The purpose of this study was to construct a recombinant Enterococcus faecalis with constitutive secretory expression of protease Ker and evaluate its application effect by solid state fermentation of soybean meal. In this study, the recombinant plasmid pSIP401-kerhds was constructed and used as the basic framework. The recombinant vector pSIP401Z-s6-kerhds was constructed by the introduction of the constitutive promoter p10 and efficient signal peptide S6. The recombinant E. faecalis was constructed by transferring the recombinant vector pSIP401Z-s6-kerhds into the excellent probiotics E. faecalis EXW27. The extracellular protease activity of the recombinant E. faecalis reached 125.37 U/mL. The optimal reaction temperature of recombinant protease Ker was 40 ℃ and the high relative activity and stability was obtained at pH 5.0~10.0. Meanwhile, protease Ker exhibited good tolerance to bile salt solution. The enzymatic properties of Ker made it applicable to the intestinal environment of animals. Compared with E. faecalis EXW27, the recombinant E. faecalis could degrade the protein of soybean meal more effectively. In this study, the recombinant E. faecalis with the characters of probiotic and high protease activity was constructed. This stain showed its potential as a novel probiotic.
GUO Jianjun
,
ZENG Jing
,
YUAN Lin
,
WEI Guowen
. Construction and application of the recombinant Enterococcus faecalis with constitutive secretion of protease[J]. Food and Fermentation Industries, 2020
, 46(3)
: 14
-21
.
DOI: 10.13995/j.cnki.11-1802/ts.022317
[1] 于书坤. 饲料蛋白酶的最新研究与应用进展[J]. 饲料工业, 2019, 40(10): 22-26.
[2] 毛银, 陆春波, 李国辉,等. 菌酶协同发酵豆粕工艺的优化[J]. 食品与发酵工业, 2019, 45(14): 108-114.
[3] 王雄, 陈清华. 饲用蛋白酶及其在肉鸡日粮中应用的研究进展[J]. 饲料研究, 2016(21): 10-14.
[4] 王勇灵. 饲用蛋白酶在动物营养与饲料生产中的应用[J]. 饲料与畜牧, 2016(1): 33-39.
[5] MUNIR K, MAQSOOD S. A review on role of exogenous enzyme supplementation in poultry production[J]. Emirates Journal of Food and Agriculture, 2013,25(7): 549-554.
[6] CHOWDHURY M A K. Optimizing performance and profit for better sustainability: a review on protease application in aquafeed[J]. Nutricion Acuicola: Investigacion & Desarrollo, 2015: 296-307.
[7] JYOTI V, MODI D R, RAJESH S, et al. Vital role of alkaline protease in bio-industries: a review[J]. Plant Archives, 2011, 11(2): 1 083-1 092.
[8] CHEN H, PARK I, ZHANG S, et al. Effect of supplemental protease on growth performance, nutrient digestibility, and gut health in nursery pigs fed diets with corn or sorghum[J]. Journal of Animal Science, 2017, 95(suppl_4):195-195.
[9] WENK C. Recent advances in animal feed additives such as metabolic modifiers, antimicrobial agents, probiotics, enzymes and highly available minerals-review[J]. Asian-Australasian Journal of Animal Sciences, 2000, 13(1): 86-95.
[10] TORRES-PITARCH A, HERMANS D, MANZANILLA E G, et al. Effect of feed enzymes on digestibility and growth in weaned pigs: a systematic review and meta-analysis[J]. Animal Feed Science and Technology, 2017, 233: 145-159.
[11] GADDE U, KIM W H, OH S T, et al. Alternatives to antibiotics for maximizing growth performance and feed efficiency in poultry: a review[J]. Animal Health Research Reviews, 2017, 18(1): 26-45.
[12] KLAENHAMMER T R. Functional activities of Lactobacillus probiotics: genetic mandate[J]. International Dairy Journal, 1998, 8(5-6): 497-505.
[13] CANDELA M, PERNA F, CARNEVALI P, et al. Interaction of probiotic Lactobacillus and Bifidobacterium strains with human intestinal epithelial cells: adhesion properties, competition against enteropathogens and modulation of IL-8 production[J]. International Journal of Food Microbiology, 2008, 125(3): 286-292.
[14] CUNNINGHAM-RUNDLES S, AHRNÉ S, BENGMARK S, et al. Probiotics and immune response[J]. The American Journal of Gastroenterology, 2000, 95(1): S22-S25.
[15] LAND M H, ROUSTER-STEVENS K, WOODS C R, et al. Lactobacillus sepsis associated with probiotic therapy[J]. Pediatrics, 2005, 115(1): 178-181.
[16] KLAENHAMMER T R. Get Cultured: Eat Bacteria[J]. Annual Review of Food Science and Technology, 2019, 10: 1-20.
[17] PETERBAUER C, MAISCHBERGER T, HALTRICH D. Food-grade gene expression in lactic acid bacteria[J]. Biotechnology Journal, 2011, 6(9):1 147-1 161.
[18] DUONG T, MILLER M J, BARRANGOU R, et al. Construction of vectors for inducible and constitutive gene expression in Lactobacillus[J]. Microbial Biotechnology, 2011, 4(3): 357-367.
[19] MATHIESEN G, SVEEN A, PIARD J C, et al. Heterologous protein secretion by Lactobacillus plantarum using homologous signal peptides[J]. Journal of Applied Microbiology, 2008, 105(1): 215-226.
[20] PENG C, SHI C, CAO X, et al. Factors influencing recombinant protein secretion efficiency in gram-positive bacteria: signal peptide and beyond[J]. Frontiers in Bioengineering and Biotechnology, 2019, 7: 139.
[21] LIU B, ZHANG J, FANG Z, et al. Functional analysis of the C-terminal propeptide of keratinase from Bacillus licheniformis BBE11-1 and its effect on the production of keratinase in Bacillus subtilis[J]. Process Biochemistry, 2014, 49(9): 1 538-1 542.
[22] MUKHERJEE R, CHAKRABORTY R, DUTTA A. Role of fermentation in improving nutritional quality of soybean meal-a review[J]. Asian-Australasian Journal of Animal Sciences, 2016, 29(11): 1523.
[23] GREEN M R, SAMBROOK J. Molecular cloning: a laboratory manual[M]. New York: Cold Spring Harbor Laboratory Press, 2012.
[24] RUD I, JENSEN P R, NATERSTAD K, et al. A synthetic promoter library for constitutive gene expression in Lactobacillus plantarum[J]. Microbiology, 2006, 152(Pt 4):1 011-1 019.
[25] BENACHOUR A, MORIN T, HÉBERT L, et al. Identification of secreted and surface proteins from Enterococcus faecalis[J]. Canadian Journal of Microbiology, 2009, 55(8):967-974.
[26] RUIZ-CRUZ S, SOLANO-COLLADO V, ESPINOSA M, et al. Novel plasmid-based genetic tools for the study of promoters and terminators in Streptococcus pneumoniae and Enterococcus faecalis[J]. Journal of Microbiological Methods, 2010, 83(2): 156-163.
[27] LAEMMLI U K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4[J]. Nature, 1970, 227: 680-685.
[28] CHAUCHEYRAS-DURAND F, DURAND H. Probiotics in animal nutrition and health[J]. Beneficial Microbes, 2009, 1(1): 3-9.