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Alkaline pullulanase transformed in site-specific mutagenesis mode, and application thereof

A technology of pullulanase and site-directed mutation, which is applied in the fields of application, enzyme, hydrolase, etc., can solve the problems of limited application, low pullulanase secretion ability, and difficulty in meeting the conditions of industrial production, and achieve improved thermal stability , Enzyme activity and stability improvement effect

Active Publication Date: 2018-11-30
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, natural strains isolated from nature have low pullulanase secretion ability and are difficult to meet the conditions of industrial production, which limits its industrial application.

Method used

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  • Alkaline pullulanase transformed in site-specific mutagenesis mode, and application thereof
  • Alkaline pullulanase transformed in site-specific mutagenesis mode, and application thereof
  • Alkaline pullulanase transformed in site-specific mutagenesis mode, and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Construction of pullulanase mutant F744A

[0018] According to the 3D structure analysis of alkaline pullulanase PulSL3△C ( figure 1 ), the 744th phenylalanine leads to a wide crack and a sharp bulge, this structure may inhibit the enzyme from hydrolyzing the second α-1, 6 glycosidic bond of pullulan, and to the branched chain The hydrolysis inhibition of densely branched sugars such as starch and glycogen was more serious. The side chain of phenylalanine is a benzene ring with a large steric hindrance, which leads to the generation of cracks and protrusions. If Phe744 is mutated into alanine with a methyl side chain, namely Ala744, the benzene ring can be greatly reduced The resulting steric hindrance. This mutant was named F744A.

[0019] Design primers containing mutation sites to recombine plasmid pET-22b(+)- pulSL3△C ( figure 2 ) as a template, the expression plasmid pET-22b(+)- F744A , transforming E. coli BL21(DE3) competent cells were used...

Embodiment 2

[0027] Example 2 Induced expression and purification of pullulanase mutant F744A

[0028] A single colony was picked from the transformation plate and inoculated into LB liquid medium containing 100 µg / mL Amp for overnight culture. Inoculate the overnight culture solution at 1% inoculum volume in 50 mL LB medium containing 100 µg / mL Amp, and cultivate to OD at 37°C at 180 rpm 600 When it reaches 0.5, add IPTG and 0.5% NaCl at a final concentration of 0.1 mmol / L, and incubate at 25°C for 32 h at 180 rpm. The fermentation broth was centrifuged at 13000 rpm for 10 min at 4°C, and the supernatant was taken as the crude enzyme solution.

[0029] The crude enzyme solution was first concentrated with a 10 kDa hollow fiber column, and then (NH 4 ) 2 SO 4 After fractionation precipitation, the dialyzed enzyme solution was filtered through a 0.22 µm membrane to prepare the loading sample. First use the prepacked column DEAE FF (Hi Trap TM , 5 mL) for purification, the equilibrium ...

Embodiment 3

[0030] Example 3 Determination of Enzyme Activity and Properties of Pullulanase Mutant F744A

[0031] 1. Enzyme activity assay of pullulanase mutant F744A

[0032]The activity of pullulanase was determined by DNS method. Incubate the mixture containing 0.9 mL 0.5% pullulan (TCI company) and 0.1 mL enzyme solution under appropriate conditions for 10 min, add 1.5 mL DNS to terminate the reaction, boil for 5 min to develop color, cool down and dilute to volume with distilled water 10 mL, measure OD 540 . Definition of enzyme activity unit: Under certain reaction conditions, the amount of enzyme required to hydrolyze pullulan to produce 1 μmol reducing sugar per minute is defined as an enzyme activity unit U.

[0033] Compared with the original enzyme PulSL3△C, the activity of pullulanase mutant F744A was increased by 32.18%.

[0034] 2. Optimum reaction pH and pH stability of pullulanase mutant F744A

[0035] Determination of the optimum reaction pH: Put the pullulanase in t...

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Abstract

The invention provides an alkaline pullulanase transformed in a site-specific mutagenesis mode, and application thereof. According to the alkaline pullulanase transformed in the site-specific mutagenesis mode, on the basis of the amino acid shown in SEQ ID NO.2, phenylalanine at the 744 position is mutated into alanine; mutant F744A transformed Escherichia coli is subjected to heterologous expression, and it is found that the enzyme activity of a mutant is improved by 32.18%; thermal stability is also improved, and T<50><30> is 55.84 DEG C and is improved by 2.94 DEG C compared with an original enzyme PulSL3 delta C; and the pH stability range of the mutant F744A is wider, and after heat preservation is conducted for one hour within the pH range from 5.0 to 10.0, 90 % or above of the enzyme activity can still be retained. The alkaline pullulanase serves as an additive to be applied to the detergent industry, and the washing effect can be improved obviously.

Description

technical field [0001] The invention relates to an alkaline pullulanase modified by site-directed mutation and an application thereof, belonging to the fields of genetic engineering and enzyme engineering. Background technique [0002] Pullulanase can specifically cut the α-1, 6 glycosidic bond of starch to obtain amylose. Applying it to the starch processing industry can significantly improve the utilization rate and production efficiency of starch. In the feed industry, corn-soybean meal-based diets are currently used as raw materials to produce feed, but corn amylopectin is easy to form "anti-nutritional factors" that are difficult to be digested by the animal intestines, so that young animals cannot eat well. Absorption and utilization of feed will also cause intestinal diseases. Therefore, adding pullulanase to feed can promote the digestion, absorption and utilization of nutrients in feed by animals, thereby improving economic benefits. As a kind of widely used enzym...

Claims

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Application Information

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IPC IPC(8): C12N9/44C12N15/56C11D3/386
CPCC11D3/38636C12N9/2457C12Y302/01041
Inventor 林娟林云王国增许鑫琦黄晗
Owner FUZHOU UNIV
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