be appreciated using triacetin or sunflower oil, while using 300mM methyl phenyl acetate, butyl butyrate or ethyl butyrate most enzyme molecules remained immobilized. The results show that the detergent properties of some reaction products increase the enzyme release from the hydrophobic support, and this problem increased if the concentration of the reactants increased. Using 500mM tributyrin, even in fully aqueous medium, some enzyme desorption from the support may be observed. Thus, the results show a limitation of this kind of biocatalysts that should be considered in the selection of an industrial lipase biocatalyst.

Zymomonas mobilis as a model system for production of biofuels and biochemicals.

Summary Zymomonas mobilis is a natural ethanologen with many desirable industrial biocatalyst characteristics. In this review, we will discuss work to develop Z. mobilis as a model system for biofuel production from the perspectives of substrate utilization, development for industrial robustness, potential product spectrum, strain evaluation and fermentation strategies. This review also encompasses perspectives related to classical genetic tools and emerging technologies in this context.

A more polar N-terminal helix releases MBP-tagged Thermus thermophilus proline dehydrogenase from tetramer-polymer self-association

Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme involved in the biosynthesis of l-glutamate. ProDH is of interest for biocatalysis because the protein might be applied in multi-enzyme reactions for the synthesis of structurally complex molecules. We recently demonstrated that the thermotolerant ProDH from Thermus thermophilus (TtProDH) is overproduced in Escherichia coli when using maltose-binding protein (MBP) as a solubility tag. However, MBP-TtProDH and MBP-clipped TtProDH are prone to aggregation through non-native self-association. Here we provide evidence that the hydrophobic N-terminal helix of TtProDH is responsible for the self-association process. The more polar MBP-tagged F10E/L12E variant exclusively forms tetramers and exhibits excellent catalytic features over a wide range of temperatures. Understanding the hydrodynamic and catalytic properties of thermostable enzymes is important for the development of industrial biocatalysts as well as for pharmaceutical applications.

Structural and functional characterization of a highly stable endo-β-1,4-xylanase from Fusarium oxysporum and its development as an efficient immobilized biocatalyst

BackgroundReplacing fossil fuel with renewable sources such as lignocellulosic biomass is currently a promising alternative for obtaining biofuel and for fighting against the consequences of climate change. However, the recalcitrant structure of lignocellulosic biomass residues constitutes a major limitation for its widespread use in industry. The efficient hydrolysis of lignocellulosic materials requires the complementary action of multiple enzymes including xylanases and β-xylosidases, which are responsible for cleaving exo- and endoxylan linkages, that release oligocarbohydrates that can be further processed by other enzymes.ResultsWe have identified the endo-β-1,4-xylanase Xyl2 from Fusarium oxysporum as a promising glycoside hydrolase family 11 enzyme for the industrial degradation of xylan. To characterize Xyl2, we have cloned the synthetic optimized gene and expressed and purified recombinant Xyl2 to homogeneity, finally obtaining 10 mg pure Xyl2 per liter of culture. The crystal structure of Xyl2 at 1.56 Å resolution and the structure of a methyl-xylopyranoside Xyl2 complex at 2.84 Å resolution cast a highly detailed view of the active site of the enzyme, revealing the molecular basis for the high catalytic efficiency of Xyl2. The kinetic analysis of Xyl2 demonstrates high xylanase activity and non-negligible β-xylosidase activity under a variety of experimental conditions including alkaline pH and elevated temperature. Immobilizing Xyl2 on a variety of solid supports enhances the enzymatic properties that render Xyl2 a promising industrial biocatalyst, which, together with the detailed structural data, may establish Xyl2 as a platform for future developments of industrially relevant xylanases.Conclusions F. oxysporum Xyl2 is a GH11 xylanase which is highly active in free form and immobilized onto a variety of solid supports in a wide pH range. Furthermore, immobilization of Xyl2 on certain supports significantly increases its thermal stability. A mechanistic rationale for Xyl2's remarkable catalytic efficiency at alkaline pH is proposed on the basis of two crystallographic structures. Together, these properties render Xyl2 an attractive biocatalyst for the sustainable industrial degradation of xylan.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0605-z) contains supplementary material, which is available to authorized users.

The role of conformational flexibility in Baeyer-Villiger monooxygenase catalysis and structure

BackgroundThe Baeyer-Villiger monooxygenases (BMVOs) are a group of microbial enzymes that have garnered interest as industrial biocatalysts. While great strides have been made in recent years to understand the mechanism of these enzymes from a structural perspective, our understanding remains incomplete. In particular, the role of a twenty residue loop (residues 487–504), which we refer to as the “Control Loop,” that is observed in either an ordered or disordered state in various crystal structures remains unclear. MethodsUsing SAXS, we have made the first observations of the Loop in solution with two BVMOs, cyclohexanone monooxygenase (CHMO) and cyclopentadecanone monooxygenase. We also made a series of mutants of CHMO and analyzed them using SAXS, ITC, and an uncoupling assay. ResultsThese experiments show that Control Loop ordering results in an overall more compact enzyme without altering global protein foldedness. We have also demonstrated that the Loop plays a critical and complex role on enzyme structure and catalysis. The Control Loop appears to have a direct impact on the organization of the overall structure of the protein, as well as in influencing the active site environment. ConclusionsThe data imply that the Loop can be divided into two regions, referred to as “sub-loops,” that coordinate overall domain movements to changes in the active site. General significanceA better understanding of the mechanistic role of the Control Loop may ultimately be helpful in designing mutants with altered specificity and improved catalytic efficiency.

Protein-directed assembly of cobalt phosphate hybrid nanoflowers

The understanding and controlling of biomimetic hybrid materials are a key objective in bio-nanotechnology, materials chemistry, and colloid science fields. Biomaterials, such as, enzyme, DNA, RNA, and proteins have become important templates for the construction of inorganic–organic hybrid nanoflowers. From this perspective, we present a simple approach to synthesize protein and metal hybrid flower-like structure using bovine serum albumin (BSA) and cobalt phosphate, and the results of our study on the formation mechanism involved. The time dependent growing stage and formation mechanism were analyzed by electron microscopes and spectroscopic techniques. The protein-directed assembly method for preparation of hybrid nanoflowers described in this work could be used to fabricate other bio-metal hybrid materials with possible applications in biosensors, bioanalytical devices, and industrial biocatalyst fields.

Cell-free synthesis of functional phospholipase A1 from Serratia sp.

BackgroundPhospholipase A1 is an enzyme that hydrolyzes phospholipids at the sn-1 position. It has potential applications across diverse fields including food, pharmaceutical, and biofuel industries. Although there has been increasing interest in the use of phospholipase A1 for degumming of plant oils during biodiesel production, production of recombinant phospholipase A1 has been hampered by low efficiency of gene expression and its toxicity to the host cell.ResultsWhile expression of phospholipase A1 in Escherichia coli resulted in extremely low productivity associated with inhibition of transformed cell growth, drastically higher production of functional phospholipase A1 was achieved in a cell-free protein synthesis system where enzyme expression is decoupled from cell physiology. Compared with expression in E. coli, cell-free synthesis resulted in an over 1000-fold higher titer of functional phospholipase A1. Cell-free produced phospholipase A1 was also used for successfully degumming crude plant oil.ConclusionsWe demonstrate successful production of Serratia sp. phospholipase A1 in a cell-free protein synthesis system. Including the phospholipase A1 investigated in this study, many industrial enzymes can interfere with the regular physiology of cells, making cellular production of them problematic. With the experimental results presented herewith, we believe that cell-free protein synthesis will provide a viable option for rapid production of important industrial biocatalysts.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-016-0563-5) contains supplementary material, which is available to authorized users.

Industrial Acetogenic Biocatalysts: A Comparative Metabolic and Genomic Analysis.

Synthesis gas (syngas) fermentation by anaerobic acetogenic bacteria employing the Wood–Ljungdahl pathway is a bioprocess for production of biofuels and biocommodities. The major fermentation products of the most relevant biocatalytic strains (Clostridium ljungdahlii, C. autoethanogenum, C. ragsdalei, and C. coskatii) are acetic acid and ethanol. A comparative metabolic and genomic analysis using the mentioned biocatalysts might offer targets for metabolic engineering and thus improve the production of compounds apart from ethanol. Autotrophic growth and product formation of the four wild type (WT) strains were compared in uncontrolled batch experiments. The genomes of C. ragsdalei and C. coskatii were sequenced and the genome sequences of all four biocatalytic strains analyzed in comparative manner. Growth and product spectra (acetate, ethanol, 2,3-butanediol) of C. autoethanogenum, C. ljungdahlii, and C. ragsdalei were rather similar. In contrast, C. coskatii produced significantly less ethanol and its genome sequence lacks two genes encoding aldehyde:ferredoxin oxidoreductases (AOR). Comparative genome sequence analysis of the four WT strains revealed high average nucleotide identity (ANI) of C. ljungdahlii and C. autoethanogenum (99.3%) and C. coskatii (98.3%). In contrast, C. ljungdahlii WT and C. ragsdalei WT showed an ANI-based similarity of only 95.8%. Additionally, recombinant C. ljungdahlii strains were constructed that harbor an artificial acetone synthesis operon (ASO) consisting of the following genes: adc, ctfA, ctfB, and thlA (encoding acetoacetate decarboxylase, acetoacetyl-CoA:acetate/butyrate:CoA-transferase subunits A and B, and thiolase) under the control of thlA promoter (PthlA) from C. acetobutylicum or native pta-ack promoter (Ppta-ack) from C. ljungdahlii. Respective recombinant strains produced 2-propanol rather than acetone, due to the presence of a NADPH-dependent primary-secondary alcohol dehydrogenase that converts acetone to 2-propanol. Furthermore, the ClosTronTM system was used to construct an adhE1 integration mutant. These results provide extensive insights into genetic features of industrially relevant bacterial biocatalysts and expand the toolbox for metabolic engineering of acetogenic bacteria able to ferment syngas.

A multi-tolerant low molecular weight mannanase from Bacillus sp. CSB39 and its compatibility as an industrial biocatalyst.

Bacillus sp. CSB39, isolated from popular traditional Korean food (Kimchi), produced a low molecular weight, thermostable mannanase (MnCSB39); 571.14U/mL using locust bean gum galactomannan as a major substrate. It was purified to homogeneity using a simple and effective two-step purification strategy, Sepharose CL-6B and DEAE Sepharose Fast Flow, which resulted in 25.47% yield and 19.32-fold purity. The surfactant-, NaCl-, urea-, and protease-tolerant monomeric protein had a mass of ∼30kDa as analyzed by SDS-PAGE and galactomannan zymography. MnCSB39 was found to have optimal activity at pH 7.5 and temperature of 70°C. The enzyme showed ˃55% activity at 5.0-15% (w/v) NaCl, and ˃93% of the initial activity after incubation at 37°C for 60min. Trypsin and proteinase K had no effect on MnCBS39. The enzyme showed ˃80% activity in up to 3M urea. The N-terminal amino acid sequence, ALKGDGX, did not show identity with reported mannanases, which suggests the novelty of our enzyme. Activation energy for galactomannan hydrolysis was 26.85kJmol(-1) with a Kcat of 142.58×10(4)s(-1). MnCSB39 had Km and Vmax values of 0.082mg/mL and 1099±1.0Umg(-1), respectively. Thermodynamic parameters such as ΔH, ΔG, ΔS, Q10, ΔGE-S, and ΔGE-T supported the spontaneous formation of products and the high hydrolytic efficiency and feasibility of the enzymatic reaction, which strengthen its novelty. MnCSB39 activity was affected by metal ions, modulators, chelators, and detergents. Mannobiose was the principal end-product of hydrolysis. Bacillus subtilis CSB39 produced a maximum of 1524.44U mannanase from solid state fermentation of 1g wheat bran. MnCSB39 was simple to purify, was active at a wide pH and temperature range, multi-stress tolerant and catalyzes a thermodynamically possible reaction, characteristics that suggests its suitability for application as an industrial biocatalyst.

Optimization of medium parameters by response surface methodology (RSM) for enhanced production of cutinase from Aspergillus sp. RL2Ct.

Cutinases are hydrolytic enzymes which catalyzes esterification and trans-esterification reactions that make them highly potential industrial biocatalyst. In the present investigation microorganisms showing cutinase activity were isolated from plant samples. The strain showing maximum cutinase activity was identified by 18S rDNA sequencing as Aspergillus sp. RL2Ct and was selected for further studies. To achieve maximum enzyme production, the medium components affecting cutinase production were screened by Plackett–Burman followed by central composite design. The results obtained suggested that cutin, temperature and CaCl2 have influenced the cutinase production significantly with very high confidence levels. Cutinase production was maximum (663 U/mg protein) when using cutin prepared from orange peel as sole source of carbon. An overall 4.33-fold increase in the production of cutinase was observed after optimization of culture conditions (including 2.5-fold increase using RSM) during 24 h of incubation. The production time of Aspergillus sp. RL2Ct cutinase is significantly lower than the most of the earlier reported cutinase-producing fungus.

Structural insights of a hormone sensitive lipase homologue Est22.

Hormone sensitive lipase (HSL) catalyzes the hydrolysis of triacylglycerols into fatty acids and glycerol, thus playing key roles in energy homeostasis. However, the application of HSL serving as a pharmaceutical target and an industrial biocatalyst is largely hampered due to the lack of high-resolution structural information. Here we report biochemical properties and crystal structures of a novel HSL homologue esterase Est22 from a deep-sea metagenomic library. Est22 prefers short acyl chain esters and has a very high activity with substrate p-nitrophenyl butyrate. The crystal structures of wild type and mutated Est22 with its product p-nitrophenol are solved with resolutions ranging from 1.4 Å to 2.43 Å. The Est22 exhibits a α/β-hydrolase fold consisting with a catalytic domain and a substrate-recognizing cap domain. Residues Ser188, Asp287, and His317 comprise the catalytic triad in the catalytic domain. The p-nitrophenol molecule occupies the substrate binding pocket and forms hydrogen bonds with adjacent residues Gly108, Gly109, and Gly189. Est22 exhibits a dimeric form in solution, whereas mutants D287A and H317A change to polymeric form, which totally abolished its enzymatic activities. Our study provides insights into the catalytic mechanism of HSL family esterase and facilitates the understanding for further industrial and biotechnological applications of esterases.

Preparation and characterization of a highly stable phenoxazinone synthase nanogel.

BackgroundPhenoxazinone synthase (PHS) is a laccase-like multicopper oxidase originating from Streptomyces with great industrial application potential. In this paper, we prepared the PHS nanogel retaining 82 % of its initial activity by aqueous in situ polymerization at pH 9.3.ResultsThe average diameter of the PHS nanogel was 50.8 nm based on dynamic light scattering (DLS) analysis. Fluorescence analysis indicated the impressive preservation of the enzyme molecular structure upon modification. The PHS nanogel exhibited the most activity at pH 4.0–4.5 and 50 °C while the corresponding values were pH 4.5 and 40 °C for the native PHS. The Km and Vmax of the PHS nanogel were found to be 0.052 mM and 0.018 mM/min, whereas those of the native PHS were 0.077 mM and 0.021 mM/min, respectively. In addition, the PHS nanogel possessed higher thermal and storage stability and solvent tolerance compared with the native one. The half-life of the PHS nanogel was 1.71 h and multiplied around ninefold compared to 0.19 h for the native one.ConclusionIn summary, the PHS nanogel could be a promising biocatalyst in industry.

High-throughput screening methods for nitrilases.

Nitrilases have been widely acknowledged as important alternatives to chemical catalysts, as they have been proved to transform an immense variety of nitriles under mild conditions and often in a stereoselective or regioselective manner. In the discovery of new nitrilases to establish viable industrial processes, screening plays an important role in identifying which subset of candidates contains a nitrilase of interest from a collection of organisms, clone banks, or enzyme libraries. However, the traditional methods for evaluating the nitrilases are a time-consuming, laborious, and costly process and have been regarded as a bottleneck in developing these nitrilases as industrial biocatalysts. In the past few years, a number of high-throughput screening methods have been developed for rapid evaluation and identification of nitrilases. Here, we review the various methodologies developed for high-throughput screening of nitrilases and focus on their advantages and limitations.

Catalytic Asymmetric Reduction of Difficult-to-Reduce Ketones: Triple-Code Saturation Mutagenesis of an Alcohol Dehydrogenase

Catalytic asymmetric reduction of prochiral ketones with the formation of enantio-pure secondary alcohols is of fundamental importance in organic chemistry, chiral man-made transition-metal catalysts, or organocatalysts and enzymes of the alcohol dehydrogenase (ADH) type. A distinct limitation is the traditional requirement that the α- and α′-moieties flanking the carbonyl function differ sterically and/or electronically. Difficult-to-reduce ketones such as tetrahydrofuran-3-one and tetrahydrothiofuran-3-one and related substrates are particularly challenging, irrespective of the catalyst type. The ADH from Thermoethanolicus brockii (TbSADH) is an attractive industrial biocatalyst, because of its high thermostability, but it also fails in the reduction of such ketones. We have successfully applied directed evolution using the previously developed concept of triple-code saturation mutagenesis at sites lining the TbSADH binding pocket with tetrahydrofuran-3-one serving as the model compound. Highly (R)- and...