Signal peptide mutant capable of improving expression quantity of recombinant pullulanase and application thereof

A pullulanase and signal peptide technology, applied in the direction of peptides, enzymes, depsipeptides, etc., can solve problems such as loss of activity, and achieve the effect of increasing the amount of extracellular expression

Active Publication Date: 2016-11-09
南宁邦尔克生物技术有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Pullulanase derived from Bacillus nagano has good pH adaptability, the optimum pH measured at 60°C is 5.0, and the optimum temperature measured at pH 4.5 is 62.5°C; the enzyme has good thermal stability At pH 4.5, 60°C and stored toget...

Method used

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  • Signal peptide mutant capable of improving expression quantity of recombinant pullulanase and application thereof
  • Signal peptide mutant capable of improving expression quantity of recombinant pullulanase and application thereof
  • Signal peptide mutant capable of improving expression quantity of recombinant pullulanase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] This example illustrates the acquisition of a signal peptide mutant derived from the α-acetolactate decarboxylase gene of Bacillus brevis, and the acquisition of amino acids comprising this mutant combined with pullulanase derived from Bacillus naganoensis A method for recombining Bacillus subtilis.

[0020] 1. Construction of recombinant plasmid pMLK83-P43

[0021] According to the promoter P43 sequence annotated in Genbank, the upstream primer was designed as 5'attgctggacgcttatggac3' and the downstream primer as 5'cgggatccattcctctcttacctataat 3'. PCR reaction system 100ul: DNA template (Bacillus subtilis 1A751 total DNA) 1ul (about 20ng), 5×PrimeSTAR Buffer 20ul, 10pmol / ul dNTP 2ul, 10pmol / ul forward and reverse primers 2ul each, 2.5U / ul PrimeSTAR HS DNA polymerase 1ul, add ddH 2 0 to 100ul. PCR reaction program: 94°C for 5min; 30 cycles of 94°C for 30s, 60°C for 30s, 72°C for 1min; 72°C for 10min; store at 4°C. The PCR fragment and plasmid pMLK83 were double dige...

Embodiment 2

[0052] This example illustrates the method for producing pullulanase using the recombinant Bacillus subtilis described in Example 1.

[0053] The operation steps are as follows:

[0054] 1) Preparation of the first-class species: A single colony of the genetically engineered strain of Bacillus subtilis was cultured overnight in 4 ml of LB liquid medium at 37° C. and 220 rpm on a shaker, and the obtained strain was the first-class species.

[0055] 2) Preparation of the secondary species: the primary species was inoculated in 800 ml LB liquid medium, and cultured on a shaker at 37° C. at 220 rpm until the OD600 was about 0.6 (about 4 to 5 hours).

[0056] 3) Preparation of the third-level seed: inoculate the second-level seed into an 80L LB liquid fermenter, control the temperature at 37°C, control the pH to 7.0 with citric acid and NaOH, ventilate and stir for 5-6 hours, and control the dissolved oxygen at 20-30 %, cultivate until OD600 is about 0.6 (about 5-6 hours).

[005...

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PUM

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Abstract

The invention discloses a signal peptide mutant capable of improving the expression quantity of recombinant pullulanase and an application thereof. The signal peptide mutant is obtained by saturation mutagenesis of an N end of a sequence of an alpha-acetolactate decarboxylase gene signal peptide derived from bacillus brevis. A specific mutation site is a seventh-site threonine replaced with valine. Compared with a no-mutation signal peptide, the signal peptide mutant can improve extracellular expression of the recombinant pullulanase derived from bacillus naganoensis in bacillus subtilis by more than or equal to 50%, and has good application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a signal peptide mutant capable of increasing the expression of recombinant pullulanase and an application thereof. Background technique [0002] Pullulanase (Pullulanase, EC 3.2.1.41) can specifically hydrolyze the α-1,6-glucosidic bonds of polysaccharides, and make the branched chains of amylopectin-type polysaccharides detach from the main chain. It plays an important role in the starch processing industry. use. When used together with α-amylase and glucoamylase to produce medical glucose, starch can be completely degraded into glucose, and the crystallization of glucose can be significantly accelerated, improving the utilization rate of equipment. Used in conjunction with β-amylase, almost 100% of starch can be converted into maltose, so as to obtain ultra-high maltose syrup above 0.8g / ml; adding pullulanase in beer production can improve the utilization rate of sugar and shorte...

Claims

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Application Information

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IPC IPC(8): C07K14/00C12N9/44C12N1/21C12R1/125
CPCC07K14/00C12N9/2457C12Y302/01041
Inventor 李晓明黄日波廖东庆梁莲华李丛韦旭钦王青艳蒙健宗
Owner 南宁邦尔克生物技术有限责任公司
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