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Enzymoimmuno screening, fermentation preparation and application of strains used for producing glucosyltransferase

A technology for producing glucosides and strains, applied in the field of fermentation, can solve the problems of low conversion rate of oligosaccharides, time-consuming, inability to correctly reflect the ability to convert glycosides, etc.

Active Publication Date: 2014-05-07
中国科学院上海生命科学研究院湖州工业生物技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the screening methods of common enzyme-producing strains at home and abroad are mostly based on the amount of enzyme activity, while the determination of glucosyltransferase activity is mostly based on the amount of glucose produced rather than the amount of trans-oligosaccharides as the basis for the level of enzyme activity, while Studies have shown that the ability to hydrolyze maltose as a substrate cannot correctly reflect the ability to transglycoside. The strains screened by this method often have high enzyme activity, but the conversion rate of oligosaccharides is low.
[0007] Another screening method is to use the HPLC method to determine the enzyme activity based on the amount of oligosaccharides such as isomaltose and panose produced, which is more scientific and accurate, but the sample size is large and it takes a lot of time
In other words, the assay is cumbersome and time-consuming, not suitable for large-scale or high-throughput screening

Method used

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  • Enzymoimmuno screening, fermentation preparation and application of strains used for producing glucosyltransferase
  • Enzymoimmuno screening, fermentation preparation and application of strains used for producing glucosyltransferase
  • Enzymoimmuno screening, fermentation preparation and application of strains used for producing glucosyltransferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] antiserum test

[0071] 1.1 Obtaining antiserum (primary antibody)

[0072] 1.1.1 Antigen preparation

[0073] The commercially available glucosidyl transferase was subjected to SDS-PAGE electrophoresis first, and the target protein band was cut off after Coomassie brilliant blue staining.

[0074] 1.1.2 Emulsified antigen

[0075] Grind the cut target protein band with a tissue grinder, add an equal amount of Freund's incomplete immune adjuvant, suck it into two syringes, connect the two syringes with a latex tube about 5 cm long, and push back and forth alternately Needle until a milky white emulsion forms. To check the emulsification, put one drop on the surface of cold water. If it does not spread on the water surface, the emulsification is complete.

[0076] 2.1.3 Immunization scheme

[0077] In this experiment, Japanese big-eared rabbits were used, and the mixed immunization method was used. The immunization procedures are as follows:

[0078] Table 1 Immuni...

Embodiment 2

[0086] Enzyme-linked detection method

[0087] 2.1 Preparation of crude antigen (glucosidyl transferase)

[0088] Take 1ml of fermentation broth, centrifuge at 10,000rpm, take the supernatant, add 2 times the volume of ice ethanol to extract, centrifuge at 10,000rpm, and take the precipitate. The precipitate was diluted with freshly prepared pH9.6 sodium carbonate buffer until the protein content was 1-10 μg / ml, and plated.

[0089] 2.2 planking

[0090] Plate the diluted antigen and dilute with sodium carbonate buffer: add 50ul antigen solution and 50ul sodium carbonate buffer to the first well, mix well, transfer 50ul to the second well, add 50ul sodium carbonate buffer, mix well , placed at 37°C for one hour.

[0091] 2.2 Blocking

[0092] Pour off the residual liquid, pat dry, add freshly prepared PBS-BSA to block, add 100ul to each well, and place at 37°C for two hours.

[0093] * PBS: Phosphate Buffered Saline (PH7.4)

[0094] * BSA: bovine serum albumin

[0095...

Embodiment 3

[0112] Screening of strains with high production of glucosidyl transferase

[0113] 3.1 Strains

[0114] More than 100 strains of industrial production of glucoamylase, acid protease, citric acid, pectinase and gluconic acid were selected, and these strains belonged to Aspergillus niger, Aspergillus foetidus, Aspergillus carbonarious, Aspergillus cinmomeus, Aspergillus usamii, Aspergillus nidulan, Aspergillus oryzae, P. chrysogenum, Aureobasidium pullulans, etc.

[0115] 3.2 Medium

[0116] Maltose 2%, yeast powder 0.5%, K 3 PO 4 0.1%, MgSO 4 ·7H 2 O 0.05%, FeCl 3 0.03%

[0117] 3.3 Fermentation conditions

[0118] 100ml of liquid in a 500ml Erlenmeyer flask, initial pH 5.5, 30°C, 250rpm, rotate for 72 hours.

[0119] 3.4 ELISA results

[0120] Crude antigen (glucosidyl transferase) is according to the method for 2.1 in embodiment 2, and detection process is according to the method described in embodiment 2, and representative result is as table 2 and figure 1 shown...

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Abstract

The invention relates to enzymoimmuno screening, fermentation preparation and application of strain for producing glucosyltransferase, in particular to a method for screening strains in a large quantity used for producing glucosyltransferase, comprising the following steps: (a) utilizing the specific antibody which resists the glucosyltransferase to detect the valence of the glucosyltransferase serving as antigen in the protein in each candidate strain; and (b) according to the detected valence, selecting the candidate strains with high valence to serve as the strains for producing the glucosyltransferase. The invention also provides a method for producing the glucosyltransferase by utilizing the strains obtained by the screening method and a method for efficiently producing oligomaltose from the glucosyltransferase. The invention not only can realize screening in a large quantity, but also greatly improves the efficiency of producing the oligomaltose by enzymatic synthesis by utilizing the glucosyltransferase.

Description

technical field [0001] The invention relates to the field of fermentation, and more specifically relates to the enzyme-linked immunological screening, fermentation preparation and application of the glucosidyl transferase (Alpha-GTase) producing strain in efficient catalytic synthesis of isomaltooligosaccharides. Background technique [0002] Glucosidase (Transglucosidase, EC 2.4.1.24, alpha-GTase) is a kind of α-D glucosidase, which can hydrolyze maltose to generate glucose, and can also specifically carry out the transfer reaction of glucosidic bonds. One of the essential enzyme preparations for polysaccharides, it produces non-fermentable oligosaccharides such as isomaltose and panose with α-1,6 bonds from maltose. [0003] In recent years, it has been found that low-molecular-weight oligosaccharides such as isomaltose and panose are good bifidus factors, which cannot be digested and absorbed by the human body after ingestion, and are not easily used by most spoilage bact...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569C12N1/14C12N9/10C12P19/18C12R1/645C12R1/80C12R1/69C12R1/66C12R1/685
Inventor 李平作江昊王莉章婷凌晨叶晴
Owner 中国科学院上海生命科学研究院湖州工业生物技术中心