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Lipase and mutant thereof

A lipase and mutant technology, applied in the field of lipase and its mutants, can solve problems such as high probability, small workload, and no change in enzymatic properties, so as to reduce feed-to-meat ratio, improve production performance, and improve utilization rate Effect

Active Publication Date: 2014-05-07
QINGDAO VLAND BIOTECH GRP
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows for creating novel types of lipases called LPA 1 (lipid ester hydrolasing proteins) which are very effective at breaking down oily or greasy fats into smaller molecules like triglycerides instead of losing them themselves during digestion process. These lipids have specific properties such as being able to absorb water vapor when heated up while still keeping it soluble even if kept dry out under normal conditions. By modifying these lipid lytic enzymes based upon their optimal acidic pH values, they may also increase their effectiveness in promoting absorption of certain nutrients found naturally occurring within animal tissues. Overall, this innovative technical solution provides an efficient way to breakdown olfactors through hydro lysis without compromising beneficial ones' health benefits over time.

Problems solved by technology

This patented technical problem addressed in this patents relates to finding and cloning high performance lipases from bacteria called Acremonium sporogenum LB-19(Asp.) However, previous attempts were limited due to lack of proper expression systems during fermentations.

Method used

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  • Lipase and mutant thereof
  • Lipase and mutant thereof
  • Lipase and mutant thereof

Examples

Experimental program
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Embodiment 1

[0040] The amplification of embodiment 1 lipase gene

[0041] (1) Extract total genomic DNA from Aspergillus tubingensis

[0042]Aspergillus tubingensis 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 minutes, and the supernatant was discarded; 400 μl of extraction buffer (100 mM TrisHCl, 100 mM EDTA, 250 mM NaCl, 1% SDS ); then add 100mg of quartz sand or glass beads, shake vigorously in a bead beating instrument for about 2min; add 200μl 10MNH4AC in a water bath for 20min in a water bath at 65°C for 10min; centrifuge at 13000rpm for 10min to take the supernatant, then add 2 times the volume of absolute ethanol, Place at -20°C for 30min; centrifuge at 13000rpm for 10min, discard the supernatant, wash twice with 70% ethanol; air dry, add appropriate amount of water to dissolve and store at -20°C.

[0043] (2) Gene cloning

[0044] Using the extracted total gen...

Embodiment 2

[0051] Construction of embodiment 2 expression vector

[0052] The gene sequence SEQ ID NO:2 cloned in Example 1 was subjected to KpnI and XbaI double enzyme digestion overnight, and then the target gene fragment was recovered from the gel; similarly, KpnI and XbaI were performed on the Trichoderma expression vector pTG (containing hygromycin hph gene). XbaI double enzyme digestion and recovery; the recovered gene fragment and the vector were ligated overnight at 16°C, and transformed into E. coli DH5a, and finally the Trichoderma recombinant expression plasmids were obtained, which were named pTG-Lip-1.

[0053] Using the same method as above, a recombinant expression plasmid carrying SEQ ID NO:4 was constructed and named pTG-Lip.

Embodiment 3

[0054] Embodiment 3 transforms and screens

[0055] (1) Protoplast preparation

[0056] Inoculate Trichoderma reesei mycelium and grow on PDA plate for 4 days; cut out a colony with a diameter of about 3 cm and place it in about 60ml of YEG (0.5% yeast powder, 1% glucose) liquid medium, shake at 200rpm at 30°C Cultivate overnight; collect the mycelium by filtering with multi-layer gauze; place the mycelia in 10-20ml of lysing enzyme solution (SigmaL1412) for 2-3 hours to enzymatically hydrolyze; take out the enzymatic hydrolysis solution, add 0.7M NaCl solution, shake gently, and pour in Filter with three layers of sterilized lens tissue, collect the filtrate, centrifuge at 3000rpm for 10min; discard the supernatant, add 10-20ml STC solution (20% sucrose, 50mM Tris-Cl, 50mM CaCl 2 ) to suspend, then centrifuge at 3000rpm for 10min; add an appropriate amount of STC to suspend and aliquot (150μl / tube, 10 8 pieces / ml).

[0057] (2) Transformation and verification

[0058] Tak...

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Abstract

The invention provides novel lipase and mutant thereof. The amino acid sequence of the lipase is SEQ ID NO.1. The novel lipase lip-1 is obtained in a cloning manner from aspergillus tubingensis, the optimal acting pH value is 6.5, and the optimal acting temperature is 40 DEG C; more than 52.3 percent of enzyme activity can be reserved in the pH value range of 4.0 to 7.0. Mutation is executed on the basis of the lipase lip-1 to obtain three single-point mutants, the tolerance of the three mutants for trypsin can be respectively improved by 34.64 percent, 36.30 percent and 53.43 percent compared with the tolerance before the mutation, so that the application of the mutant in the feed additive field can be favored. By adopting the lipase lip-1 and the three mutants, the utilization rate of grease substances in the feed can be effectively increased, so that the food consumption and daily weight increment of broiler chicken can be improved, the feed-to-meat ratio can be reduced, the productivity of the cultured animal can be improved, and the application prospect is wide.

Description

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Claims

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Application Information

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Owner QINGDAO VLAND BIOTECH GRP
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