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A kind of β-galactosidase gala and its application

A galactosidase and lactose technology, which is applied to beta-galactosidase GalA and its application fields, can solve the problems of unsatisfactory application, single variety, high price of beta-galactosidase, etc., and achieves good industrial application potential , sequence novelty, good thermal stability and storage stability

Active Publication Date: 2021-09-17
QINGDAO UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Moreover, the β-galactosidase currently on the market is expensive and single in variety, which cannot meet the application under specific conditions, so it is urgent to develop a new β-galactosidase

Method used

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  • A kind of β-galactosidase gala and its application
  • A kind of β-galactosidase gala and its application
  • A kind of β-galactosidase gala and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Sequence analysis of β-galactosidase GalA

[0026] Enzyme-producing gene of β-galactosidase GalA of the present invention galA for artificially synthesized sequences. In previous studies, the inventors found that the deep-sea bacterium Q15 has a high lactose-degrading activity. When sequencing its entire gene, it was found that it contained a predicted β-galactosidase sequence, and it was named as galA . Under the condition that the amino acid sequence remains unchanged, the inventors optimized the base sequence of the gene sequence according to the codon preference of the host (Escherichia coli), so as to facilitate its high-efficiency expression in Escherichia coli. The β-galactosidase of the present invention contains 2007 base sequences and encodes 669 amino acid sequences.

[0027] Multiple sequence comparison Basic Local Alignment Search Tool (Blast) found that in the "Genbank" database, the amino acid sequence similarity with the β-galactosidase de...

Embodiment 2

[0036] Example 2 Preparation and purification method of β-galactosidase GalA

[0037] Cultivate the recombinant strain BL21(DE3) / pET22b-GalA in 100 mL of LB liquid medium (50 μg / mL ampicillin) in a shaker at 37 °C at 180 rpm to OD 600 =0.6, add the inducer isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.1 mM, and induce at 20 ℃ for 20 h. The substrate 4-Nitrophenyl-β-d-glucopyranoside, ONPG (purchased from Sigma Alddrich, USA) was used for the determination of β-galactosidase activity. The standard method for measuring the enzyme activity of β-galactosidase GalA is: add 900 μL 0.3% ONPG substrate (20 mMTris-HCl buffer, pH 7.0) to 100 μL enzyme solution, react at 50 °C for 10 min, add 1 M Na 2 CO 3 The reagent terminates the reaction, according to the ONP standard curve at OD 420 Activity was determined at nm. The unit (U) of β-galactosidase is defined as the amount of enzyme required to release 1 µmol ONP per minute under the assay conditions.

[0038]...

Embodiment 3

[0039] Example 3 Stability of β-galactosidase GalA

[0040] The β-galactosidase GalA purified in Example 2 was tested for enzyme activity under different conditions to detect the effects of different temperatures and pH on the enzyme activity. React at different temperatures (0-70°C) for 10 min, detect the effect of different reaction temperatures on enzyme activity, take the highest enzyme activity as 100%, calculate the relative enzyme activity of β-galactosidase GalA at different temperatures, β- The optimal reaction temperature of galactosidase GalA is 50 ℃. The β-galactosidase GalA was incubated at 50 °C for different times (6, 12, 18, 24, 30, 36 h), and the remaining enzyme activity was detected at the optimal reaction temperature (50 °C) to determine the enzyme activity before incubation as 100%. Such as figure 2 As shown, β-galactosidase GalA has good thermal stability, and 89.7% of the enzyme activity is still retained after continuous incubation at 50° C. for 24 ...

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Abstract

The invention relates to a β-galactosidase GalA and its application. The amino acid sequence of the β-galactosidase GalA is shown in SEQ ID NO.1. The invention provides a preparation method for recombinant expression and purification of the β-galactosidase GalA, the enzyme activity of the fermentation broth can reach 3795.6 U / mL, the purity of one-step purification is >97%, and the recovery rate is 79.5%. The β-galactosidase GalA of the present invention has good thermal stability and storage stability, and the optimum reaction temperature is 50°C, and it still retains 89.7% of the enzyme activity after continuous incubation at 50°C for 24 h. At room temperature (30 ℃) stored for 30 days, still can retain 81.5% of the enzyme activity. The β-galactosidase GalA of the invention has high activity, good stability, strong ability to degrade lactose, and good application potential.

Description

technical field [0001] The invention relates to a beta-galactosidase GalA and application thereof, belonging to the field of biotechnology. Background technique [0002] β-Galactosidase (EC 3.2.1.23) is a glycoside hydrolase that catalyzes the hydrolysis and transgalactosylation of lactose. As a commonly used medical enzyme, β-galactosidase has been widely used in the dairy industry to decompose lactose into galactose and glucose. In addition, β-galactosidase can also generate galactooligosaccharides through transglycosylation during the hydrolysis of lactose or other structurally related galactosides. β-galactosidase can be widely used in nutrition and food processing. [0003] The hydrolytic activity of β-galactosidase has been used in the food industry for decades to aid in the absorption of undigested lactose. Worldwide, especially in East Asia, many children and adults are suffering from lactose indigestion and intolerance caused by lactase deficiency. The reason be...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/38C12N15/56A23L5/20A23C7/04
CPCA23C7/043A23L5/25C12N9/2471C12Y302/01023
Inventor 陈豪董娟陈鹏狄国虎吴祥根
Owner QINGDAO UNIV
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