In Vitro Stability of Phytase from Recombinant Bacteria E. Coli BL21 (DE3) EAS1-AMP

The objective of the research was to inquire the Km, Vm, activity, intracellular phytase stability exposed to pH variation, temperature variation and protease (pepsin and pancreas) in vitro. The phytase was produced from recombinant bacteria E. coli BL21(DE3) EAS1-AMP using 1.5 mM IPTG as inducer. Intracellular enzyme was extracted via freeze shock and centrifugation. Pure enzyme was acquired through NI-NTA agarose column. The enzyme was then tested for Km, Vm, phytase activity and stability against pH, temperature and protease. Treatment levels for stability against protease were P0: without protease, P1: addition of pepsin, P2: addition of pepsin and pancreas, and the data were statistically analyzed using analysis of variance of one-way Completely Randomized Design. Crude intracellular phytase had Vm 6.39 υM/sec, Km 34.82 υM, and 277 units activity. Intracellular phytas was stable at pH 4–6 and 0–550 C. Protease level influenced the activity of intracellular phytase (P<0.05). Intracellular phytase was stable against pepsin but not pancreas. Keywords: Km, Vm, activity, intracellular phytase, pH, temperature, protease

PRODUCTION AND CHARACTERIZATION OF CRUDE INTRACELLULER PHYTASE FROM RECOMBINANT BACTERIA pEAS1AMP

This research was aimed at producing a crude intracellular phytase characterized from recombinant bacteria. The recombinant bacteria (pEAS1AMP) was produced by way of transforming pET-22b(+) +pEAS1 into competent E. coli BL21 and E. coli BL21(DE3) cells. Crude intracellular phytase production was induced using 1,5 mM Isopropyl-β-D-thiogalactopyranosid (IPTG). Recombinant bacteria product and enzyme activity test followed the Sajidan method. E. coli BL21(+)pEAS1 and E. coli BL21 (DE3)(+)pEAS1 recombinant bacteria showed growth after 20 hours and 10 hours of transformation. Phytase activity of E. coli BL21 (DE3)(+)+pEAS1 showed higher than those of E. coli BL21(+)+pEAS1. Crude intracellular phytase of pEAS1AMP recombinant bacteria has an optimum activity at pH 5, 40o C, incubation period of 60 minutes, substrate concentration of 2%, molecular weight (MW) of 47.3 kDa, Km = 15.91 υM and Vm = 2.41 υM/second. Mg2+ acts as a cofactor but Fe3+ (10-4 M) acts as an inhibitor. Keywords: bacteria recombinant pEAS1AMP, competent cells, crude intracellular phytas

Dietary Phytase: an ideal approach for a cost effective and low-polluting aquafeed

Global fishmeal production from wild-catch sources cannot continue to increase indefinitely; suitable alternatives have to be found for sustainable aquaculture. Plant-based aquafeed seems to be the ideal alternative to this, but has its own limitations. Plant ingredients are rich in phytic acid, which reduces the bioavailability of nutrients like minerals and protein to the fish, thereby causing aquaculture pollution. Dietary phytase treatment reduces the aquaculture pollution by improving the bioavailability of nutrients, and reduces the feed cost as evident from poultry and piggery. Phytase activity is highly dependent upon the pH of the gut. Unlike mammals, fish are either gastric or agastric, and hence, the action of dietary phytase varies from species to species. In this article, the authors attempt to summarise various effects of phytase on nutrient utilization, growth of fish and aquatic pollution

Phosphorus in pig diets

Pig feed is mainly based on cereals where phosphorus (P) is mostly present in inositol hexaphosphate (IP6), which is not readily available to monogastric animals. More available P sources are often added to ensure that pigs’ requirements are fulfilled; this results in high excretion levels of P. The digestibility of P depends on phytase activity and amount of IP6 in feedstuffs. The overall aim was to study effects of liquid feeding, P levels and phytase supplementation on digestibility and performance. Effects of soaking and P levels on digestibility were studied by total collection in metabolic cages, and effects on performance were studied in 192 growing pigs. Effects of soaking fermentation and phytase supplementation on ileal and total tract digestibility were studied with indicator technique on pigs surgically fitted with PVTC cannulas. P levels and phytase supplementation were studied in 104 pregnant sows for two reproduction cycles. All diets were cereal based and included wheat. Basic properties of a cereal mix fermented with whey, wet wheat distillers’ grain and water in different temperatures were also studied. Soaking reduced the level of IP6, whereas apparent digestibility of P was not significantly improved. Soaking increased average daily weight gain, carcass weights and improved the energy conversion ratio in pigs fed a low P diet to the same level as pigs fed high P diets. Low P diets resulted in lower femur density than high P diets. However, soaking of a low P diet resulted in increased femur density. Fermentation degraded IP6 efficiently and improved ileal apparent digestibility of P, organic matter, nitrogen, amino acids and total tract apparent digestibility of organic matter. Microbiological and biochemical properties of fermented liquid diets are strongly affected by feed components and temperature used. Phytase supplementation slightly affected apparent digestibility of P. Supplementing a low P gestation diet with phytase did not significantly affect sow performance. The slight effects of phytase supplementations found may depend on high levels of intrinsic phytase in the diets, and possibly suggest that the provided P level in the sows may have been sufficient. Under typical Swedish conditions of sow management, reduced total P level in gestation diets seems not to negatively affect performance

Citric Acid and Microbial Phytase Inclusion in the Diet to Improve Utilization Phytate Phosporus and Growth of Broiler

Penelitian ini menggunakan anak ayam umur 3 sampai dengan 38 hari (180 ekor) dan 7 sampai dengan 42 hari (180 ekor). Ayam diberi ransum A, B, dan C yang berbahan dasar jagung (CSM) dan ransum E, F, dan G yang berbahan dasar gandum (WSM) yang diberi perlakuan penambahan steam pada suhu 1000C selama 10 menit. Semua ransum ditambah enzim fitase (SP-1002 ct) sebanyak 500 U/kg dan dibuat dalam bentuk pelet. Khusus ransum C dan G ditambah asam sitrat. Kandungan fosfor total ransum adalah 4,5 g/kg dan fosfor tersedia adalah 1,5 g/kg (defisien P). Bobot badan harian adalah 36, 34, dan 45 g/hari untuk ayam yang diberi ransum A, B, dan C dan 54, 46, dan 55 g/hari untuk ransum E, F, dan G. Deposisi fosfor total adalah 5,75; 5,36; dan 6,99 g berturut-turut untuk ransum A, B dan C dan 8,15; 6,90; dan 7,77 g untuk E, F, dan G. Penambahan asam sitrat dalam ransum CSM yang mengandung enzim fitase rendah mampu meningkatkan pertumbuhan dan deposisi fosfor. Pada ransum WSM, perlakuan panas menyebabkan penurunan aktifitas fitase dan penurunan ini tidak dapat digantikan dengan penambahan enzim fitase (microbial phytase) akan tetapi dapat digantikan dengan penambahan asam sitrat. Hasil penelitian ini dapat disimpulkan bahwa asam sitrat berpengaruh terhadap efisiensi enzim fitase (microbial phytase) dalam mendegradasi senyawa fitat di dalam saluran pencernaan

Stability of commercial phytase products stored under different environmental conditions

Citation: De Jong, J. A., DeRouchey, J. M., Tokach, M. D., Goodband, R. D., Woodworth, J. C., Jones, C. K., . . . Bergstrom, J. R. (2016). Stability of commercial phytase products stored under different environmental conditions. Journal of Animal Science, 94, 114-114. doi:10.2527/msasas2016-241A 300-d study evaluated the stability of 4 phytase products stored under varied environmental conditions. The 4 products were: 1) Quantum Blue 5G (AB Vista, Marlborough, United Kingdom); 2) Ronozyme HiPhos GT 2700 (DSM Nutritional Products, Parsippany, NJ); 3) Axtra Phy TPT (Dupont, Wilmington, DE); and 4) Microtech 5000 Plus (Guangdong VTR Bio-Tech Co., Ltd., Guangdong, China). Products were stored as pure forms at ?20, 4, 22, or 35°C (75% humidity), or in a vitamin or vitamin trace mineral (VTM) premix at 22 and 35°C (75% humidity). Samples were stored in paper bags and sampled on d 30, 60, 90, 120, 210, and 300. Stability was determined as amount of residual phytase activity (% of initial). For pure forms, all interactive and main effects of product, time, and temperature were significant (P < 0.05). From d 30 to 300, products had similar reductions in phytase activity at the 3 highest temperatures; however, Quantum Blue 5G, Ronozyme HiPhos GT 2700, and Axtra Phy TPT had reduced (P < 0.05) phytase activity compared to Microtech 5000 Plus at ?20°C. As storage time increased, residual phytase activity was reduced (P < 0.05) regardless of product and storage temperature. Also, when product was stored at 4 and 22°C, phytase activity was improved compared to ?20 and 35°C. For vitamin and VTM premixes, a time×temperature × product interaction (P < 0.05) was observed as a result of, Axtra Phy TPT and Microtech 5000 Plus having reduced residual phytase activity (P < 0.05) compared to the other 2 products when stored at 22°C, while activity of Axtra Phy TPT was reduced (P < 0.05) even further than the other 3 products when stored at 35°C regardless of form. From d 30 to 300, Axtra Phy TPT and Microtech 5000 Plus had the lowest (P < 0.05) residual phytase activity compared to the other 2 products. The VTM had decreased (P < 0.05) residual phytase activity compared to the pure product and vitamin premixes. In conclusion, phytase stored for longer than 90–120 d at 35°C or ?20°C in pure form, or when stored as a VTM premix had reduced residual phytase activity

A note on the effect of supplementation with microbial phytase and organic acids on feed intake and growth performance of growing pigs

peer-reviewedThis experiment was designed to investigate the effects of supplementation with phytase, either alone or in combination with organic acids, on feed intake and growth of pigs from 8 to 89 kg live weight. Some 240 pigs were used in four experimental treatments comprising: (1) control, (2) control plus phytase, (3) control plus phytase plus liquid organic acids (formic, propionic), and (4) control plus phytase plus powdered organic acids (formic, fumaric, propionic). Feed intake and growth rate in the weaner stage were increased (P < 0.05) by phytase supplementation, with some additional benefits from organic acid inclusion. Interval to slaughter was reduced (P < 0.05) by phytase supplementation

Phytase activity in lichens

Phytase activity was investigated in 13 lichen species using a novel assay method. The work tested the hypothesis that phytase is a component of the suite of surface-bound lichen enzymes that hydrolyse simple organic forms of phosphorus (P) and nitrogen (N) deposited onto the thallus surface. Hydrolysis of inositol hexaphosphate (InsP6, the substrate for phytase) and appearance of lower-order inositol phosphates (InsP5–InsP1), the hydrolysis products, were measured by ion chromatography. Phytase activity in Evernia prunastri was compared among locations with contrasting rates of N deposition. Phytase activity was readily measurable in epiphytic lichens (e.g. 11.3 lmol InsP6 hydrolysed g-1 h-1 in Bryoria fuscescens) but low in two terricolous species tested (Cladonia portentosa and Peltigera membranacea). Phytase and phosphomonoesterase activities were positively correlated amongst species. In E. prunastri both enzyme activities were promoted by N enrichment and phytase activity was readily released into thallus washings. InsP6 was not detected in tree canopy throughfall but was present in pollen leachate. Capacity to hydrolyse InsP6 appears widespread amongst lichens potentially promoting P capture from atmospheric deposits and plant leachates, and P cycling in forest canopies. The enzyme assay used here might find wider application in studies on plant root–fungal–soil systems

Effect of phosphorus level and phytase inclusion on the performance, bone mineral concentration, apparent nutrient digestibility, and on mineral and nitrogen utilisation in finisher pigs.

peer-reviewedFunding for this research was provided under the National Development Plan, through the Research Stimulus Fund, administered by the Irish Department of Agriculture, Fisheries and Food.Two experiments were conducted to investigate the interaction between dietary P concentration and phytase (PHY) inclusion in the diet of finisher pigs. In Experiment 1, the growth performance and bone analysis experiment, pigs (6 replicate groups of 14 pigs each per treatment; initial body weight (BW) = 45.2 kg) were allocated to one of six dietary treatments (for 74 days) in a 3 × 2 factorial arrangement: T1 – available P in the diet = 1.5 g/kg; T2 = T1 with 500 units of phytase (FTU)/kg; T3 – available P = 2.0 g/kg; T4 = T3 with 500 FTU/kg; T5 – available P = 2.5 g/kg; T6 = T5 with 500 FTU/kg. Experiment 2 consisted of a digestibility and a P, Ca and N balance study, and pigs (6 per treatment; initial BW = 67.3 kg) were offered identical diets to those offered in Experiment 1. There was an interaction between dietary P level and PHY inclusion for average daily gain (ADG) and carcass weight (CW; P < 0.05) in Experiment 1. Pigs offered the low P diet supplemented with PHY had a higher ADG and CW than pigs offered the non-PHY, low P diet. However, there was no effect (P > 0.05) of PHY inclusion on ADG or CW with the medium or high P diets. Higher concentrations of ash, P and Ca in bone were noted in pigs offered the medium and high P diets (P < 0.001) and PHY (P < 0.01) diets when compared to pigs offered the low P without PHY. Pigs offered diets supplemented with PHY had lower faecal P output (P < 0.01) and a higher P digestibility (P < 0.001) and P retention (P < 0.05) than pigs offered diets without added PHY. In conclusion, supplementation of a low-P finisher diet with PHY resulted in pigs that had a similar carcass weight, but weaker bones than pigs offered a medium or high P diet.Department of Agriculture, Food and the Marin

Contribution of intestinal- and cereal-derived phytase activity on phytate degradation in young broilers

There is little consensus as to the capability of poultry to utilize dietary phytate without supplemental phytase. Therefore, an experiment was conducted to examine the extent to which endogenous phytase of intestinal and cereal origin contributes to phytate degradation in birds aged 0 to 14 d posthatch. Ross 308 broilers (n = 720) were fed one of 4 experimental diets with differing dietary ingredient combinations and approximate total phytate levels of 10 g/kg, dietary phytase activity analyzed at 460 U/kg, dietary calcium (Ca) levels of 11 g/kg, and nonphytate-phosphorus (P) levels of 4 g/kg. Broiler performance, gizzard, duodenum, jejunum and ileum pH, Ca and P digestibility and solubility, amount of dietary phytate hydrolyzed in the gizzard, jejunum, and ileal digesta phytase activity were analyzed at d 4, 6, 8, 10, 12, and 14 posthatch. Intestinal endogenous phytase activity increased significantly (P < 0.001) between d 4 and 6, resulting in increased phytate hydrolysis in the gizzard (P = 0.003), jejunum (P < 0.001), and ileum (P < 0.001). Phytase activity and phytate hydrolysis continued to increase with age, with a greater phytase activity and associated increase in phytate hydrolysis and mineral utilization between d 10 and 12. Gizzard and jejunum Ca and P solubility and ileal Ca and P digestibility increased significantly (P < 0.001), and gastrointestinal pH decreased significantly (P < 0.001) between d 4 and 6. By d 14, phytase activity recovered in the ileum was approximately 45 U/kg. There were strong correlations between phytase activity measured in the ileum and phytate hydrolyzed in the gizzard (r = 0.905, P < 0.001), jejunum (r = 0.901, P = 0.023), and ileum (r = 0.938, P = 0.042). This study shows intestinal- and dietary-derived endogenous phytase activity is responsible for phytate-P hydrolysis in broilers