Method for increasing lipase expression through glycosylation modification as well as mutant enzyme and application thereof

A lipase and Rhizopus oryzae lipase technology, applied in the field of enzyme engineering, can solve the problems of low expression and enzyme activity, and achieve the effect of improving enzyme activity and secretion.

Active Publication Date: 2015-07-08
TAIXING YIMING BIOLOGICAL PRODS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to overcome the defects of the existing Rhizopus oryzae lipase expression level and low enzyme activity, the present invention provides

Method used

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  • Method for increasing lipase expression through glycosylation modification as well as mutant enzyme and application thereof
  • Method for increasing lipase expression through glycosylation modification as well as mutant enzyme and application thereof
  • Method for increasing lipase expression through glycosylation modification as well as mutant enzyme and application thereof

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Effect test

Embodiment 1

[0033] Example 1: Introduction of N-glycosylated mutant enzymes and construction of genetic engineering

[0034] In the present invention, three kinds of N-glycosylation site mutants are designed in the leader peptide of Rhizopus oryzae lipase ROL, that is, SAS and NT amino acids are transformed into N-glycosylation sites NGT and NLT respectively, and the mutant enzymes are named as proROLA (amino acid sequence as shown in SEQ ID NO.1) and proROLB (amino acid sequence as shown in SEQ ID NO.2); naming of mutant enzymes that simultaneously transform SAS and NT into N-glycosylation sites It is proROLAB (the amino acid sequence is shown in SEQ ID NO.3). The specific mutation method of the leader peptide is as follows: figure 1 shown.

[0035] The specific construction method is as follows:

[0036] (1) With the carrier pPIC9K-proROL, pGAPZα of the Aspergillus oryzae lipase gene proROL containing the amino acid sequence shown in SEQ ID NO.4 as a template, simultaneously use rest...

Embodiment 2

[0041] Embodiment 2: the growth comparison of the genetically engineered bacteria expressing mutant enzyme and wild enzyme

[0042] Using GS115 / pGAPZα-proROL as a control, the growth of genetically engineered bacteria expressing the lipase mutant of Rhizopus oryzae was compared.

[0043] Shake flask fermentation conditions: Streak positive transformants on YPD-G418 plate ((w / v): yeast extract 1%, glucose 2%, agar powder 2%, tryptone 2%, G4180.025%), culture at 30°C 3d, pick a single clone and inoculate it into 100mLYPD liquid medium ((w / v): yeast extract 1%, glucose 2%, tryptone 2%), 30°C 200rpm·min -1 Shake flask culture, sampling every 12h or 24h.

[0044] Strain growth curve as figure 2 shown. The results showed that the growth of the mutant strains was similar to that of the control. The strains grew rapidly within 48 hours, and after 72 hours, the growth of each strain became slow and tended to be stable. It shows that the introduction of glycosylation site into the ...

Embodiment 3

[0045] Example 3: Effect of N-glycosylation on the Secretion Level of Rhizopus oryzae Lipase

[0046] The present invention compares the lipase secretion situation of genetically engineered bacteria expressing mutant enzymes and wild enzymes.

[0047] Each genetically engineered bacteria was fermented and cultured, and the specific culture conditions were: Streak the positive transformants on the YPD-G418 plate, culture at 30°C for 3 days, pick a single clone and inoculate it into 100mL YPD liquid medium, 30°C, 200rpm·min -1 Shake flask culture, sampling every 12h or 24h.

[0048] The result is as image 3 As shown, the protein concentration of rhizopus oryzae lipase proROL without glycosylation sites was the lowest, while the extracellular protein concentration of rhizopus oryzae lipase proROLAB with two glycosylation sites was slightly higher than that with only one sugar The lipases proROLA and proROLB at the sylation site were preliminarily judged that the introduced gly...

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Abstract

The invention discloses a method for increasing lipase expression through glycosylation modification as well as a mutant enzyme and an application thereof and belongs to the field of enzyme engineering. The N-glycosylation mutation is performed on a leading peptide sequence of rhizopus oryzae lipase, the SAS and/or NT amino acid are/is respectively modified to an N-glycosylation site NGT and/or NLT, the extracellular protein concentrations of the obtained mutant enzyme proROLA, proROLB and proROLAB are increased by 211%, 188% and 233% compared with those of the un-glycosylated proROL, the enzyme activities when culturing in a fermentation tank are respectively 8210 U.mL<-1>, 8457 U.ml<-1> and 9366 U.mL<-1>, and the un-mutated proROL extracellular enzyme activity is almost zero. The rhizopus oryzae lipase provided by the invention has obviously increased lipase enzyme activity and can be used in the fields such as food, chemical engineering and biological energy source.

Description

technical field [0001] The invention relates to a method for improving the expression of lipase through glycosylation modification, a mutant enzyme and an application thereof, belonging to the field of enzyme engineering. Background technique [0002] Rhizopus oryzae lipase has good 1,3-position specificity, preferentially catalyzes medium and long chain fatty acids, and has important applications in food, chemical industry and bioenergy. Utilizing the activities of enzymes such as hydrolysis, synthesis and transesterification, Rhizopus oryzae lipase has different applications. [0003] At present, the whole gene sequence of Rhizopus oryzae lipase has been published on NCBI. The enzyme consists of 26 amino acid signal sequence (presequsece), 97 amino acid leader peptide sequence (prosequsence) and 269 amino acid mature peptide sequence (mROL). Previous studies have shown that the leader peptide plays a very important role in the secretion and folding of Rhizopus oryzae lip...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12N15/81C12N1/19C12R1/845C12R1/84
CPCC12N9/20C12Y301/01003C12N15/81C12N1/145C12N1/165C12R2001/84C12R2001/845
Inventor 喻晓蔚徐岩
Owner TAIXING YIMING BIOLOGICAL PRODS
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