Aquaculture nutrition, tập 19, số 2, 2013

Aquaculture Nutrition 2013 19; 117–127 doi: 10.1111/j.1365-2095.2012.00946.x 1 2 Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung, Taiwan; Food Science, National Pingtung University of Science and Technology, Pingtung, Taiwan To produce a thermostable and neutral phytase (phy) of Bacillus subtilis E20 in Escherichia coli HMS174 and evaluate its efficiency in improving growth performance The phy C of B subtilis E20 was expressed in E coli HMS 174, and then the 42-kDa recombinant phy C was purified by Ni-NAT and analysed by SDS–PAGE The recombinant phy C had optimal ranges of pH of ~ and temperature of 50 ~ 60 °C A thermostability analysis showed that the enzyme is a thermostable phytase, and around 33% of residual activity was detected after being incubated at 90 ~ 100 °C for 10 The recombinant phy C-pretreated soybean meal for feed preparation improved white shrimp, Litopenaeus vannamei, growth and feed efficiency Overall, the neutral and thermostable phy C is suitable for aquafeed, and it is able to improve the nutritional utilization, resulting in enhanced shrimp growth and reduced feed costs KEY WORDS: Bacillus subtilis E20, growth performance, Litopenaeus vannamei, phytase, soybean meal Received August 2011; accepted January 2012 Correspondence: Chun-Hung Liu, Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan E-mail: chliu@mail.npust.edu.tw There is continuing interest in identifying and developing ingredients from plants as alternatives to the high cost of ª 2012 Blackwell Publishing Ltd Department of fish meal for use in aquafeed (Hardy 1995; Tacon et al 1998) Nowadays, concern is raised about the negative impacts on global fish meal production of overfishing and natural disasters, such as the tsunami in Chile Among plant feedstuffs being investigated to replace fish meal, soybean meal is considered a promising protein source because of its stable supply, price and nutritional composition One of the major problems associated with the use of plant proteins in aquafeed is the presence of anti-nutritional factors, such as phytate (myo-inositol-1,2,3,4,5,6hexakisphosphates), which is the main storage form of phosphate (P) in plant feedstuff Up to 80% of the total P content in plants may be present in the form of phytate (Lall 1991; Eeckhout & De Paepe 1994) and is not practically available for monogastric or agastric aquatic animals (National Research Council (NRC) 1993) In addition to phytate-P, the absorption and bioavailability of indispensable minerals such as calcium, zinc, iron and magnesium can be negatively affected by forming insoluble chelated complexes with phytase (Papatryphon et al 1999) In addition, phytate can also combine with proteins and vitamins in insoluble complexes (Liu et al 1998; Sugiura et al 2001), resulting in their decreased utilization, activity and digestibility because of interference with lipid and starch digestibility (Cosgrove 1996) Therefore, the issue of phytate must be dealt with to improve nutrition utilization of soybean meal and increase substitution ratios of fish meal to bring down the consequent costs of aquafeed Microbial phytase is widely used in feed for animal production to minimize the problem of phytate and is included in aquafeed (Cao et al 2007) to improve the nutritive value (Forster et al 1999), the digestibility of nutrients and minerals (Cheng & Hardy 2003; Yoo et al 2005), growth performance (Sajjadi & Carter 2004) and reduce phosphorus discharge (Forster et al 1999; Sajjadi & Carter 2004) On the contrary, dietary phytase supplementation had no effect on the growth of tiger shrimp, Penaeus monodon (Biswas et al 2007) Actually, most commercial phytase, adapted for digestive systems of livestock animals, may differ in its efficiency of phytate degradation in fish (Liebert & Portz 2005) because of the varying physiological conditions in different species because of its great dependence on the gut pH In addition, native phytase activity may also be inactivated by heat treatment during aquafeed processing Therefore, developing a neutral phytase with improved thermal stability is important and suitable for some fish and/or shrimps with near-neutral digestive tracts Shen et al (2004) reported that the pH values in the intestine, liver, and stomach of Litopenaeus vannamei were 5.9 ~ 6.1 (6.08 ± 0.04), 6.7 ~ 7.0 (6.82 ± 0.04) and 5.1 ~ 5.3 (5.22 ± 0.04), respectively Phytase with a beta-propeller structure is mainly isolated from Bacillus sp The phytase (phy C) of Bacillus is a nonglycosylated protein with higher thermal stability and an optimal pH in the neutral range (Gulati et al 2007; Rao et al 2008; Guerrero-Olazara´n et al 2010), in contrast to commercial production of phytase, which is currently focused on fungal histidine acid phytase from Aspergillus sp (Ha et al 2000; Cao et al 2007) Therefore, we report the expression of phy C from Bacillus subtilis E20 in Escherichia coli HMS147, and the biochemical properties of the recombinant phytase The enzyme was subsequently used to pretreat soybean meal for experimental diet preparation to evaluate the improvement in the growth performance of white shrimp, L vannamei juveniles Escherichia coli XL-1 Blue was used in the cloning steps and routinely grown with shaking in Luria-Bertani (LB) broth (L3022; Sigma, St Louis, MO, USA) at 37 °C Bacillus subtilis E20 isolated from natto (Liu et al 2009) was used as a source of the phy C gene and grown in nutrient broth (NB) (BD, Sparks, MD, USA) at 40 °C with shaking Escherichia coli HMS 147 (DE3) (Navagen, Madison, WI, USA) was used for phytase expression and grown in LB broth with shaking The LB broth for growing both of the E coli strains was supplemented with ampicillin (100 lg mLÀ1) when appropriate The cultivation and genomic DNA isolation of B subtilis E20 were performed according to Liu et al (2009) The phy C nucleotide sequence data reported in this article were deposited in the GenBank nucleotide sequence database under accession no FJ541287 (Liu et al 2009) The phy C gene fragment encoding the mature enzyme was amplified by a polymerase chain reaction (PCR) with the 6XHis phyF (5′-CAACATATGCACCACCACCACCACCAAAT CATCAAAAACACTTTTGT-3′) primer with insertions of the Nde I site and His-tag encoding sequence, and phy-R (5′-TTGGATCCTTATTTTCCGCTTCTGTC-3′) primer with insertion of the Bam HI site The following PCR protocol was used: initial denaturation at 95 °C for and 20 s; followed by 30 cycles of denaturation at 95 °C for min, annealing at 54 °C for min, and extension at 72 °C for 1.5 min; followed by an additional incubation at 72 °C for 10 and then °C for 30 s at the end of the final cycle After the PCR, samples were analysed by electrophoresis on 20 lg mLÀ1 agarose gels, and then the amplified PCR fragment was purified by a Gel Extraction System (GE10200; ALS, Kaohsiung, Taiwan) The purified PCR fragments were cloned into the PCRII TOPO vector of the TOPO TA cloning system (Invitrogen, Carlsbad, CA, USA) and transferred into E coli XL-1 Blue according to the manufacturer’s protocol and then subjected to a sequence analysis The PCRII TOPO vector containing the phy C gene was digested with Nde I and Bam HI and then analysed by electrophoresis on 20 lg mLÀ1 agarose gels, and the phy C gene was purified using the Gel Extraction System (GE10200; ALS) Thereafter, the purified phy C gene was ligated with the Nde I/Bam HI-digested pET 25b(+) vector to generate 6XHis-phytase DNA-pET 25b(+) The ligation mixture was used to transform competent E coli HMS 174 cells by electroporation (Micro PulserTM; Bio-Rad, CA, USA) using 0.1-cm cuvettes and a 1.8-kV pulse and then checked for protein expression The 6XHis-phytase DNA-pET 25b(+) transformant was grown in LB medium containing 100 lg mLÀ1 ampicillin and agitated at 200 rpm at 37 °C until the OD600 reached 0.5 Isopropyl-b-D-thiogalactopyranoside (IPTG at 0.1 mM) was then added, and the culture was shifted to 23 °C for 16 h because the production levels of the Aquaculture Nutrition, 19; 117–127 ª 2012 Blackwell Publishing Ltd recombinant phy C was found to be higher at 23 than at 20 and 28 °C After 16 h of incubation, the sample was collected for electrophoresis and phytase activity analysis Cells harvested from the above induced culture were subjected to centrifugation at 5000 xg and °C for 10 The pelleted cells were resuspended in 10 mM Tris buffer (pH 8) containing mM CaCl2 and sonicated on ice using MicrosonTM XL 2000 (Misonix, New York, NY, USA) Subsequently, they were centrifuged at 11 000 g for 30 at °C The suspension was used for the phytase activity assay The enzyme assay method was modified from Shimizu (1992) Phytase activity was assayed by measuring the rate of increase in inorganic orthophosphate cleaved from phytate A reaction mixture containing 100 lL of the enzyme preparation and 300 lL of mg LÀ1 phytate in 10 mM Tris buffer (pH 8) containing mM CaCl2 was incubated at 50 °C for 15 Then, the reaction was stopped by adding 400 lL of 50 lg mLÀ1 trichloroacetic acid The liberated phosphate was measured at 700 nm by following the production of phosphomolybdate with 1.5 mL of colour reagent (freshly prepared by mixing four volumes of 30 lg mLÀ1 ammonium molybdate solution in 50 lg mLÀ1 sulphuric acid and one volume of 50 lg mLÀ1 ferrous sulphate solution) One unit of phytase activity was defined as the amount of enzyme hydrolysis of lmmol phosphate minÀ1 under the assay conditions The protein concentration in the enzyme preparations was determined using the protein assay dye (Bio-Rad, ST, USA) Samples were fractionated by sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS–PAGE) using a 0.12 g mLÀ1 SDS–PAGE gel Proteins in the resolved gel were detected by Coomassie Brilliant Blue R250 For the phytase zymogram analysis, the gel was soaked in 10 lg mLÀ1 Triton X-100 for a period of h at room temperature, and then it was moved to 0.1 M sodium acetate buffer (pH 5) at a temperature of °C for h Thereafter, the gel was changed to 0.1 M sodium acetate buffer (pH 5) containing lg mLÀ1 phytate at °C for 16 h for phytase activity detection Activity bands were visualized after the gel was immersed in a 20 lg mLÀ1 cobalt chloride solution for at room temperature, and then the cobalt chloride solution was replaced with a freshly prepared solution Aquaculture Nutrition, 19; 117–127 ª 2012 Blackwell Publishing Ltd containing equal volumes of a 62.5 lg mLÀ1 ammonium molybdate solution and 4.2 lg mLÀ1 ammonium vanadate solution Phytase activity was evident as zones of clearing on an opaque background (Bae et al 1999) The recombinant phy C was purified using an Ni-nitrilotriacetic acid (NTA) column A sample in 10 mM Tris buffer containing 150 mM NaCl and mM CaCl2 at pH 7.6 was applied to the Ni-NTA matrix and then washed with the above buffer containing 20 mM imidazole and then eluted with the above buffer except that the imidazole concentrations used were 50 and 100 mM The effect of pH on the enzyme activity at 50 °C was determined Purified recombinant phytase was replaced with buffer using Amicon YM-10 (Millipore, Bedford, MA, USA) with various pH buffers of 200 mM sodium acetate (pH ~ 6) or 100 mM Tris buffer (pH ~ 10) Thereafter, enzyme assays were performed as described earlier with phytate in different buffers at pH values of ~ 10 Temperature effects on enzyme activity were determined in this study The enzyme and substrate, phytate, were first mixed; then the reaction mixtures were placed at various temperatures of 20 ~ 80 °C, and the enzyme activity was analysed as described above Metal requirements for the active conformation of the enzyme were tested by analysing the enzyme in 100 mM Tris buffer (pH 8.0) only or supplemented with mM CaCl2 or mM CaCl2 plus 10 mM EDTA Enzyme assays were performed as described earlier For analysis of the thermal stability, purified recombinant phytase was incubated at various temperatures of 10, 20, 30, 40, 50, 60, 70, 80, 90 and 100 °C for 10 min, followed by cooling to 28 °C Phytase activity was detected by the method described earlier Recombinant phy C preparation and use for soybean meal pretreatment Recombinant phy C was produced and plement and enzyme-pretreated soybean meal, and the diets without phosphorus supplement but with enzyme-pretreated soybean meal were prepared, respectively The formulation of the experimental diets is presented, respectively, in Tables and The moisture, ash, crude protein, ether extract and total phosphate of key ingredients including fishmeal and soybean meal were 100.8, 148.2, 663.2, 80.7 and 20.1 g kgÀ1, and 100.3, 73.2, 421.9, 30 and 6.9 g kgÀ1, respectively Briefly, the ingredients except enzyme-pretreated soybean meal were ground up in a Hammer mill to pass through a 60-mesh screen The experimental diets were prepared by mixing the dry ingredients and enzyme-pretreated soybean meal, and then adding water until a stiff dough resulted Each diet was then passed through a mincer with a die, and the resulting spaghetti-like strings were dried in an incubator with a dehumidifier at °C until the moisture was [...]... Penaeus monodon Aquaculture, 264, 353–362 Tave, D (1988) Genetics and breeding of Tilapia: a review In: Proceedings of the 2nd International Symposium on Tilapia in Aquaculture (Pullin, R.S.V., Bhukaswan, T.Tonguthai, K & Maclean, J.L eds), pp 285–294 16–20 March 1987 Bangkok, Thailand Aquaculture Nutrition, 19; 128–138 ª 2012 Blackwell Publishing Ltd Aquaculture Nutrition 2013 19; 139–147... practical diets without fish meal for Aquaculture Nutrition, 19; 139–147 ª 2012 Blackwell Publishing Ltd sunshine bass (Mornone chrysops 9 M saxatilis) Aquaculture, 188, 299–309 Willson, R.P & Poe, W.E (1985) Apparent digestibility of protein and energy in feed ingredients for channel catfish Prog FishCult., 47, 154–158 Aquaculture Nutrition, 19; 139–147 ª 2012 Blackwell Publishing... levels) or high-range (three highest ration levels) in each case The overall effect was examined on a nonlinear basis as per Glencross (2008): Aquaculture Nutrition, 19; 128–138 ª 2012 Blackwell Publishing Ltd Aquaculture Nutrition, 19; 128–138 ª 2012 Blackwell Publishing Ltd 75 50 25 100 75 50 25 100 75 50 25 100 75 50 25 0 0 Wild Wild Wild Wild G8 G8 G8 G8 Wild Wild Wild Wild... exhibited the highest dry matter ADCs in animal and plant ingredients, while meat and bone meal and rapeseed meal were the lowest, respectively Aquaculture Nutrition, 19; 139–147 ª 2012 Blackwell Publishing Ltd Aquaculture Nutrition, 19; 139–147 ª 2012 Blackwell Publishing Ltd ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 81.5 92.8 93.9 65.2 86.3 92.1 89.9 92.1 90.3 93.7 92.2 87.3 92.4 91.8... Oncorhynchus mykiss Aquaculture, 179, 95–107 Sara, J.P., Gous, R.M & Bureau, D.P (2009) Describing growth and predicting feed intake in the marine prawn Fenneropenaeus indicus Part I: theoretical and practical aspects of measuring and predicting genetic parameters Aquaculture, 287, 402–413 Smith, D.M & Tabrett, S.J (2004) Accurate measurement of in vivo digestibility in shrimp feeds Aquaculture, 2 32, 563–580... 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Biotechnol Biochem., 56, 1266–1269 Aquaculture Nutrition, 19; 117–127 ª 2012 Blackwell Publishing Ltd Sugiura, S.H., Gabaudan, J., Dong, F.M & Hardy, R.W (2001) Dietary microbial phytase supplementation and the utilization of phosphorus, trace minerals and protein by rainbow trout Oncorhynchus mykiss (Walbaum) fed soybean meal-based diets Aquacult Res., 32, 583–592 Tacon, A.G.J., Dominy, W.G &... Publishing Ltd Zhou, Q.C., Tan, B.P., Mai, K.S & Liu, Y.J (2004) Apparent digestibility of selected feed ingredients for juvenile cobia (Rachycentron canadum) Aquaculture, 241, 441–451 Aquaculture Nutrition doi: 10.1111/j.1365-2095.2012.00950.x 2013 19; 148–162 School of Animal Production Technology, Institute of Agricultural Technology, Suranaree University of Technology, Muang, Thailand