Study of Extraction and Enzymatic Properties of Cell-Envelope Proteinases from a Novel Wild Lactobacillus plantarum LP69

Abstract

Lactobacilli cell-envelope proteinases (CEPs) have been widely used in the development of new streams of blockbuster nutraceuticals because of numerous biopharmaceutical potentials; thus, the development of viable methods for CEP extraction and the improvement of extraction efficiency will promote their full-scale application. In this study, CEP from a novel wild Lactobacillus plantarum LP69 was released from cells by incubating in calcium-free buffer. The extraction conditions of CEP were optimized by response surface methodology with the enzyme activity and specific activity as the detective marker. The optimal extraction conditions were: time of 80 min, temperature of 39 • C and buffer pH of 6.5. Under these conditions, enzyme activity and specific activity were (23.94 ± 0.86) U/mL and (1.37 ± 0.03) U/mg, respectively, which were well matched with the predicted values (22.12 U/mL and 1.36 U/mg). Optimal activity of the crude CEP occurred at pH 8.0 and 40 • C. It is a metallopeptidase, activated by Ca 2+ , inhibited by Zn 2+ and ethylene-diamine-tetra-acetic acid, and a serine proteinase which is inhibited by phenylmethylsulfonyl fluoride. Kinetic studies showed that CEP from LP69 could hydrolyze whey protein, lactoglobulin and casein. Our study improves the extraction efficiency of CEPs from LP69, providing the reference for their industrial development.CEPs have many biotechnological potentials. The development of efficient and commercially feasible methods for CEP extraction and the optimization of the extraction conditions, including pH, conductivity and temperature are therefore essential. Several methods have been confirmed for releasing CEPs from lactobacilli [4][5][6]. The most commonly used approach of extracting CEPs from lactobacilli is by washing the cells with a calcium-free buffer [4,7]. However, this method could not release CEP from some lactobacilli species [8][9][10]. Other methods have also been used to extract cell-surface-associated proteins, such as the use of muramidases, lithium chloride, guanidine hydrochloride, glycine, and urea. A study found that the presence of urea led to many cell surface proteins released from Lactobacillus rhamnosus [6]. Bhowmik et al. [11] also observed that guanidine hydrochloride solution was the most effective extraction agent on Lactobacillus acidophilus, since it could release abundant cell surface proteins. The proteinases from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 were released by the use of lysozyme/mutanolysin treatment with cold temperature shock [12]. Agyei [13] found that some extraction methods did not extract CEPs from certain lactobacillis, which depend on the differences in the localization of CEPs on the bacterial cell-envelope. Therefore, the method for the release of CEP is dependent on the species and/or strain, and no single method is suitable for all lactobacilli.In the past few decades, the proteolytic systems of Lactococcus have been widely studied, especially regarding biochemical and genetic aspects. Unlike the lactococ...

Cell‐envelope proteinases from lactic acid bacteria: Biochemical features and biotechnological applications

“…plantarum , and Lb. sanfrancisco (Chen et al., 2018; Fernández de Palencia, Pelaez, Romero, & Martin‐Hernandez, 1997; Fira et al., 2001; Gobbetti, Smacchi, & Corsetti, 1996; Zhang, Tu, Sadiq, Zhang, & Wang, 2019).…”

“…helveticus CNRZ303, Lb. plantarum LP69, and S. thermophilus CNRZ 385 having optimum pH of 7.0 to 8.0 show an adaptation to weak alkaline conditions (Chen et al., 2018; Fernandez‐Espla et al., 2000; Villegas et al., 2015; Zevaco & Gripon, 1988). Notably, optimal pH for CEP activities also depends on the type of substrate being hydrolyzed.…”

Effect of prebiotics, inorganic salts and amino acids for cell envelope proteinase production from Lactobacillus plantarum LP69 [pdf]

“…Enzyme activity was measured according to a previous study, which based on the Folin method (Chen et al, 2018).…”

“…The production of CEPs from Lactobacilli is affected not only by the medium compositions but also by temperature and pH, and these fermentation parameters may determine the yield and activity of the enzymes produced (Espeche Turbay et al, 2009). Most studies have delineated the isolation, purification and properties of CEP from some lactobacilli species (Chen et al, 2018;Laloi et al, 1991;Scolari et al, 2006). Agyei et al (2012) optimized the batch culture conditions for CEP production from Lactobacillus delbrueckii subsp.…”

Screening of the main nutrients for cell envelope proteinases production by Lactobacillus plantarum LP69 using Plackett-Burman design

“…At present, many studies have described that L. plantarum has multiple physiological characters including immune adjustment and gastrointestinal disease regulation, and thus have a widespread use in food, feed, medical care and other fields (Wu et al, 2019). Previously, our laboratory had elucidated that L. plantarum LP69 had great potential to produce angiotensin-I-converting enzyme (ACE, peptidyldipeptide hydrolase)-inhibitory peptides during goat milk fermentation (Chen et al, 2018a), and also CEPs (Chen et al, 2018b). In previously work, our lab screened substances that have important impact on CEP production of L. plantarum LP69 from carbon sources, nitrogen sources, prebiotics, inorganic salts, amino acids, and determined the optimal concentration of these substances by single-factor experiments.…”