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Raw starch-digesting glucoamylase, preparation method thereof and application of raw starch-digesting glucoamylase to raw starch hydrolysis and preparation of ethanol by simultaneous saccharification and fermentation of raw starch

A raw starch saccharifying enzyme and raw starch technology, which is applied in the directions of biochemical equipment and methods, glycosylating enzymes, and botanical equipment and methods, can solve the problems of increased production and application costs, low activity of raw starch saccharifying enzymes, etc.

Inactive Publication Date: 2016-09-07
GUANGXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the activity of the reported raw starch glucoamylase is not high, and they have a preference for the type of hydrolyzed raw starch (Li HF, Chi ZM, Wang XH, Duan XH, Ma LY, Gao LM. Purification and characterization of extracellular amylase from the marine yeast Aureobasidiumpullulans N13d and its raw potato starch digestion.Enzyme and Microbial Technology,2007,40:1006-1012;Sun H,Zhao P,Ge X,Xia Y,Hao Z,Liu J,Peng M.Recent advances in microbial raw starch degrading enzymes.Applied Biochemistry and Biotechnology, 2010,160:988-1003), which increases the cost of production and application, so it is necessary to develop excellent raw starch glucoamylases with a wide range of starch substrates and high hydrolytic activity

Method used

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  • Raw starch-digesting glucoamylase, preparation method thereof and application of raw starch-digesting glucoamylase to raw starch hydrolysis and preparation of ethanol by simultaneous saccharification and fermentation of raw starch
  • Raw starch-digesting glucoamylase, preparation method thereof and application of raw starch-digesting glucoamylase to raw starch hydrolysis and preparation of ethanol by simultaneous saccharification and fermentation of raw starch
  • Raw starch-digesting glucoamylase, preparation method thereof and application of raw starch-digesting glucoamylase to raw starch hydrolysis and preparation of ethanol by simultaneous saccharification and fermentation of raw starch

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0098] Example 1, Separation and Purification of Raw Starch Glucoamylase Produced by Penicillium GXU20 and Determination of Enzymatic Properties

[0099] 1. Separation and purification of raw amylase produced by Penicillium GXU20

[0100] Penicillium GXU20 was fermented and cultured with the enzyme-producing medium according to the method of Example 2 of the Chinese invention patent with the authorized announcement number 101955887B, and the crude enzyme liquid of Penicillium GXU20 was obtained. Among them, Penicillium GXU20 is a biological material authorized in the Chinese invention patent with the authorization announcement number 101955887B.

[0101] 1. Ethanol graded precipitation

[0102]Ethanol precipitation was performed using a two-step method. Add an appropriate amount of Penicillium GXU20 crude enzyme solution concentrated by a rotary evaporator into a 500mL Erlenmeyer flask, place it on ice and slowly stir the crude enzyme solution with a magnetic stirrer, slowly...

Embodiment 2

[0154] Embodiment 2, cloning, expression and identification of raw starch glucoamylase gene

[0155] 1. Cloning of the gene encoding raw starch glucoamylase

[0156] 1. Mass spectrometry analysis of raw starch glucoamylase PoGA15A

[0157] Perform SDS-PAGE electrophoresis on the raw starch glucoamylase PoGA15A obtained in Example 1, decolorize after staining with Coomassie Brilliant Blue R-250, wear clean PE gloves on both hands, wipe the sterilized scalpel with ethanol to lightly remove the target protein band Gently cut and take it out (try to cut off the excess protein glue on the edge of the target band to facilitate subsequent trypsin hydrolysis), put it into an EP tube, trypsinize it at 37°C overnight, and perform ion orbital hydrazine mass spectrometry (LTQ-Orbitrap LC / MS / MS) detection.

[0158] Three internal amino acid polypeptide sequences highly matching with the glucoamylases from Penicillium marneffei and Penicillium oxalicum were obtained (results are shown in ...

Embodiment 3

[0187] Example 3, Application of Recombinant Raw Starch Glucoamylase rPoGA15A in Hydrolysis of Raw Starch and Simultaneous Saccharification and Fermentation of Raw Meal to Produce Alcohol

[0188] 1. Recombinant raw starch glucoamylase rPoGA15A efficiently and rapidly hydrolyzes raw starch

[0189] 1. Hydrolysis of different types of raw starch by recombinant raw starch glucoamylase rPoGA15A

[0190] Determination of total starch content: Weigh 1g Co 60 Put irradiated sterilized rice, corn, cassava, and potato raw starch into finger bottles, add 10mL of 2M concentrated hydrochloric acid solution, place in boiling water bath for 20min, and neutralize with 2M sodium hydroxide solution to pH 7.0 , 100mL volumetric flask to constant volume, DNS method to measure the glucose content in the supernatant, convert it into the corresponding starch amount, and calculate the total starch content (Moon SK, Kim SW, Choi GW.Simultaneous saccharification and continuous fermentation of sludge...

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Abstract

The invention discloses a preparation method of raw starch-digesting glucoamylase and application of the raw starch-digesting glucoamylase to raw starch hydrolysis and preparation of ethanol by simultaneous saccharification and fermentation of raw starch. The raw starch-digesting glucoamylase is obtained after codon optimization, and a nucleotide sequence of the raw starch-digesting glucoamylase is shown as any of from A1) to A5): A1), sequence 3 in a sequence table; A2), nucleotides from 1st position to 1878th position in the sequence 3 in the sequence table; A3), DNA molecules shown by nucleotides in 19th position to 1878th position in the sequence 3 in the sequence table; A4), DNA molecules shown by nucleotides in 19th position to 1857th position in the sequence 3 in the sequence table; A5), a protein sequence shown in sequence 4 in a coding sequence table and showing 98% identity with the sequence limited by A1), A2), A3) or A4). Through experiments, it is proven that raw starch-digesting glucoamylase genes can be effectively expressed in a heterologous expression system to obtain the raw starch-digesting glucoamylase rPoGA15A, and the raw starch-digesting glucoamylase rPoGA15A and alpha-amylase act synergistically to hydrolyze the raw starch rapidly and efficiently and prepare the ethanol by simultaneous saccharification and fermentation of the raw starch.

Description

technical field [0001] The invention relates to a preparation method of raw starch glucoamylase in the field of biotechnology, in particular to a preparation method of raw starch glucoamylase rPoGA15A and the application of the raw starch glucoamylase rPoGA15A in the hydrolysis of raw starch and synchronous saccharification and fermentation of raw materials to produce alcohol. Background technique [0002] Starch synthesized by plants through photosynthesis is one of the most important storage carbohydrates in plants in nature. Starch is more abundant in seeds (such as rice, wheat, corn, sorghum, etc.) and roots (such as cassava, potatoes, etc.) of plants. High, its yield is second only to cellulose. [0003] Starch is a polymer of glucose molecules, including a straight chain connected by α-1,4-glucosidic bonds, and α-1,6-glycosidic bonds form random and variable branch points, and the length of the branch points can be connected different straight chains. Starch can be d...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N9/34C12N15/81C12P19/14C12P7/06
CPCC12N9/2428C12N15/815C12N2800/102C12N2800/22C12P7/06C12P19/14C12Y302/01003Y02E50/10
Inventor 冯家勋徐强胜闫语丝
Owner GUANGXI UNIV
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