Protein Engineering of Substrate Specificity toward Nitrilases: Strategies and Challenges.

Nitrilase is extensively applied across diverse sectors owing to its unique catalytic properties. Nevertheless, in industrial production, nitrilases often face issues such as low catalytic efficiency, limited substrate range, suboptimal selectivity, and side reaction products, which have garnered heightened attention. With the widespread recognition that the structure of enzymes has a direct impact on their catalytic properties, an increasing number of researchers are beginning to optimize the functional characteristics of nitrilases by modifying their structures, in order to meet specific industrial or biotechnology application needs.

Regulatory significance of terminator: A systematic approach for dissecting terminator-mediated enhancement of upstream mRNA stability.

The primary function of terminators is to terminate transcription in gene expression. Although some studies have suggested that terminators also contribute positively to upstream gene expression, the extent and underlying mechanism of this effect remain largely unexplored. Here, the correlation between terminating strength and upstream mRNA stability was investigated by constructing a terminator mutation library through randomizing 5 nucleotides, assisted by FlowSeq technology, terminator variants were categorized based on the downstream fluorescence intensity, followed by high-throughput sequencing.

A comprehensive review on microbial hyaluronan-degrading enzymes: from virulence factors to biotechnological tools.

Hyaluronan (HA), a natural high molecular weight polysaccharide, has extensive applications in cosmetology and medical treatment. Hyaluronan-degrading enzymes (Hyals) act as molecular scissors that cleave HA by breaking the glucosidic linkage. Hyals are present in diverse organisms, including vertebrates, invertebrates and microorganisms, and play momentous roles in biological processes.

Unlocking Green Biomanufacturing Potential: Superior Heterologous Gene Expression with a T7 Integration Overexpression System in .

Industrial biotechnology employs cells for producing valuable products and serving as biocatalysts sustainably, addressing resource, energy, and environmental issues. is a preferred host for creating microbial chassis cells and producing industrial enzymes and functional nutritional products. In this study, a dual-module T7 integration expression system in was established.

Protein Engineering of Nicotinamide Riboside Kinase Based on a Combinatorial Semirational Design Strategy for Efficient Biocatalytic Synthesis of Nicotinamide Mononucleotides.

Industrial biosynthesis of β-nicotinamide mononucleotide (β-NMN) lacks a highly active nicotinamide riboside kinase for the phosphorylation process. Cumbersome preprocessing steps and excessive ATP addition contribute to its increased cost. To tackle these challenges, a docking combination simulation (DCS) semirational mutagenesis strategy was designed in this study, combining various modification strategies to obtain a mutant NRK-TRA with 2.

Protective effect of ferulic acid-hyaluronic acid copolymer against UVB irradiation in a human HaCaT cell line.

Excessive UVB exposure increased the production of reactive oxygen species (ROS), leading to oxidative damage and epidermal inflammation. To enhance UVB protection effect, a strong phenolic antioxidant, ferulic acid (FA) was designed onto HA via a free radical mediated method. Our previous work has confirmed its structural characterization and in vitro antioxidant.

Multilevel Systematic Optimization To Achieve Efficient Integrated Expression of .

Genomic integration of heterologous genes is the preferred approach in industrial fermentation-related strains due to the drawbacks associated with plasmid-mediated microbial fermentation, including additional growth burden, genetic instability, and antibiotic contamination. Synthetic biology and genome editing advancements have made gene integration convenient. Integrated expression is extensively used in the field of biomanufacturing and is anticipated to become the prevailing method for expressing recombinant proteins.

Unraveling the intricacies of glycosaminoglycan biosynthesis: Decoding the molecular symphony in understanding complex polysaccharide assembly.

Glycosaminoglycans (GAGs) are extensively utilized in clinical, cosmetic, and healthcare field, as well as in the treatment of thrombosis, osteoarthritis, rheumatism, and cancer. The biological production of GAGs is a strategy that has garnered significant attention due to its numerous advantages over traditional preparation methods. In this review, we embark on a journey to decode the intricate molecular symphony that orchestrates the biosynthesis of glycosaminoglycans.

Engineering protein translocation and unfolded protein response enhanced human PH-20 secretion in Pichia pastoris.

Hyaluronidases catalyze the degradation of hyaluronan (HA), which is finding rising applications in medicine, cosmetic, and food industries. Recombinant expression of hyaluronidases in microbial hosts has been given special attention as a sustainable way to substitute animal tissue-derived hyaluronidases. In this study, we focused on optimizing the secretion of hyaluronidase from Homo sapiens in Pichia pastoris by secretion pathway engineering.

Technology and functional insights into the nicotinamide mononucleotide for human health.

Nicotinamide mononucleotide (NMN), a naturally occurring biologically active nucleotide, mainly functions via mediating the biosynthesis of NAD. In recent years, its excellent pharmacological activities including anti-aging, treating neurodegenerative diseases, and protecting the heart have attracted increasing attention from scholars and entrepreneurs for production of a wide range of formulations, including functional food ingredients, health care products, active pharmaceuticals, and pharmaceutical intermediates. Presently, the synthesis methods of NMN mainly include two categories: chemical synthesis and biosynthesis.

Improving the soluble expression of difficult-to-express proteins in prokaryotic expression system via protein engineering and synthetic biology strategies.

Solubility and folding stability are key concerns for difficult-to-express proteins (DEPs) restricted by amino acid sequences and superarchitecture, resolved by the precise distribution of amino acids and molecular interactions as well as the assistance of the expression system. Therefore, an increasing number of tools are available to achieve efficient expression of DEPs, including directed evolution, solubilization partners, chaperones, and affluent expression hosts, among others. Furthermore, genome editing tools, such as transposons and CRISPR Cas9/dCas9, have been developed and expanded to construct engineered expression hosts capable of efficient expression ability of soluble proteins.

Geometric Remodeling of Nitrilase Active Pocket Based on ALF-Scanning Strategy To Enhance Aromatic Nitrile Substrate Preference and Catalytic Efficiency.

Nitrilase can catalyze nitrile compounds to generate corresponding carboxylic acids. Nitrilases as promiscuous enzymes can catalyze a variety of nitrile substrates, such as aliphatic nitriles, aromatic nitriles, etc. However, researchers tend to prefer enzymes with high substrate specificity and high catalytic efficiency.

Protein engineering of NADH pyrophosphatase for efficient biocatalytic production of reduced nicotinamide mononucleotide.

NADH pyrophosphatase, a hydrolase catalyzing the phosphate bond of NADH to reduced nicotinamide mononucleotide, has potential applications in the food, cosmetic and pharmaceutical industry. Here, we investigated the effects of vector screening, promoter and RBS strategies on NADH pyrophosphatase expression and protein engineering on its enzymatic activity and thermal stability. In this study, we describe a NADH pyrophosphatase derived from ().

Efficient secretory expression of phospholipase D for the high-yield production of phosphatidylserine and phospholipid derivates from soybean lecithin.

Phospholipase D (PLD) is an essential biocatalyst for the biological production of phosphatidylserine and phospholipid modification. However, the efficient heterologous expression of PLD is limited by its cell toxicity. In this study, a PLD was secretory expressed efficiently in with an activity around 100 U/mL.

High-level expression and characterization of a highly active hyaluronate lyase HylC with significant potential in hyaluronan oligosaccharide preparation.

Hyaluronate lyases (HA lyases) have been proved to distribute widely among microorganisms, with large potential in hyaluronan processing. Here, a highly active HA lyase HylC from Citrobacter freundii strain Cf1 is reported. HylC was expressed in Escherichia coli BL21(DE3) under the regulation of T7 promoter, and purified to electrophoretic homogeneity for enzymatic characterization, which suggested its suitable thermo- and pH stability under 45 °C and pH rang of 4-8, and high halotolerancy in 1.

Development of a Growth-Dependent System to Regulate Cell Growth and Keratinase Production in .

Keratinases specifically degrade insoluble keratin waste, thus contributing to environmental protection and sustainable biomass development. However, their industrial application is hindered by inefficient enzyme production and poor biomass generation. In this study, the heterologous expression of keratinase was found to have cytotoxicity and might block host cell growth due to its proteolytic property.

High-efficiency secretory expression and characterization of the recombinant type III human-like collagen in Pichia pastoris.

Collagen, the highest content protein in the body, has irreplaceable biological functions, and it is widespread concerned in food, beauty, and medicine with great market demand. The gene encoding the recombinant type III human-like collagen α1 chain fragment was integrated into P. pastoris genome after partial amino acids were substituted.