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A kind of mutant enzyme for synthesizing monoglucose diglyceride and its preparation method and application

A technology of diglyceride and mutant is applied in the field of bioengineering, which can solve the problems of low synthesis reaction efficiency, low content of glyceroglycolipids, and many synthesis steps, and achieve the effects of good industrial application prospect, high conversion rate and high purity.

Active Publication Date: 2022-04-15
ZHENGZHOU UNIVERSITY OF LIGHT INDUSTRY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

The common extraction method is liquid-liquid extraction (organic solvent is chloroform / methanol (2:1, v / v)), the extracted lipids not only contain glyceroglycolipids, but also neutral lipids, free fatty acids and Phospholipids need to be further separated and purified by thin-layer chromatography / solid-phase extraction / high-performance liquid chromatography. This method has low content of glyceroglycolipids in plant resources, low yield (no more than 20%) and low yield, etc. Problems (The Journal of Biological Chemistry, 1957, 226:497-509; Journal of Lipid Research, 2006, 47:804-814.); chemical synthesis of glyceroglycolipids has side reactions, more synthesis steps, and lower synthesis reaction efficiency Low-level issues (Chinese Journal of Chemistry, 2009, 27: 2217-2222; Li Chunxia, ​​Preliminary study on the synthesis and structure-activity relationship of glyceroglycolipids [D], Qingdao Ocean University, 2002)

Method used

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  • A kind of mutant enzyme for synthesizing monoglucose diglyceride and its preparation method and application
  • A kind of mutant enzyme for synthesizing monoglucose diglyceride and its preparation method and application
  • A kind of mutant enzyme for synthesizing monoglucose diglyceride and its preparation method and application

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Experimental program
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Effect test

Embodiment 1

[0028] A synthetic marine bacterium ( Candidatus pelagibacter sp. HTCC7211) A method for preparing mutant enzymes of glyceroglycolipid synthase Agt and monoglucose diglyceride, the steps are as follows

[0029] (1) Strain, enzyme and culture medium:

[0030] Escherichia coli Escherichia coli DH5α, restriction endonuclease Nde I and Sal I, Pyrobest DNA polymerase, DNA ligation kit kit ver.2.1, bacterial genome DNA extraction kit ver.3.0 were purchased from TAKARA company; Escherichia coli Escherichia coli BL21-CodonPlus(DE3)-RIL was purchased from Novagen; LB medium: containing 10 g of Tryptone, 5 g of Yeast extract, and 10 g of NaCl per liter; the concentration of ampicillin was 100 mg / L, and the concentration of chloramphenicol was 34 mg / L L; Cloning and expression vector pET15b is preserved in our laboratory.

[0031] (2) Marine bacteria C. pelagibacter sp. HTCC7211 culture

[0032] marine bacteria C. pelagibacter sp. HTCC7211 was cultured according to the ref...

Embodiment 2

[0040] Activity analysis of glyceroglycolipid synthase

[0041] The enzymatic standard reaction system (1 mL) of glyceroglycolipid synthase is as follows: 0.1 mM UDP-glucose, 0.1 mM diglyceride (sn-glycerol-1-stearate-2-oleate), 2.0 μM purified Protein and 50 mM Tris-HCl (pH 8.5) reaction buffer solution. The above reaction system without enzyme solution was used as the control group, the reaction temperature was 35°C, and the reaction temperature was incubated at 200 rpm for 20 hours with constant shaking, and the enzyme-catalyzed reaction products were detected by liquid chromatography-mass spectrometry (LC-MS). One unit of enzyme activity: the amount of enzyme required to catalyze the synthesis of 1 µmol of monoglucose and diglyceride per minute under standard conditions.

[0042] UDP-glucose and diglyceride (sn-glycerol-1-stearate-2-oleate) were used as substrates to compare and analyze the catalytic activity of glyceroglycolipid synthase Agt and mutant T257S. The Michae...

Embodiment 3

[0049] Establishment of Optimum Process Conditions for Synthesis of Monoglucose Diglyceride Catalyzed by Glyceroglycolipid Synthase Mutant T257S

[0050] Using UDP-glucose and diglyceride (sn-glycerol-1-stearate-2-oleate) as substrates, optimize the reaction temperature, reaction pH, mutant T257S enzyme concentration and diglyceride concentration Process conditions for the synthesis of monoglucose and diglycerides catalyzed by the glyceroglycolipid synthase mutant T257S. After optimization, the optimal reaction conditions for the synthesis of monoglucose and diglycerides catalyzed by the mutant T257S of glyceroglycolipid synthase were enzyme concentration 8 U / mL, UDP-glucose concentration 500 mg / L, reaction temperature 35°C and reaction pH 8.5. The result is as image 3 As shown, after reacting for 8 h, 424.5 mg / L monoglucose diglyceride was produced, while 365 mg / L diglyceride was consumed, according to the catalytic reaction formula Figure 4 , the calculated conversion ra...

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Abstract

The invention belongs to the field of bioengineering, in particular to a mutant enzyme for synthesizing monoglucose and diglyceride, a preparation method and application thereof. The nucleotide sequence of the mutant is shown in SEQ ID NO.1, and the amino acid sequence edited by nucleotides is shown in SEQ ID No.2. The marine bacterial glyceroglycolipid synthase mutant T257S prepared by the present application has good transglycosylation catalytic activity, and can efficiently convert UDP-glucose and diglyceride (sn-glycerol-1-stearate-2-oleate ), the conversion rate of monoglucose diglyceride reaches 68.5%, and the prepared monoglucose diglyceride has a high purity of more than 93.8%. It is suitable for the bioenzymatic preparation of glycolipid compound monoglucose diglyceride and has good industrial application prospect.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a mutant enzyme for synthesizing monoglucose and diglyceride, a preparation method and application thereof. Background technique [0002] Glyceroglycolipids are compounds in which sugars are linked to glycerolipids by glycosidic bonds, and mainly exist in seaweeds, cyanobacteria and higher plants. The basic structure of glyceroglycolipids consists of a 1,2-diacyl-sn-glycerol moiety, and its monosaccharide or oligosaccharide is attached to the sn-3 position of the glycerol backbone, mainly including monogalactosyl diacylcerol (monogalactosyl diacylcerol, MGal-DAG), monoglucosyl diacylcerol (MGlc-DAG), monogalactosyl monoacylglycerol (MGMG), digalactosyldiacylglycerol (DGDG) and thioisorhamna Sugar diglycerol (sulfoquinovosy ldiacylglycerol, SQDG), etc. Glyceroglycolipids have important biological activities, such as antitumor, antiviral and anti-inflammatory activities, and related...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/70C12P19/18C12P19/02
CPCC12N9/1051C12P19/18C12P19/02
Inventor 魏涛张志平杨旭赵彩梦赵雨哲黄申毛多斌段乃心王敏孟祥玉
Owner ZHENGZHOU UNIVERSITY OF LIGHT INDUSTRY
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