Dataset Information

Efficient Phytase Secretion and Phytate Degradation by Recombinant Bifidobacterium longum JCM 1217.

ABSTRACT: Genetic engineering of probiotics, like bifidobacteria, may improve their microbial cell factory economy. This work designed a novel shuttle plasmid pBPES, which bears exogenous appA and is stable within Bifidobacterium longum JCM 1217. Cloning of three predicted promoters into pBPES proved that all of them drive appA expression in B. longum JCM 1217. Transformation of plasmids pBPES-tu and pBPES-groEL into B. longum JCM1217 resulted in much more phytase secretion suggests P tu and P groEL are strong promoters. Further in vitro and in vivo experiments suggested B. longum JCM 1217/pBPES-tu degrades phytate efficiently. In conclusion, the study screened two stronger promoters and constructed a recombinant live probiotic strain for effectively phytase secretion and phytate degradation in gut. The strategy used in the study provided a novel technique for improving the bioaccessibility of phytate and decreasing phosphorus excretion.

SUBMITTER: Sun Z

PROVIDER: S-EPMC6476914 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Efficient Phytase Secretion and Phytate Degradation by Recombinant Bifidobacterium longum JCM 1217.

Sun Zhongke Z Yue Zonghao Z Yang Xingdong X Hao Xinqi X Song Maoping M Li Lili L Chen Can C Chu Cuiwei C Li Chengwei C

Frontiers in microbiology 20190416

Genetic engineering of probiotics, like bifidobacteria, may improve their microbial cell factory economy. This work designed a novel shuttle plasmid pBPES, which bears exogenous appA and is stable within Bifidobacterium longum JCM 1217. Cloning of three predicted promoters into pBPES proved that all of them drive appA expression in B. longum JCM 1217. Transformation of plasmids pBPES-tu and pBPES-groEL into B. longum JCM1217 resulted in much more phytase secretion sug ...[more]

PMID: 31040837

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Project description:This study was conducted to determine effects of high phytase use on growth performance, amino acid (AA) digestibility, intestinal phytate breakdown, and nutrient transporter expression in starter broiler chickens. Male Ross 308 chicks were allocated to 24 pens, at 15 birds/pen and assigned to one of 4 dietary treatments. Treatments were: a control diet (PCa+) that contained adequate levels of calcium (Ca) and phosphorus (P) for growing broiler chicks; a reduced Ca and P diet (PCa-:-1.5 g P/kg and -1.6 g Ca/kg), and 2 additional diets in which phytase was supplemented in the PCa- diet at 1,500 (PCa-Phy1500) and 3,000 (PCa-Phy3000) FTU/kg feed. A common starter diet was fed from day 1 to 8. From day 8 to 22, birds were fed the 4 experimental diets. On day 22, birds were killed for sample collection. From day 8 to 15, average daily gain and average daily feed intake were not different across treatments (P < 0.05) but gain-to-feed ratio (G:F) was reduced (P < 0.006) in the PCa- treatment compared with other treatments. There were no further performance differences, but a tendency of phytase treatments improving the overall G:F (P = 0.079; day 8-22). Up to both the duodenum-jejunum and ileum, phytate, P, and Ca disappearance were increased (P < 0.05) in the PCa-Phy1500 and PCa-Phy3000 treatments compared with PCa- treatment. Phytase dose dependently increased myoinositol (MI) concentration in the digesta from both the duodenum-jejunum and ileum (P < 0.001). The highest concentration of MI was found in the PCa-Phy3000 treatment. Plasma MI concentration was increased by phytase supplementation (P < 0.001). Prececal disappearance of Cys was lower (P < 0.05) in the PCa- treatment than in PCa1and PCa-Phy3000 treatment. Expression of MUC2 in the duodenum-jejunum was higher (P < 0.05) in the PCa-Phy3000 treatment than in other treatments. Phytase-induced hydrolysis of phytate led to elevated digesta and plasma MI concentrations and reduced digesta concentrations of phytate breakdown intermediates.

Project description:BackgroundThere is good evidence for a substantial endogenous phytase activity originating from the epithelial tissue or the microbiota resident in the digestive tract of broiler chickens. However, ionophore coccidiostats, which are frequently used as feed additives in broiler diets to prevent coccidiosis, might affect the bacterial composition and the abundance of phytase producers in the gastrointestinal tract. The aim of the present study was to investigate whether supplementation of a frequently used mixture of the coccidiostats Narasin and Nicarbazin alone or together with a phytase affects microbiota composition of the digestive tract of broiler chickens, characteristics of phytate breakdown in crop and terminal ileum, and precaecal phosphorus and crude protein digestibility.ResultsLarge differences in the microbial composition and diversity were detected between the treatments with and without coccidiostat supplementation. Disappearance of myo-inositol 1,2,3,4,5,6-hexakis(dihydrogen phosphate) (InsP6) in the digestive tract, precaecal P digestibility, inorganic P in blood serum, and the concentration of inositol phosphate isomers in the crop and ileum digesta were significantly affected by phytase supplementation, but not by coccidiostat supplementation. Crude protein digestibility was increased by coccidiostat supplementation when more phosphate was available. Neither microbial abundance and diversity nor any other trait measured at the end of the experiment was affected by coccidiostat when it was only supplemented from day 1 to 10 of age.ConclusionsThe coccidiostats used herein had large effects on overall microbiota composition of the digestive tract. The coccidiostats did not seem to affect endogenous or exogenous phytase activity up to the terminal ileum of broiler chickens. The effects of phytase on growth, phosphorus digestibility, and myo-inositol release were not altered by the presence of the coccidiostats. The effects of phytase and coccidiostats on nutrient digestibility can be of significant relevance for phosphorus and protein-reduced feeding concepts if confirmed in further experiments.

Project description:BackgroundMicrobial phytase is one of the most widely used enzymes in food industries like cattle, poultry, and aquaculture food. Therefore, understanding the kinetic properties of the enzyme is very important to evaluate and predict its behavior in the digestive system of livestock. Working on phytase is one of the most challenging experiments because of some problems, including free inorganic phosphate (FIP) impurity in phytate (substrate) and interference reaction of the reagent with both phosphates (product and phytate impurity).ObjectiveIn the present study, FIP impurity of phytate was removed, and then it was shown that the substrate (phytate) has a dual role in enzyme kinetics: substrate and activator.Material and methodsphytate impurity was decreased by two-step recrystallization prior to the enzyme assay. The impurity removal was estimated by the ISO30024:2009 method and confirmed by Fourier-transform infrared (FTIR) spectroscopy. The kinetic behavior of phytase activity was evaluated using the purified phytate as substrate by non-Michaelis-Menten analysis, including Eadie-Hofstee, Clearance, and Hill plots. The possibility of an allosteric site on phytase was assessed by molecular docking.ResultsThe results showed a 97.2% decrease in FIP due to recrystallization. The phytase saturation curve had a sigmoidal appearance, and Lineweaver-Burk plot with a negative y-intercept indicated the positive homotropic effect of the substrate on the enzyme activity. A right-side concavity of Eadie-Hofstee plot confirmed it. Hill coefficient was calculated to be 2.26. Molecular docking also showed that Escherichia coli phytase molecule has another binding site for phytate very close to the active site, called "allosteric site".ConclusionsThe observations strongly propose the existence of an intrinsic molecular mechanism in Escherichia coli phytase molecules to be promoted for more activity by its substrate, phytate (positive homotropic allosteric effect). In silico analysis showed that phytate binding to the allosteric site caused new substrate-mediated inter-domain interactions, which seems to lead to a more active conformation of phytase. Our results provide a strong basis for animal feed development strategies, especially in the case of poultry food and supplements, regarding a short food passage time in their gastrointestinal tract and variable concentration of phytate along with it. Additionally, the results strengthen our understanding of phytase auto-activation as well as allosteric regulation of monomeric proteins in general.

Dataset Information

Efficient Phytase Secretion and Phytate Degradation by Recombinant Bifidobacterium longum JCM 1217.

Publications

Efficient Phytase Secretion and Phytate Degradation by Recombinant <i>Bifidobacterium longum</i> JCM 1217.

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