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Candida tropicalis mutant strain and its application in the preparation of bcaa

A technology of Candida and mutant strains, applied in the direction of fermentation, fungi, and microorganism-based methods, etc., can solve the problems that enzymes cannot be recycled and reused, and L-alanine cannot be completely removed, so as to simplify separation and purification and Refining process, high product purity and high yield

Active Publication Date: 2022-01-14
INNOBIO CORP LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In order to simplify the separation, purification and refining process of the target amino acid, the patent CN200910047124.4 uses a combination of enzymes to catalyze the removal of alanine and some chiral amino acids in the reaction system, but the results cannot completely remove L-alanine acid, and enzymes cannot be recycled

Method used

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  • Candida tropicalis mutant strain and its application in the preparation of bcaa
  • Candida tropicalis mutant strain and its application in the preparation of bcaa
  • Candida tropicalis mutant strain and its application in the preparation of bcaa

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1. Acquisition and cultivation of yeast

[0031] Using Candida tropicalis (CICC No. 1253) as the starting strain, a mutant strain of Candida tropicalis IBEC-16 was obtained through ultraviolet mutagenesis and domestication screening. This yeast can specifically remove L-Ala and L-Glu , and in the presence of L-Ala and L-Glu, L-Ile, L-Val, and L-Leu cannot be utilized; under the culture conditions of adding certain inorganic salts, the bacteria can completely consume 30g / L L-propane contained in the medium acid.

[0032] The medium composition used was as follows:

[0033] Slant medium: glucose 5g / L, yeast extract 10g / L, peptone 10g / L, 15-20g / L agar, sterilized at 121°C for 20min.

[0034] Plate medium: glucose 5g / L, yeast extract 10g / L, peptone 10g / L, 15-20g / L agar, sterilized at 121°C for 20min.

[0035] Acclimatization medium: L-alanine 10-40g / L, inorganic salt (KH 2 PO 4 ·3H 2 O 2.5g / L, MgSO 4 ·7H 2 O1.0g / L, KCl 0.5g / L).

[0036] Shake flask mediu...

Embodiment 2

[0048] The BCAA fermented liquid used in this example is obtained by the following method: prepare L-valine fermented liquid according to the method described in the article "Research on L-valine Fermentation Technology", "Chinese Pharmaceutical Association Academic Annual Meeting", 2004.

[0049] Take a circle of activated mutant strain IBEC-16 and insert it into a Erlenmeyer flask containing YPD medium, place it in a shaker at 30°C and 220rpm and culture it for 16 hours; insert 10% of the inoculum into a 5L fermentation medium containing YPD medium In the tank, the initial ventilation rate is 0.7VVM, cultivated at 30°C, 200-700rpm, DO is controlled at 20%, pH is controlled at 5.0, and cultivated for about 6 hours. Adjust the pH of 3L BCAA fermentation broth to 6.0, insert Candida tropicalis, and react the initial OD 620 =5 or so, initial aeration volume 0.7VVM, cultivate under 30°C, 300-600rpm conditions, DO control at 30-50%, pH control at 5.5-6.0, sampling and measuring a...

Embodiment 3

[0053] Utilization of L-alanine and L-valine by Candida tropicalis CICC NO.1253 and its mutant strain IBEC-16

[0054] Take a circle of activated two kinds of bacteria and put them into Erlenmeyer flasks filled with 100ml YPD medium respectively, place them in a shaker at 30°C and 220rpm and culture them for 20h. The thalli were collected by centrifugation, and then respectively inserted into shake flask medium (L-valine 40g / L, L-alanine 30g / L, KH 2 PO 4 ·3H 2 O 2.5g / L, MgSO 4 ·7H 2 O 1.0g / L, KCl 0.5g / L), the reaction initial OD 620 = about 10, cultivated at 30°C and 220rpm for 24 hours, controlled the pH to about 5.5-6.0, took samples every 8 hours and analyzed the content of amino acids in the reaction solution by liquid chromatography, the results were as follows figure 1 shown. From figure 1 According to the results, the starting strain CICC NO.1253 can utilize L-alanine (L-Ala) and L-valine (L-Val) to varying degrees; L-valine will be utilized, and L-alanine wil...

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Abstract

A mutant strain of Candida tropicalis and its application in the preparation of BCAA. The preservation number of the mutant strain of Candida tropicalis is CGMCC No.17928. The yeast can specifically remove L-Ala, L-Glu, and does not utilize L-Ile, L-Val and L-Leu in the presence of L-Ala and L-Glu. The yeast is inserted into the BCAA fermentation broth for cultivation, and the filtrate is decolorized, desalted, concentrated and crystallized, and dried to obtain a high-purity BCAA product, and the product yield reaches more than 85%. Through a simple co-cultivation process, it can remove miscellaneous acids without affecting the yield of target amino acids, and the mutant strain cells can be reused. The whole process is environmentally friendly and easy to control, and effectively simplifies the separation, purification and refining process. The product has high purity and high yield, and the product can be directly used in the preparation of downstream products, which is suitable for large-scale industrial applications.

Description

Background technique [0001] L-leucine (L-Leu), L-valine (L-Val) and L-isoleucine (L-Ile) belong to branched-chain amino acids, and are also widely used in food, health products, feed, etc. Applications. In terms of sports nutrition, branched-chain amino acids help promote muscle recovery after training, and are especially suitable for bodybuilders and athletes as dietary supplements. [0002] In the process of producing branched-chain amino acids by fermentation, ion exchange method and multi-step crystallization method are often used to remove impurity acids. Due to the similar physical and chemical properties of impurity acids and products, the yield of purification and refining is affected. Among the impurity acids that are difficult to remove L-alanine (L-Ala) accounts for a large proportion. Patent CN201510744314.7 uses an ion exchange column to separate L-valine and impurity amino acids, the extraction yield of L-valine is about 68%, and the production wastewater is re...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/16C12P13/08C12P13/06C12R1/74
CPCC12N1/16C12P13/08C12P13/06C12R2001/74C12N1/165
Inventor 范超洪皓刘军孙博齐佳琨吴文忠
Owner INNOBIO CORP LTD
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