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Marine-microbe-sourced low-temperature beta-galactosidase, and coding gene thereof and application thereof

A technology of galactosidase and marine microorganisms, applied in the direction of plant genetic improvement, application, genetic engineering, etc., can solve problems such as limited application, high optimum temperature, and rapid inactivation

Inactive Publication Date: 2013-06-05
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the β-galactosidase that has been used in the milk processing industry has Kluyveromyces from the Dutch DSM company. The molecular weight of the enzyme is 135kDa, and the optimum pH is pH6.8, which is close to the natural pH of fresh milk; There are some significant disadvantages, such as the enzyme has a narrow pH stability range, and it is rapidly inactivated when the pH is greater than 7.0 or less than pH 6.2; the optimum temperature for action is higher, which is 45°C, and the activity at low temperatures is low, and Ca 2+ It has a significant inhibitory effect on its enzyme activity, and these shortcomings greatly limit its application in the dairy industry

Method used

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  • Marine-microbe-sourced low-temperature beta-galactosidase, and coding gene thereof and application thereof
  • Marine-microbe-sourced low-temperature beta-galactosidase, and coding gene thereof and application thereof
  • Marine-microbe-sourced low-temperature beta-galactosidase, and coding gene thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Embodiment 1: A kind of preparation method of low-temperature β-galactosidase derived from marine microorganisms

[0053] 1. Fermentation of Halomonas sp.P6009-1

[0054] Inoculate the Halomonas sp.P6009-1 strain in Zobell2216E liquid medium (peptone 5g, yeast powder 1g, iron phosphate 0.1g, dissolved in 1L artificial seawater, pH7.4), 28°C, shaker (130rpm) for 24h to activate. The formula of artificial seawater is: NaCl25.0g, Na 2 SO 4 4.0g, KCl0.7g, NaHCO 3 0.20g, KBr0.10g, H 3 BO 3 0.03g, NaF0.003g, 53mL1.0mol / L MgCl 2 Solution, 10mL1.0mol / l CaCl 2 Solution, 0.90ml0.1mol / L SrCl 2 Solution, distilled water 1000ml. Then take 500 μL of the activated bacterial liquid and inoculate it into 100 mL of Zobell 2216E medium containing 2% lactose, and cultivate it at 28°C with shaking at 130 rpm for 4 days.

[0055] 2. Preparation of β-galactosidase crude enzyme solution of Halomonas sp.P6009-1

[0056] After the fermentation, the fermentation broth was centrifuged a...

Embodiment 2

[0059] Embodiment 2: The thermostability and optimal reaction temperature of the low-temperature β-galactosidase prepared in embodiment 1

[0060] at 74 μl K 2 HPO 4 -KH 2 PO 4 (pH7.2, 0.1mol / L) buffer solution, add 1μL of LP6009-BGalH, 40mmol / LONPG or 25μl of lactose, place at 0-60℃ for 10min, then add 100μL of 1mol / L Na 2 CO 3 The reaction was terminated, and the absorbance value of ONP was measured at 410 nm or the amount of lactose was measured by HPLC. In thermostability experiments, the enzymes were pre-incubated at different temperatures of K 2 HPO 4 -KH 2 PO 4 (pH7.2, 0.1mol / L) buffer solution for 1 hour, and then carry out the above reaction. The ability to hydrolyze ONPG and lactose at the optimum temperature was determined. the result shows( figure 2 ), when ONPG was used as the substrate, the optimal reaction temperature of BGalH was 40°C; when lactose was used as the substrate, the optimal reaction temperature was 35°C. At 0°C, BGalH can maintain 5% a...

Embodiment 3

[0061] Embodiment 3: pH stability and optimum pH of the low-temperature β-galactosidase prepared in embodiment 1

[0062] Take 1 μL of P6009-BGalH, 74 μL of pH3.0-9.5 buffer (pH3.0-5.5 is 0.1mol / L citric acid-sodium citrate buffer; pH6.0-8.0 is 0.1mol / L K 2 HPO 4 -KH 2 PO 4 Buffer solution; pH8.5-9.5 is 0.1mol / L Tris-HCl buffer solution), 25μL of 40mmol / L ONPG dissolved in each of the above buffer solutions, mix well, keep the reaction solution containing BGalH at 30 ℃, and react 10min, then add 100μL of 1mol / L Na 2 CO 3 The reaction was terminated, and the absorbance value of ONP was measured at 410 nm or the amount of lactose was measured by HPLC. In the pH stability experiment, P6009-BGalH was pre-incubated in different pH buffers at 4°C for 1h, and then the above decomposition reaction was carried out at 30°C. The experimental results show( Figure 4 ), when ONPG was used as substrate, the optimum reaction pH of BGalH was 8.0; when lactose was used as substrate, the o...

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Abstract

The invention relates to the field of marine biotechnology, and provides a marine-microbe-sourced low-temperature beta-galactosidase, which is obtained by fermentation culturing and purification of a marine halomonas sp. strain P6009-1 obtained by the applicant previously. The marine halomonas sp. strain P6009-1 is cultured and separated from seawater in East China Sea areas, and has a collection number of CCTCC No: M2011001. Under a temperature of 0 DEG C, a capacity of the enzyme in catalyzing lactose hydrolysis is 20% of a maximal activity, such that good low-temperature performance is shown. The invention further provides a coding gene of beta-galactosidase, and an application of beta-galactosidase in decomposing milk lactose.

Description

technical field [0001] The invention relates to the technical field of marine organisms, in particular to a low-temperature beta-galactosidase derived from marine microorganisms, its coding gene and its application. Background technique [0002] β-galactosidase (scientific name β-D-galactoside galactohydrolase, β-D-galactoside galatohydrolase, EC3.2.1.23) is a specific hydrolysis of β-1,4-glucoside It is a key hydrolase that can hydrolyze lactose into glucose and galactose, and also has the function of galactoside transfer. [0003] β-galactosidase is widely found in organisms in nature, such as plants, bacteria, fungi (Aspergillus oryzae, Aspergillus niger, Saccharomyces brittle, lactic acid yeast), actinomycetes, and animal intestines (especially infant intestines) middle. [0004] Judging from the catalytic reaction, β-galactosidase belongs to glycosyl hydrolase (abbreviated as GH). According to its sequence and structure, β-galactosidase is divided into four families, ...

Claims

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Application Information

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IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N5/10C12N1/21C12P19/14
Inventor 刘小宇王国祥卢小玲缪明永高云焦炳华胡波刘军华
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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