Method for preparing optical pure L-tertiary leucine from active inclusion body

A technology of tert-leucine and inclusion bodies, applied in the field of bioengineering, to achieve the effects of simple process flow, reduced production cost, and easy separation and purification

Active Publication Date: 2018-03-30
XIAMEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no precedent for preparing L-Tle or other products by constructing bifunctional enzymes to prepare active inclusion bodies. Therefore, genetic engineering methods a

Method used

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  • Method for preparing optical pure L-tertiary leucine from active inclusion body
  • Method for preparing optical pure L-tertiary leucine from active inclusion body
  • Method for preparing optical pure L-tertiary leucine from active inclusion body

Examples

Experimental program
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Effect test

Embodiment 1

[0043] Construction of FDH-LeuDH Bifunctional Enzyme Recombinant Strain

[0044] Overlap extension polymerase chain reaction (OE-PCR) was used to construct the FDH-LeuDH fusion enzyme mediated by different connecting peptides, and the construction of the FDH-R1-LeuDH fusion enzyme gene was taken as an example to describe the construction process. First, design primers based on LeuDH, FDH, connecting peptide sequence and restriction site on pET28a plasmid:

[0045] P1: 5'-GGAATTC CATATG AAAATTGTCCTGGTCCTGT-3' (SEQ ID NO 05), the underline is the NdeI restriction site sequence.

[0046] Connecting peptide primers:

[0047] 5’-GCCTATGGCAAACACGATAAAAAG XXX ATGACATTGGAAATCTTCGA-3', XXX refers to the connecting peptide sequence, see Table 1 for details.

[0048] P3: 5'-ATGACATTGGAAATCTTCGAATAT-3' (SEQ ID NO 06).

[0049] P4: 5'-CCG CTCGAG TTACCGGCGACTAATGATGT-3' (SEQ ID NO 07), the underline is the XhoI restriction site sequence.

[0050] Using FDH and LeuDH genes as templ...

Embodiment 2

[0058] Preparation of Bifunctional Enzyme Active Inclusion Body

[0059] The bifunctional enzyme recombinant strain was inoculated in LB medium, activated overnight at 37°C, 200rpm, then transferred to Lb medium with an inoculum size of 1%, cultivated at 37°C, 200rpm until the OD600 was about 0.5, and added a final concentration of 0.2 mM IPTG, 16°C, 200rpm induced expression for 24h. After the cultivation was completed, the bacterial cells were collected, washed twice with PBS buffer (pH=7.2), and stored at -80°C until use. Whole-cell SDS-PAGE was used to verify whether the recombinant bifunctional enzyme was successfully expressed, and the results were as follows: figure 2 As shown, where M represents the protein marker, bands 1-9 are FDH single enzyme, LeuDH single enzyme, FDH-DL-LeuDH, FDH-S1-LeuDH, FDH-S2-LeuDH, FDH-S3-LeuDH, FDH-R1 -LeuDH, FDH-R2-LeuDH and FDH-R3-LeuDH, it can be seen that the bifunctional enzymes mediated by the seven connecting peptides were all suc...

Embodiment 3

[0062] Characterization of Bifunctional Enzyme Active Inclusion Body

[0063] The morphology of inclusion bodies was directly observed by scanning electron microscopy. The sample preparation method is as follows: 5 μL of the inclusion body sample was dropped on a single crystal silicon wafer, air-dried overnight, and then coated with about 2 nm thick platinum in a JFC-1600 (JEOL, Tokyo, Japan) sputtering apparatus (sputtering condition : 10mA, 30s), and then put the coated sample into a field emission Sigma scanning electron microscope (Carl-Zeiss AG, Germany) for observation. image 3 It is the SEM structure diagram of partial inclusion body, where A is FDH-R1-LeuDH, B is FDH-R2-LeuDH, C is FDH-S1-LeuDH, D is FDH-S2-LeuDH, it can be seen that the rigid linker peptide mediates The inclusion body with bifunctional enzyme activity showed a sheet structure, while the inclusion body with bifunctional enzyme activity mediated by a flexible linker peptide presented an irregular bal...

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Abstract

The invention discloses a method for preparing optical pure L-tertiary leucine from an active inclusion body. The method comprises the following steps: (1) preparing a difunctional enzymatic activityinclusion body, wherein the difunctional enzymatic activity inclusion body comprises an active component, namely a fused difunctional enzyme; the fused difunctional enzyme comprises leucine dehydrogenase, LeuDH part connected and a polymerase part which are connected through a connecting peptide; the polymerase part is used for coenzyme NAD+ (Nicotinamide Adenine Dinucleotide+) regeneration; (2) putting the difunctional enzymatic activity inclusion body into a reaction mixed liquid with the pH value of 6.0-10.0, re-suspending, performing a reaction at 20-40 DEG C, and controlling the pH valueto be 6.0-10.0 in the reaction period, wherein the reaction mixed liquid comprises 50-1000mM of trimethyl pyroracemic acid, 50-1000mM of ammonium formate and 0.05-5mM of coenzyme NAD+.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for preparing optically pure L-tert-leucine by utilizing active inclusion bodies. Background technique [0002] The tert-butyl group in the structure of L-tert-leucine amino acid (L-Tle) is beneficial to the reaction from the back due to the large steric hindrance, so L-Tle and its derivatives are often used as catalysts for inducing asymmetric reactions, so The generated products are characterized by high selectivity, and are often used as templates for inducing asymmetric synthesis reactions, and are widely used in asymmetric synthesis. In addition, the tert-butyl structure of L-Tle has strong hydrophobicity, which can effectively control the molecular configuration; in polypeptide components, L-Tle is gradually replacing Val, Leu and Ile, because it can enhance the hydrophobicity and Stability against enzymatic degradation. [0003] L-Tle has a wi...

Claims

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Application Information

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IPC IPC(8): C12P13/04C12N9/06C12N9/02
CPCC07K2319/00C12N9/0008C12N9/0016C12P13/04C12Y102/01002C12Y104/01009
Inventor 方柏山张永辉
Owner XIAMEN UNIV
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