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Method for producing amylase by utilizing transgenic maize

A technology of transgenic corn and amylase, which is applied in the fields of biochemical equipment and methods, glycosylase, genetic engineering, etc., can solve the problems of high cost and low enzyme activity, and achieve the effect of high activity and low production cost.

Pending Publication Date: 2021-03-19
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the enzyme activity per unit weight of amylase expressed in plant bioreactors is still relatively low, and it is necessary to greatly increase the expression of amylase in plants to improve the commercial value of this technology
At present, amylase used in industry is still mainly produced through microbial fermentation, and the cost is relatively high

Method used

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  • Method for producing amylase by utilizing transgenic maize
  • Method for producing amylase by utilizing transgenic maize
  • Method for producing amylase by utilizing transgenic maize

Examples

Experimental program
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Effect test

Embodiment 1

[0023] Embodiment 1, construction maize genetic transformation vector

[0024] The coding genes involved in the present invention are all artificially synthesized by Shanghai Sangong Company. The maize genetic transformation vector is constructed based on the pCambia1300 (NCBI sequence number AF234296) vector, the only difference is that the hygromycin selection gene hptII (located between the two XhoI sites) on the original pCambia1300 is replaced with the self-developed The glyphosate-resistant selection gene g10-evo (only used for the screening of positive maize transformation lines) (Zhao, Qc., Liu, Mh., Zhang, Xw. et al. Generation of insect-resistant and glyphosate-tolerant rice by introduction of a T-DNA containing two Bt insecticidal genes and an EPSPSgene.J.Zhejiang Univ.Sci.B 16,824–831(2015)), the vector was named 1300-g10evo, and stored in a -80°C refrigerator. The above-mentioned vectors are just examples, rather than the content of the present invention. Researc...

Embodiment 2

[0028] Embodiment 2, the acquisition of transgenic corn

[0029] Maize transformation methods have been relatively mature, for example, Frame et al. described the method of using Agrobacterium to transform maize (Frame et al., (2002) Plant Physiol, 129:13-22). Take the Agrobacterium tumefaciens LBA4404 strain containing the vector p1300-GT1-AS constructed by the method in Example 1, draw a plate, pick a single colony for inoculation, and prepare the Agrobacterium for transformation. Ears of Nongda 178 corn 8-10 days after pollination were taken. All immature embryos (1.0-1.5 mm in size) were collected. The above-mentioned Agrobacterium tumefaciens and immature embryos were co-cultured for 2-3 days (shading, 22° C.). Then transfer the immature embryos to the callus induction medium (containing 200mg / LTimentin, used to kill Agrobacterium, refer to (Frame et al., (2002) Plant Physiol, 129:13-22)), culture in the dark at 28°C 10-14 days. All calli were then transferred to sele...

Embodiment 3

[0032] Example 3, Determination of Amylase Activity Expressed in Transgenic Corn Seeds

[0033] Amylase activity was determined by the DNS method (Miller, 1959). The specific experimental method is as follows:

[0034] Maltose standard solution: prepare 10mM maltose standard solution with deionized water;

[0035] To make the maltose standard curve, add different reagents according to the following table 1.

[0036] The making of table 1 maltose standard curve

[0037]

[0038]

[0039] Shake well and boil in water for 5 minutes, take it out and cool it down, measure the absorbance at 540nm wavelength, draw the standard curve with the maltose content as the abscissa and the absorbance as the ordinate, such as figure 2 shown.

[0040] Preparation of enzyme solution: take 10 mg of corn seeds expressing amylase and add 1 ml of buffer solution (20 mM sodium acetate, pH 5.4, 250 mM NaCl) to grind, and the mixed solution after grinding is the enzyme solution.

[0041] Su...

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Abstract

The invention discloses a method for producing amylase by using transgenic maize. The method comprises the following steps of transferring an encoding gene of amylase into a maize genome under the action of a GT1 promoter to construct amylase-producing transgenic maize. According to the transgenic maize, high expression of amylase is realized in endosperm, and the activity of the produced amylaseis extremely high and reaches 8000 units per gram of seeds or above; the amylase produced by the transgenic maize does not need to be separated and concentrated, and can be directly applied to the production processes of feed production, food processing, textile industry and the like; and a maize bioreactor is used for producing amylase, the production cost is low, and the method is an environment-friendly production mode.

Description

[0001] (1) Technical field [0002] The invention relates to a method for producing amylase by using transgenic corn. [0003] (2) Background technology [0004] Amylase (Amylase) is widely used in grain processing, food industry, brewing, textile and pharmaceutical fields, and is currently the most widely used enzyme in the fermentation industry. Amylase is a hydrolytic enzyme that can hydrolyze starch into maltodextrin, glucose syrup and other substances. It is an essential enzyme in the production of sugar industrial products such as glucose and fructose. Amylase is also an important component of feed enzymes, which can degrade macromolecular polysaccharides, so that animals can better digest and absorb nutrients, which is of great significance for improving animal production performance and saving feed resources. In addition, amylase is also an essential enzyme preparation in the production of bioethanol. After starch is degraded into six-carbon sugar by amylase, it can be...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/26C12N15/56C12N15/113C12N15/84C12N5/10
CPCC12N9/2414C12N15/8257C12Y302/01001
Inventor 林海燕许超沈志成
Owner ZHEJIANG UNIV
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