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Modified threonine transaldolase and application thereof

A threonine and transaldolase technology, applied in the field of enzyme engineering, can solve the problems of low conversion rate, difficult product separation and purification, poor stereoselectivity, etc.

Pending Publication Date: 2021-11-02
SUZHOU LEAD BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, this method still has defects such as low conversion rate, poor stereoselectivity, and difficult separation and purification of products, and does not have the conditions for industrialization

Method used

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  • Modified threonine transaldolase and application thereof
  • Modified threonine transaldolase and application thereof
  • Modified threonine transaldolase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0156] Example 1: Materials and methods

[0157] Unless otherwise specified, the experimental methods used in the present invention are all conventional methods, and details of gene cloning operations can be found in the aforementioned Sambrook et al., 1989.

[0158] i) Reagents and instruments:

[0159]DNA polymerase (PrimeSTAR Max DNAPolymerase) and DpnI endonuclease were purchased from TaKaRa Company;

[0160] The plasmid extraction kit was purchased from Axygen;

[0161] P-thymphenylbenzaldehyde was purchased from Aladdin, product number M185093, with a purity of 98%;

[0162] L-threonine was purchased from McLean, product number C10393311, analytically pure;

[0163] Pyridoxal phosphate was purchased from Aladdin, product number P136795, purity ≥ 98%;

[0164] Magnesium chloride was purchased from Aladdin, product number A2006034, analytically pure;

[0165] Oxidized nicotinamide adenine dinucleotide (NAD) was purchased from Aladdin, product number N196974, with a pu...

Embodiment 2

[0194] Embodiment 2, preparation and detection of the mutant of LTTA (LTTA01) of Pseudomonas fluorescens

[0195] Using the nucleic acid encoding LTTA01 (SEQ ID NO: 1) as a template, the mutant was prepared according to the method of Example 1. The obtained mutants are shown in Table 1. According to the method of Example 1, LTTA01 and its mutants were expressed by shaking flask culture, and enzyme powder was prepared. The activity of LTTA01 and its mutants was determined according to method 2 in item vi) of Example 1, and the results are shown in Table 1, wherein the relative activity refers to the activity of the mutant / the activity of LTTA01.

[0196] Table 1

[0197]

[0198]

Embodiment 3

[0199] Example 3: Preparation and detection of mutants of L-threonine transaldolase from different sources

[0200] According to the method of Example 1, a mutant comprising the substitution combination N35S-C57M-F70H was prepared based on the following LTTA-encoded nucleic acid:

[0201] - LTTA of Pseudomonas sp.34E 7: LTTA02, encoding nucleic acid SEQ ID NO: 3;

[0202] - LTTA of Pseudomonas sp. Irchel s3a18: LTTA03, encoding nucleic acid SEQ ID NO: 5; and

[0203] - LTTA of Chitiniphilus shinanonensis: LTTA04, encoding nucleic acid SEQ ID NO:7.

[0204] According to the method of Example 1, LTTA01 and its mutants were expressed by shaking flask culture, and enzyme powder was prepared. The activity of each wild-type LTTA and its mutants was measured according to method 2 in item vi) of Example 1, and the results are shown in Table 2, wherein the relative activity refers to the activity of the mutant / the activity of LTTA01.

[0205] Table 2

[0206] LTTA or mutan...

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Abstract

The present invention relates to a modified L-threonine transaldolase (LTTA). In particular, the modified LTTAs of the present invention have improved activity of catalyzing the reaction of contacting benzaldehyde or a derivative thereof with L-threonine compared to its beginning. The invention also relates to polynucleotides encoding the modified LTTAs of the invention, vectors and host cells expressing the modified LTTAs of the invention, and methods of producing 3-phenyl-L-serine and derivatives thereof using the modified LTTAs and host cells of the invention.

Description

technical field [0001] The present invention relates to the field of enzyme engineering. In particular, the present invention relates to a modified L-threonine transaldolase (LTTA) and its use in the production of 3-phenyl-L-serine and its derivatives. Background technique [0002] 3-phenyl-L-serine and its derivatives, the general structure of which is as follows: [0003] [0004] 3-Phenyl-L-serine and its derivatives are very important intermediates in organic synthesis, which are widely used in drug synthesis. For example: (2S,3R)-p-thymphenylphenylserine is a key intermediate for the synthesis of veterinary drugs florfenicol and thiamphenicol, and (2S,3R)-p-nitrophenylserine can be used as a compound for the antibacterial drug chloramphenicol key intermediates. In addition, 3-phenyl-L-serine and derivatives thereof can also prepare chiral aziridines (David Tanner, ChiralAziridines-Their Synthesis and Use in Stereoselective Transformations, Angew.Chem., Int.Ed. En...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/70C12N1/21C12P13/04C12R1/19
CPCC12N9/1022C12N15/70C12P13/04C12Y202/01C12N9/10C12N15/74
Inventor 谢新开梁晓亮黄晓飞
Owner SUZHOU LEAD BIOTECH CO LTD
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