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Method for expressing high-enzyme-activity amylase

A technology of amylase and high enzyme activity, applied in the field of enzyme engineering, can solve the problem that the effect is not obvious and so on

Inactive Publication Date: 2018-11-13
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The bottleneck in the production of Aspergillus oryzae amylase is that there is anti-inhibition regulation in the later stage of Aspergillus oryzae cultivation, which will inhibit the production of Aspergillus oryzae amylase. Increased the amylase activity in the late stage of high concentration culture, but the effect was not obvious

Method used

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  • Method for expressing high-enzyme-activity amylase
  • Method for expressing high-enzyme-activity amylase
  • Method for expressing high-enzyme-activity amylase

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Experimental program
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Effect test

Embodiment 1

[0035] A method for expressing high enzyme activity amylase, the flow chart is as figure 1 As shown, the AodevR gene in Aspergillus oryzae is replaced by the selectable marker gene pyrG by homologous recombination, thereby obtaining the AodevR knockout strain, and then using the strain to express amylase, a high-enzyme amylase can be obtained, the marked in the figure The SmaI restriction site and probe probe position were used for Southem blot verification. Specific steps are as follows:

[0036] 1) Utilize PrimeStar PCR polymerase (TaKaRa, Japan) to amplify the fragments on both sides of the AodevR gene (AodevRL, the gene sequence is shown in SEQ ID NO.1; AodevR R, the gene sequence is shown in SEQ ID NO.2) and pyrG gene (The gene sequence is shown in SEQ ID NO.3), the main process is as follows:

[0037] PCR system: 25 μL of primestar Mix enzyme; 2 μL of upstream and downstream primers (see Table 1 for specific primer sequences); 1 μL of A.oryzae RIB40 (gifted by Noda Ins...

Embodiment 2

[0060] Embodiment 2 Amylase activity assay

[0061] The No. 2 transformed strain prepared in the example, with Con: the strain obtained by introducing pyrG into E-F1 (Δku70::ptrA, ΔAF, ΔpyrG) as a reference strain, was inserted into YPD liquid medium, 45mL / bottle; conidia Number: 10 5 , 30° C., 150 rpm, and cultured in 3 repetitions.

[0062] Using the amylase (AMS) test box starch-iodine colorimetric method (Shanghai Ji Ning), samples were taken on the 2d, 3d, 4d, and 5d of culture, and the amylase activity in the YPD medium was determined.

[0063] see results Figure 5 , Con: the strain obtained by introducing pyrG into E-F1 (Δku70::ptrA, ΔAF, ΔpyrG) as a reference strain; ΔdevR: AodevR knockout strain. The deletion of AodevR significantly increased the amylase activity of Aspergillus oryzae, and the amylase activity of the AodevR knockout strain was 3 times that of the reference strain on day 2, and increased to 7 times that of the reference strain on day 5.

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Abstract

The invention discloses a method for expressing high-enzyme-activity amylase. The method comprises the following steps: firstly, knocking out an AodevR gene in aspergillus oryzae; then expressing theamylase by utilizing the strain to obtain the high-enzyme-activity amylase. The AodevR gene in the aspergillus oryzae is replaced with a selective marker gene pyrG through homologous recombination, sothat an AodevR knock-out strain is obtained; then the amylase is expressed by utilizing the strain and the high-enzyme-activity amylase can be obtained; a test result proves that the activity of theaspergillus oryzae amylase is remarkably improved through the deficiency of AodevR; at the second day, the activity of the amylase of the AodevR knock-out strain is 3 times of that of a reference strain; at the fifth day, the activity of the amylase of the AodevR knock-out strain is increased to 7 times of that of the reference strain.

Description

technical field [0001] The invention belongs to the technical field of enzyme engineering, and in particular relates to a method for expressing amylase with high enzyme activity. Background technique [0002] Starch, one of the most abundant raw materials in nature, is an important component of human food and has a wide range of uses in the food field. Starch is easy to obtain from plants, and the price is low. The industrial application of starch is the key to alleviating environmental pollution and realizing sustainable development of human beings. The key to starch utilization is to degrade starch into fermentable sugar - glucose. Amylase can directly convert uncooked raw starch into fermentable sugars such as glucose. It is the earliest industrial production and the most widely used enzyme preparation with the largest output so far. According to the mode of action, it can be divided into α-amylase and β-amylase. α-amylase is widely distributed in animals (saliva, panc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/26C12N15/80C12N1/15C12R1/69
CPCC07K14/38C12N9/2411C12N9/242C12N15/80C12N15/902
Inventor 金锋杰庄淼张智敏王宝腾
Owner NANJING FORESTRY UNIV