Monoacyl-diacyl lipase, and coding gene and application thereof

A mono- and di-acyl lipase and gene technology, applied in the field of biology, can solve the problems of low enzyme activity, limited application, and small number of mono- and di-acyl lipases, and achieve high enzyme activity and reduce the cost of separation and purification.

Inactive Publication Date: 2010-11-03
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

This patented technology allows creation of specialized vectors that allow certain types or forms of lipid biosynthesis called diacylslipases (Bs) from yeast cells expressing them at very low levels. These Bs are then separated into their components through various methods like filtration techniques such as ultrafiltrations, centrifuges, gel electrophoresis, etc., which reduces costs compared to other ways they were previously used. Overall this process makes it possible to produce large amounts of these compounds efficiently while minimizing any side reactions during production.

Problems solved by technology

This patented technical solution involves developing a type called lipid oxidation enzymatics or LPE hereinafter referred to collectively as “lipases” based on similar feature(s). These enzymats are able to break down long chain triglycerides found naturally occurring within animal tissues like bone marrow extractives during manufacturing processes. They help reduce odors caused by oily ingredients while still giving good results when applied to other products made without these harmful effects.

Method used

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  • Monoacyl-diacyl lipase, and coding gene and application thereof
  • Monoacyl-diacyl lipase, and coding gene and application thereof
  • Monoacyl-diacyl lipase, and coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Preparation of Penicillium arcuates cDNA

[0026] 1.1 Extraction of total RNA from Penicillium arcuatens

[0027] (1) Take an appropriate amount of arctic Penicillium filaments, absorb the water with filter paper, grind with liquid nitrogen, add 1 ml of Trizol reagent (Invitrogen), shake on a shaker for 5 min, and let stand for 1 min at room temperature;

[0028] (2) add 0.2ml of chloroform, shake for 15 seconds, and let stand for 2min;

[0029] (3) 4℃, 12000rpm, 15min;

[0030] (4) Aspirate the supernatant, add an equal volume of isopropanol, and precipitate at -20°C for 30min;

[0031] (5) 4℃, 12000rpm, 15min;

[0032] (6) Pour off the supernatant, wash the precipitate with 1 ml of 75% ethanol, 7500rpm, 4°C, 5min;

[0033] (7) Repeat (6) step once;

[0034] (8) pour off the supernatant and dry for 10min;

[0035] (9) adding an appropriate amount of DEPC water to dissolve to obtain total RNA;

[0036] 1.2 Preparation of the first strand of Penicillium ...

Embodiment 2

[0045] Embodiment 2 Penicillium arcus mono-diacyl lipase gene mdl (9) primer design

[0046] 2.1 Primer design

[0047] According to the sequence of the mdl gene in GenBank (GenBank accession number is AF288219), the following pair of primers were designed and synthesized:

[0048] FW(P1): 5'-GACC GATGTTTCGACCAGCGAACT-3'

[0049] REV (P2): 5'-ATTT TTAAACCCTCTTGAATGGCA-3'

[0050] SnaBI and NotI restriction sites are designed at both ends of P1 and P2 respectively (see the italicized and underlined part in the above sequence)

[0051] 2.2 Penicillium arcuum mono-diacyl lipase gene mdl (9) PCR amplification

[0052] Using P1 and P2 primers, using Penicillium arcuatus (purchased from the Institute of Microbiology, Chinese Academy of Sciences, No. 3.4515) cDNA as a template, the PCR reaction system is:

[0053] Template DNA (10μg / μl) 1μl

[0054] 10×Buffer 2μl

[0055] dNTP (2.5mmol / μl) 0.5μl

[0056] primer (P1) (10pmol / μl) 0.8μl

[0057] primer (P2) (10pmol / μl) 0.8...

Embodiment 3

[0070] Embodiment 3 mono-diacyl lipase gene mdl (9) Secretory expression in Pichia pastoris GS115

[0071] 3.1 Preparation of Pichia pastoris GS115 (Invitrogen) Electroporation Competent Cells and Electroporation Transformation

[0072] (1) Pick a fresh single colony and put it in 5ml YPD liquid medium, cultivate it at 30°C and 250rpm for 12-14 hours;

[0073] (2) Inoculate 0.1% of the inoculum into a 2-liter Erlenmeyer flask containing 500 ml of YPD medium, and cultivate at 30° C. and 250 rpm for 12-14 hours to make OD600=1.3-1.5;

[0074] (3) Centrifuge at 1500 rpm for 5 minutes at 4°C to collect the cells;

[0075] (4) Wash the cells twice with 500-250ml ice-cold sterile water;

[0076] (5) Wash the cells once with 20ml of ice-cold 1M sorbitol solution;

[0077] (6) Resuspend the cells with 1ml of ice-cold 1M sorbitol solution to a final volume of about 1.5ml, and dispense 80μl into small centrifuge tubes;

[0078] 3.2 Electric shock transformation of Pichia pastoris y...

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Abstract

The invention provides a novel monoacyl-diacyl lipase Mdl (9), and a gene mdl (9) for coding the monoacyl-diacyl lipase. The amino acid sequence of the monoacyl-diacyl lipase is shown in SEQ ID No.2, and the nucleotide sequence for coding the monoacyl-diacyl lipase gene is shown in SEQ ID No.1. An experiment indicates that the monoacyl-diacyl lipase Mdl (9) has high activity of hydrolyzing monoacylglyceride and diacylglyceride. The invention also provides a method for secreting an expression carrier and effectively expressing the lipase in Pichia pastoris. By using the method, the expressed monoacyl-diacyl lipase can be effectively secreted out of cells, thereby lowering the cost for separation and purification and enhancing the expression efficiency. Under the condition of shake culture, when methanol is used as an inducer for induction culture, the exocytosis quantity can reach 660 mg/L; and when glycerin monostearate is used as a substrate, the enzyme activity reaches 2700 U/ml. The invention lays foundation for industrial application of the monoacyl-diacyl lipase.

Description

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Claims

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Application Information

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Owner CHINA AGRI UNIV
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