enzyme engineering

Molybdenum- or tungsten-dependent formate dehydrogenases have emerged as significant catalysts for the chemical reduction of CO2 to formate, with biotechnological applications envisaged in climate-change mitigation. The role of Met405 in the active site of Desulfovibrio vulgaris formate dehydrogenase AB (DvFdhAB) has remained elusive. However, its proximity to the metal site and the conformational change that it undergoes between the resting and active forms suggests a functional role. In this work, the M405S variant was engineered, which allowed the active-site geometry in the absence of methionine Sδ interactions with the metal site to be revealed and the role of Met405 in catalysis to be probed...

Along with an ever-deepening understanding of the catalytic principle of natural enzymes, the rational design of high-activity biomimetic nanozymes has become a hot topic in current research. Inspired by the active centers of natural enzymes consisting of catalytic sites and binding pockets, a Cu-doped CoS2 hollow nanocube (Cu/CoS2 HNCs) nanozyme integrating substitution defects and vacancies is developed through a defect engineering strategy. It is shown that the vacancies and substitution defects in the developed Cu/CoS2 HNC nanozymes serve as binding pockets and catalytic sites, respectively...

Engineered nanoparticles (ENPs) have been increasingly used in agricultural operations, leading to an urgent need for robust methods to analyze co-occurring ENPs in plant tissues. In response, this study advanced the simultaneous extraction of coexisting silver, cerium oxide, and copper oxide ENPs in lettuce shoots and roots using macerozyme R-10 and analyzed them by single-particle inductively coupled plasma-mass spectrometry (ICP-MS). Additionally, the standard stock suspensions of the ENPs were stabilized with citrate, and the long-term stability (up to 5 months) was examined for the first time...

Spermidine is a naturally occurring polyamine widely utilized in the prevention and treatment of various diseases. Current spermidine biosynthetic methods have problems such as low efficiency and complex multi-enzyme catalysis. Based on sequence-structure-function relationships, we engineered the widely studied homospermidine synthase from Blastochloris viridis ( Bv HSS) and obtained mutants that could catalyze the production of spermidine from 1,3-diaminopropane and putrescine. The specific activities of Bv HSS and the mutants D361E and E232D + D361E (E232D-D) were 8...

Sequencing of biomacromolecules is a crucial cornerstone in life sciences. Glycans, one of the fundamental biomolecules, derive their physiological and pathological functions from their structures. Glycan sequencing faces challenges due to its structural complexity and current detection technology limitations. As a highly sensitive sensor, nanopores can directly convert nucleic acid sequence information into electrical signals, spearheading the revolution of third-generation nucleic acid sequencing technologies...

Bioluminescent indicators are power tools for studying dynamic biological processes. In this study, we present the generation of novel bioluminescent indicators by modifying the luciferin molecule with an analyte-binding moiety. Specifically, we have successfully developed the first bioluminescent indicator for potassium ions (K+ ), which are critical electrolytes in biological systems. Our approach involved the design and synthesis of a K+ -binding luciferin named potassiorin. Additionally, we engineered a luciferase enzyme called BRIPO (bioluminescent red indicator for potassium) to work synergistically with potassiorin, resulting in optimized K+ -dependent bioluminescence responses...

Uric acid is the end product of purine metabolism in humans due to inactivation of the uricase determined by the mutated uricase gene. Uricase catalyzes the conversion of uric acid into water-soluble allantoin that is easily excreted by the kidneys. Hyperuricemia occurs when the serum concentration of uric acid exceeds its solubility (7 mg/dl). However, modifications to improve the uricase activity is under development for treating the hyperuricemia. Here we designed 7 types of human-porcine chimeric uricase by multiple sequence comparisons and targeted mutagenesis...

D-allulose, a highly desirable sugar substitute, is primarily produced using the D-allulose 3-epimerase (DAE). However, the availability of usable DAE enzymes is limited. In this study, we discovered and engineered a novel DAE Rum55, derived from a human gut bacterium Ruminococcus sp. CAG55. The activity of Rum55 was strictly dependent on the presence of Co2+ , and it exhibited an equilibrium conversion rate of 30.6 % and a half-life of 4.5 h at 50 °C. To enhance its performance, we engineered the interface interaction of Rum55 to stabilize its tetramer structure, and the best variant E268R was then attached with a self-assembling peptide to form active enzyme aggregates as carrier-free immobilization...

Rieske non-heme iron oxygenases (ROs) are redox enzymes that are essential for microbial biodegradation and natural product synthesis. These enzymes utilize molecular oxygen for oxygenation reactions, making them very useful in applied enzymology due to their broad reaction scope and high selectivities. The mechanism of oxygen activation in ROs involves electron transfers between redox centers of associated protein components, forming an electron transfer chain (ETC). Although the ETC is essential for electron replenishment, it carries the risk of reactive oxygen species (ROS) formation due to electron loss during oxygen activation...

Uricase (EC 1.7.3.3) is an oxidoreductase enzyme which is widely exploited for diagnostic and treatment purposes in medicine. This study has been focused on producing recombinant uricase from E. coli BL21 in a bubble column bioreactor (BCB) and finding the optimal conditions for maximum uricase activity. The three most effective variables on uricase activity were selected through Plackett-Burman design from 8 different variables and were further optimized by central composite design of RSM. The selected variables included the inoculum size (%v/v), IPTG (Isopropyl β-d-1-thiogalactopyranoside) concentration (mM) and the initial pH of culture medium...

The yeast endoplasmic reticulum sequestration and screening (YESS) system is a generalizable platform that has become highly useful to investigate post-translational modification enzymes (PTM-enzymes). This system enables researchers to profile and engineer the activity and substrate specificity of PTM-enzymes and to discover inhibitor-resistant enzyme mutants. In this study, we expand the capabilities of YESS by transferring its functional components to integrative plasmids. The YESS integrative system yields uniform protein expression and protease activities in various configurations, allows one to integrate activity reporters at two independent loci and to split the system between integrative and centromeric plasmids...

Klebsiella oxytoca JCM 1665 was subjected to extracellular rhodanese production using a submerged fermentation technique. The organism was further engineered for higher cyanide tolerance and rhodanese yield using ethylmethanesulfonate as a mutagen. Mutagenesis resulted in an improved mutant with high cyanide tolerance (100 mM) and rhodanese yield (26.7 ± 0.67 U/mL). This yield was 4.34-fold higher than the wild strain (6.15 ± 0.65 U/mL). At temperatures ranging from 30 to 80 °C, the first-order thermal denaturation constant ( Kd ) for free enzyme increases from 0...

The microtubule-associated Tau protein is found in the central nervous system (CNS) in six major isoforms. Neurodegenerative diseases have been linked to post-translational changes of Tau, most notably phosphorylation. Tau protein's molecular diversity is highly helpful in the identification of neurodegenerative illnesses. Nonetheless, one major obstacle to the early detection of brain illness is the nanoscale identification of tau proteins. The standard methods for identifying tau protein include western blotting, polymerase chain reaction (PCR), and real-time PCR...

Salt stress is one of the major factors limiting plant growth and productivity. Many studies have shown that serine hydroxymethyltransferase (SHMT) gene play an important role in growth, development and stress response in plants. However, to date, there have been few studies on whether SHMT3 can enhance salt tolerance in plants. Therefore, the effects of overexpression or silencing of CsSHMT3 gene on cucumber seedling growth under salt stress were investigated in this study. The results showed that overexpression of CsSHMT3 gene in cucumber seedlings resulted in a significant increase in chlorophyll content, photosynthetic rate and proline (Pro) content, and antioxidant enzyme activity under salt stress condition; whereas the content of malondialdehyde (MDA), superoxide anion (H2 O2 ), hydrogen peroxide (O2 ·- ) and relative conductivity were significantly decreased when CsSHMT3 gene was overexpressed...

Enzymatic cascades have become a green and sustainable approach for the synthesis of valuable chemicals and pharmaceuticals. Using sequential enzymes to construct a multienzyme complex is an effective way to enhance the overall performance of biosynthetic routes. Here we report the design of an efficient in vitro hybrid biocatalytic system by assembling three enzymes that can convert styrene to ( S )-1-phenyl-1,2-ethanediol. Specifically, we prepared the three enzymes in different ways, which were cell surface-displayed, purified, and cell-free expressed...

Antimicrobial resistance poses a significant global threat, reaching dangerously high levels as reported by the World Health Organization. The emergence and rapid spread of new resistance mechanisms, coupled with the absence of effective treatments in recent decades, have led to thousands of deaths annually from infections caused by drug-resistant microorganisms. Consequently, there is an urgent need for the development of new compounds capable of combating antibiotic-resistant bacteria. A promising class of molecules exhibiting potent bactericidal effects is peptidoglycan hydrolases...

The 3-ketoacyl-CoA thiolase is the rate-limiting enzyme for linear dicarboxylic acids production. However, the promiscuous substrate specificity and suboptimal catalytic performance have restricted its application. Here we present both biochemical and structural analyses of a high-efficiency 3-ketoacyl-CoA thiolase Tfu_0875. Notably, Tfu_0875 displayed heightened activity and substrate specificity for succinyl-CoA, a key precursor in adipic acid production. To enhance its performance, a deep learning approach (DLKcat) was employed to identify effective mutants, and a computational strategy, known as the greedy accumulated strategy for protein engineering (GRAPE), was used to accumulate these effective mutants...

INTRODUCTION: Administration of adipose-derived stem cells (ADSCs) into the joint cavity has been shown to alleviate the symptoms of knee osteoarthritis (OA) by releasing exosomes and anti-inflammatory cytokines. However, the therapeutic effect of these cells is limited by their rapid disappearance after administration. Thus, it is necessary to prolong cell survival in the joint cavity. This study aimed to investigate the potential application of ADSCs adhered to atelocollagen microspheres (AMSs) for cell therapy of knee OA...

Photobiocatalysis-where light is used to expand the reactivity of an enzyme-has recently emerged as a powerful strategy to develop chemistries that are new to nature. These systems have shown potential in asymmetric radical reactions that have long eluded small-molecule catalysts1 . So far, unnatural photobiocatalytic reactions are limited to overall reductive and redox-neutral processes2-9 . Here we report photobiocatalytic asymmetric sp3 -sp3 oxidative cross-coupling between organoboron reagents and amino acids...

Filamentous fungi are important cell factories for the production of high-value enzymes and chemicals for the food, chemical, and pharmaceutical industries. Under submerged fermentation, filamentous fungi exhibit diverse fungal morphologies that are influenced by environmental factors, which in turn affect the rheological properties and mass transfer of the fermentation system, and ultimately the synthesis of products. In this review, we first summarize the mechanisms of mycelial morphogenesis and then provide an overview of current developments in methods and strategies for morphological regulation, including physicochemical and metabolic engineering approaches...