High-thermal-stability mutant of D-allulose-3-epimerase and application thereof

A technology of epimerase and high thermal stability, applied in the field of genetic engineering, can solve problems such as unfavorable industrial application and promotion of D-psicose, and achieve the effects of improving thermal stability and prolonging the service life.

Active Publication Date: 2017-01-25
LIVINGZONE SHANGHAI BIO CHEM TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the resources of DPE enzymes with good thermal stability are still relatively scarce, which is not conducive to the industrial application of D-psicose

Method used

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  • High-thermal-stability mutant of D-allulose-3-epimerase and application thereof
  • High-thermal-stability mutant of D-allulose-3-epimerase and application thereof
  • High-thermal-stability mutant of D-allulose-3-epimerase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1. Establishment of a mutant library of D-psicose-3-epimerase

[0023] The BsDPE-pET29a (+) plasmid synthesized with the whole gene (synthesized by Changzhou Jiyu Biotechnology Co., Ltd., the nucleotide shown in SEQ ID NO.2 encoding the amino acid sequence shown in SEQ ID NO.1 is cloned on the plasmid sequence) as a template, using primers F1 and R1 as forward and reverse primers, respectively, to perform error-prone PCR to construct a mutant library.

[0024] The primer sequence is as follows: F1: 5'-GGAATTC CATATG AACAAAGTGGGC-3';R1:5'-CGC GGATCC TTATGCCAGTTTTTC-3', with NdeI and BamHI restriction enzyme sites at both ends.

[0025] The error-prone PCR reaction system is as follows: 10* PCR buffer 2.5μL, 10mM dGTP 0.5μL, 10mM dTTP 0.5μL, 10mM dCTP 2.5μL, 10mM dATP 2.5μL, 1mM MnCl 2 2.5 μL, 55 mM MgCl 2 2.5 μL, 10 μM F11 μL, 10 μM R1 1 μL, Template (10ng / μL) 1 μL, Taq DNA polymerase (5U / μL, takara) 0.2 μL, ddH 2 O make up to 25 μL.

[0026] Error-pron...

Embodiment 2

[0028] Example 2 Activation and induced expression of mutants

[0029] Pick a single clone from the plate cultured overnight to a 96-deep well plate filled with 1 mL of LB liquid medium with a final concentration of 50 μg / mL kanamycin sulfate, and culture overnight at 37°C and 220 rpm with shaking. On the next day, draw 100 μL of the culture solution from the 96 empty plates of the above overnight culture, and add it to a fresh 1 mL 96 deep-well plate containing LB liquid medium with a final concentration of 50 μg / mL kanamycin sulfate, at 37 ° C, 220 rpm After 4 hours of shaking culture, IPTG with a final concentration of 1 mM was added for induction, and then culture was continued at 30° C. for 20 hours. A total of 400 single clones were picked for activity screening.

[0030] Centrifuge the induced bacterial solution at 4°C and 4000 rpm for 15 min, and discard the supernatant. The cells were suspended in 100 μL of 50 mM sodium phosphate buffer (pH 7.0), added with lysozyme...

Embodiment 3

[0031] Example 3 Establishment of High Performance Liquid Chromatography HPLC High Throughput Screening Method

[0032]High performance liquid chromatography HPLC was carried out according to the following conditions: Shimadzu SHIMADZU LC-20A HPLC with RID detectoror equivalent; analytical column: Waters Sugar-Pak I, 6.5×300mm column; mobile phase: water; flow rate: 0.6mL / min; column temperature : 80°C; detector: RID, detector temperature 60°C.

[0033] Pure D-fructose and D-psicose produced by Sigma were used as standard products, and the sample volume was 20 μL. Chromatographic analysis results see figure 1 and 2 , the retention time of D-fructose is 9.389min ( figure 1 ), the retention time of D-psicose is 12.233min ( figure 2 ), the two have a high degree of separation, and can be used as a method for screening enzyme mutants.

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Abstract

The invention discloses a high-thermal-stability mutant of D-allulose-3-epimerase and an application of the high-thermal-stability mutant of D-allulose-3-epimerase. The 116th bits of serine of the mutant at D-allulose-3-epimerase is mutated to isoleucine, and the 251st bits of lysine is mutated to leucine, and a double mutant enzyme S116I/K251L is obtained. According to the invention, the 116th bit of serine (Ser, S) of D-allulose-3-epimerase BsDPE of Burkholderia sp. MR1 is mutated to isoleucine (Ile, I), and the 251st bits of lysine (Lys, K) is mutated to leucine (Leu, L), thus the thermal stability of the double mutant enzyme S116I/K251L obtained therefrom is significantly improved; and the half-life period at 60 DEG C is improved from the wild type 337.3 min to 417.9 min at present, and is 1.24 times o the wild type; the using cycle of the enzyme in compounding D-allulose is largely prolonged; the high-thermal-stability mutant of D-allulose-3-epimerase has important industrial application value.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a mutant of D-psicose-3-epimerase (DPE) with high thermal stability and its application. Background technique [0002] D-psicose-3-epimerase (D-Psicose-3-Epimerase, abbreviated as DPE) belongs to the class of epimerases, which can catalyze the epimerization of C3 hydroxyl groups of various ketoses. For example, catalyzing D-fructose to generate D-psicose is a good biocatalyst for producing rare sugar D-psicose. Since the content of D-psicose in nature is very small and it is extremely difficult to obtain, the biological preparation of D-psicose is a research hotspot in recent years. [0003] After protein directed evolution technology becomes an effective new strategy for transforming protein molecules, it can quickly produce new enzymes with great industrial application value without knowing the three-dimensional structure of proteins, which greatly promo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/90C12P19/24C12P19/02
Inventor 余允东祝俊
Owner LIVINGZONE SHANGHAI BIO CHEM TECH CO LTD
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