Lipase mutant with improved thermal stability and application of lipase mutant

A lipase and mutant technology, applied in the fields of application, enzyme, hydrolase, etc., can solve the problems of unsatisfactory industrial production, poor heat resistance of Rhizopus sinica lipase, etc., and achieve the effect of reducing loss rate and improving heat resistance

Active Publication Date: 2019-01-08
YUNNAN NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The object of the present invention is to provide a kind of lipase mutant and application thereof, aim to ...

Method used

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  • Lipase mutant with improved thermal stability and application of lipase mutant
  • Lipase mutant with improved thermal stability and application of lipase mutant
  • Lipase mutant with improved thermal stability and application of lipase mutant

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Embodiment 1

[0049] Preparation of embodiment 1 lipase mutant

[0050] like figure 1 Shown, the obtaining method of the lipase mutant of the embodiment of the present invention comprises the following steps:

[0051] (1) Site-directed mutagenesis: the recombinant plasmid in which the Rhizopus sinensis lipase gene was linked to the carrier was used as a template, and the PCR amplification system for mutation was prepared according to the kit instructions to prepare a 50 μL mutation system.

[0052] (2) PCR verification of the mutation: 10 μL of the mutation product was taken and detected by 0.8% agarose gel electrophoresis. After the band is correct, add 1μDMT digestive enzyme to the mutant product, flick and mix well, and incubate at 37°C for 70min.

[0053] (3) Transformation: Add 5 μL of the mutated product to 50 μL DMT competent cells, flick and mix well, and place in ice bath for 30 min; heat shock at 42°C for 45 s and immediately cool on ice for 10 min; add 500 μL LB medium, 180 Tr...

Embodiment 2

[0060] Embodiment 2 lipase optimum pH is measured

[0061] Adjust the pH of the buffer solution to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, dilute the enzyme solution to an appropriate multiple, and measure the optimum pH at 37°C according to the lipase activity assay method. After the pH, add half points on both sides of the maximum value and continue to detect the optimum value (for example, if the optimum pH is 9, then take pH8, 8.5, 9, 9.5 and 10 to measure according to the lipase activity assay method).

[0062] The optimal pH value of the lipase enzymatic reaction is as follows: figure 2 shown. The optimum of mutant and lipase is 9. No significant changes.

Embodiment 3

[0063] Embodiment 3 lipase optimum temperature is measured

[0064] According to the assay method of lipase activity, under the conditions of the above-mentioned optimum pH, the reactants were placed at different temperatures to react at 0°C, 10°C, 20°C, 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, after measuring the optimum temperature, add half points on both sides of the maximum value (for example, if the optimum temperature is 40°C, add 30°C, 35°C, 40°C, 45°C, 50°C to determine the lipase activity method) .

[0065] Optimum temperature for lipase-catalyzed reaction image 3 As shown, the optimum temperature of the mutant and lipase were 35°C and 40°C respectively; the optimum temperature of the mutant was 5°C lower than that of the lipase.

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Abstract

The invention belongs to the technical field of bioengineering, and discloses a lipase mutant with improved thermal stability and application of the lipase mutant. An amino acid sequence of the lipasemutant is shown as SEQ ID NO.2, the lipase mutant is obtained by converting valine at position 268 in the amino acid sequence 2 coding rhizopus chinensis lipase into cysteine, and coding genes of themutant are shown as SEQ ID NO.3. The mutant has relatively desirable heat resistance characteristics, and is therefore particularly suitable for industrial mass production.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and relates to a lipase mutant, in particular to a lipase mutant with improved thermostability and application thereof. Background technique [0002] Lipase has been widely used in bioenergy, oil processing, food processing, leather processing and feed addition due to its special physiological and biochemical characteristics. It is worth noting that lipase plays a vital role in the biodiesel production industry, the development of renewable energy and environmental protection. In the industrial production process, enzyme preparations often experience high temperature environments, while natural lipases generally have poor heat resistance, and the loss rate in the production process is as high as 70%-80%. The shortcoming of the inactivation of natural lipase at high temperature leads to a substantial increase in the cost of industrial production and limits the application of lipase in indu...

Claims

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Application Information

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IPC IPC(8): C12N9/20C12N15/55C12N15/81C12N1/19A23K20/189
CPCA23K20/189C12N9/20C12Y301/01003
Inventor 韩楠玉黄遵锡姜占宝苗华彪
Owner YUNNAN NORMAL UNIV
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