Lipase and engineering strain of recombinant expression thereof

A technology of lipase and engineering bacteria, applied in the field of microbial engineering, can solve the problems of hidden safety hazards and low expression level of Pichia pastoris, and achieve the effect of strong tolerance

Inactive Publication Date: 2013-03-20
QINGDAO VLAND BIOTECH GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, lipase products in the domestic market are mostly produced by heterologous expression of Pichia pastoris, which has the advantage of mature fermentation process;

Method used

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  • Lipase and engineering strain of recombinant expression thereof
  • Lipase and engineering strain of recombinant expression thereof
  • Lipase and engineering strain of recombinant expression thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 Construction of Aspergillus niger lipase gene expression vector

[0021] 1.1 Extraction of total genomic DNA from Aspergillus niger

[0022] Aspergillus niger (Aspergillus niger) was inoculated with shake flask medium for overnight culture, and an appropriate amount of bacteria was placed in a centrifuge tube, centrifuged at 13,000 rpm for 5 min, and the supernatant was discarded; 400 μl of extraction buffer (100 mM TrisHCl, 100 mM EDTA, 250 mM NaCl, 1% SDS); then add 100 mg of quartz sand or glass beads, shake vigorously in a bead beater for about 2 min; add 200 μl 10M NH4AC in a water bath for 20 min at 65°C and ice bath for 10 min; centrifuge at 13000 rpm for 10 min to take Add 2 times the volume of absolute ethanol to the supernatant and place at -20°C for 30 min; centrifuge at 13,000 rpm for 10 min to discard the supernatant, wash twice with 70% ethanol; air dry, add appropriate amount of water to dissolve and store at -20°C.

[0023] 1.2 Gene cloning

...

Embodiment 2

[0029] Example 2 Transformation and screening

[0030] 1.1 Protoplast preparation

[0031] Inoculate Trichoderma reesei mycelia and grow on PDA plates for 4 days; cut colonies with a diameter of about 3 cm and place them in liquid medium of about 60 ml YEG (0.5% yeast powder, 1% glucose) at 30°C, Shake culture at 200 rpm overnight; collect the mycelium by filtering with multi-layer gauze; place the mycelia in a 10-20 ml lysing enzyme solution (Sigma L1412) for 2-3 hours; take out the enzymatic solution, add 0.7 M NaCl solution, Shake lightly, pour on three layers of sterilized lens tissue and filter, collect the filtrate, centrifuge at 3000 rpm for 10 min; discard the supernatant, add 10-20 ml STC solution (20% sucrose, 50mM Tris-Cl, 50mM CaCl 2 ) suspension, 3000 rpm, centrifuged for 10 min; add an appropriate amount of STC suspension (150 μl / tube, 10 8 pieces / ml).

[0032] 1.2 Transformation and verification

[0033] Take 2 μg pKDN-ANL DNA and add it to 150 μl protoplast...

Embodiment 3

[0035] Example 3 Fermentation Verification and Enzymatic Properties Determination

[0036] The positive transformant Trichoderma reesei ZQ-1 and the starting strain (negative control) were inoculated in MM fermentation medium (1.5% glucose, 1.7% lactose, 2.5% corn steep liquor, 0.44% (NH 4 ) 2 SO 4 , 0.09%MgSO 4 , 2%KH 2 PO 4 , 0.04%CaCl 2 , 0.018% Tween-80, 0.018% trace elements, 0.018% polypropylene glycol-2000) culture, culture at 28°C for 48 hours, then culture at 25°C for 48 hours, take the supernatant for SDS-PAGE electrophoresis detection, the results are as follows figure 1 As shown, wherein the place indicated by the arrow is the recombinantly expressed lipase, indicating that the Trichoderma reesei engineered bacterium constructed in the present invention can recombinantly express the lipase of Aspergillus niger.

[0037] Optimum action pH analysis: the results of dilution test with buffers with pH values ​​of 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 showed that the op...

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Abstract

The invention provides an engineering strain of recombinant expression lipase. Lipase gene obtained by cloning in Aspergillus niger is transferred into Trichoderma reesei, so that Trichoderma reesei engineering strain can be constructed and has the preservation number of China Center for Type Culture Collection (CCTCC) No.: M2012483. The recombinant expression lipase has the optimal action pH of 5.0 and the optimal action temperature of 30 DEG C, and the tolerance of the recombinant expression lipase to gastric juice, pepsase and artificial intestinal juice can be enhanced. The lipase remarkably improves the utilization rate of oil matter in feed, thus remarkably reducing the addition of oil in the feed and reducing the cost of the feed.

Description

technical field [0001] The invention belongs to the technical field of microbial engineering, and in particular relates to a lipase gene and its recombinant expression engineering strain. Background technique [0002] Lipase is triacylglycerol acyl hydrolase, which catalyzes the hydrolysis of natural substrate oils to generate fatty acids, glycerol and mono- or di-glycerides. The basic unit of lipase is only amino acids, usually only one polypeptide chain. Its catalytic activity is only determined by its protein structure (Schmid et al., 1998). [0003] Lipase widely exists in animals, plants and microorganisms. Plants that contain more lipase are the seeds of oil crops, such as castor bean and rapeseed. When the oil seeds germinate, lipase can work synergistically with other enzymes to catalyze the decomposition of oils and fats to generate sugars, providing seeds for rooting Nutrients and energy necessary for germination; the pancreas and adipose tissue of higher animal...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12N15/55C12N1/15A23K1/165C12R1/885
Inventor 黄亦钧张青徐娟许丽红王华明吴秀秀
Owner QINGDAO VLAND BIOTECH GRP
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