Perkinsburnett6184

47 μM, which was an increase of approximately 21.4 %, and the yield of Ala-Gln was approximately 2.59 mM-1 L-1 mg-1. Collectively, these findings suggest the potential practical application of this method in the rational design of Lals for increased catalytic performance.Inspired by the cascades performed in vivo, the assembly of multiple enzymes in vitro has strongly moved into the focus of researchers in the field of biocatalysis. In this study, a new, mild and accurate enzyme cross-linking method is revealed. Microbial transglutaminase (MTG) acts as a "cross-linking medium" by identifying the amide group of the glutamine and the primary amine group of lysine in the artificial peptide tags specifically to form an iso-peptide bond. Here, carbonic anhydrase (CA) and formate dehydrogenase (FDH) with different peptide tags that can be recognized by MTG were linked together to obtain different proportions of cross-linked enzymes for efficient conversion of greenhouse gas carbon dioxide to formic acid. After cross-linking, we obtained "one-to-one" and "one-to-more" cross-linked enzyme aggregates. There is a minor residual loss of the two enzymes, the remaining enzyme activity of CA is more than 93%, and the remaining enzyme activity of FDH is more than 84%. In particular, the overall catalytic efficiency of the cross-linked enzyme is increased by 5.8 times compared with free enzymes and the thermal stability of FDH at different temperatures is improved. The applied strategy demonstrates the potential application of MTG in multi-enzyme assembly and synthetic biology.Lactobacillus acidophilus is an important probiotic. The β-glucosidase produced by L. acidophilus GIM1.208 can transform quercetin glycosides of Rosa roxburghii Tratt to release quercetin and improve the functional activity of raw materials. Understanding the interaction and the characteristics of the two will lay a theoretical foundation for the site-directed transformation and functional application of the catalytic active site of enzymes. In our study, using the heterologously expressed and highly stable, purified L. acidophilus GIM1.208 BGL as the strain, the representative quercetin in β-glucosidase and Rosa roxburghii Tratt was preliminarily predicted and explored using ultraviolet-visible absorption spectroscopy. Lipofermata in vivo Fluorescence spectroscopy combined with molecular docking was used to determine the interaction characteristics of the glycoside substrates, rutin (Rut) and isoquercitrin (Iso). Results from molecular docking showed that Asp159, Arg56, Iso294, Phe292, and Gly25 were the main residues of β-glucosidase and Rut. Arg56 was found to be the most crucial residue of β-glucosidase and isoquercitrin; the interaction between Rut and Iso and β-glucosidase was mainly driven by hydrogen bonding. The combined free energy of β-glucosidase and Iso was found to be -182.10 kcal/mol, while that of β-glucosidase and Rut was -32.37 kcal/mol. The results of fluorescence spectroscopy showed that the fluorescence intensity of β-glucosidase decreased with an increase in Rut and Iso concentrations. This interaction made β-glucosidase quench endogenous fluorescence, which was static quenching. The binding constants of Rut and Iso with β-glucosidase were determined to be 0.50×107 and 0.31×107 L/mol, respectively, indicating that rutin had a stronger affinity when interacting with β-glucosidase. These findings were consistent with our prediction results determined using molecular docking studies.LinB is an important haloalkane dehalogenase involved in the degradation pathway of different isomers of hexachlorocyclohexane (HCH), mainly in catalyzing degradation of the notorious β-HCH. The HCH isomers are known to have neurotoxic, carcinogenic and estrogenic effects. Enzymatic bioremediation for decontamination of β- as well as other HCH isomers can prove to be a potential remediation strategy. For any bioremediation technology that is to be developed, apart from having high turnover number, the candidate enzyme must also be available in sufficient amounts. In this direction, the LinB variants reported in database were tested in laboratory studies. The variant LinBSSO4-3 however could not be obtained in soluble fraction by using standard procedures. The protein LinBSSO4-3 was cloned in pDEST17 vector and codon optimized for better expression in Escherichia coli BL21AI using a strong T7 promoter. However, the over-expression of this protein in ectopic host E. coli, led to aggregation of the protein in fo development for organochlorines, specifically HCH. Such a protocol for refolding of haloalkane dehalogenases from inclusion bodies has not been developed or reported before.Hyaluronidases are low expressed toxins of brown spider venoms, but, as highly active molecules, they present an important role as spreading factors. By degrading extracellular matrix components, these enzymes favor the diffusion of toxins in the affected tissue and at systemic level. Here, a novel isoform of hyaluronidase of Loxosceles intermedia Mello-Leitão (1934) venom was cloned, expressed in a baculovirus-insect cell expression system and fully active purified. This recombinant enzyme, named LiHyal2 (Loxosceles intermedia Hyaluronidase isoform 2), shares high identity with hyaluronidases of other spiders and scorpions. The catalytic and sugar binding amino acid residues are conserved in LiHyal2, human, and honeybee venom hyaluronidases and the molecular model of LiHyal2 shares major similarities with their crystal structures, including the active site. LiHyal2 was expressed as a 45 kDa protein and degraded hyaluronic acid (HA) and chondroitin sulphate as demonstrated by HA zymography and agarose gel electrophoresis. Lectin blot analysis revealed that LiHyal2 is post-translationally modified by the addition of high mannose N-linked carbohydrates. In vivo experiments showed that LiHyal2 potentialize dermonecrosis and edema induced by a recombinant phospholipase-D (PLD) of L. intermedia venom, as well as enhance the increase in capillary permeability triggered by this PLD, indicating that these toxins act synergistically during envenomation. Altogether, these results introduce a novel approach to express spider recombinant toxins, contribute to the elucidation of brown spider venom mechanisms and add to the development of a more specific treatment of envenomation victims.