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Immobilized beta-glucosaccharase and preparation method and application thereof

A glucosidase and reaction technology, applied in the fields of enzyme engineering and bioengineering applications, can solve the problem of high use cost, and achieve the effects of improving catalytic efficiency, improving pH stability, and improving glucose tolerance

Inactive Publication Date: 2017-11-17
NANJING FORESTRY UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these enzymes cannot meet the needs of industrial applications, mainly in terms of pH stability, sugar tolerance, temperature stability and other properties that need to be improved, and the cost of using enzymes is relatively high

Method used

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  • Immobilized beta-glucosaccharase and preparation method and application thereof
  • Immobilized beta-glucosaccharase and preparation method and application thereof
  • Immobilized beta-glucosaccharase and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The β-glucosidase clone and recombinant enzyme preparation of the GH3 family of embodiment 1.T.petrophila DSM 13995 origin

[0032] 1.1 Culture of T.petrophila DSM 13995

[0033] Thermotoga petrophila DSM 13995 was purchased from the DSMZ Culture Collection Center (www.dsmz.de) with the number 13995. The medium formula is: 10g / L starch, 5g / L tryptone, 3g / L yeast extract, 5g / L meat extract, 10g / L2‐morpholinoethanesulfonic, 10mg / L FeSO 4 ×7H 2 O, 1mg / Lresazurin, adjust the pH to 7.2. Inoculate with a syringe according to 0.5% inoculation amount, culture statically at 85° C. for 24 hours, and collect cells.

[0034] 1.2 Genomic DNA extraction

[0035] (1) Statically culture Thermotoga petrophila DSM 13995 for about 24 hours, take 30 mL of bacterial liquid and centrifuge at 4,000 g for 10 min to collect cells.

[0036] (2) Resuspend the bacteria in 9.5 mL TE buffer, add 0.5 mL 10% sodium dodecyl sulfate (SDS) and 50 μL proteinase K (20 mg / mL), mix well, and incubate at...

Embodiment 2

[0050] Example 2. Preparation of immobilized enzyme (NKA-9II) by immobilizing β-glucosidase derived from T.petrophila DSM 13995GH3 family using macroporous adsorption resin

[0051] 2.1 Macroporous resin pretreatment

[0052] In order to remove impurities and pollutants in the macroporous resin, and more effectively maximize the amount of enzyme protein adsorbed by the macroporous adsorption resin carrier, the experiment pretreated the macroporous resin by adding ethanol, hydrochloric acid and sodium hydroxide respectively. First, the macroporous resin (20 g) was added to 100 mL of absolute ethanol and soaked for 24 hours, and then washed with deionized water repeatedly for 3 times until no ethanol smell remained. Then the treated macroporous resin was soaked in 100 mL solution containing 5% hydrochloric acid for 4 h, and then the resin was washed to neutral pH with deionized water. Then soak the cleaned resin in a solution containing 1M sodium hydroxide (100 mL) for 4 hours,...

Embodiment 3

[0097] Example 3. Zn 2+ Effects on the enzymatic properties of the immobilized enzyme NKA‐9II

[0098] 3.1. Metal cation Zn 2+ Effect on the catalytic activity of the prepared immobilized enzyme NKA-9II

[0099] Experiment by comparing different metal cations Fe 2+ , Li 2+ , Al 3+ , K + , Ni 2+ , Mn 2+ , Ca 2+ , Zn 2+ and Mg 2+ The impact on the catalytic activity of the immobilized enzyme NKA-9II, the results are shown in Table 2, the metal ion Mn 2+ and Zn 2+ The catalytic efficiency of the immobilized enzyme can be significantly improved. mn 2+ It can increase the enzyme activity of immobilized enzyme by 45%; Zn 2+ It can increase the enzyme activity of immobilized enzyme by 92%, the results show that Zn 2+ effect is more obvious.

[0100] 3.2. Zn 2+ Effect on the reproducibility of the prepared immobilized enzyme NKA-9II

[0101] In the presence or absence of 2mM Zn 2+ In the case of participation, 10 repeated reactions were carried out, the results are ...

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Abstract

The invention provides immobilized beta-glucosaccharase and a preparation method and application thereof. The method comprises the following steps of (1) macroporous resin pre-treatment; (2) macroporous adsorption resin NKA-9 polyetherimide surface modification; (3) macroporous resin absorption beta-glucosaccharase; (4) glutaric dialdehyde cross linking reaction modification. The metal ion Zn<2+> is used as an activating agent and a heat stabilizer of the immobilized beta-glucosaccharase for using macroporous adsorption resin for the first time. Through the participation of Zn<2+>, the catalysis efficiency of the beta-glucosaccharase is improved by 20 percent or higher; the catalysis efficiency of the immobilized enzyme NKA-9II is improved by 92 percent or higher. Relative to the initial enzyme activity, through the participation of Zn<2+>, after the incubation for 7h under the condition of 85 DEG C, the enzyme activity of the immobilized enzyme NKA-9II is maintained at a value being 80 percent or higher; after the incubation for 7h under the condition of 90 DEG C, the enzyme activity of the immobilized enzyme NKA-9II is maintained at a value being 54 percent or higher.

Description

technical field [0001] The invention belongs to the field of enzyme engineering and bioengineering applications, in particular to an immobilized enzyme and its preparation method and application, especially to the immobilization method of β‐glucosidase and the use of Zn 2+ The application of the method for improving the catalytic ability and enzymatic characteristics of the immobilized enzyme. Background technique [0002] β‐glucosidase (β‐glucosidase, EC 3.2.1.21) belongs to the class of exohydrolase, also known as β‐D‐glucoside hydrolase, which can break β‐glucosidase in short chains of cellooligosaccharides or cellobiose. 1,4‐glycosidic bonds, can also break β‐1,4‐glycosidic bonds between residues of carbohydrates with aromatic groups or alkyl groups, and thus are widely used in cellulose hydrolysis, flavonoids and saponins Biocatalysis and transformation of natural active substances such as ketones and other fields. However, the main obstacle restricting the large-scal...

Claims

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Application Information

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IPC IPC(8): C12N11/08C12P19/14
CPCC12N9/2445C12N11/08C12P19/14C12Y302/01021Y02P20/50
Inventor 赵林果石学佳萧伟王振中李琦
Owner NANJING FORESTRY UNIV
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