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Method for preparing glucose from starch sugar by using acidothermus sp.-derived debranching enzyme having heat resistance and acid resistance, and glucose prepared thereby

A technology for preparing starch sugar and glucose, which is applied to biochemical equipment and methods, using carriers to introduce foreign genetic material, enzymes, etc., can solve the problem that microorganisms cannot be used directly, and achieve the effect of improving yield

Inactive Publication Date: 2018-10-23
CJ CHEILJEDANG CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the high molecular weight of starch, starchy biomass such as corn biomass cannot be directly used by microorganisms

Method used

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  • Method for preparing glucose from starch sugar by using acidothermus sp.-derived debranching enzyme having heat resistance and acid resistance, and glucose prepared thereby
  • Method for preparing glucose from starch sugar by using acidothermus sp.-derived debranching enzyme having heat resistance and acid resistance, and glucose prepared thereby
  • Method for preparing glucose from starch sugar by using acidothermus sp.-derived debranching enzyme having heat resistance and acid resistance, and glucose prepared thereby

Examples

Experimental program
Comparison scheme
Effect test

example 1

[0036] Example 1: Gene isolation and recombinant protein preparation

[0037] 1) Preparation of recombinant Escherichia coli

[0038] For the PCR, a forward primer sequence and a reverse primer sequence respectively comprising: both the terminal sequence of the debranching enzyme gene derived from the thermotolerant and acid-tolerant A. cellulolyticus and the recognition sequences of the restriction enzymes NdeI and HindIII were used .

[0039] (forward) 5'GAA-TTC-ATG-CCG-GAA-3'

[0040] (reverse) 5'GAA-TTC-TTA-GAG-GAC-3'

[0041] As a result, a PCR product of 2.1 kb was obtained. The obtained DNA fragment was inserted into pET-28a(+) vector, and transfected into Escherichia coli BL21(DE3). The DNA has the sequence described in SEQ ID NO: 1. The transformed E. coli was spread on plate medium containing kanamycin. Firstly, the kanamycin-resistant strains were screened out and cultured separately in liquid medium. DNA from kanamycin-resistant strains was purified and dige...

example 2

[0045] Example 2: Measuring Enzymatic Activity by HPLC

[0046] 1) The activity of a debranching enzyme (isoamylase) derived from an acid-resistant / heat-resistant microorganism (Acidothermus cellulolyticus) was measured by HPLC under the following conditions. Activity measurement HPLC analysis conditions are as follows.

[0047] HPLC analysis conditions

[0048] - Detector: RID

[0049] -Flow rate: 0.6ml / min

[0050] - Sample injection volume: 10 μl

[0051] - Analytical column: Biot-Rad HPX 87C

[0052] -Solvent: deionized water

[0053] The total time for the analysis was set at 20 minutes.

[0054] The activity of a debranching enzyme (isoamylase) derived from an acid-resistant / heat-resistant microorganism (Acidothermus cellulolyticus) was confirmed by multiple enzymatic reactions with glucoamylase (glucoamylase) using corn starch as a raw material Facilitate the reaction to produce glucose. The enzymatic reaction conditions are as follows.

[0055] First, the gluc...

example 3

[0056] Example 3: Comparison of the yield of glucose prepared using a debranching enzyme (isoamylase) derived from Acidothermus sp. and a commercially available debranching enzyme (pululanase)

[0057] The yield of glucose from starch was investigated using a debranching enzyme derived from A. cellulolyticus and a commercially available debranching enzyme (Pululanase, Novozymes). The enzymatic reaction conditions are as follows.

[0058] First, glucoamylase (glucoamylase, DuPont) and each debranching enzyme (isoamylase, pullulanase) were used at a total unit ratio of 1:4. Multiple enzymatic reactions with liquefied starch (DE 9.5) were carried out at a temperature of 60°C for 72 hours. The reaction was terminated by heating at 100°C for 5 minutes. Then, the amount of glucose produced was measured by HPLC according to the ratio between the areas of the peaks corresponding to the products of the enzymatic reaction. As a result, the reaction using the debranching enzyme (isoam...

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Abstract

The present application relates to improving the production yield of glucose to be prepared by using starch sugar as a raw material and using an Acidothermus sp.-derived enzyme. The present application can replace an acid saccharification method, which produces a bitter taste through a hydrolysis reverse reaction, and, compared to a conventional enzymatic saccharification method in which a debranching enzyme is used, has an advantage of enabling costs to be reduced since a relatively high yield can be ensured by reducing side reactions of a saccharification reaction.

Description

technical field [0001] The present invention relates to a method for preparing glucose from starch sugars using debranching enzymes and glucoamylases derived from Acidothermus species. Background technique [0002] Biomass can be broadly classified into sugar (sugar) biomass, starch (starch) biomass, and cellulose (cellulose) biomass according to main components. Sugars are readily available from sugar cane and can be taken up directly by organisms without additional pretreatment or glycosylation. However, starchy biomass such as corn biomass cannot be directly used by microorganisms due to the high molecular weight of starch. Therefore, starch needs to be broken down into glucose. This process requires water and is therefore called hydrolysis. [0003] Starch is mainly hydrolyzed by enzymes, just as we digest rice. The cooked rice we eat is degraded into glucose in the stomach by enzymes secreted from the salivary glands, and the glucose is transferred to body cells and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/02C12N15/70C12N9/44
CPCC12N15/70C12P19/02C12Y302/01068C12N9/246C12P19/16C08L3/02C08L3/12
Inventor 安准甲尹烂媖金成俌朴承源
Owner CJ CHEILJEDANG CORP