Exploration of GH94 Sequence Space for Enzyme Discovery Reveals a Novel Glucosylgalactose Phosphorylase Specificity

The substantial increase in DNA sequencing efforts has led to a rapid expansion of available sequences in glycoside hydrolase families. The ever-increasing sequence space presents considerable opportunities for the search for enzymes with novel functionalities. In this work, the sequence-function space of glycoside hydrolase family 94 (GH94) was explored in detail, using a combined approach of phylogenetic analysis and sequence similarity networks. The identification and experimental screening of unknown clusters led to the discovery of an enzyme from the soil bacterium Paenibacillus polymyxa that acts as a 4-O-β-d -glucosyl-d -galactose phosphorylase (GGalP), a specificity that has not been reported to date. Detailed characterization of GGalP revealed that its kinetic parameters were consistent with those of other known phosphorylases. Furthermore, the enzyme could be used for production of the rare disaccharides 4-O-β-d -glucosyl-d -galactose and 4-O-β-d -glucosyl-l -arabinose. Our current work highlights the power of rational sequence space exploration in the search for novel enzyme specificities, as well as the potential of phosphorylases for rare disaccharide synthesis.     

Hydrophobic Tagged Dihydrofolate Reductase for Creating Misfolded Glycoprotein Mimetics

In the endoplasmic reticulum (ER), nascent glycoproteins that have not acquired the native conformation are either repaired or sorted for degradation by specific quality‐control systems composed by various proteins. Among them, UDP‐glucose:glycoprotein glucosyltransferase (UGGT) serves as a folding sensor in the ER. However, the molecular mechanism of its recognition remains obscure. This study used pseudo‐misfolded glycoproteins, comprising a modified dihydrofolate reductase with artificial pyrene–cysteine moiety on the protein surface (pDHFR) and Man₉GlcNAc₂‐methotrexate (M9‐MTX). All five M9‐MTX/pDHFR complexes, with a pyrene group at different positions, were found to be good substrates of UGGT, irrespective of the site of pyrene modification. These results suggest UGGT's mode of substrate recognition is fuzzy, thus allowing various glycoproteins to be accommodated in the folding cycle.     

Intracellular Delivery of Proteins with Cell-Penetrating Peptides for Therapeutic Uses in Human Disease

Protein therapy exhibits several advantages over small molecule drugs and is increasingly being developed for the treatment of disorders ranging from single enzyme deficiencies to cancer. Cell-penetrating peptides (CPPs), a group of small peptides capable of promoting transport of molecular cargo across the plasma membrane, have become important tools in promoting the cellular uptake of exogenously delivered proteins. Although the molecular mechanisms of uptake are not firmly established, CPPs have been empirically shown to promote uptake of various molecules, including large proteins over 100 kiloDaltons (kDa). Recombinant proteins that include a CPP tag to promote intracellular delivery show promise as therapeutic agents with encouraging success rates in both animal and human trials. This review highlights recent advances in protein-CPP therapy and discusses optimization strategies and potential detrimental effects.     

Bioactive properties of enzymatic hydrolysates from abdominal skin gelatin of yellowfin tuna (Thunnus albacares)

Gelatin (90.6 ± 0.1%) was optimally prepared by response surface methodology from yellowfin tuna (Thunnus albacares, YT) abdominal skin. To investigate bioactive properties of enzymatic hydrolysates from the abdominal skin gelatin (ASG), ASG was hydrolysed with alcalase, protamex, neutrase and flavourzyme as affected by hydrolysis time. Antioxidant, nitrite scavenging and angiotensin‐I converting enzyme (ACE) inhibitory activities of the hydrolysates were determined. Antioxidant activities of the hydrolysates were found through linoleic acid peroxidation inhibitory effects. Alcalase‐derived hydrolysates (AHs) were more effective than others in metal ions chelating, superoxide anion scavenging and hydroxyl radical scavenging activities (P < 0.05). AHs showed significantly stronger nitrite scavenging activities (44.4–60.7%) than others (P < 0.05). Fraction A from AH showed strong ACE inhibitory activity (IC₅₀ of 0.75 mg mL^?1). These results suggest that YT ASG and its enzymatic hydrolysates could be functional food and/or pharmaceutical ingredients with potent antioxidant, anticarcinogenic and antihypertensive benefits.     

Chemical Reactions Directed Peptide Self-Assembly

Fabrication of self-assembled nanostructures is one of the important aspects in nanoscience and nanotechnology. The study of self-assembled soft materials remains an area of interest due to their potential applications in biomedicine. The versatile properties of soft materials can be tuned using a bottom up approach of small molecules. Peptide based self-assembly has significant impact in biology because of its unique features such as biocompatibility, straight peptide chain and the presence of different side chain functionality. These unique features explore peptides in various self-assembly process. In this review, we briefly introduce chemical reaction-mediated peptide self-assembly. Herein, we have emphasised enzymes, native chemical ligation and photochemical reactions in the exploration of peptide self-assembly.     

Flavourzyme酶水解蛋清蛋白的工艺研究

以水解度和蛋白质残留量为指标,研究温度、pH、酶用量及蛋清浓度对Flavourzyme酶解蛋清蛋白的影响,并通过正交试验来确定其最佳工艺。结果是温度55℃、pH6.5、加酶量为质量分数6%、蛋清料液质量体积比1 g:5 m L为最佳工艺参数。在该条件下,6 h酶解物的蛋白质残留率为27.68%,水解度为38.75%,水解物的相对分子质量大部分集中在300以下,即主要以游离氨基酸及二肽形式存在。     

2D Enzyme Cascade Network with Efficient Substrate Channeling by Swinging Arms

In living cells, compartmentalized or membrane‐associated enzymes are often assembled into large networks to cooperatively catalyze cascade reaction pathways essential for cellular metabolism. Here, we report the assembly of an artificial 2D enzyme network of two cascade enzymes—glucose‐6‐phosphate dehydrogenase (G6PDH) and lactate dehydrogenase (LDH)—on a wireframe DNA origami template. Swinging arms were used to facilitate the transport of the redox intermediate of NAD⁺/NADH between enzyme pairs on the array. The assemblies of 2D enzyme networks were characterized by gel electrophoresis and visualized by atomic force microscopy (AFM). The spatial arrangements of multiple enzyme pairs were optimized to facilitate efficient substrate channeling by exploiting the programmability of DNA origami to manipulate the key parameters of swinging arm length and stoichiometry. Compared with a single enzyme pair, the 2D organized enzyme systems exhibited higher reaction efficiency due to the promoted transfer of intermediates within the network.     

Iron-Based Nanozymes in Disease Diagnosis and Treatment

Iron-based nanozymes are currently one of the few clinical inorganic nanoparticles for disease diagnosis and treatment. Overcoming the shortcomings of natural enzymes, such as easy inactivation and low yield, combined with their special nanometer properties and magnetic functions, iron-based nanozymes have broad prospects in biomedicine. This minireview summarizes their preparation, biological activity, catalytic mechanism, and applications in diagnosis and treatment of diseases. Finally, challenges to their future development and the trends of iron-based nanozymes are discussed. The purpose of this minireview is to better understand and reasonably speculate on the rational design of iron-based nanozymes as an increasingly important new paradigm for diagnostics.