Improving Catalytic Efficiency of L-Arabinose Isomerase from Lactobacillus plantarum CY6 towards D-Galactose by Molecular Modification.

L-Arabinose isomerase (L-AI) has been commonly used as an efficient biocatalyst to produce D-tagatose via the isomerization of D-galactose. However, it remains a significant challenge to efficiently synthesize D-tagatose using the native (wild type) L-AI at an industrial scale. Hence, it is extremely urgent to redesign L-AI to improve its catalytic efficiency towards D-galactose, and herein a structure-based molecular modification of Lactobacillus plantarum CY6 L-AI (LpAI) was performed. Among the engineered LpAI, both F118M and F279I mutants showed an increased D-galactose isomerization activity. Particularly, the specific activity of double mutant F118M/F279I towards D-galactose was increased by 210.1% compared to that of the wild type LpAI (WT). Besides the catalytic activity, the substrate preference of F118M/F279I was also largely changed from L-arabinose to D-galactose. In the enzymatic production of D-tagatose, the yield and conversion ratio of F118M/F279I were increased by 81.2% and 79.6%, respectively, compared to that of WT. Furthermore, the D-tagatose production of whole cells expressing F118M/F279I displayed about 2-fold higher than that of WT cell. These results revealed that the designed site-directed mutagenesis is useful for improving the catalytic efficiency of LpAI towards D-galactose.

The Discovery of Selective Protein Arginine Methyltransferase 5 Inhibitors in the Management of ß-Thalassemia through Computational Methods.

ß-Thalassemia is an inherited genetic disorder associated with ß-globin chain synthesis, which ultimately becomes anemia. Adenosine-2,3-dialdehyde, by inhibiting arginine methyl transferase 5 (PRMT5), can induce fetal hemoglobin (HbF) levels. Hence, the materialization of PRMT5 inhibitors is considered a promising therapy in the management of ß-thalassemia. This study conducted a virtual screening of certain compounds similar to 5'-deoxy-5'methyladenosine (3XV) using the PubChem database. The top 10 compounds were chosen based on the best docking scores, while their interactions with the PRMT5 active site were analyzed. Further, the top two compounds demonstrating the lowest binding energy were subjected to drug-likeness analysis and pharmacokinetic property predictions, followed by molecular dynamics simulation studies. Based on the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) score and molecular interactions, (3R,4S)-2-(6-aminopurin-9-yl)-5-[(4-ethylcyclohexyl)sulfanylmethyl]oxolane-3,4-diol (TOP1) and 2-(6-Aminopurin-9-yl)-5-[(6-aminopurin-9-yl)methylsulfanylmethyl]oxolane-3,4-diol (TOP2) were identified as potential hit compounds, while TOP1 exhibited higher binding affinity and stabler binding capabilities than TOP2 during molecular dynamics simulation (MDS) analysis. Taken together, the outcomes of our study could aid researchers in identifying promising PRMT5 inhibitors. Moreover, further investigations through in vivo and in vitro experiments would unquestionably confirm that this compound could be employed as a therapeutic drug in the management of ß-thalassemia.

D-allose, a typical rare sugar: properties, applications, and biosynthetic advances and challenges.

D-allose, a C-3 epimer of D-glucose and an aldose-ketose isomer of D-allulose, exhibits 80% of sucrose's sweetness while being remarkably low in calories and nontoxic, making it an appealing sucrose substitute. Its diverse physiological functions, particularly potent anticancer and antitumor effects, render it a promising candidate for clinical treatment, garnering sustained attention. However, its limited availability in natural sources and the challenges associated with chemical synthesis necessitate exploring biosynthetic strategies to enhance production. This overview encapsulates recent advancements in D-allose's physicochemical properties, physiological functions, applications, and biosynthesis. It also briefly discusses the crucial role of understanding aldoketose isomerase structure and optimizing its performance in D-allose synthesis. Furthermore, it delves into the challenges and future perspectives in D-allose bioproduction. Early efforts focused on identifying and characterizing enzymes responsible for D-allose production, followed by detailed crystal structure analysis to improve performance through molecular modification. Strategies such as enzyme immobilization and implementing multi-enzyme cascade reactions, utilizing more cost-effective feedstocks, were explored. Despite progress, challenges remain, including the lack of efficient high-throughput screening methods for enzyme modification, the need for food-grade expression systems, the establishment of ordered substrate channels in multi-enzyme cascade reactions, and the development of downstream separation and purification processes.

A comprehensive review of recent advances in the characterization of L-rhamnose isomerase for the biocatalytic production of D-allose from D-allulose.

D-Allose and D-allulose are two important rare natural monosaccharides found in meager amounts. They are considered to be the ideal substitutes for table sugar (sucrose) for, their significantly lower calorie content with around 80 % and 70 % of the sweetness of sucrose, respectively. Additionally, both monosaccharides have gained much attention due to their remarkable physiological properties and excellent health benefits. Nevertheless, D-allose and D-allulose are rare in nature and difficult to produce by chemical methods. Consequently, scientists are exploring bioconversion methods to convert D-allulose into D-allose, with a key enzyme, L-rhamnose isomerase (L-RhIse), playing a remarkable role in this process. This review provides an in-depth analysis of the extractions, physiological functions and applications of D-allose from D-allulose. Specifically, it provides a detailed description of all documented L-RhIse, encompassing their biochemical properties including, pH, temperature, stabilities, half-lives, metal ion dependence, molecular weight, kinetic parameters, specific activities and specificities of the substrates, conversion ratio, crystal structure, catalytic mechanism as well as their wide-ranging applications across diverse fields. So far, L-RhIses have been discovered and characterized experimentally by numerous mesophilic and thermophilic bacteria. Furthermore, the crystal forms of L-RhIses from E. coli and Stutzerimonas/Pseudomonas stutzeri have been previously cracked, together with their catalytic mechanism. However, there is room for further exploration, particularly the molecular modification of L-RhIse for enhancing its catalytic performance and thermostability through the directed evolution or site-directed mutagenesis.

In Silico Molecular Docking and Dynamics Simulation Analysis of Potential Histone Lysine Methyl Transferase Inhibitors for Managing ß-Thalassemia.

A decreased hemoglobin synthesis is contemplated as a pathological indication of ß-thalassemia. Recent studies show that EPZ035544 from Epizyme could induce fetal hemoglobin (HbF) levels due to its proven capability to inhibit euchromatin histone lysine methyl transferase (EHMT2). Therefore, the development of EHMT2 inhibitors is considered promising in managing ß-thalassemia. Our strategy to find novel compounds that are EHMT2 inhibitors relies on the virtual screening of ligands that have a structural similarity to N2-[4-methoxy-3-(2,3,4,7-tetrahydro-1H-azepin-5-yl) phenyl]-N4,6-dimethyl-pyrimidine-2,4-diamine (F80) using the PubChem database. In silico docking studies using Autodock Vina were employed to screen a library of 985 compounds and evaluate their binding ability with EHMT2. The selection of hit compounds was based on the docking score and mode of interaction with the protein. The top two ranked compounds were selected for further investigations, including pharmacokinetic properties analysis and molecular dynamics simulations (MDS). Based on the obtained docking score and interaction analysis, N-(4-methoxy-3-methylphenyl)-4,6-diphenylpyrimidin-2-amine (TP1) and 2-N-[4-methoxy-3-(5-methoxy-3H-indol-2-yl)phenyl]-4-N,6-dimethylpyrimidine-2,4-diamine (TP2) were found to be promising candidates, and TP1 exhibited better stability in the MDS study compared to TP2. In summary, our approach helps identify potential EHMT2 inhibitors, and further validation using in vitro and in vivo experiments could certainly enable this molecule to be used as a therapeutic drug in managing ß-thalassemia disease.

Rewiring Bacillus subtilis and bioprocess optimization for oxidoreductive reaction-mediated biosynthesis of D-tagatose.

D-tagatose holds significant importance as a functional monosaccharide with diverse applications in food, medicine, and other fields. This study aimed to explore the oxidoreductive pathway for D-tagatose production, surpassing the contemporary isomerization-mediated biosynthesis approach in order to enhance the thermodynamic equilibrium of the reactions. Initially, a novel galactitol dehydrogenase was discovered through biochemical and bioinformatics analyses. By co-expressing the galactitol dehydrogenase and xylose reductase, the oxidoreductive pathway for D-tagatose synthesis was successfully established in Bacillus subtilis. Subsequently, pathway fine-tuning was achieved via promoter regulation and dehydrogenase-mediated cofactor regeneration, resulting in 6.75-fold higher D-tagatose compared to that produced by the strain containing the unmodified promoter. Finally, optimization of fermentation conditions and medium composition produced 39.57 g/L D-tagatose in a fed-batch experiment, with a productivity of 0.33 g/L/h and a yield of 0.55 mol/mol D-galactose. These findings highlight the potential of the constructed redox pathway as an effective approach for D-tagatose production.

d-Arabitol Ameliorates Obesity and Metabolic Disorders via the Gut Microbiota-SCFAs-WAT Browning Axis.

d-Arabitol, which is typically found in mushrooms, lichens, and higher fungi, might play an effective role in alleviating visceral fat accumulation and insulin resistance particularly for its low calorie and glycemic index. However, the regulatory mechanisms of d-arabitol for alleviating obesity and associated metabolic disorders remain poorly understood. This study aimed to investigate and analyze the underlying relationship between d-arabitol-mediated gut microbiota and obesity. The results showed that d-arabitol dramatically ameliorated body weight gain, fat accumulation, and insulin resistance in HFD-fed rats. Likewise, d-arabitol remarkably increased the relative abundance of the genera Blautia, Anaerostipes, and Phascolarctobacterium and decreased the genera Romboutsia and Clostridium_sensu_stricto_1. Furthermore, these alterations in gut microflora increased SCFAs, which in turn indirectly promoted AMPK-PGC-1α-related white adipose tissue (WAT) browning. Therefore, d-arabitol would have the potential to alleviate obesity through the gut microbiota-SCFAs-WAT browning axis. It could be considered as a sugar substitute for the obese population and diabetic patients.

High-level production of d-arabitol by Zygosaccharomyces rouxii from glucose: Metabolic engineering and process optimization.

d-Arabitol is a top value-added compound with wide applications in the food, pharmaceutical and biochemical industries. Nevertheless, sustainable biosynthesis of d-arabitol is limited by lack of efficient strains and suitable fermentation process. Herein, metabolic engineering and process optimization were performed in Zygosaccharomyces rouxii to overcoming these limitations. Adopting systems metabolic engineering include enhancement of innate biosynthetic pathway, supply of precursor substrate d-ribulose-5P and cofactors regeneration, a novel recombinant strain ZR-5A with good performance was obtained, which boosted d-arabitol production up to 29.01 g/L, 59.31 % higher than the parent strain. Further with the optimum medium composition and fed-batch fermentation, the strain ZR-5A finally produced 149.10 g/L d-arabitol with the productivity of 1.04 g/L/h, which was the highest titer ever reported by Z.rouxii system. This is the first report on the use of metabolic engineering to construct Z. rouxii chassis for the sustainable production of d-arabitol.

Biocatalytic conversion of a lactose-rich dairy waste into D-tagatose, D-arabitol and galactitol using sequential whole cell and fermentation technologies.

Dairy industry waste has been explored as a cheap and attractive raw material to produce various commercially important rare sugars. In this study, a lactose-rich dairy byproduct, namely cheese whey powder (CWP), was microbially converted into three low caloric sweeteners using whole-cell and fermentation technologies. Firstly, the simultaneous lactose hydrolysis and isomerization of lactose-derived D-galactose into D-tagatose was performed by an engineered Escherichia coli strain co-expressing ß-galactosidase and L-arabinose isomerase, which eventually produced 68.35 g/L D-tagatose during sequential feeding of CWP. Subsequently, the mixed syrup containing lactose-derived D-glucose and residual D-galactose was subjected to fermentation by Metschnikowia pulcherrima E1, which produced 60.12 g/L D-arabitol and 28.26 g/L galactitol. The net titer of the three rare sugars was 156.73 g/L from 300 g/L lactose (equivalent to 428.57 g/L CWP), which was equivalent to 1.12 mol product/mol lactose and 52.24% conversion efficiency in terms of lactose.

Functional and Structural Roles of the Dimer Interface in the Activity and Stability of Clostridium butyricum 1,3-Propanediol Oxidoreductase.

Biosynthesis of 1,3-propanediol (1,3-PD) by 1,3-propanediol oxidoreductase (PDOR) is often limited by the stability issues. To address this issue, the goal of the present study was to engineer the Clostridium butyricum PDOR dimeric interface. The interface exists between the chains and plays a role in the synthesis of 1,3-PD, which is hindered by the increased temperature and pH. Herein, we engineered PDOR by HotSpot Wizard 3.0 and molecular dynamics simulations, improving its thermal stability, pH tolerance, and catalytic properties with respect to the wild-type PDOR activity at 37 °C. Compared to the activity of the wild-type PDOR, the N298C mutant showed 0.5-fold greater activity at pH 8.0, while the P299E mutant showed significantly increased activity of over five fold at pH 4.0. Further structural comparisons between the wild-type and P299E mutant revealed that the extraordinary stability of the P299E mutant could be due to the formation of additional hydrogen bonds and salt bridges. The N298C mutant also exhibits thermal stability at a broad range of temperature at pH 8 with respect to wild-type PDOR and other mutants. The molecular dynamics simulations revealed that stability profiles of P299E mutants at pH 4.0 are attributed to identical root mean square deviation values and stable conformations in the motif region present in the dimer interface of the enzyme. These findings suggest that the dimer interface motifs are essential for the compactness and stability of the PDOR enzyme; therefore, engineering the PDOR using a structure-guided approach could aid in improving its activity and stability under various physiological conditions (pH and temperature).

Enhanced Biosynthesis of D-Arabitol by Metschnikowia reukaufii Through Optimizing Medium Composition and Fermentation Conditions.

D-Arabitol is an important functional sugar alcohol, which can be used in the preparation of foods, chemicals, and medicines. Despite biological production of D-arabitol from low-cost substrates has recently been the focus of research, low yield of this technology has limited its large-scale exploitation. Optimization of this bioprocess could be a promising option to improve the yield of D-arabitol. In this study, one-factor-at-a-time (OFAT) strategy and Box-Behnken design (BBD) were used to increase D-arabitol production by Metschnikowia reukaufii CICC 31,858 through optimizing the fermentation conditions and medium composition. The OFAT optimization provided the optimal conditions for temperature, agitation speed, and fermentation time of 30℃, 220 rpm, and 6 days, respectively. Likewise, the optimum concentrations of peptone, ammonium sulfate, KH2PO4, MgSO4·7H2O, and fumaric acid in the fermentation medium were (g/L) 7.5, 1, 2, 0.5, and 7.5, respectively. Under these optimum conditions, 80.43 g/L of D-arabitol was produced from 200 g/L of glucose, with a productivity of 0.56 g/L/h. The BBD optimization with three important components of fermentation medium (KH2PO4, MgSO4·7H2O, and fumaric acid) showed that the predicted titer of D-arabitol varied from 47.21 to 89.27 g/L, and the actual titer of D-arabitol ranged from 47.36 to 89.83 g/L. The optimum concentrations (g/L) of KH2PO4, MgSO4·7H2O, and fumaric acid in the fermentation medium were found to be 1.0, 0.5, and 4.7 g/L, respectively. Under the optimum conditions, 92.45 g/L of D-arabitol was finally produced with the yield and productivity of 0.46 g/g and 0.64 g/L/h, respectively.

The Characterization of a Novel D-allulose 3-Epimerase from Blautia produca and Its Application in D-allulose Production.

D-allulose is a natural rare sugar with important physiological properties that is used in food, health care items, and even the pharmaceutical industry. In the current study, a novel D-allulose 3-epimerase gene (Bp-DAE) from the probiotic strain Blautia produca was discovered for the production and characterization of an enzyme known as Bp-DAE that can epimerize D-fructose into D-allulose. Bp-DAE was strictly dependent on metals (Mn2+ and Co2+), and the addition of 1 mM of Mn2+ could enhance the half-life of Bp-DAE at 55 °C from 60 to 180 min. It exhibited optimal activity in a pH of 8 and 55 °C, and the Km values of Bp-DAE for the different substrates D-fructose and D-allulose were 235.7 and 150.7 mM, respectively. Bp-DAE was used for the transformation from 500 g/L D-fructose to 150 g/L D-allulose and exhibited a 30% of conversion yield during biotransformation. Furthermore, it was possible to employ the food-grade microbial species Bacillus subtilis for the production of D-allulose using a technique of whole-cell catalysis to circumvent the laborious process of enzyme purification and to obtain a more stable biocatalyst. This method also yields a 30% conversion yield.

Conversion of Agroindustrial Wastes to Rhamnolipid by Enterobacter sp. UJS-RC and Its Role against Biofilm-Forming Foodborne Pathogens.

Rhamnolipid is the main group of biosurfactants predominantly produced by Pseudomonas aeruginosa, a ubiquitous and opportunistic pathogen, which limits its large-scale exploitation. Thus, cost-effective rhamnolipid production from a newly isolated nonpathogenic Enterobacter sp. UJS-RC was investigated. The highest rhamnolipid production (4.4 ± 0.2 g/L) was achieved in a medium constituting agroindustrial wastes (sugarcane molasses and corn steep liquor) as substrates. Rhamnolipid exhibited reduced surface tension to 72-28 mN/m with an emulsification index of 75%. The structural analyses demonstrated the presence of methoxyl, carboxyl, and hydroxyl groups in rhamnolipid. Mass spectra indicated eight rhamnolipid congeners, where dirhamnolipid (m/z 650.01) was the dominant congener. Rhamnolipid inhibited biofilm formation of Staphylococcus aureus in a dose-dependent manner, supported by scanning electron microscopy disclosing the disruption of the microcolony/exopolysaccharide matrix. Rhamnolipid's ability to generate reactive oxygen species has thrown light on the mechanism through which the killing of test bacteria may occur.

Biocatalytic Synthesis of D-Allulose Using Novel D-Tagatose 3-Epimerase From Christensenella minuta.

D-allulose, which is one of the important rare sugars, has gained significant attention in the food and pharmaceutical industries as a potential alternative to sucrose and fructose. Enzymes belonging to the D-tagatose 3-epimerase (DTEase) family can reversibly catalyze the epimerization of D-fructose at the C3 position and convert it into D-allulose by a good number of naturally occurring microorganisms. However, microbial synthesis of D-allulose is still at its immature stage in the industrial arena, mostly due to the preference of slightly acidic conditions for Izumoring reactions. Discovery of novel DTEase that works at acidic conditions is highly preferred for industrial applications. In this study, a novel DTEase, DTE-CM, capable of catalyzing D-fructose into D-allulose was applications. In this study, a novel DTEase, DTE-CM, capable of catalyzing D-fructose into D-allulose was DTE-CM on D-fructose was found to be remarkably influenced and modulated by the type of metal ions (co-factors). The DTE-CM on D-fructose was found to be remarkably influenced and modulated by the type of metal ions (co-factors). The 50°C from 0.5 to 3.5 h at a concentration of 0.1 mM. The enzyme exhibited its maximum catalytic activity on D-fructose at pH 6.0 and 50°C from 0.5 to 3.5 h at a concentration of 0.1 mM. The enzyme exhibited its maximum catalytic activity on -fructose at pH 6.0 and 50°C with a K cat /K m value of 45 mM-1min-1. The 500 g/L D-fructose, which corresponded to 30% conversion rate. With these interesting catalytic properties, this enzyme could be a promising candidate for industrial biocatalytic applications.

Recent developments in Polyhydroxyalkanoates (PHAs) production - A review.

Polyhydroxyalkanoates (PHAs) are inevitably a key biopolymer that has the potential to replace the conventional petrochemical based plastics that pose jeopardy to the environment globally. Even then the reach of PHA in the common market is so restricted. The economy of PHA is such that, even after several attempts the overall production cost seems to be high and this very factor surpasses PHAs usage when compared to the conventional polymers. The major focus of the review relies on the synthesis of PHA from Mixed Microbial Cultures (MMCs), through a 3-stage process most probably utilizing feedstocks from waste streams or models that mimic them. Emphasis was given to the works carried out in the past decade and their coherence with each and every individual criteria (Aeration, Substrate and bioprocess parameters) such that to understand their effect in enhancing the overall production of PHA.

Age-dependent antioxidant responses to the bioconcentration of microcystin-LR in the mysid crustacean, Neomysis awatschensis.

Microcystins (MCs) are naturally occurring algal toxins in the aquatic environment and pose a serious threat to the ecosystem. In general, aquatic populations are structured by organisms of different ages, with varying degrees of biochemical and physiological responses. In this study, juvenile and adult marine mysids (Neomysis awatschensis) were exposed to MC-Leucine Arginine (MC-LR) (0.1, 1, and 10 µg L-1) for 7 days, and the bioconcentration dynamics and responses of antioxidant defense system were measured during the exposure and additional depuration periods (7 days). MC-LR bioconcentrated in a dose-dependent manner, from a threshold concentration of 1 µg L-1 in both stages, and the levels reduced gradually during the depuration phase. Bioconcentration patterns of MC-LR were highly age-specific, as juvenile mysids showed peaks during the exposure period, whereas adults exhibited a peak on the first day of depuration. After exposure to 10 µg L-1 concentration, elevated levels of malondialdehyde (MDA) and glutathione (GSH) were observed during the late (days 5 and 7) exposure and early (days 1 and 3) depuration periods in juvenile mysids, while adult mysids showed a peak on day 7 of the exposure period. Age-specific responses were also observed in the enzymatic activities of glutathione S-transferase (GST), catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), and glutathione reductase (GR). Juvenile mysids showed a significant elevation in all enzymatic activities during the exposure and/or depuration phase upon exposure to 10 µg L-1 MC-LR, but only CAT and SOD enzymes showed significant changes during the exposure and/or depuration periods in adults. Overall, our results indicate the bioconcentration potential of MC-LR and its threshold in the marine mysid, in addition to age-specific MC-LR dynamics and subsequent biochemical responses.

Engineering an FMN-based iLOV protein for the detection of arsenic ions.

Over the past few decades, genetically encoded fluorescent proteins have been widely used as efficient probes to explore and investigate the roles of metal ions in biological processes. The discovery of small FMN-based fluorescent proteins, such as iLOV and FbFP, has enabled researchers to exploit these fluorescent reporter proteins for metal-sensing applications. In this study, we report the inherent binding properties of iLOV towards arsenic ions. The fluorescence quenching of iLOV was linearly related to the concentration of arsenic ions, and engineered proteins showed better sensitivity than the wild-type protein. Engineering key residues around the chromophore converted the iLOV protein into a highly sensitive sensor for As3+ ions. iLOVN468S exhibited an improved binding affinity with a dissociation constant of 1.5 µM. Furthermore, the circular dichroism spectra indicated that the fluorescence quenching mechanism might be related to arsenic-protein complex formation. Thus, the reagentless sensing of arsenic can potentially be exploited to determine intracellular or environmental arsenic using a genetically encoded biosensing approach.

FMN-Based Fluorescent Proteins as Heavy Metal Sensors Against Mercury Ions.

Bacterial light-oxygen-voltage-sensing photoreceptor-derived flavin mononucleotide (FMN)- based fluorescent proteins act as a promising distinct class of fluorescent proteins utilized for various biomedical and biotechnological applications. The key property of its independency towards oxygen for its chromophore maturation has greatly helped this protein to outperform the other fluorescent proteins such as GFP and DsRed for anaerobic applications. Here, we describe the feasibility of FMN-containing fluorescent protein FbFP as a metal-sensing probe by measuring the fluorescence emission changes of a protein with respect to the concentration of metal ions. In the present study, we demonstrated the mercury-sensing ability of FbFP protein and the possible amino acids responsible for metal binding. A ratiometric approach was employed here in order to exploit the fluorescence changes observed at two different emission maxima with respect to Hg(2+) at micromolar concentration. The engineered variant FbFPC56I showed high sensitivity towards Hg(2+) and followed a good linear relationship from 0.1 to 3 µM of Hg(2+). Thus, further engineering with a rational approach would enable the FbFP to be developed as a novel and highly selective and sensitive biosensor for other toxic heavy metal ions as well.

Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications.

The bioprocess engineering with biocatalysts broadly spans its development and actual application of enzymes in an industrial context. Recently, both the use of bioprocess engineering and the development and employment of enzyme engineering techniques have been increasing rapidly. Importantly, engineering techniques that incorporate unnatural amino acids (UAAs) in vivo has begun to produce enzymes with greater stability and altered catalytic properties. Despite the growth of this technique, its potential value in bioprocess applications remains to be fully exploited. In this review, we explore the methodologies involved in UAA incorporation as well as ways to synthesize these UAAs. In addition, we summarize recent efforts to increase the yield of UAA engineered proteins in Escherichia coli and also the application of this tool in enzyme engineering. Furthermore, this protein engineering tool based on the incorporation of UAA can be used to develop immobilized enzymes that are ideal for bioprocess applications. Considering the potential of this tool and by exploiting these engineered enzymes, we expect the field of bioprocess engineering to open up new opportunities for biocatalysis in the near future.

Unnatural amino acid mutagenesis-based enzyme engineering.

Traditional enzyme engineering relies on substituting one amino acid by one of the other 19 natural amino acids to change the functional properties of an enzyme. However, incorporation of unnatural amino acids (UAAs) has been harnessed to engineer efficient enzymes for biocatalysis. Residue-specific and site-specific in vivo incorporation methods are becoming the preferred approach for producing enzymes with altered or improved functions. We describe the contribution of in vivo UAA incorporation methodologies to enzyme engineering as well as the future prospects for the field, including the integration of UAAs with other new advances in enzyme engineering.