Protein, method for manufacturing same, and method for evaluating protein activity

Patent metadata

Field	Value
Publication number	US-2016102301-A1
Application number	US-201414895356-A
Country	US
Kind code	A1
Filing date	Aug 7, 2014
Priority date	Aug 9, 2013
Publication date	Apr 14, 2016
Grant date	—

Abstract

The present invention relates to a method for producing a protein, comprising an inspection process, wherein the inspection process comprises: a step of approximating an infrared absorption band derived from a protein appearing around 1500 to 1600 cm −1 or around 1600 to 1700 cm −1 in an infrared absorption spectrum of the protein, by one or more normal distributions, a step of calculating an index value indicating a degree of broadening of the infrared absorption band based on the normal distributions, and a step of comparing the index value with a predetermined threshold to select, as a good-quality product, a protein having a degree of broadening of the infrared absorption band that is smaller than the threshold.

First claim

1 . A method for producing a protein, comprising an inspection process, wherein the inspection process comprises:
a step of approximating an infrared absorption band derived from a protein appearing around 1500 to 1600 cm −1 or around 1600 to 1700 cm −1 in an infrared absorption spectrum of the protein, by one or more normal distributions, a step of calculating an index value indicating a degree of broadening of the infrared absorption band based on the normal distributions, and a step of comparing the index value with a predetermined threshold to select, as a good-quality product, a protein having a degree of broadening of the infrared absorption band that is smaller than the threshold.

2 . The method according to claim 1 , wherein the index value is a half-value width of a single normal distribution, when the infrared absorption band is approximated by the single normal distribution.

3 . The method according to claim 1 , wherein the index value is a value which is obtained by subjecting the infrared absorption band to waveform separation to obtain a plurality of normal distributions, and then dividing a sum of areas of one or more normal distributions around a peak top position of the infrared absorption band by a sum of areas of one or more normal distributions around an end of the infrared absorption band.

4 . The method according to claim 1 , wherein the index value is a value which is obtained by subjecting the infrared absorption band to waveform separation to obtain two normal distributions each having a peak around a peak top position of the infrared absorption band and having a different half-value width, and then dividing an area of a normal distribution having a smaller half-value width among the two normal distributions by an area of a normal distribution having a larger half-value width.

5 . The method according to claim 1 , wherein
the index value is a value obtained by subjecting the infrared absorption band to waveform separation to obtain an n number of normal distributions A 1 to A n (wherein n is an integer of 3 or greater), and when the number n is an even number, by dividing a sum of area(s) of at least one or both of A n/2 and A n/2+1 by a sum of an area of at least one selected from the group consisting of A 1 to A n/2−1 and A n/2+2 to A n , or when the number n is an odd number, by dividing an area of A (n−1)/2 by a sum of an area of at least one selected from the group consisting of A 1 to A (n−1)/2−1 and A (n−1)/2+2 to A n .

6 . The method according to claim 1 , wherein the protein is an immobilized lipase formed by immobilizing a lipase on a resin carrier.

7 . The method according to claim 6 , wherein
the index value is a half-value width of a single normal distribution, when an infrared absorption band derived from a lipase appearing around 1600 to 1700 cm −1 is approximated by the single normal distribution, and in the selection step, an immobilized lipase in which the index value is 70 cm −1 or less is selected as a good-quality product.

8 . The method according to claim 6 , wherein
the index value is a value obtained by subjecting an infrared absorption band derived from a lipase appearing around 1600 to 1700 cm −1 to waveform separation to obtain two normal distributions A 1 and A 2 , and then dividing an area of A 1 by an area of A 2 , with regard to the waveform separation, the infrared absorption band is subjected to the waveform separation to obtain the two normal distributions, A 1 (peak position: 1656 cm −1 , half-value width: 47 cm −1 ) and A 2 (peak position: 1656 cm −1 , half-value width: 82 cm −1 ), so that an absolute value of a difference between an area of the infrared absorption band derived from the lipase and a sum of the areas of the two normal distributions becomes a minimum, and in the selection step, an immobilized lipase in which the index value is 0.27 or more is selected as a good-quality product.

9 . The method according to claim 6 , wherein
the index value is a value obtained by subjecting an infrared absorption band derived from a lipase appearing around 1600 to 1700 cm −1 to waveform separation to obtain three normal distributions A 1 , A 2 and A 3 , and then dividing an area of A 2 by a sum of areas of A 1 and A 3 , with regard to the waveform separation, the infrared absorption band is subjected to the waveform separation to obtain the three normal distributions, A 1 (peak position: 1680 cm −1 , half-value width: 50 cm −1 ), A 2 (peak position: 1656 cm −1 , half-value width: 50 cm −1 ) and A 3 (peak position: 1631 cm −1 , half-value width: 50 cm −1 ), so that an absolute value of a difference between an area of the infrared absorption band derived from the lipase and a sum of the areas of the three normal distributions becomes a minimum, and in the selection step, an immobilized lipase in which the index value is 0.9 or more is selected as a good-quality product.

10 . The method according to claim 6 , wherein
the index value is a value obtained by subjecting an infrared absorption band derived from a lipase appearing around 1600 to 1700 cm −1 to waveform separation to obtain five normal distributions A 1 , A 2 , A 3 , A 4 and A 5 , and then dividing an area of A 3 by a sum of areas of A 1 , A 2 , A 4 and A 5 , with regard to the waveform separation, the infrared absorption band is subjected to the waveform separation to obtain the five normal distributions, A 1 (peak position: 1685 cm −1 , half-value width: 30 cm −1 ), A 2 (peak position: 1670 cm −1 , half-value width: 30 cm −1 ), A 3 (peak position: 1656 cm −1 , half-value width: 30 cm −1 ), A 4 (peak position: 1641 cm −1 , half-value width: 30 cm −1 ) and A 5 (peak position: 1626 cm −1 , half-value width: 30 cm −1 ), so that an absolute value of a difference between an area of the infrared absorption band derived from the lipase and a sum of the areas of the five normal distributions becomes a minimum, and in the selection step, an immobilized lipase in which the index value is 0.35 or more is selected as a good-quality product.

11 . The method according to claim 6 , wherein
the index value is a value obtained by subjecting an infrared absorption band derived from a lipase appearing around 1600 to 1700 cm −1 to waveform separation to obtain eight normal distributions A 1 , A 2 , A 3 , A 4 , A 5 , A 6 , A 7 and A 8 , and then dividing a sum of areas of A 4 and A 5 by a sum of areas of A 1 , A 2 , A 3 , A 6 , A 7 and A 8 , with regard to the waveform separation, the infrared absorption band is subjected to the waveform separation to obtain the eight normal distributions, A 1 (peak position: 1692 cm −1 , half-value width: 19 cm −1 ), A 2 (peak position: 1682 cm −1 , half-value width: 19 cm −1 ), A 3 (peak position: 1670 cm −1 , half-value width: 19 cm −1 ), A 4 (peak position: 1658 cm −1 , half-value width: 19 cm −1 ), A 5 (peak position: 1648 cm −1 , half-value width: 19 cm −1 ), A 6 (peak position: 1638 cm−1, half-value width: 19 cm −1 ), A 7 (peak position: 1629 cm −1 , half-value width: 19 cm −1 ) and A 8 (peak position: 1619 cm −1 , half-value width: 19 cm −1 ), so that an absolute value of a difference between an area of the infrared absorption band derived from the lipase and a sum of the areas of the eight normal distributions becomes a minimum, and in the selection step, an immobilized lipase in which the index value is 0.6 or more is selected as a good-quality product.

12 . The method according to claim 6 , wherein
the index value is a value obtained by subjecting an infrared absorption band derived from a lipase appearing around 1600 to 1700 cm −1 to waveform separation to obtain ei

Assignees

• Ube Industries

Inventors

• Suzuki Katsuki
• Yoshida Youichi

Classifications

• C07K16/00 Primary

  Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies · CPC title

• C07K1/00

  General methods for the preparation of peptides {, i.e. processes for the organic chemical preparation of peptides or proteins of any length} · CPC title

• G01N21/35

  using infrared light (G01N21/39 takes precedence) · CPC title

• C12N9/20

  Triglyceride splitting, e.g. by means of lipase · CPC title

• C12N11/14

  Enzymes or microbial cells immobilised on or in an inorganic carrier · CPC title