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Screening method for mutated strains capable of realizing high yield of amylase

A screening method and amylase technology, applied in the field of biology, can solve the problems of inability to screen out bacterial cells, large randomness, time-consuming and labor-intensive, etc., and achieve the effects of simple and easy operation and low screening cost in the screening process.

Inactive Publication Date: 2016-10-26
HUNAN UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But its disadvantage is that the randomness is very large, and the ideal bacteria cannot be screened out in many cases.
Plate colony transparent circle method is an existing conventional method for screening high amylase-producing strains after mutation

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] 1 soil sampling

[0031] Samples were collected using the five-point sampling method. Select representative soil at the destination, with an area of ​​2m×2m. First, take the midpoint of the diagonal as the central sampling point, and then select four points on the diagonal that are 0.5m away from the central sampling point as sampling points.

[0032] 2 Primary screening and re-screening of strains

[0033] The colonies in the sampled soil were enriched and preliminarily screened by iodine staining.

[0034] Amylase-producing bacteria can produce amylase excreted from the body and decompose starch around the colony. The iodine solution will appear blue when it meets starch. When the screened colonies are amylase-producing bacteria, the surrounding starch is decomposed, and the iodine solution does not develop color, so that an obvious transparent circle will be formed around the colonies.

[0035] step:

[0036] (1) Weigh and number 0.5g of each sample, fully dilu...

Embodiment 2

[0049] 1 soil sampling

[0050] Samples were collected using the five-point sampling method. Select representative soil at the destination, with an area of ​​2m×2m. First, take the midpoint of the diagonal line as the central sampling point, and then select four points on the diagonal line that are 1m away from the central sampling point as sampling points.

[0051] 2 Primary screening and re-screening of strains

[0052] The colonies in the sampled soil were enriched and cultured and preliminarily screened by iodine staining method:

[0053] (1) Weigh and number 0.5g of each sample, fully dilute and dissolve with sterile water, then take an equal amount and transfer to 200mL enriched liquid medium, and culture in a 37°C incubator at 200r / min;

[0054] (2) After 24 hours, each sample was diluted 10 times with sterile water, and the absorption concentration was 10 -6 100 μL of the culture medium was evenly spread on the screening plate, and cultured in a 37°C incubator for ...

Embodiment 3

[0063] 1 soil sampling

[0064] Samples were collected using the five-point sampling method. Select representative soil at the destination, with an area of ​​2m×2m. First, take the midpoint of the diagonal as the central sampling point, and then select four points on the diagonal that are 2m away from the central sampling point as sampling points.

[0065] 2 Primary screening and re-screening of strains

[0066] The colonies in the sampled soil were enriched and cultured and preliminarily screened by iodine staining method:

[0067] (1) Weigh and number 0.5g of each sample, fully dilute and dissolve with sterile water, then transfer an equal amount to 200mL enriched liquid medium, and culture in a 37(±1)°C incubator at 100r / min;

[0068] (2) After 24 hours, each sample was diluted 10 times with sterile water, and the absorption concentration was 10 -6 Spread 100 μL of the culture medium evenly on the screening plate, and incubate in the incubator at 37 (±1) °C for 50 h;

...

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Abstract

The invention relates to a screening method for mutated strains capable of realizing high yield of amylase. The method comprises a step of screening of mutated strains, i.e., screening of first-generation strains having undergone mutation; the step comprises a substep of primary screening and a substep of secondary screening; the secondary screening is carried out in a manner the same as the manner used in primary screening so as to obtain second-generation mutated starting strains; then mutation and screening are carried out again; and the screening method for the generation II and generation III to generation n is same, and mutation and screening are carried out until high-amylase-yield stains meeting requirements are bred. With the screening method, the starting strains having undergone mutation treatment undergoes primary screening and secondary screening again so as to obtain the second-generation starting strains; the screening method is simple and easily practicable, and can obtain the high-amylase-yield stains in a short time; the whole screening process is low in investment and high in targeting performance; and orientated production of the amylase strains with high enzyme activity can be realized, and the method has large-scale promotion value.

Description

technical field [0001] The invention relates to the field of biology, in particular to a screening method for bacterial strains, in particular to a screening method for high-yielding amylase strains after mutagenesis. Background technique [0002] Amylases refer to enzymes capable of hydrolyzing O-glucose linkages in starch, glycogen and related polysaccharides. Generally, it refers to an enzyme that acts on α-1,4-glucans such as soluble starch, amylose, and glycogen and hydrolyzes α-1,4-glucosidic bonds. Amylase can be divided into two types according to the mode of action: α-amylase (EC3.2.1.1.) and β-amylase (EC3.2.1.2.). α-amylase is widely distributed in animals (saliva, pancreas, etc.), plants (malt, wild gooseberry) and microorganisms. Most of the microbial enzymes are secreted. Amylase with Ca 2+ As an essential factor and as a stabilizing factor, it acts on both amylose and amylopectin, and cuts the α-1,4-chain with the same function. Therefore, the reaction ha...

Claims

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Application Information

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IPC IPC(8): C12N1/02C12Q1/04
Inventor 金元昌凌玲吴银亮蔡英桂苏壬香张建贺
Owner HUNAN UNIV OF SCI & TECH
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