Construction of heat-resistant beta-galactosidase mutant

A technology of galactosidase and mutants, which is applied in the field of protein engineering, can solve the problems of not being able to cooperate with pasteurization, milk flavor and color changes, etc., and achieve the effects of improving flavor and taste, saving sucrose consumption, and increasing sweetness

Inactive Publication Date: 2011-11-23
JIANGNAN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when hydrolyzing lactose in milk, the optimum reaction temperature of β-galactosidase is not as high as possible. It is generally believed that 55-65°C is a more suitable process condition, and too high hydrolysis temperature will lead to milk flavor. and color

Method used

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  • Construction of heat-resistant beta-galactosidase mutant
  • Construction of heat-resistant beta-galactosidase mutant
  • Construction of heat-resistant beta-galactosidase mutant

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Identification of remodeling sites for thermostable β-galactosidase BgaB.

[0035] (1) Test method:

[0036] Construction of BgaB molecular model and substrate complex model

[0037] All calculations were completed by the CDOCKER calculation program provided by Accelerys DiscoveryStudio 2.1 software (Accelerys Software Inc., Accelrys Discovery Studio 2.1, San Diego, 2008; G.Wu, D.H.Robertson, C.L.Brooks, M.Vieth, Detailed analysis of grid-based molecular docking: a case study of CDOCKER-A CHARMm-based MD docking algorithm, J. Comp. Chem, 24(2003) 1549-1562).

[0038] Determination method of Michaelis constant Km and inhibition constant Ki

[0039] The concentration of ONPG is prepared from 0.8-10mmol / L, and different concentrations of lactose, galactose and glucose are added to the substrate analogue inhibition experiment. The reaction system is the same as the enzyme activity test, and the enzyme solution purified by a nickel column is added, and the difference is me...

Embodiment 2

[0052] Construction of Thermostable β-Galactosidase BgaB Mutant

[0053] 1. Site-directed modification of the active site of the heat-resistant β-galactosidase gene bgab gene

[0054] The plasmid pKK223-3-bgab (Dong Y. N., Liu X.M., Chen H.Q., et al..Enhancement of the hydrolysis activity of beta-galactosidase from Geobacillus stearothermophilus by saturation mutagenesis.Journal of Dairy Science, 2011, 94(3):1176-1184) was used as a template, and a pair of primers (Sequence 1 and Sequence 2) were used to carry out site-specific modification of the bgab gene by the method of full plasmid amplification , the PCR program is: 95°C for 30s, 55°C for 30s, 68°C for 6.6min, 16 cycles. PCR reaction system: 5 μl dNTPs (2mM), 5 μl 10×Buf., 1 μl KOD plus (high-fidelity polymerase, Toyobo), 2 μl MgSO 4 (25mM), 1.5 μl each of upstream and downstream primers, and 1 μl template.

[0055] 2. Screening of Clones

[0056] The amplified PCR product was digested with endonuclease Dpn I (Fermen...

Embodiment 3

[0058] Inoculate the transformant of the mutant enzyme in 250mL LBA liquid medium and culture at 200r / min at 37°C until OD 600When it reaches 0.6~0.8, add IPTG (isopropyl-β-D-thiogalactoside, final concentration 1mM), induce for 20h, collect the bacteria by centrifugation, resuspend with 50mM phosphate buffer (pH 6.5), and place in ice bath Ultrasound was used to break the wall, centrifuged at 10000r / min for 30min, and water-bathed at 60°C for 30min to remove some heat-labile foreign proteins, and the supernatant was purified by Ni-NTA agarose column (see Example 3 for details);

[0059] 4. Dialysis, freeze drying

[0060] The eluate was collected, and purified enzyme was obtained after dialysis and freeze-drying.

[0061] Example 3

[0062] Expression and purification of mutant BgaB-F341T of thermostable β-galactosidase BgaB

[0063] 1. Experimental method: The recombinant mutant enzyme contains 6 histidine tags at the C-terminus. After prokaryotic expression, the purified...

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Abstract

The invention relates to construction of a heat-resistant beta-galactosidase mutant. When the heat-resistant beta-galactosidase (BgaB) mutant (BgaB-F341T) provided by the invention is used for a lactose hydrolysis process to produce low-lactose products, the inhibition action of the hydrolysis products to the hydrolytic activity of the enzyme can be effectively removed, so that the hydrolysis reaction is thoroughly carried out and the hydrolysis efficiency is improved; and when the heat-resistant beta-galactosidase (BgaB) mutant (BgaB-F341T) is used to replace medium-temperature lactase, the low-temperature hydrolysis process of low-lactose milk can be innovated. The hydrolysis temperature can be set at 55-60 DEG C, at the temperature, not only is the hydrolysis reaction speed high, but also common impure bacteria in the milk has stopped growing, therefore the product hygiene and quality problems caused by impure bacterial contamination can be effectively controlled; and in addition, by the high-temperature hydrolysis process using the heat-resistant lactase, the lactose hydrolysis and pasteurization processes can be combined, not only is the production cycle shortened, but also the energy consumption is reduced by taking full use of the waste heat from the pasteurization process, thus significantly saving production cost.

Description

technical field [0001] The invention relates to the construction of a thermostable beta-galactosidase mutant. Belongs to the field of protein engineering. Background technique [0002] β-galactosidase (β-galactosidase) full name β-D-galactoside galactohydrolase (β-D-galactoside galacto hydrolase, EC3.2.1.23), commercial β-galactosidase commonly known as Lactase can catalyze the hydrolysis reaction of β-1,4-galactosidic bonds in lactose or lactose analogues. Lactase is widely used in the fields of food industry, medicine, and biochemical analysis, among which the largest application is in the dairy industry. It can degrade lactose into glucose and galactose to produce low-lactose milk, thereby effectively solving problems such as lactose intolerance. According to epidemiological surveys and studies, lactose intolerance is mainly caused by a higher proportion of people of color, with the highest incidence rates among Asians and African Americans. About 75% to 95% of Chinese...

Claims

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Application Information

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IPC IPC(8): C12N9/38C12N15/56C12N15/10C12N15/70A23C9/12C12R1/07C12R1/19
Inventor 董艺凝王领陈卫徐峻张灏陈海琴
Owner JIANGNAN UNIV
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