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ENGINEERED ROBUST HIGH Tm-PHYTASE CLADE POLYPEPTIDES AND FRAGMENTS THEREOF

A phytase, engineering technology, applied in the direction of enzymes, hydrolases, biochemical equipment and methods, etc., can solve problems such as lack of robustness

Pending Publication Date: 2021-08-24
DUPONT NUTRITION BIOSCIENCES APS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, none of these phytases are suitably robust to withstand high levels of stress under commercially relevant feed pelleting conditions when applied in feed in liquid form prior to conditioning and pelleting

Method used

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  • ENGINEERED ROBUST HIGH Tm-PHYTASE CLADE POLYPEPTIDES AND FRAGMENTS THEREOF
  • ENGINEERED ROBUST HIGH Tm-PHYTASE CLADE POLYPEPTIDES AND FRAGMENTS THEREOF
  • ENGINEERED ROBUST HIGH Tm-PHYTASE CLADE POLYPEPTIDES AND FRAGMENTS THEREOF

Examples

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example

[0462] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton et al., DICTIONARY OFMICROBIOLOGY AND MOLECULAR BIOLOGY, 2nd ed., John Wiley and Sons, New York (1994), and Hale and Marham, THE HARPER COLLINSDICTIONARY OF BIOLOGY [ HarperCollins Biological Dictionary], Harper Perennial, New York (1991) provides the skilled artisan with a general dictionary of many of the terms used in this disclosure.

[0463] The disclosure is further defined in the examples below. It should be understood that these Examples, while indicating certain embodiments, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this disclosure, and without departing from the spirit and scope of this disclosure, can make various changes and modifications to ada...

example 1

[0465] Production of Phytase Molecule

[0466] DNA manipulations were performed using molecular biology techniques known in the art to generate the phytase gene. Polynucleotide fragments corresponding to the coding sequences of various phytases were synthesized using preferred codons of the fungal expression host Trichoderma reseei (T. reesei), and randomly reassembled using PCR technology. The signal sequence of the pepl aspartic protease from Trichoderma reesei (SEQ ID NO: 63), artificially interrupted by the pepl intron, was introduced into the N-terminus (5' end) of each phytase gene sequence. As recommended by the supplier, use BP recombinant technology introduced the gene into pDonor221 vector (Invitrogen, USA). The resulting entry plasmid (entry plasmid) was recombined with the destination vector pTTTpyr2 to obtain the final expression vector. pTTTpyr2 is similar to the previously described pTTTpyrG vector (PCT Publication WO 2011 / 063308), except that the pyrG gene ...

example 2

[0470] Preparation and Characterization of Phytase

[0471] Protein Purification and Standardization

[0472]The T. reesei strain encoding the recombinant phytase was grown as described above, and the clarified supernatant was used to purify the phytase. The filtered culture supernatant was diluted 5-fold with wash buffer (25 mM sodium acetate, pH 5.5), and loaded onto an MTP filter plate (Millipore (Millipore) Multiscreen Solvinert deep-well filter plate 96-well MTP, 0.45 uM Aqueous membrane, #MDRLN0410) on cation exchange resin (WorkBeads 40S from Bio-Works) equilibrated with purified water. The MTP was placed in a centrifuge and the effluent was discarded during the 1 minute centrifugation (100 x g). Phytase protein samples were eluted using elution buffer (25 mM sodium acetate, 0.5 M NaCl, pH 5.5) during 1 min of centrifugation (100 x g). Samples from the protein purification step were diluted 5-fold with sodium acetate buffer (25 mM sodium acetate, 0.5 M NaCl, pH 5.5...

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Abstract

Engineered robust high Tm-phytase clade polypeptides and fragments thereof are described herein. Also described are methods of making such engineered robust high Tm- phytase clade and fragments thereof and use thereof in enhancing animal performance.

Description

[0001] Cross References to Related Applications [0002] This application claims U.S. Provisional Patent Application No. 62 / 769,713, filed November 20, 2018, U.S. Provisional Patent Application No. 62 / 851,122, filed May 22, 2019, and U.S. Provisional Patent Application No. Priority to Provisional Patent Application No. 62 / 887,714, the disclosure of each of the above is incorporated herein by reference in its entirety. [0003] incorporated by reference [0004] The Sequence Listing, which will be created and submitted concurrently on November 15, 2019, is hereby incorporated by reference in its entirety, provided as a file of 324 KB in size and titled "20191115_NB41175-WO-PCT_Sequence Listing_ST25". technical field [0005] The field relates to engineered robust high Tm phytase clade polypeptides and fragments thereof, methods of producing such engineered robust high Tm phytase clade polypeptides and fragments thereof, and such engineered robust Use of high Tm phytase clade ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A23K20/189C12N9/16
CPCC12N9/16C12Y301/03A23K20/189A23K50/30A23K10/16A23K50/75A23K40/10C12N15/63A23K50/10C12N15/80C12Y301/03026
Inventor L·M·贝比T·克里斯滕森S·哈宁金慧淑R·梅耶达尔I·尼古拉耶夫J·C·普拉萨德S·普莱斯鲁斯J·F·瑟伦森R·A·索尔格
Owner DUPONT NUTRITION BIOSCIENCES APS
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