Method for catalyzing and synthesizing maltose laurate through yeast show lipase
A technology of laurate ester and lipase, which is applied in the field of bioengineering, can solve the problems of high production cost, limited commercial application, lengthy protection and deprotection steps, and achieve the effect of inhibiting side reactions and improving operational stability
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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,
[0022] Upstream primer: 5'-AAGGAAAAAAAGAATTCGTTCCAGTTTCTGG-3';
[0023] Downstream primer: 5'-TTTTCCTTTTGCGGCCGCTAATGAAACG-3'
[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 shows lipase catalyzed synthesis of maltose laurate
example 1
[0029] Example 1 Take 0.2g of maltose and 0.5g of lauric acid, add them to a stoppered Erlenmeyer flask containing 10mL of acetone, mix and preheat for 10min, then add 0.01g of yeast-displayed lipase, place in a water bath shaker to start the reaction, and the rotation speed is 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 and crystallize at 4°C to obtain maltose laurate product, which can be dried and pulverized.
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