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  • Tn5 DNA Library for 1 ng DNA br Acknowledgement We acknowled

    2020-08-04


    Acknowledgement We acknowledge for funding support from Maryada Foundation, a DoD/Army Contract No. W911NF-06-1-0095, and National Institutes of Health through the New England Center of Excellence for Biodefense (Grant AI057159-01).
    Introduction The Gram-negative bacterium Lysobacter sp. XL1 secretes a number of proteins into the environment, including bacteriolytic enzymes (L1–L5) that hydrolyse peptidoglycan, the main structural component of the cell walls in competitive bacteria [1], [2], [3], [4], [5]. The bacteriolytic enzymes of this bacterium are the basis of the antimicrobial preparation of lysoamidase, which possesses a broad range of action and, unlike antibiotics, causes no resistance in pathogenic bacteria [6]. Lysoamidase is approved for external use. Each particular lytic enzyme of the complex can be the basis of new-generation antimicrobial drugs for internal use. This fact determines the significance of multifaceted research into proteins of the complex, including the development of exact methods of their quantitation. The homologous (61.5%) lytic proteases AlpA (L1) and AlpB (L5) have been studied the most. They are the most active enzymes of the complex. AlpA and AlpB are also homologous to the well-investigated α-lytic protease from Lysobacter enzymogenes (78% and 56% homology, respectively) [7]. The enzymes hydrolyse cells of Gram-positive and Gram-negative bacteria and exhibit a protease activity on casein. With respect to staphylococcal peptidoglycan, AlpA has been shown to exhibit an endopeptidase activity, that is, to hydrolyse the peptide bond in the peptidoglycan interpeptide bridge, and an amidase activity, that is, to break down the amide bond between N-acetylmuramic Tn5 DNA Library for 1 ng DNA and the first amino acid of the peptidoglycan peptide subunit [8]. AlpA and AlpB are synthesized as inactive large precursors, preproproteins [7]. The pre-part (signal peptide) provides for the translocation through the cytoplasmic membrane. Secretion of AlpB through the outer membrane from the periplasm proceeds by means of outer-membrane vesicles [9], and AlpA apparently makes use of a type II secretion apparatus the way the α-lytic protease from L. enzymogenes does [10], [11]. Despite the current knowledge on these enzymes, quantitation of complex protein mixtures has been challenging, for example, isolating them from the culture liquid or as part of lysoamidase.
    Materials and methods
    Results and discussion
    Acknowledgement This study was supported by the Russian Foundation for Basic Research (project No. 13-04-00644).
    Introduction Plant species such as papaya and pineapple are important sources of endopeptidases (Salas et al., 2008, Soares et al., 2012), which are important inputs into the food industry. Papain (EC 3.4.22.2), chymopapain (EC 3.4.22.6), glycyl endopeptidase (EC 3.4.22.25) and caricain (EC 3.4.22.30) are among the proteases identified and characterized in papaya, mainly in latex (Azarkan et al., 2003) which have been widely used for meat tenderization, edema treatment and shrink proofing of wool (Braia et al., 2013). In pineapple, stem bromelain (EC 3.4.22.32) and fruit bromelain (EC 3.4.22.33) have been reported, and comosain (EC 3.4.22.31) in a lesser extent (Larocca et al., 2010), which have anti-inflammatory, antithrombotic and fibrinolytic effects (Baez et al., 2007). Bromelain extracted from fruit and other crop residues has also been used for meat tenderization, beverage clarification and in baking (Ketnawa et al., 2010, Ketnawa and Rawdkuen, 2011).