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Strain for producing alpha-transglucosidase and application thereof

A glucosidase and production technology, which is applied in the field of microbial screening, can solve the problems of dependence on imports, low conversion rate of production process, restriction of isomaltose oligosaccharide production, etc., and achieves the effect of broad market prospect and improved glycosidation efficiency.

Active Publication Date: 2015-03-18
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, there is a process of producing isomaltooligosaccharide by enzymatic method using starch or maltose as raw material in industry, but the conversion rate of the existing production process is not high
In addition, although the production of isomaltose in my country is very high, α -Transglucosidase, as the most critical enzyme preparation in the production of isomaltooligosaccharide, is used in a large amount, but industrial production has not been realized in China, and the enzyme still relies on imports. These limiting factors have greatly restricted domestic isomaltooligosaccharide production

Method used

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  • Strain for producing alpha-transglucosidase and application thereof
  • Strain for producing alpha-transglucosidase and application thereof
  • Strain for producing alpha-transglucosidase and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: UV mutagenesis and screening

[0025]Starting strain: Asperillus niger (Asperillus niger) CGMCC No.8245 is constructed by transforming the α-transglucosidase gene from Aspergillus fumigatus into Aspergillus niger host strain G1, which can express α- Aspergillus niger engineered bacteria that transfer glucosidase.

[0026] 1.1 UV mutagenesis method

[0027] (1) Inoculate the starting strain Asperillus niger CGMCC No.8245 on a CMA slant for activation, and culture it at 37°C for 4 days;

[0028] (2) Wash the activated fresh slant with 3ml of sterile 0.1% Tween-80 to prepare spore suspension, draw 10ul and place it on a hemocytometer for counting, dilute the spore suspension until the number of spores is about 10 according to the counting results 6 pcs / ml or so;

[0029] (3) Take 5ml of the diluted spore suspension and place it in a 9cm petri dish, and carry out ultraviolet mutagenesis under the conditions of 30w of ultraviolet lamp, irradiation distance o...

Embodiment 2

[0047] Embodiment 2: the shake flask fermentation verification of mutant strain Aspergillus niger Afm78

[0048] The mutant strain Aspergillus niger Afm78 (CGMCC No.8470) and the starting strain were inoculated on the CMA plate for 4-5 days, and their respective spore suspensions were inoculated in 20ml of CLS fermentation medium, under the conditions of 30°C and 200rpm Cultivate for 48 hours; then inoculate 2ml of CSL culture broth into 20mL of TSB fermentation medium according to 10% inoculum amount, culture at 30°C and 200rpm for 5 days, and collect the fermentation supernatants of Afm78 and starting strains respectively; The supernatant was desalted and desugared with Econo-Pac10DG Columns (BOI-RAD), and then subjected to 12% SDS-PAGE electrophoresis detection and analysis, the results were as follows figure 1 As shown, two strains all have obvious protein band at 109kDa place, illustrate that the mutant strain Aspergillus niger Afm78 that the present invention obtains als...

Embodiment 3

[0049] Embodiment 3: enzymatic property analysis

[0050]With the buffer solution dilution that pH value is 2.0,3.0,4.0,5.0,6.0,7.0,8.0,9.0,10.0,11.0 the fermentation supernatant of starting bacterium described in embodiment 2 and mutant bacterium, measure its enzyme activity respectively, with The highest enzyme activity is 100%, calculate the relative enzyme activity, and make the pH-relative enzyme activity curve. The result is as figure 2 As shown, the α-transglucosidase expressed by the mutant Aspergillus niger Afm78 of the present invention has no change in the action pH-relative enzyme activity curve compared with the original strain, and the optimum action pH value is 5.0.

[0051] At 30°C, 40°C, 50°C, 60°C, 70°C, 80°C, 90°C, and pH 5.0, the enzyme activities of the fermentation supernatants of the starting bacteria and mutant bacteria described in Example 2 were measured, and the highest enzyme activity If it is 100%, calculate the relative enzyme activity and make...

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Abstract

The invention provides Asperillus niger Afm78 for producing alpha-transglucosidase with high yield. The strain is preserved in the China General Microbiological Culture Collection Center (CGMCC) of the Institute of Microbiology of Chinese Academy of Science in Datun Road, Chaoyang District in Beijing on November 12th, 2013, and the preservation number of the strain is CGMCC No.8470. According to the strain, Aspergillus niger CGMCC No.8245 is used as an original strain, the Asperillus niger Afm78 for producing alpha-transglucosidase with high yield is obtained by using an ultraviolet mutagenesis method, and the fermenting enzyme activity of the strain is as high as 6699U / mL, that is, is 54% higher than that of the original strain. The alpha-transglucosidase expressed by the Asperillus niger Afm78 can be widely applied to the field of food machining. By adding the alpha-transglucosidase provided by the invention in production of isomalto oligosaccharide, the glycoside transferring efficiency can be greater than 50%, the mass volume ratio of the isomalto oligosaccharide in an obtained product can be greater than 92%, and wide market prospect is achieved.

Description

technical field [0001] The invention belongs to the technical field of microbial screening, and in particular relates to a production strain of α-transglucosidase and its application. Background technique [0002] α-transglucosidase (α-transglucosidase E.C.2.4.1.24) can cut α-1, 4 glycosidic bonds from the non-reducing end of the oligosaccharide substrate to release glucose, or free glucose residues Transfer to another carbohydrate substrate with α-1, 6 glycosidic bonds, so as to obtain non-fermentable isomaltose oligosaccharides (referred to as IMO, mainly including isomaltose, panose, isomaltotriose and tetrasaccharides or more) Glycans), glycolipids or glycopeptides, etc. The enzyme not only has hydrolysis ability, but also can specifically carry out the transfer reaction of glucosidic bonds, and is one of the necessary enzyme preparations for the production of isomaltooligosaccharides. [0003] α-transglucosidase is widely distributed in nature with various types and d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/14C12N9/10C12R1/685
Inventor 王华明訾祯祯
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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