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Method for producing maltooligosyl trehalose synthase and maltooligosyl trehalose hydrolase and method for producing trehalose

A technology of trehalose synthase and trehalose hydrolase, which is applied in the fields of bioengineering and enzyme engineering, to achieve the effects of increased yield, high enzyme activity titer, and high cell density

Inactive Publication Date: 2017-07-28
山东福洋生物制造工程研究院
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  • Abstract
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  • Application Information

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Problems solved by technology

[0012] The object of the present invention is to provide a method for producing malto-oligosaccharide-based trehalose synthase or malto-oligosaccharide-based trehalose hydrolase, which uses the carbon dioxide release rate as a fermentation parameter to control it, and then realizes the control of each enzyme in the fermentation process. The effective control of various parameters overcomes the defects of repeatedly adjusting various parameters in the existing fermentation technology process, so as to achieve the purpose of simple operation, high cell density and high enzyme activity titer

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  • Method for producing maltooligosyl trehalose synthase and maltooligosyl trehalose hydrolase and method for producing trehalose

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Experimental program
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Embodiment 1

[0059] The method for producing maltooligosaccharide-based trehalose synthase provided in this embodiment comprises the following steps:

[0060] 1.1 Inoculate the transgenic Escherichia coli capable of expressing maltooligosaccharide-based trehalose synthetase into the primary seed shake flask (containing liquid LB medium) according to 1% inoculum amount, place it at 37°C, 220rpm, and culture it on a shaking table for 8h, OD 600 Cultivate to 2.1 to obtain the primary seed activation solution.

[0061] 1.2 Transfer the first-level seed activation liquid to a 15L second-level seed tank (containing LB liquid medium) according to the inoculum amount of 4%, control the reaction temperature at 37°C, DO greater than 10%, pH7.0 (adjusted with 20% ammonia water), and carry out Strain culture, growth 10.5 hours, OD 600 If the value is greater than 4.4, the secondary seed activation solution is obtained.

[0062] Among them, the formula of LB liquid medium: 1% peptone, 0.5% yeast powd...

Embodiment 2

[0073] The method for producing maltooligosaccharyl trehalose hydrolase provided in this example is basically the same as the method in Example 1, except that the transgenic Escherichia coli capable of expressing maltooligosaccharyl trehalose hydrolase is used instead of the expression Transgenic Escherichia coli for maltooligosaccharide-based trehalose synthetase to obtain crude enzyme solution containing maltooligosaccharide-based trehalose hydrolase.

Embodiment 3

[0075] The method for producing trehalose provided in this embodiment comprises the following steps:

[0076] 3.1 Add 9kg of pure starch to the reaction tank (BLBIO-50-FMT, Shanghai Bailun Biotechnology Co., Ltd.), control the pH to 5.5, add 27g of high-temperature amylase, control the temperature at 105°C, and treat for 3.6 hours to liquefy the starch into DE5 % maltodextrin.

[0077] Among them, the activity unit of the high-temperature amylase is 6500U / mL, and the dosage thereof is: 3.0 g of high-temperature amylase corresponds to 1.0 kg of starch.

[0078] 3.2 Maltodextrin Control the temperature at 55°C, use 0.2mol / L phosphoric acid and 0.2mol / L sodium hydroxide to adjust the pH to 6.0, add pullulanase and maltooligosaccharyl trehalose synthase obtained in Example 1 The crude enzyme solution of the maltooligosaccharide-based trehalose hydrolase obtained in Example 2 was treated for 12 hours.

[0079] Among them, the activity unit of pullulanase is 1000-1500U / mL, and the...

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Abstract

The invention discloses a method for producing maltooligosyl trehalose synthase and maltooligosyl trehalose hydrolase and a method for producing trehalose, and relates to the field of biological engineering and enzyme engineering. The method takes recombinant engineering strains capable of expressing the maltooligosyl trehalose synthase or the maltooligosyl trehalose hydrolase as starting strains, various parameters in the fermentation process are controlled through feedback and adjustment of carbon dioxide excretion rate, so that operations are simple, microbial density is high and enzyme activity titer is high, and the yield of the trehalose can be imprtoved by 80% or more when the trehalose is produced through the maltooligosyl trehalose synthase or the maltooligosyl trehalose hydrolase prepared by the method.

Description

technical field [0001] The invention relates to the fields of bioengineering and enzyme engineering, in particular to a method for producing maltooligosaccharide-based trehalose synthetase, maltooligosaccharide-based trehalose hydrolase and a method for producing trehalose. Background technique [0002] Trehalose is a non-reducing sugar composed of two glucose molecules bonded by α,α-1,1 glycosidic bonds. It widely exists in bacteria, yeast, algae, fungi, insects and some stress-resistant plants. Trehalose has a non-specific protective effect on biological macromolecules such as cell membranes, proteins, and nucleic acids, and has good water absorption, stability, and is not easy to caramelize. Exogenous trehalose also has a non-specific protective effect on organisms and biological macromolecules. The unique biological properties of trehalose make it have important applications in food, medicine, cosmetics, vaccines and other fields. [0003] The production methods of treh...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/26C12N9/90C12P19/12C12P19/14C12P19/24C12N15/70
Inventor 赵伟曹大鹏段莹莹任峰范秀旺高文学
Owner 山东福洋生物制造工程研究院
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