p-Hydroxybenzoate hydroxylase (PHBH) catalyzes the ortho-hydroxylation of 4-hydroxybenzoate (4-HB) to protocatechuate (PCA). PHBHs are commonly known as homodimers, and the prediction of pyridine nucleotide binding and specificity remains an ongoing focus in this field. Therefore, our study aimed to determine the dimerization interface in AspPHBH from Arthrobacter sp. PAMC25564 and identify the canonical pyridine nucleotide-binding residues, along with coenzyme specificity, through site-directed mutagenesis. The results confirm a functional dimeric assembly from a tetramer that appeared in the crystallographic asymmetric unit identical to that established in previous studies. Furthermore, AspPHBH exhibits coenzyme versatility, utilizing both NADH and NADPH, with a preference for NADH. Rational engineering experiments demonstrated that targeted mutations in coenzyme surrounding residues profoundly impact NADPH binding, leading to nearly abrogated enzymatic activity compared to that of NADH. R50, R273, and S166 emerged as significant residues for NAD(P)H binding, having a near-fatal impact on NADPH binding compared to NADH. Likewise, the E44 residue plays a critical role in determining coenzyme specificity. Overall, our findings contribute to the fundamental understanding of the determinants of PHBH\'s active dimeric conformation, coenzyme binding and specificity holding promise for biotechnological advancements.

This study introduces (S)-Opto-prop-2, a second-generation photoswitchable ligand designed for precise modulation of β2-adrenoceptor (β2AR). Synthesised by incorporating an azobenzene moiety with propranolol, (S)-Opto-prop-2 exhibited a high PSScis (photostationary state for cis isomer) percentage (∼90 %) and a favourable half-life (>10 days), facilitating diverse bioassay measurements. In vitro, the cis-isomer displayed substantially higher β2AR binding affinity than the trans-isomer (1000-fold), making (S)-Opto-prop-2 one of the best photoswitchable GPCR (G protein-coupled receptor) ligands reported so far. Molecular docking of (S)-Opto-prop-2 in the X-ray structure of propranolol-bound β2AR followed by site-directed mutagenesis studies, identified D1133.32, N3127.39 and F2896.51 as crucial residues that contribute to ligand-receptor interactions at the molecular level. In vivo efficacy was assessed using a rabbit ocular hypertension model, revealing that the cis isomer mimicked propranolol\'s effects in reducing intraocular pressure, while the trans isomer was inactive. Dynamic optical modulation of β2AR by (S)-Opto-prop-2 was demonstrated in two different cAMP bioassays and using live-cell confocal imaging, indicating reversible and dynamic control of β2AR activity using the new photopharmacology tool. In conclusion, (S)-Opto-prop-2 emerges as a promising photoswitchable ligand for precise and reversible β2AR modulation with light. The new tool shows superior cis-on binding affinity, one of the largest reported differences in affinity (1000-fold) between its two configurations, in vivo efficacy, and dynamic modulation. This study contributes valuable insights into the evolving field of photopharmacology, offering a potential avenue for targeted therapy in β2AR-associated pathologies.

Among the myriad of existing tyrosine kinase receptors, the TAM family-abbreviated from Tyro3, Axl, and Mer tyrosine kinase (MerTK)-has been extensively studied with an outstanding contribution from the team of Prof. Greg Lemke. MerTK activity is implicated in a wide variety of functions involving the elimination of apoptotic cells and has recently been linked to cancers, auto-immune diseases, and atherosclerosis/stroke. In the retina, MerTK is required for the circadian phagocytosis of oxidized photoreceptor outer segments by the retinal-pigment epithelial cells, a function crucial for the long-term maintenance of vision. We previously showed that MerTK ligands carry the opposite role in vitro, with Gas6 inhibiting the internalization of photoreceptor outer segments while Protein S acts conversely. Using site-directed mutagenesis and ligand-stimulated phagocytosis assays on transfected cells, we presently demonstrate, for the first time, that Gas6 and Protein S recognize different amino acids on MerTK Ig-like domains. In addition, MerTK\'s function in retinal-pigment epithelial cells is rhythmic and might thus rely on the respective stoichiometry of both ligands at different times of the day. Accordingly, we show that ligand bioavailability varies during the circadian cycle using RT-qPCR and immunoblots on retinal and retinal-pigment epithelial samples from control and beta5 integrin knockout mice where retinal phagocytosis is arrhythmic. Taken together, our results suggest that Gas6 and Protein S might both contribute to refine the acute regulation of MerTK in time for the daily phagocytic peak.

Aflatoxin B1 (AFB1) contamination is a serious threat to nutritional safety and public health. The CotA-laccase from Bacillus licheniformis ANSB821 previously reported by our laboratory showed great potential to degrade AFB1 without redox mediators. However, the use of this CotA-laccase to remove AFB1 in animal feed is limited because of its low catalytic efficiency and low expression level. In order to make better use of this excellent enzyme to effectively degrade AFB1, twelve mutants of CotA-laccase were constructed by site-directed mutagenesis. Among these mutants, E186A and E186R showed the best degradation ability of AFB1, with degradation ratios of 82.2% and 91.8% within 12 h, which were 1.6- and 1.8-times higher than those of the wild-type CotA-laccase, respectively. The catalytic efficiencies (kcat/Km) of E186A and E186R were found to be 1.8- and 3.2-times higher, respectively, than those of the wild-type CotA-laccase. Then the expression vectors pPICZαA-N-E186A and pPICZαA-N-E186R with an optimized signal peptide were constructed and transformed into Pichia pastoris GS115. The optimized signal peptide improved the secretory expressions of E186A and E186R in P. pastoris GS115. Collectively, the current study provided ideal candidate CotA-laccase mutants for AFB1 detoxification in food and animal feed and a feasible protocol, which was desperately needed for the industrial production of CotA-laccases.

α-arbutin has important applications in cosmetics and medicine. However, the extraction yield from plant tissues is relatively low, which restricts its application value. In this study, we investigated the synthesis of α-arbutin using maltodextrin as the donor and hydroquinone as the acceptor, using a cyclodextrin glucosyltransferase (CGTase) from Anaerobranca gottschalkii. We performed site-saturated and site-directed mutagenesis on AgCGTase. The activity of the variant AgCGTase-F235G-N166H was 3.48 times higher than that of the wild type. Moreover, we achieved a conversion rate of 63% by optimizing the reaction pH, temperature, and hydroquinone addition amount. Overall, this study successfully constructed a strain with improved conversion rate for the synthetic production of α-arbutin and hydroquinone. These findings have significant implications for reducing the industrial production cost of α-arbutin and enhancing the conversion rate of the product.

Protein engineering mechanisms can be an efficient approach to enhance the biochemical properties of various biocatalysts. Immobilization of biocatalysts and the introduction of new-to-nature chemical reactivities are also possible through the same mechanism. Discovering new protocols that enhance the catalytic active protein that possesses novelty in terms of being stable, active, and, stereoselectivity with functions could be identified as essential areas in terms of concurrent bioorganic chemistry (synergistic relationship between organic chemistry and biochemistry in the context of enzyme engineering). However, with our current level of knowledge about protein folding and its correlation with protein conformation and activities, it is almost impossible to design proteins with specific biological and physical properties. Hence, contemporary protein engineering typically involves reprogramming existing enzymes by mutagenesis to generate new phenotypes with desired properties. These processes ensure that limitations of naturally occurring enzymes are not encountered. For example, researchers have engineered cellulases and hemicellulases to withstand harsh conditions encountered during biomass pretreatment, such as high temperatures and acidic environments. By enhancing the activity and robustness of these enzymes, biofuel production becomes more economically viable and environmentally sustainable. Recent trends in enzyme engineering have enabled the development of tailored biocatalysts for pharmaceutical applications. For instance, researchers have engineered enzymes such as cytochrome P450s and amine oxidases to catalyze challenging reactions involved in drug synthesis. In addition to conventional methods, there has been an increasing application of machine learning techniques to identify patterns in data. These patterns are then used to predict protein structures, enhance enzyme solubility, stability, and function, forecast substrate specificity, and assist in rational protein design. In this review, we discussed recent trends in enzyme engineering to optimize the biochemical properties of various biocatalysts. Using examples relevant to biotechnology in engineering enzymes, we try to expatiate the significance of enzyme engineering with how these methods could be applied to optimize the biochemical properties of a naturally occurring enzyme.

In recent years, a convenient phosphatase-coupled sulfotransferase assay method has been proven to be applicable to most sulfotransferases. The central principle of the method is that phosphatase specifically degrades 3\'-phosphoadenosine-5\'-phosphate (PAP) and leaves 3\'-phosphoadenosine-5\'-phosphosulfate (PAPS). Our group previously acquired a yeast 3\',5\'-bisphosphate nucleotidase (YND), which showed a higher catalytic activity for PAP than PAPS and could be a potential phosphatase for the sulfotransferase assay. Here, we obtained a beneficial mutant of YND with markedly improved substrate specificity towards PAP via rational design. Of 9 chosen mutation sites in the active site pocket, the mutation G236D showed the best specificity for PAP. After optimization of the reaction conditions, the mutant YNDG236D displayed a 4.8-fold increase in the catalytic ratio PAP/PAPS compared to the wild-type. We subsequently applied YNDG236D to the assay of human SULT1A1 and SULT1A3 with their known substrate 1-naphthol, indicating that the mutant could be used to evaluate sulfotransferase activity by colorimetry. Analysis of the MD simulation results revealed that the improved substrate specificity of the mutant towards PAP may stem from a more stable protein conformation and the changed flexibility of key residues in the entrance of the substrate tunnel. This research will provide a valuable reference for the development of efficient sulfotransferase activity assays.

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.

BACKGROUND: Recently, new and advanced techniques have been adopted to design and produce nanobodies, which are used in diagnostic and immunotherapy treatments. Traditionally, nanobodies are prepared from camelid immune libraries that require animal treatments. However, such approaches require large library sizes and complicated selection procedures. The current study has employed CDR grafting and site-directed mutagenesis techniques to create genetically engineered nanobodies against the tumor marker CD20 (anti-CD20 nanobodies) used in leukemia treatment.
RESULTS: In this study, we utilized the swapping method to graft CDRs from the VH Rituximab antibody to VHH CDRs. We aimed to enhance the binding affinity of the nanobodies by substituting the amino acids (Y101R-Y102R-Y107R) in the VHH-CDR3. To assess the binding capacity of the mutated nanobodies, we conducted an ELISA test. Moreover, through flow cytometry analysis, we compared the fluorescence intensity of the grafted CD20 and mutant nanobodies with that of the commercially available human anti-CD20 in Raji cells. The results showed a significant difference in the fluorescence intensity of the grafted nanobodies and mutant nanobodies when compared to the commercially available human anti-CD20.
CONCLUSIONS: The approach we followed in this study makes it possible to create multiple anti-CD20 nanobodies with varying affinities without the need for extensive selection efforts. Additionally, our research has demonstrated that computational tools are highly reliable in designing functional nanobodies.

Daily agro-industrial waste, primarily cellulose, lignin, and hemicellulose, poses a significant environmental challenge. Harnessing lignocellulolytic enzymes, particularly endo-1,4-β-xylanases, for efficient saccharification is a cost-effective strategy, transforming biomass into high-value products. This study focuses on the cloning, expression, site-directed mutagenesis, purification, three-dimensional modeling, and characterization of the recombinant endo-1,4-β-xylanase (XlnA) from Aspergillus clavatus in Escherichia coli. This work includes evaluation of the stability at varied NaCl concentrations, determining kinetic constants, and presenting the heterologous expression of XlnAΔ36 using pET22b(+). The expression led to purified enzymes with robust stability across diverse pH levels, exceptional thermostability at 50 °C, and 96-100% relative stability after 24 h in 3.0 M NaCl. Three-dimensional modeling reveals a GH11 architecture with catalytic residues Glu 132 and 22. XlnAΔ36 demonstrates outstanding kinetic parameters compared to other endo-1,4-β-xylanases, indicating its potential for industrial enzymatic cocktails, enhancing saccharification. Moreover, its ability to yield high-value compounds, such as sugars, suggests a promising and ecologically positive alternative for the food and biotechnology industries.