A kind of production technology of double-enzyme compound production gamma-cyclodextrin

A production process and cyclodextrin technology, applied in the field of cyclodextrin production, can solve the problems of long reaction period, low substrate concentration, low enzyme activity, etc., and achieve low production cost, short reaction period and high raw material conversion rate. Effect

Active Publication Date: 2014-10-01
JIANGNAN UNIV
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  • Summary
  • Abstract
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  • Application Information

AI Technical Summary

Problems solved by technology

However, these processes also have serious problems such as low substrate concentration and long reaction cycle.
There is no report on the combined use of isoamylase and γ-CGTase to produce γ-cyclodextrin, because the enzyme activity of γ-CGTase is very low under neutral conditions, only about 10% of the highest enzyme activity; and isoamylase The enzyme activity is very low or even inactivated under alkaline conditions

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Embodiment 1: Construction and cultivation of genetically engineered bacteria

[0025] According to the γ-CGTase sequence in NCBI, the sequence of γ-CGTase in Alkalophilic Bacillus 7364 was used as a template to design primers with the amino acid at position 223 mutated from alanine (A) to lysine (K), and obtained after PCR amplification. fragment. The target fragment and the carrier pET24a were digested by double enzymes and recovered from the gel, connected overnight at 16°C and then transferred into E.coli JM109 competent cells, spread on LB / Kana plates and cultured at 37°C for 8 hours, picked single clone colonies to LB / Kana In Kana liquid medium, cultured at 37°C, 200rpm for 8h, extracted the plasmid and verified by enzyme digestion, obtained the recombinant expression vector pET24a(+)-γ-γ-CGTase / A223K, and transferred it into E.coli BL21(DE3) competent cells, Obtain the recombinant bacteria E.coli BL21(DE3) / pET24a(+)-γ-CGTase / A223K, inoculate it in TB medium, cul...

Embodiment 2

[0027] Raw material pretreatment:

[0028] According to the concentration of 5-20%, the starch slurry is adjusted, and the temperature is kept at 60-90°C for 5-15 minutes, so that the starch granules can fully absorb water and swell;

[0029] Enzymatic production process:

[0030] After pretreatment, set the temperature to 40-60°C, adjust the pH to 7.0-8.0, add 10-100 units of γ-CGTase / A223K and 10-100 U of isoamylase per gram of starch at the same time, and then After adding 5% cyclododecanone by volume, fully react for 6-12 hours;

[0031] The extraction process of γ-cyclodextrin:

[0032] After killing the enzyme in a boiling water bath, filter the reaction liquid, wash the filter cake 2-3 times with distilled water, collect the filter cake, including the precipitate formed by cyclododecanone and γ-cyclodextrin and unreacted starch; Redissolve in water, and then use steam distillation to remove cyclododecanone. After the distillation is over, filter the distillate to rem...

Embodiment 3

[0035] Raw material pretreatment:

[0036] Same as Example 2

[0037] Enzymatic production process:

[0038] After pretreatment, set the temperature to 40-60°C, adjust the pH to 7.0-8.0, add isoamylase at a rate of 10-100 units per gram of starch, and react for 2-6 hours; then add 10-100 units One unit of γ-CGTase / A223K and 5% cyclododecanone are added according to the mass volume ratio, and fully reacted for 4-10 hours;

[0039] The extraction process and detection method of γ-cyclodextrin:

[0040] Same as Example 2

[0041] The total conversion rate of starch is as high as 68.8%, the proportion of γ-cyclodextrin in the product is 64.3%, and the proportion of β-cyclodextrin is 4.5%.

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PUM

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Abstract

The invention relates to a production process of gamma-cyclodextrin through double-enzyme compounding and belongs to the technical field of cyclodextrin production. The production process of the gamma-cyclodextrin through the double-enzyme compounding lowers the production cost of the gamma-cyclodextrin and adopts the technical scheme that the process comprises the steps of utilizing gamma-CGTase (cyclodextrin glycosyltransferase) (A223K) from basophilic bacillus and isoamylase to produce the gamma-cyclodextrin, preparing starch slurry according to a concentration of 5-20%, keeping warm at 60-90 DEG C for 5-15min, setting the temperature to be 40-60 DEG C, regulating pH to be 7.0-8.0, adding 10-100U of gamma-CGTase (A223K) and 10-100U of isoamylase to starch per gram, adding 5%(w / v) of organic solvent, conducting sufficient reaction for 6-12h, recovering the organic solvent, and obtaining the gamma-cyclodextrin by a crystallization method.

Description

technical field [0001] The invention relates to a production process for producing gamma-cyclodextrin by compounding double enzymes, and belongs to the technical field of cyclodextrin production. Background technique [0002] Cyclodextrins (Cyclodextrins, usually abbreviated as CDs) is a general term for a class of cyclic compounds produced by starch or its derivatives under the action of cyclodextrin glucosyltransferase (CGTase). CDs are composed of D-glucopyranose as a unit, connected by α-1,4-glycosidic bonds, and common cyclodextrins are composed of 6, 7 and 8 glucose units, which are called α-, β- and gamma-cyclodextrin. Due to the special structure of cyclodextrin itself, it can embed many inorganic and organic compounds, such as drug carriers, quality improvers, stabilizers and adsorbents, so it is widely used in food, chemistry, medicine, agricultural environmental protection, biotechnology, etc. And textile technology and other fields have a wide range of applicat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/18C08B37/16
Inventor 吴敬王磊吴丹陈坚
Owner JIANGNAN UNIV
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