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Microbulbifer arenaceous beta-galactosidase as well as encoding gene and application thereof

A technology of galactosidase and gene, which is applied in the field of Microvesicle β-galactosidase and its coding gene and its application, can solve the problems that Microvesicle β-galactosidase has not yet been seen.

Active Publication Date: 2020-05-15
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

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  • Microbulbifer arenaceous beta-galactosidase as well as encoding gene and application thereof
  • Microbulbifer arenaceous beta-galactosidase as well as encoding gene and application thereof
  • Microbulbifer arenaceous beta-galactosidase as well as encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1, the cloning of Microvesicle sandum β-galactosidase and its coding gene

[0076] A large number of sequence analyzes and functional verifications were carried out on Microvesicle sandum BH1, from which a gene encoding β-galactosidase was cloned, with a full length of 2388bp, as shown in sequence 1 of the sequence list. The DNA molecule shown in sequence 1 of the sequence listing encodes the β-galactosidase shown in sequence 2, named MaBgal2A. MaBgal2A consists of 795 amino acids, of which the predicted 1st to 18th amino acids from the N-terminal are the signal peptide sequence.

Embodiment 2

[0077] Embodiment 2, the construction of engineered bacteria expressing Microvesicle sandum β-galactosidase

[0078] 1. Construction of vector for recombinant expression of Microvesicle sandum β-galactosidase

[0079] 1. Use the DNA fragment shown in the 55th-2388th position of the sequence 1 from the 5' end to replace the fragment between the NheI and XhoI restriction sites of the pET-28a (+) vector to obtain the recombinant expression vector pET-28a (+)-MaBgal2A (verified by sequencing).

[0080] The exogenously inserted DNA molecule is fused with part of the nucleotides on the carrier backbone to form a fusion gene shown in sequence 3 of the sequence listing, which encodes a fusion protein shown in sequence 4 of the sequence listing. In sequence 4 of the sequence listing, the 5th-10th position is a 6×His tag, and the 24th-800th position is MaBgal2A without signal peptide.

[0081] 2. Use the DNA fragment shown in the 55th-2388th position of the sequence 1 from the 5' end ...

Embodiment 3

[0089] Example 3, preparation and purification of Microvesicle sandum β-galactosidase and its enzymatic properties

[0090] 1. Preparation of recombinant protein MaBgal2A

[0091] 1. Inoculate the 4 kinds of recombinant bacteria prepared in Example 2 into LB liquid medium containing 50 μg / mL kanamycin respectively, and cultivate them with shaking at 37°C and 200 rpm until the bacterial liquid OD 600nm Reach between 0.6-0.8, add isopropyl-β-D-thiogalactopyranoside (IPTG) in the culture system, the concentration of IPTG in the culture system is 1mmol / L, 30 ℃, 200rpm induce and cultivate overnight, then The culture system was centrifuged at 11510g to collect bacterial precipitates, resuspended in buffer A and then sonicated (250W, 20min). After sonication, centrifuged at 11510g for 10min, and the supernatant was collected as the crude enzyme solution. After fragmentation, the target protein did not form inclusion bodies in Escherichia coli containing various expression vectors, ...

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Abstract

The invention discloses microbulbifer arenaceous beta-galactosidase as well as an encoding gene and application thereof. The invention provides the microbulbifer arenaceous beta-galactosidase which isa protein of which the sequence is shown in a sequence 2 in the description. The microbulbifer arenaceous beta-galactosidase provided by the invention is excellent in enzymatic property, and the specific enzyme activity to a natural substrate, namely lactose, is 38.69U / mg. Meanwhile, the lactose can be efficiently hydrolyzed, 80% or greater of the lactose in milk can be hydrolyzed with an enzymeaddition amount of 1U / mL at a refrigeration temperature of 4 DEG C after 36 hours of a reaction, after 48 hours of the reaction, 90% or greater of the lactose can be hydrolyzed, after 72 hours of thereaction, all lactose in the milk can be hydrolyzed. In addition, after 84 hours of the reaction, 90% or greater of the lactose in a 5% (w / v) whey solution can be hydrolyzed. The protein disclosed bythe invention has great application value for production of low-lactose or lactose-free milk products in the food industry.

Description

technical field [0001] The invention relates to a microvesicle sandy mildew beta-galactosidase, its coding gene and application. Background technique [0002] β-galactosidase (EC3.2.1.23, β-D-galactoside galactosyl hydrolase) belongs to the family of glycoside hydrolases, which catalyze the hydrolysis of glycosidic bonds between non-reducing terminal β-D-galactosides and transgalactoside function. At present, it is mainly used in food, medicine, analysis and other fields. [0003] Lactose is a disaccharide composed of one molecule of galactose and one molecule of glucose linked by β-1,4 glycosidic bonds. Lactose mainly exists in dairy products, and milk contains about 4.5% lactose. Adults cannot digest lactose due to lack of β-galactosidase activity in the gut. Gut microbes can ferment lactose and cause a series of symptoms such as bloating, abdominal pain and diarrhea, which is called lactose intolerance. Treating dairy products with β-galactosidase can hydrolyze lacto...

Claims

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Application Information

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IPC IPC(8): C12N9/38C12N15/56C12P19/14C12P19/02
CPCC12N9/2471C12Y302/01023C12P19/14C12P19/02
Inventor 江正强姚宇晨温永平刘瑜孙健闫巧娟
Owner CHINA AGRI UNIV
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