Fusion protein for catalyzing glucose to synthesize D-psicose and construction method thereof

A technology of psicose and fusion protein, applied in chemical instruments and methods, methods based on microorganisms, biochemical equipment and methods, etc., can solve the problems of high cost, difficult process and complicated purification of chemical synthesis methods, and achieve The effect of reducing production cost, not easy to pollute, process safety and environmental protection

Pending Publication Date: 2022-06-07
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At first, D-psicose was synthesized by chemical method. With the deepening of research, the chemical synthesis method has the disadvantages of high cost, difficult process, complicated purification, easy to cause environmental pollution, etc., and is gradually replaced by biotransformation method

Method used

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  • Fusion protein for catalyzing glucose to synthesize D-psicose and construction method thereof
  • Fusion protein for catalyzing glucose to synthesize D-psicose and construction method thereof
  • Fusion protein for catalyzing glucose to synthesize D-psicose and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Functional verification of glucose isomerase and D-psicose isomerase

[0028] Escherichia coli BL21 / AcceGI and D-psicose 3-epimerase encoding genes (CcDPEase, SEQ ID NO: 16) containing the gene encoding glucose isomerase (AcceGI, SEQ ID NO: 15) and D-psicose 3-epimerase, respectively BL21 / CcDPEase was cultured overnight in LB medium at 37°C and 200rpm, and transferred to a 250mL conical flask containing 25mL of fresh LB medium at an inoculum of 4% (v / v) at 37°C and 200rpm. Cultured to log phase. IPTG with a final concentration of 500 μM was added, and the culture was continued at 25° C. and 200 rpm for 8 h to express the gene of glucose isomerase and D-psicose 3-epimerase. Glucose and D-fructose were added respectively to verify the functions of glucose isomerase and D-psicose isomerase. E. coli BL21 / pET28a(PB)N with empty plasmid pET28a(PB)N was used as blank control, and other operating conditions were the same.

[0029] Add glucose at a final concentrat...

Embodiment 2

[0033] Example 2: Construction and functional analysis of fusion proteins

[0034] The gene encoding glucose isomerase (AcceGI, SEQ ID NO: 15) derived from Acidothermus cellulolyticus 11B and D-psicose 3-epimerase (CcDPEase, SEQ ID NO: 16) derived from Clostridium cellulolyticum H10 ), respectively cloned into the vector pET28a(PB)N between the BamHI and HindIII sites of the vector pET28a(PB)N or the BamHI site of the vector pET28a(PB)N-CcDPEase (see primers) Table 1) to obtain recombinant plasmids pET28a(PB)N-GP, pET28a(PB)N-GS1P, pET28a(PB)N-GS2P, pET28a(PB)N-GS3P, pET28a(PB)N-GE1P, pET28a(PB)N-GE1P ) N-GE2P and pET28a(PB)N-GE3P (see Table 2 for the linking peptides used, and the plasmid map is shown in image 3 shown). Among them, the expression of all pathway genes is controlled by T7 promoter and T7 terminator. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) to obtain recombinant Escherichia coli BL21 / GP, BL21 / GS1P, BL21 / GS2P, BL21 / GS3P, BL21 / GE1...

Embodiment 3

[0045] Example 3: Optimization of reaction conditions for the generation of D-psicose from glucose catalyzed by fusion proteins GE3P and GS3P

[0046] In order to improve the yield and conversion rate of the one-step conversion of glucose to D-psicose catalyzed by the fusion protein, the present invention further optimizes the reaction conditions for converting D-psicose.

[0047] The invention optimizes the reaction conditions for the conversion of glucose to D-psicose: the temperature range of the reaction is 45-85° C., the pH range of the reaction is 4.5-8.5, and the concentration of the substrate glucose is 100 g / L. The results showed that the optimum reaction temperature was 65°C ( Image 6 A), the optimum reaction pH is 7.5 ( Image 6 B).

[0048] Under the above optimal conditions, the yield of D-psicose can reach 5.69 g / L. The results show that the method for catalyzing the generation of D-psicose from glucose by using the fusion protein constructed in the present i...

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Abstract

The invention provides a fusion protein for catalyzing glucose to synthesize D-psicose and a construction method of the fusion protein, and relates to the field of biochemical engineering. In order to avoid the problems of low conversion efficiency and the like caused by diffusion of an intermediate product in a double-enzyme catalysis system, the fusion protein connected by connecting peptides with different lengths is constructed, and the conversion efficiency is improved by constructing a substrate channel. Specifically, a glucose isomerase gene and D-psicose 3-epimerase are connected by using a flexible connecting peptide and a rigid connecting peptide with different lengths, and a result shows that the synthesis efficiency of D psicose is improved along with the increase of the length of the connecting peptide, and the rigid connecting peptide is superior to the flexible connecting peptide. According to the fusion protein provided by the invention, an intermediate product is transferred to a next enzyme through a substrate channel, so that diffusion of the intermediate product is reduced, and the synthesis efficiency of D-psicose is improved.

Description

technical field [0001] The invention belongs to the field of biochemical industry, and in particular relates to a fusion protein that catalyzes the synthesis of D-psicose from glucose and a construction method thereof. Background technique [0002] D-psicose (D-Psicose or D-Allulose) is a six-carbon sugar with very low content in nature and is an epimer of D-fructose at the C-3 site. D-psicose is difficult to be digested and absorbed, and provides little energy for life activities, so it is a very useful low-calorie sweetener. In the field of medicine and health, D-psicose can inhibit fatty liver enzymes and intestinal α-glucosidase, thereby reducing the accumulation of body fat and inhibiting the rise of blood sugar concentration. In the field of food application, D-psicose has the advantages of high sweetness, good solubility, low calorie and low blood sugar response, and is considered as one of the most ideal sucrose substitutes. At present, D-psicose has been certified...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/92C12N9/90C12N15/62C12N15/70C12N1/21C12P19/24C12P19/02C12R1/19
CPCC12N9/92C12N9/90C12N15/70C12P19/24C12P19/02C12Y503/01018C12Y501/03C07K2319/00Y02E50/10
Inventor 许敬亮吕永坤贾箐朱丽娟张辉赵安琪熊文龙阿拉牧屈凌波应汉杰王诗元
Owner ZHENGZHOU UNIV
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