br Acknowledgement We acknowledge for funding support from M
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 , , , , . 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 . 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) . 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 mibefradil and the first amino acid of the peptidoglycan peptide subunit . AlpA and AlpB are synthesized as inactive large precursors, preproproteins . 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 , and AlpA apparently makes use of a type II secretion apparatus the way the α-lytic protease from L. enzymogenes does , . 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 22.214.171.124), chymopapain (EC 126.96.36.199), glycyl endopeptidase (EC 188.8.131.52) and caricain (EC 184.108.40.206) 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 220.127.116.11) and fruit bromelain (EC 18.104.22.168) have been reported, and comosain (EC 22.214.171.124) 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). The production of papaya and pineapple around the world, is characterized by generating a volume of waste close to 25% for papaya (seeds and peels) and 50% for pineapple (Ketnawa et al., 2012, Ordoñez et al., 2016), not counting the harvest surpluses and the rejected fruits (green and ripe). In Colombia both species are cultivated, despite not being native fruits. “Honey gold” pineapple is grown in the Orinoquía, Pacific and Amazon regions, with a total production of 619.048 tons in 2015, with the main producers being Santander and Valle del Cauca (MADR, 2017a). The production of papaya in 2015 was 176.226 Ton (MADR, 2017b), representing 1% of world production (Dane, 2016). Although it is a low figure, the annual increase in cultivated hectares, as well as the increase in demand, has led to an increase in the generation of waste. In the case of pineapple, the waste generated by the fruits and juice processing industries are mainly peel, core, stem, crown and leaves. In recent years, interest in agro-industrial waste has increased, as these generate economic and environmental problems for the different production chains. In this sense, the integral management of these can be applied to the production of biofuels and active compounds with high added value, contributing not only to the development of sustainable businesses but also to those cataloged under the concept of bio refinery (Gopinath et al., 2016).