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Method for increasing the yield of monosaccharide production in agar by using buffer pretreatment

A pretreatment and buffer technology, applied in the direction of biochemical equipment and methods, enzymes, hydrolytic enzymes, etc., can solve the problems of expensive, low production yield of monosaccharides, etc.

Active Publication Date: 2021-07-06
KOREA UNIV RES & BUSINESS FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this method, although pretreatment is necessary to increase the reactivity of Aga50D, the currently used mild chemical pretreatment method with weak acid requires neutralization or lyophilization for the subsequent enzymatic reaction, resulting in a large amount of neutralization Salt generated in and a considerable amount of processing fees
Furthermore, the α-1,3 bond between 3,6-anhydro-L-galactose and D-galactose is mainly degraded when chemical pretreatment is performed, and when exo-type β-agarase is treated, the final remaining Agarotriose is not degraded into 3,6-anhydro-L-galactose and D-galactose, which also becomes the cause of lower production yield of monosaccharides
Furthermore, until now, in the production of 3,6-anhydro-L-galactose and D-galactose, agarose has been used as an initial substrate and produced from red algae by repeating the isolation and purification process several times, therefore expensive

Method used

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  • Method for increasing the yield of monosaccharide production in agar by using buffer pretreatment
  • Method for increasing the yield of monosaccharide production in agar by using buffer pretreatment
  • Method for increasing the yield of monosaccharide production in agar by using buffer pretreatment

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Production of Recombinase

[0057] Using pET-21a as a vector and Aga50D (EMBL ID: ADB81904), NABH (EMBL ID: ADB81917) and ABG (EMBL ID: CP003241) enzymes in Escherichia coli (E.coli) BL21 (DE3 ) to produce recombinant proteins. Colonies obtained by transforming host cells plated on solid medium supplemented with 50 μg / mL ampicillin were re-inoculated on Luria broth (LB) plates containing 50 μg / mL ampicillin, and then incubated at 37 °C and 220 rpm 12 hours (20 mL×2) for seed culture. Then, the cells were seeded in two 3L Erlenmeyer flasks each containing 1L LB broth, cultured with shaking under the same conditions for 3 hours (OD=0.8) and cooled on ice for 1 hour, and then added 0.1mM IPTG to inoculate at 16 Expression was induced at 120 rpm for 12 hours. The resulting culture was centrifuged (6000rpm, 4°C, 15 minutes) to collect the cells, and then the resulting cells were suspended in 20mM Tris buffer (Tris-HCl, 1M NaCl pH8), lysed with a sonicator, and centrifuge...

Embodiment 2

[0058] pretreatment agar with buffer solution

[0059] In order to compare glycation after agar pretreatment with 20 mM Tris-HCl (pH 7.5) buffer and agarose pretreatment with 3% acetic acid, two types of pretreatment were performed. 1 g (5%) agar was mixed with 20 mL of 20 mM Tris-HCl (pH 7.5) buffer at 170 °C for 5, 10, 15 and 20 min, and 1.4 g (7%) agarose was mixed with 20 mL of 3% acetic acid And microwave treatment at 130° C. for 30 minutes to carry out hydrolysis. Then, the resulting product was neutralized to pH 7 using 1M NaOH, and the final concentration of agar and agarose was adjusted to 4.5% using distilled water.

[0060] Table 1 shows the final production yields of 3,6-anhydro-L-galactose and D-galactose, the amount of HMF produced and the amount of neutralizing solvent according to the pretreatment conditions and initial substrates.

[0061] [Table 1]

[0062]

[0063]

[0064]As shown in Table 1, in agar pretreatment (170°C, 10 minutes) with 20 mM Tri...

Embodiment 3

[0065] with the enzymatic saccharification method of the pretreatment product of buffer pretreatment

[0066] 5 mL of the pretreated product of Example 2 was reacted with the enzymes Aga50D (19.0 U), ABG (4.6 U) and NABH (14.5 U) prepared in Example 1 in sequence. Each enzyme was reacted at 30°C for 12 hours and then inactivated in boiling water for 5 minutes. To confirm the effect of ABG in this process, only two enzymes such as Aga50D and NABH (excluding ABG) were reacted under the same conditions as described above.

[0067] Final reaction products such as 3,6-anhydro-L-galactose and D-galactose were quantified by HPLC (Agilent 1100, Agilent, Waldbronn, Germany) using an Aminex HPX-87H column (Bio-Rad, Richmond, CA). The mobile phase was 40% (v / v) acetonitrile (CH 3 CN) in water and analyzed at 65 °C and a flow rate of 0.5 mL / min.

[0068] The unit of Aga50D is defined as the amount of agarobiose produced from agarose in 20 mM Tris-HCl buffer (pH 7) at 30° C. for 1 minu...

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Abstract

The present invention relates to a method for improving the production yield of monosaccharides in agar by using a buffer pretreatment, and by using agar instead of agarose as an initial substrate, using a buffer as a pretreatment solution, and sequentially using agarase, Agarose-decomposing β-galactosidase and α-neo-agarobiohydrolase are used as enzymes in enzymatic hydrolysis, and have the effect of improving the production of 3,6-anhydro-L-galactose and galactose as monosaccharides. rate effect.

Description

technical field [0001] The present invention relates to a method for improving the production yield of monosaccharides such as 3,6-anhydro-L-galactose and galactose by enzymatically producing monosaccharides from agar using a buffer as a pretreatment solvent. Background technique [0002] Compared with lignocellulosic or herbaceous biomass currently used in various fields, seaweed has a low content of unsuitable components such as lignin, etc., and thus can be converted into simple sugars, which are used for easier production of bioenergy and biochemical raw materials. Furthermore, since seaweeds do not use biomass resources, they also do not have problems due to the energization of biomass resources. For such reasons, seaweed has received attention as an important biomass in biochemistry and production of alternative energy (Wi et al., Bioresour Technol, 100 (2009), pp. 6658-6660). [0003] In particular, it has been reported that red algae (for example, Gelidium amansii)...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/02C12N9/24
CPCC12N9/24C12P19/02C12N9/00C12P19/00
Inventor 金京宪崔仁杰李赞炯尹银珠
Owner KOREA UNIV RES & BUSINESS FOUND
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