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Acid glucanase CELA and gene and application thereof

A technology of acid glucanase and glucanase, applied in the field of genetic engineering, can solve the problems of viscosity increase, decomposition and early gel precipitation of saccharified mash

Active Publication Date: 2011-09-21
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

During the barley germination process, the dextran in the barley cannot be completely decomposed by its own endogenous glucanase, usually only 30%-70% can be decomposed. The residual glucan increases the viscosity of the mash and reduces the filtration rate. The finished beer is prone to haze or early gel precipitation

Method used

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  • Acid glucanase CELA and gene and application thereof
  • Acid glucanase CELA and gene and application thereof
  • Acid glucanase CELA and gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0097]Example 1 Isolation and purification of Alicyclobacillus peridum A4 (CGMCC No.3147) and its enzyme-producing properties

[0098] Inoculate Alicyclobacillus speridum A4 on the enzyme-producing medium plate (yeast extract 1.0g, tryptone 2.0g, oat dextran 5.0g, agar 30, Congo red 0.5g, adjust the pH to 3.0 with hydrochloric acid) at 60°C After culturing for 72 hours, it was stained with 0.5% Congo red, and it was preliminarily verified that it had glucanase activity according to the presence or absence and size of the transparent circle. Alicyclobacillus speridum A4 was cultured at 60°C for 48 hours in an enzyme-producing medium (1.0 g of yeast extract, 2.0 g of tryptone, 5.0 g of oat dextran, adjusted to pH 3.0 with sulfuric acid), and the glucose content of the supernatant was determined. Glycanase activity. Proved to have dextranase activity.

Embodiment 2

[0099] Example 2 Cloning of Alicyclobacillus peridum A4 (CGMCC No.3147) Glucanase Encoding Gene CELA

[0100] Acquisition of gene sequence

[0101] According to the conserved (YWEIGNE and AMKAVD) sequence of the 51st family glucanase gene, degenerate primers Cel51F and Cel51R were designed and synthesized as shown in Table 1

[0102] Alicyclobacillus hesperidum A4 (CGMCC No.3147) total DNA was used as template for PCR amplification. The PCR reaction parameters are: denaturation at 94°C for 5 min; the first 10 cycles, denaturation at 94°C for 30 sec, touchdown (0.5°C / cycle) at 50°C-55°C for 30 sec, extension at 72°C for 0.5 min, and the last 25 cycles at 94°C Denaturation for 30 sec, annealing at 55°C for 30 sec, extension at 72°C for 1.5 min. Finally, keep warm at 72°C for 10 minutes. A fragment of about 180bp was obtained, which was recovered and connected with the pEASY-T3 vector and sent to Sanbo Biotechnology Co., Ltd. for sequencing.

[0103] According to the nucleoti...

Embodiment 3

[0113] Example 3 Preparation of recombinant dextranase.

[0114] The expression vector pPIC9 is subjected to double digestion (SnaBI and NotI), and the gene CelA encoding glucanase is double-digested (SnaBI and NotI) at the same time, and the gene fragment encoding glucanase is cut out and connected to the expression vector pPIC9 to obtain The recombinant plasmid pPIC9-CelA containing the glucanase gene CelA was transformed into Pichia pastoris GS115 to obtain the recombinant Pichia pastoris strain GS115 / CelA.

[0115] The GS115 strain containing the recombinant plasmid was inoculated in 400mL of BMGY culture medium, shaken at 250rpm at 30°C for 48h, and then collected by centrifugation. Then resuspend in 200mL BMMY medium, shake culture at 250rpm at 30°C. After 48 hours of induction, the supernatant was collected by centrifugation. Determination of dextranase activity. The expression level of recombinant glucanase was 20.41U / mL. SDS-PAGE results ( figure 1 ) showed that ...

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Abstract

The invention relates to the field of genetic engineering, in particular to acid glucanase CELA and gene and application thereof. The invention provides the glucanase CELA from Alicyclobacillus hesperidumA4 (CGMCC No. 3147), the amino acid sequence of the glucanase CELA is expressed as SEQ ID No.1, and the invention provides the gene CelA for coding the glucanase. The glucanase of the invention has the following properties of: optimal pH of 3.4, optimal temperature of 65 DEG C, and good thermal stability. The acid glucanase CELA serving as a novel enzyme preparation can be widely applied to industries of animal feed, food, energy and the like.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular, the invention relates to an acid glucanase CELA and its gene and application. Background technique [0002] β-glucan is widely found in the aleurone layer and endosperm cell wall of cereals (barley, oats, rye and wheat). It belongs to the structural non-starch polysaccharide in the plant cell wall. , the content and proportion of β-glucan are also different. Among them, barley and oats contain the highest proportion. Glucanase is a general term for a class of enzymes that can degrade dextran into oligosaccharides and glucose. At present, dextranase has been widely used in food, feed, beer, medicine and other fields. At present, barley is mainly used in beer brewing and feed, so the related glucanase has been most widely used in beer brewing and feed industry, especially in Europe, in the feed formula with barley and soybean meal as the main raw materials, The addition of dext...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/42C12N15/56C12N15/63C12N1/15C12N1/19C12N1/21A23K1/165
Inventor 姚斌柏映国孟昆罗会颖石鹏君黄火清王亚茹袁铁铮杨培龙
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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