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Dextranase and application of same in preparation of low molecular dextran

A technology of dextranase and dextran, which is applied in the direction of enzymes, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems of non-concentrated product molecular weight distribution, unfavorable environmental protection, low-carbon and other problems, and achieve The effect of lower production process cost, lower production cost, and simple process

Active Publication Date: 2014-06-04
HEFEI UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] In the traditional industry, the production of dextran is mainly carried out through the fermentation of Leuconostoc mesenteroides combined with the acid hydrolysis process. Due to the random disorder of acid hydrolysis, the molecular weight distribution of the obtained product is not concentrated, which affects the quality of the product; in addition, due to the use of Hydrochloric acid hydrolysis requires harsh conditions such as high temperature and high acid, high energy consumption, not conducive to environmental protection, low carbon, and the product yield needs to be improved

Method used

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  • Dextranase and application of same in preparation of low molecular dextran
  • Dextranase and application of same in preparation of low molecular dextran
  • Dextranase and application of same in preparation of low molecular dextran

Examples

Experimental program
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Effect test

Embodiment 1

[0027] A method for isolating strain F1008 producing dextranase high enzyme activity, which comprises the following steps:

[0028] Add 1g of soil samples (soil samples were taken from Hefei, Anhui, Bengbu, Anhui, and Chengdu, Sichuan) to 99ml sterile water to prepare soil dilution, add penicillin solution (50μl to 100ml) into the PDA medium, pour it into a petri dish, and let it stand Let cool into a flat plate. Use the dilution coating method to apply the diluted soil solution on the plate respectively, and then place it upside down in a constant temperature incubator at 30°C for cultivation. After the bacteria grow out, use the inoculation needle to pick the strain into another sterile screening culture base (Formulation: Dextran T20001g, NaNO 3 0.3g, K 2HPO 4 ·3H 2 O0.4g, MgSO 4 ·7H 2 O0.02g, KCl0.02g, FeSO 4 ·7H 2 (0.001g, agar 1.6g, water 100ml, pH5.0~5.5) were streaked and separated, and then the single colonies that could grow on the screening medium were picke...

Embodiment 2

[0031] The technical scheme of using Penicillium aculeatus F1008 to produce dextranase:

[0032] 1) Strain activation: Use sterilized solid PDA medium to inoculate the F1008 strain on the slant medium of the test tube, cultivate it at 28-30°C for 48-60 hours, and then use it to prepare the strain seed solution;

[0033] 2) Strain seed fermentation culture: the composition of the seed medium is 1.5g of Dextran100kDa per 100ml of water, 0.4g of peptone, K 2 HPO 4 ·3H 2 O0.1g, NaNO 3 0.2g, MgSO 4 ·7H 2 O0.05g, KCl0.05g, FeSO 4 ·7H 2 O0.001g, pH5.0-5.5, inoculate the strain in step 1) into the seed medium, place it on a constant temperature shaker at 30-32°C, and cultivate it for 48-72 hours at a speed of 220r / min as seeds liquid, and then use the seed liquid for fermentation to prepare dextranase;

[0034] 3) Preparation of dextranase by fermentation: the components of the fermentation medium are 1.5g of Dextran100kDa per 100ml of water, 0.4g of peptone, K 2 HPO 4 ·3H ...

Embodiment 3

[0038] Application of Penicillium aculeatus F1008 in the preparation of low molecular weight dextran:

[0039] Add 240g of sucrose and 2000mL of buffer solution to an electric mechanical stirring device and a temperature-controlled beaker in a water bath. After stirring to dissolve fully, control the temperature of the water bath at 30°C, and add 4000U of dextran sucrase and 5000U of dextranase at the same time, and stir for 22- After 24 hours, raise the temperature of the water bath to 80°C and keep it warm for 1 hour to inactivate dextran sucrase. After filtering the denatured enzyme protein with gauze, place it in a boiling water bath for 20 minutes to inactivate the residual dextranase. Then vacuum filter to obtain a clear and transparent filtrate. , which is low molecular weight dextran solution.

[0040] The solution was concentrated by rotary evaporation at 70°C to remove part of the water, concentrated to half of the original volume and subjected to fractional alcohol ...

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Abstract

The invention discloses a preparation method for dextranase and application of the dextranase in preparation of low molecular dextran. The invention is characterized in that a producing bacterial strain for the dextranase is Penicillium aculeatum F1008 separated from soil and the bacterial strain is preserved in China General Microbiological Culture Collection Center with an accession number of CGMCC No. 8135. According to the invention, the dextranase with high enzyme activity is successfully prepared through fermentation culture of Penicillium aculeatum F1008; the dextranase has catalysis temperature of 25 to 35 DEG C, a catalysis pH value of 5.0 to 6.0 and average enzyme activity of higher than 600 U / mL, gives preference to reaction with a high-molecular-weight substrate in a multi-substrate solution and is especially applicable to preparation of low-molecular-weight dextran; low molecular dextran varieties prepared through catalysis of the Penicillium aculeatum F1008 dextranase comprise micromolecular dextran with weight average molecular weight of 10000 Da, 6000 to 8000 Da and 3000 to 5000 Da. The dextranase has the advantages of mild catalytic reaction condition, good substrate specificity, low energy consumption and capacity of achieving the objects consisting of yield increase, low carbon and low cost in production of micromolecular dextran.

Description

technical field [0001] The invention relates to the fields of biopharmaceuticals and enzyme engineering, in particular to a preparation method of dextranase and its application in preparing low-molecular-weight dextran. Background technique [0002] Dextran (dextran) is a chain glucan, which is composed of glucose units connected by α-1,6-glycosidic bonds, and also contains a small amount of branched chain structures composed of α-1,3-glycosidic bonds. Dextran and its derivatives have been widely used in many fields such as medicine, industry, and food because of their safety and non-toxicity, and the market demand is huge (the global annual demand is nearly tens of billions of dollars). Dextran with different molecular weights has different medicinal values ​​and biological functions. The middle molecular weight dextran with a weight average molecular weight of 20-70kDa is the most clinically used pharmaceutical grade dextran. It is mainly used as a plasma substitute for th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/14C12N9/44C12P19/16C12R1/79
Inventor 张洪斌甘微苇张宇琪胡雪芹
Owner HEFEI UNIV OF TECH
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