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Immobilized recombinant high temperature beta-galactosidase preparation method

A technology of galactosidase and high temperature, which is applied in the direction of fixing on/in the organic carrier to achieve good thermal stability, stability in use, and convenient preparation

Inactive Publication Date: 2007-05-09
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no use of trihydroxymethylphosphine as a chitosan cross-linking agent to immobilize high-temperature enzymes, especially for the immobilization of recombinant high-temperature β-galactosidase derived from Bacillus stearothermophilus.

Method used

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  • Immobilized recombinant high temperature beta-galactosidase preparation method
  • Immobilized recombinant high temperature beta-galactosidase preparation method
  • Immobilized recombinant high temperature beta-galactosidase preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] The chitosan vector was prepared according to the above-mentioned conditions in the instructions, and the vector was activated with THP. The recombinant Bacillus subtilis WB600 / pMA5-bgaB derived from the β-galactosidase gene of Bacillus stearothermophilus was selected (see "Industrial Microbe "Journal of 2004 Vol. 34 No. 1) high-temperature β-galactosidase produced by immobilized enzyme treatment.

[0027] The factors affecting the recovery rate of immobilized enzyme activity include crosslinking agent concentration, activation time, immobilization buffer pH, ionic strength, crosslinking temperature, immobilization time, order and so on. The optimal parameter conditions for immobilized enzyme obtained from single factor test are: THP concentration 0.625mg / mL, activation time 5-10min, immobilization buffer pH6.5-7.2, ionic strength 0.1-0.2mol / L, cross-linking temperature 20-30℃, immobilization time 5-10min. In addition, THP concentration, immobilization buffer pH, and ionic ...

Embodiment 2

[0035]Taking the immobilization parameter conditions in Example 1 as a reference, the Thermus sp.A4 β-galactosidase expressed in recombinant Escherichia coli (Escherichia coliMV1184) (using the plasmid pUC118 / 119 as a vector, the strain and For plasmids, see "Bioscience Biotechnology & Biochemistry" in 1998, Vol. 62, No. 8, pages 1539-1545) for enzyme immobilization. Because the optimum temperature of the enzyme is 85°C, it is also a high-temperature β-galactosidase. The optimal immobilization conditions of the high-temperature recombinase are: THP concentration is 0.625 mg / mL, immobilization buffer pH is 6.8, concentration is 0.1 mol / L, cross-linking temperature is room temperature, and immobilization time is 10 min. The activity recovery rate of the immobilized enzyme prepared under this condition is 92%, the optimal temperature is 85℃, and the enzyme activity is almost not lost after being incubated at 70℃ for 5 hours. The enzyme activity remains 80% after the lactose hydrolysi...

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Abstract

The invention relates to a method of preparing immobilization reconstructing high-heat beta-galactosidases, belonging to the immobilization and appliance technique field thereof of high-heat beta-galactosidase. Said invention takes chitose as carriers, THP as gellants, and conducts immobilization process to high-heat beta-galactosidase , takes the permanent order of activating chitose carriers with gellants firstly, then fixing the enzymes on the carriers which have been well activated to gain immobilization reconstructing high-heat beta-galactosidases. Taking THP as gellants, the preparing of immobilized enzymes is convenient, compact; the recovery rate of enzyme activities could reach higher than 90% with good heat and using stability, and the activities of immobilized enzymes could remain over 80% after heat preservation for 5 hours at the temperature of 70 DEG C; taking lactoses as hydrolytic substrates and proceeding 9 cycles at the temperature of 70 DEG C, the activities of immobilized enzymes could remain over 60%.

Description

Technical field [0001] A preparation method of immobilized recombinant high-temperature β-galactosidase belongs to the technical field of immobilization of high-temperature β-galactosidase and its application. Background technique [0002] β-galactosidase (EC3.2.1.23), commonly known as lactase, can hydrolyze β-1,4-galactosidase bonds and has important industrial uses. Heat-resistant β-galactosidase, or high-temperature lactase, is superior to room-temperature lactase due to its good thermal stability and hydrolysis properties at higher temperatures. Compared with the immobilization of normal temperature enzymes, high temperature enzymes are more difficult to select crosslinking agents, immobilization parameters and conditions because they are used at higher operating temperatures. [0003] β-galactosidase mainly comes from plants, animal organs, bacteria, yeast (intracellular), fungi or mold (extracellular). High-temperature β-galactosidase generally refers to β-galactosidase wh...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N11/10C12N11/04
Inventor 陈卫田丰伟黄文利张灏赵建新杨静
Owner JIANGNAN UNIV