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Method for catalyzing and synthesizing glucose myristate through yeast show lipase
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A technology of myristate and lipase, which is applied in the field of bioengineering, can solve the problems of limited commercial application, high production cost, complicated separation work, etc., and achieve the effect of improving operational stability and inhibiting side reactions
Inactive Publication Date: 2013-08-14
ZHEJIANG UNIV
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[0003] The currently commercially available sugar ester products are basically chemically synthesized, and the products are complex mixtures. Complicated separation work is required to obtain high-purity products
Lipase is currently the most widely used enzyme in the synthesis of sugar esters, but its commercial application is greatly limited by the high production cost and complicated and time-consuming immobilization process.
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Embodiment 1
[0019] Example 1 Preparation of Yeast Displaying Lipase
[0020] Synthesize the lipase gene (Genbank number: AF229435) of Rhizopus oryzae and the cell wall α-lectin gene of Pichia pastoris GS115 (Genbank number: M28164) by artificial synthesis, and add Connect the peptide sequence GSSGGSGGSGGSGGSGS(linker), and get the nucleotide sequence pro-ROL-linker-α-agglutinin after connection, and add EcoR I and Not I restriction sites at both ends of the sequence, where pro-ROL is the lipase gene , α-agglutinin is the cell wall α lectin gene.
[0021] Using the above artificially synthesized sequence as a template, PCR amplification was performed using the following primer pair,
[0024] The PCR reaction system is: 1 μl of template DNA, 0.5 μl of high-fidelity DNA polymerase, 0.4 μl of dNTP (50 mM), 0.5 μl of upstream and downstream primers, 5 μl of 10×PCR ...
Embodiment 2
[0028] Example 2 yeast display lipase catalyzed synthesis of glucose myristate
example 1
[0029] Example 1 Take 0.2 g of glucose and 0.5 g of myristic acid, add them to a stoppered Erlenmeyer flask containing 10 mL of acetone, mix and preheat for 10 minutes, then add 0.01 g of yeast-displayed lipase, and place in a water bath shaker to start the reaction at a speed of 200 rpm Minutes, the reaction temperature was kept at 50°C, after 12 hours of reaction, 0.5g molecular sieve (pore size less than 2nm) was added, after the reaction was continued for 12 hours, the yeast display lipase and molecular sieve were removed by centrifugation, and the supernatant was removed by rotary evaporation to remove the organic solvent, washed with water for 3 After the second time, add n-hexane to crystallize at 4°C to obtain the glucomyristate product, which can be dried and pulverized.
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Abstract
The invention discloses a method for catalyzing and synthesizing glucose myristate through yeast show lipase, comprising the following steps of: dissolving glucose and myristic acid in an organic solvent; adding the yeast show lipase; reacting at 50-60 degrees centigrade for 10-14 h; separating and purifying to obtain the glucose myristate; transforming linearly treated recombinant plasmids into pichia pastoris GS115; inoculating the obtained transformant into a BMMY (buffered methanol-complex medium) culture medium; after inducing and culturing for 72-144 h, centrifugally collecting thallus;and washing, biologically imprinting, freezing and drying the thallus so as to obtain the yeast show lipase. By showing lipase outside cells, the glucose myristate is catalyzed and synthesized through the lipase preparation. By means of the method disclosed by the invention, the transformation efficiency is improved; furthermore, the reaction time is shortened; and the production cost is reduced.
Description
technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for synthesizing glucomyristate catalyzed by yeast display lipase. Background technique [0002] Sugar esters are non-ionic surfactants with carbohydrates as hydrophilic groups and fatty acids as hydrophobic groups. They have the advantages of non-toxicity, easy biodegradation, wide range of HLB values (1-16) and good surface activity, and It has the functions of dispersing and lubricating, decontaminating, foaming, adjusting viscosity, preventing aging, preventing crystallization, and antibacterial properties, so it is widely used in food, medicine, cosmetics and other industries. It is the United Nations Food and Agriculture Organization (FAO) and the World Health Organization ( WHO) recommended food additives. [0003] The currently commercially available sugar ester products are basically chemically synthesized, and the products are complex mixtures. Compli...
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