Optimum initial ph of the medium required for maximum phytase production by the strain bacillus subtilis subsp. Development of an efficient autoinducible expression system. Phytase genes of several bacillus species have recently been cloned and characterized as single genes apparently not involved in operon structures 2326, 48. Bacillus subtilis in broiler diets with different levels.
It is capable of producing endospores resistant to adverse environmental. A read is counted each time someone views a publication summary such as the title, abstract, and list of authors, clicks on a figure, or views or downloads the fulltext. Transition state regulator abrb inhibits transcription of. Sequence analysis revealed a monocistronic operon encoding a 95. Evaluation of phytase production by fish gut bacterium, bacillus subtilis, for processing ofipomea aquaticaleaves as probable aquafeed ingredient. This gene, together with a phytase gene 168phya iden tified in the b. Its cell envelope consists of a thick peptidoglycan wall and and cell membrane.
Molecular analysis of phytase gene cloned from bacillus subtilis. The majority of orfs have been amplified in their entirety. The natto phytase gene was cloned into strain rik1285, a proteasedefective derivative of 168, to construct a random library of. Phytase enzymes can increase the nutritional value of food and feed by liberating inorganic phosphate from phytate, the major storage form of phosphorus in plants. We observed that, in contrast to the phytase genes of bacillus wildtype strains, the phytase gene of bacillus subtilis 168. The alkaline pectate lyase pel168 of bacillus subtilis.
Read enhanced activity of an alkaline phytase from bacillus subtilis 168 in acidic and neutral environments by directed evolution, biochemical engineering journal on deepdyve, the largest. Thus, pel168p is a potential candidate for further improving the specific activity and reducing the cost so that it can be used in biodegumming industry. Two strains of bacillus subtilis ncdc070 and ncim2712 were selected for cloning and sequencing of phy gene. Oct 31, 2003 read the introduction of a phytase gene from bacillus subtilis improved the growth performance of transgenic tobacco, biochemical and biophysical research communications on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Published phy gene gene sequences were used to design specific primers for amplification. P420941 fasta add to basket added to basket hide 10. The introduction of a phytase gene from bacillus subtilis improved the growth performance of transgenic tobacco article in biochemical and biophysical research communications 3104. Molecular cloning and the biochemical characterization of two. Scholars research library extracellular production of. Expression of bacillus subtilis phytase in lactobacillus. Evaluation of phytaseproducing ability by a fish gut. Molecular cloning and the biochemical characterization of two novel. The phytase of bacillus licheniformis atcc 14580 gathers the best features to be. Expression of a bacillus phytase c gene in pichia pastoris.
The isolated bacillus subtilis produces significant amount of phytase. One primary emphasis is sporulation, an archetypical form of cell development 25, 58, 66. Comparative analysis of the complete genome sequence of the. Subsequently, the combination of corn flour with nutritive supplements was used for further ssf study by bacillus sp. Bacillus genetic stock center catalog of strains, seventh.
Sambrook j, fritsch ef, maniatis t 1989 molecular cloning. Study of the optimum growth conditions of bacillus. Isolation, purification and characterization of phytase. Apr 25, 2016 bacillus subtilis, a grampositive organism, has been developed to be an attractive expression platform to produce both secreted and cytoplasmic proteins owing to its prominent biological characteristics. Bacillus subtilis cell factory converting phytic acid into scylloinos. From the 21 bacterial isolates, one bacillus subtilus strain bptk4 with high potential for phytase production was selected. To combine the advantages of different expression systems, mutants of the alkaline phytase originated from bacillus subtilis 168 phy168 were first generated via directed evolution in e. While the monocistronic phyc gene is silent in the laboratory strain bacillus subtilis 168. Extracellular phytase activity of bacillus amyloliquefaciens fzb45 contributes to its plantgrowthpromoting effectaathe genbank accession numbers for the sequences determined in this work are ay055219 to ay055226. According to our results, the optimum ph for the growth of bacillus subtilis strain wt 168 is 8. Presence of the thya gene thya and the amylaseencoding genes amys from b. Isolation, characterization, molecular gene cloning, and. Smf by bacillus subtilis powar and jagannathan, 1982.
The origins of 168, w23, and other bacillus subtilis legacy. Two bacillus subtilis strains namely ncdc070 and ncim 2712 were procured to isolate phytase phy gene from national. Several bacillus species produce phytases, extracellular degradative enzymes, which release free phosphates from myoinositol hexakisphosphate, the main storage form of phosphate in plants. The bacillus subtilis strain vtt e680 was chosen for puri. Prepare an on culture of bacillus subtilis 168 lb media with appropriate antibiotic incubate at 37degc. Isolation, characterization, molecular gene cloning, and sequencing of a novel phytase from bacillus subtilis. To attempt costeffective production of us417 phytase in bacillus subtilis, we developed an efficient system for its largescale production in the generally recognized as safe microorganism b. Extracellular phytase activity of bacillus amyloliquefaciens fzb45. Molecular cloning and production of recombinant phytase from bacillus subtilis asuia243 in pichia pastoris. Catalytic activity in acidic and neutral environments was successfully improved. Underlying mechanism for the enhanced activity was revealed by molecular docking. Phytic acid, known as myoinositol mi hexakisphosphate, is the principal storage form of phosphorus in many plant tissues, especially bran and seeds. The igem team 2016 bonn and freiburg both struggled with some experiments concerning bacillus.
The enzyme proved to be highly specific since, of the substrates tested, only phytate, adp, and atp were hydrolyzed. The bacillus subtilis strain vtt e680 was chosen for purification and characterization of its excreted phytase. An extracellular phytase from bacillus subtilis natto n77 was purified 322fold to homogeneity with the specific activity of 8. The protein structure information of pectate lyase pel168 of bacillus subtilis 168 is available now, which makes the enzyme rational design possible. It is capable of producing endospores resistant to adverse environmental conditions such as heat and desiccation and is widely used for the production of enzymes and specialty chemicals. A novel phytase gene phyl was cloned from bacillus. Enhanced secretion of natto phytase by bacillus subtilis. Bacillus subtilis is an intensively studied grampositive bacterium that has become one of the models for biofilm development.
We observed that, in contrast to the phytase genes of bacillus wildtype strains, the phytase gene of bacillus subtilis 168 is cryptic, most likely due to. An alkaline phytase from bacillus subtilis 168 was engineered using directed evolution. Bacillus subtilis, known also as the hay bacillus or grass bacillus, is a grampositive, catalasepositive bacterium, found in soil and the gastrointestinal tract of ruminants and humans. Pdf molecular cloning and production of recombinant. We previously developed an autoinducible expression system containing the srfa promoter p srfa which was activated by the signal molecules acting in the quorumsensing pathway for. Purification and characterization of phytase from bacillus. How to bacillus subtilis a collaboration bonn freiburg this manual should serve as an assistance while starting to work with the versatile bacterium bacillus subtilis. In this invention, two phytase genes from two generallyregardedassafe microorganisms, bacillus licheniformis and bacillus subtilis 168, were cloned and characterized. Twostep bacillus subtilis transformation procedure based on molecular biological methods for bacillus preparation of bacillus subtilis competent cell 1. Specific bacillus subtilis 168 variants form biofilms on. Enhanced activity of an alkaline phytase from bacillus.
The origins of 168, w23, and other bacillus subtilis. Among them, bacillus subtilis has been developed as an attractive host. Use of a promiscuous glycosyltransferase from bacillus. Isolated bacillus subtilis strain 3302 and its antagonistic. An alkaline phytase from bacillus subtilis 168 was engineered using directed evolution catalytic activity in acidic and neutral environments was successfully improved. Purified enzyme had maximal phytase activity at ph 7 and 55c. Phytases comprise a group of phosphatases that can trim inorganic phosphates from phytic acid. Hence, the phy us417 corresponding gene was cloned in the pmsp3535 vector, and for the first time for a plasmid carrying the pamb1. At present, about 60% of the commercially available enzymes are produced by bacillus species. Pdf extracellular phytase activity of bacillus amyloliquefaciens. Onepot synthesis of ginsenoside rh2 and bioactive unnatural ginsenoside by coupling promiscuous glycosyltransferase from bacillus subtilis 168 to sucrose synthase. The 42 d experiment consisted of 6 experimental diets, a diet with standard nutrient content, and 2 diets with different levels of energy.
Isolation, purification and characterization of phytase from. Molecular cloning and the biochemical characterization of. Phytases catalyze the hydrolysis of phytic acid in a stepwise manner to lower inositol. Bacillus subtilis, a model organism for grampositive bacteria, is the focus of diverse research interests in both academic and industrial settings 54, 63, 64.
This study has indicated that directed evolution can be an efficient method to improve the activity of alkaline phytase from b. The coding region of the phyl gene was 1,146 bp in size and a promoter region of approximately 300 bp was identified at the upstream sequence. Bacillus subtilis cell factory converting phytic acid into. Obtaining materials from the bacillus genetic stock center what is the bacillus genetic stock center. The annotation score provides a heuristic measure of the annotation content of a uniprotkb entry or. To examine the physiological effects of a neutral phytase on plant metabolism, a plant expression vector that expresses a phytase from bacillus subtilis strain 168 168phya was created. Phytase production at different periods of fermentation.
The 168phya gene, with its native signal peptide excluded, was subcloned into the binary vector pcambia0 to generate the recombinant clone pcx168phya. Phytase production by high cell density culture of recombinant bacillus subtilis. Purified enzyme had maximal phytase activity at ph 7 and 55 degrees c. The genome of bacillus subtilis strain 168 has been sequenced although the current part collection for b. Bacillus subtilis in broiler diets with different levels of. Isolated enzyme required calcium for its activity andor stability and was readily inhibited by edta. Molecular cloning of partial phytase gene from bacillus. The phytase producing bacteria were isolated using phytate screening agar media psm with only 1. The present study evaluated the impacts of bacillus subtilis bas inclusion in broiler diets with standard nutrient content or nutrient deficiency nd on growth performance gp. Structure of urate oxidase from bacillus subtilis 168. However, the enzyme was also highly stable at 40 c and 50 c after 168 h 7. Bacillus subtilis cell factory converting phytic acid. Jun 15, 2015 read enhanced activity of an alkaline phytase from bacillus subtilis 168 in acidic and neutral environments by directed evolution, biochemical engineering journal on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. Molecular analysis of phytase gene cloned from bacillus.
Phytase, overexpression, bacillus subtilis, multimeric dna forms. In this paper, we succeeded in the creation and screening of alkaline phytase mutants with enhanced specific activity in acidic and neutral environments by directed evolution. This gene, together with a phytase gene 168phya identified in the b. Aug 22, 2008 bacillus subtilis, a model organism for grampositive bacteria, is the focus of diverse research interests in both academic and industrial settings 54, 63, 64. The displayed sequence is further processed into a mature form. A novel phytase gene phyl was cloned from bacillus licheniformis by multiple steps of degenerate and inverse pcr. In the present study, phytase activity of bacillus subtilis b. The introduction of a phytase gene from bacillus subtilis. Original open access heterologous expression and optimization.
Hence, the phy us417 corresponding gene was cloned in the pmsp3535 vector, and for the first time for a plasmid carrying the pam. Characterization and production of the enzyme janne kerovuo danisco cultor innovation, kantvik, finland academic dissertation to be presented with the permission of. Molecular cloning and the biochemical characterization of two novel phytases from b. Under most conditions, however, it is not biologically active and is present in the spore form. Three out of the four strains investigated were identified as b. Heterologous expression and optimization using experimental. Molecular analysis of phytase 105 materials and method bacterial strains. Calciumdependent catalytic activity of a novel phytase. Pdf phytase production by high cell density culture of. Bacillus subtilis is a ubiquitous naturally occurring saprophytic bacterium that is commonly recovered from soil, water, air and decomposing plant material. The time course of phytase synthesis by bacillus sp. Extracellular phytase activity of bacillus amyloliquefaciens.
643 252 1228 941 1139 404 700 792 1163 412 1175 1513 343 860 916 790 726 431 427 559 1456 698 863 1283 869 761 652 1296 1486 1273 530 808 695 925 1145 32 735 1309 1480 1341 1056 1426 176 273 22 511 1203 649