KR0129471B1 - Preparation method of amylase producing maltotetraose - Google Patents

Abstract

This invention provides the method of preparing maltotetrose forming amylase. KFCC-10846 of streptomyces species is isolated from compost, and cultivated in an aerobic condition, i.e. the fermented culture medium mainly composed of cationic starch and oxidized starch. In this invention, the condensed maltotetrose forming amylase is produced by inexpensive carbon provider, i.e. the starch.

Description

Method for preparing maltotetraose producing amylase

The present invention relates to a method for producing maltotetraose producing amylase, and more particularly, to a method for producing maltotetraose producing amylase using microorganisms.

Maltotetraose-producing amylase (G4-amylase below 3,2,1,60 or less) produces maltotetraose, the main ingredient of malto-oligosaccharide, which is mainly used as a food additive and is used as a food additive. It is effective in controlling the physical properties such as freezing point drop or osmotic pressure as well as controlling sweetness and preventing browning. It is also used as a substrate for analysis of serum amylase.

G4-amylase is a type of alpha amylase that degrades alpha 1,4-glucoside bonds and is generally characterized in that it is degraded into an exo form. The exo-type alpha amylases produced by some microorganisms have the specificity of producing maltose, maltotriose, maltotetraose, maltopentaose, maltohexaose, etc. .

Known G4-amylase-producing strains include Bacillus circulans J62025978A, maltose, which produces maltotetraose producing enzymes based on amylose, amylopectin, glycogen, and the like. Bacillus megaterium J01202294A, Alkalophilic Bacillus sp. J630077754A and Maltotetraose and Maltopentaose, which mainly produce Bacillus strains such as Bacillus sp. J60227676A, which simultaneously produce amylase and pullulanase, which produce similar yields, and Micrococcus sp. J01218598A, which simultaneously produce amylase and flulanase. Pseudomonas stutzeri.

The present invention is to produce malto terraose as a main product in addition to the conventional strain and to isolate the amylase-producing strain suitable for industrialization and to apply to industrialization.

Microorganisms isolated by the inventors of the present invention according to the object of the present invention to produce G4-amylase is a strain of the genus Streptomyces, which is a kind of actinomycetes is isolated and identified in the compost of northern Gyeonggi-do. KSM-35 from the isolated strains was cultured and observed under an optical microscope. As a result of mycelial formation, the chain of spores appeared to be long. Particularly, the spiral chain was clear and one spore chain was generally 12-15. It was confirmed that the dog consists of spores. In addition, LL-DAP (Rf: 0.3) and texts were analyzed by TLC (Thin Layer Chromatography) by acid hydrolysis to determine the type of diaminopimelic acid of the whole cell components through which the microorganism passes. Lylic acid (glycine Rf: 0.47) was detected, and alanine (alanin Rf: 0.67) and glutamic acid (glutamic acid Rf: 0.58) were detected. No specific sugars were detected.

These results indicate that the strain is classified into Streptomyces according to the classification by Lechevalier Lechvalier. Furthermore, the comparison of morphological, cultural and physiological characteristics has identified the species as a related species of albus, but further research is required.

The morphological, culture, physiological and biochemical properties of this strain are as follows.

(1) glycosylation and G4-amylase production ability

Maltotetraose productivity amylase produced by the novel microorganism Streptomyces genus KSM35 (KFCC-10846) acts on liquefied starch under certain conditions to produce maltotetraose as a main product and other maltotriose and maltose Produced. The enzyme has a strong ability to liquefy starch, so unlike the known G4-amylase, it is considered to be an endo-type enzyme. The content of maltotetraose in the final product is about 25% of the product, and maltotriose and The maltose content was 17% and 16%, respectively.

Figure 1 Analysis of reaction product of G4-amylase and liquefied starch (HPLC analysis)

On the other hand, the separated microorganisms are not only soluble starch, but also oxidized starch and cationic starch, which are modified starch of corn starch and corn starch, compared to other G4-amylase producing microorganisms mainly using soluble starch as a carbon source. In particular, the enzyme titer of G4-producing amylase is greatly increased when cationic starch and oxidized starch are used as main carbon sources.

Table 1 Comparison of G4-amylase enzyme titers according to carbon source

Cationic starch used by the present inventors is treated with 3-chloro-2-hydroxypropyl trimethyl ammonium chloride in ordinarystarch such as corn starch. The starch has a positive cationic form (Figure 2) .Hypochloride-oxidized starch is treated with sodium chlorine (NaOC1) in general starch, and chlorine ion (Cl). Starch, which has a concentration of 0.1%, has a negative charge under weakly acidic conditions, and the structural formula is shown in Figure 3.

Figure 2 Molecular formula of quaternary ammonium starch ether (positive starch)

Figure 3 Structural formula of starch oxide

The specific mechanisms of increased starch and oxidized starch have not been found to be obvious mechanisms, but they are industrially advantageous because they are much cheaper and more readily available than soluble starch in accumulating enzymes in culture.

Thus, the present inventors have completed the present invention by discovering that the active starch and the oxidized starch are highly effective in enzymatic activity while isolating microorganisms that produce maltoterraise-producing amylase and studying alternative carbon sources.

Hereinafter, the present invention will be described in detail in Examples and Examples, but is not limited thereto.

Experimental Example

1) Selection method

Dilutions of the compost were plated on agar solid medium containing inorganic salts and starch, incubated at 37 ° C. for 3 days, and colonies were separated.

The isolated strains were transferred to starch casein KNO ₃ agar plate and incubated at 37 ° C. for 5 days, and the first strain was selected to show a relatively large diameter of transparent rings formed by adding iodine solution. These primary screening strains were incubated for 3 days at 37 ° C. and 130 rpm in an Ellenmayer flask containing 100 ml of starch casein potassium nitrate agar medium. The precipitate formed by adding milk to the culture filtrate at 80% saturation was recovered after centrifugation and dissolved in 0.04M phosphate buffer solution (pH 5.9) to prepare amylase and compared amylase activity. Strains with relatively high primary screening activity were again isolated to examine starch degradation products by TLC, and among them, strains with high maltotetraose productivity were finally selected.

2) Enzyme Activity Analysis Method of Produced Amylase

0.5 ml of gelatinized 1% soluble starch solution and 0.1 ml of phosphate buffer solution with 0.04M and pH 5.9 were added to the test tube, preheated in a 37 ℃ constant temperature water bath, and 0.1 ml of enzyme solution was added.The reaction was stopped for 30 minutes and boiled for 10 minutes to stop the reaction. . 0.5 ml of this reaction was taken and added to 5 ml of an iodine solution (0.5 g of iodine and 5 g of potassium iodide dissolved in 100 ml of distilled water 200-fold prior to use) and absorbance was measured at 700 nm. The enzyme activity was measured by measuring the amount of starch remaining after the reaction. One unit of enzyme was defined as the ability to degrade 1 mg of starch in one minute.

3) analysis of reaction products

Centrifuge the resulting precipitate by gently incubating the culture solution obtained by culturing for 3 days at 37 ℃, 400rpm, 1vvm in 5L fermenter as coenzyme solution. After separation, the mixture was dissolved in 50 mM phosphate buffer and concentrated. The concentrated enzyme solution was added to 10% of the soluble starch solution and reacted at 60 ° C. for 30 hours, and then maltotetraose produced using high performance liquid chromatography (HPLC) was analyzed.

Example 1

Owner: Streptomyces sp. KFCC-10846 KSM35

Spawn medium: 1% soluble starch, 0.5% polypeptone, 0.5% yeast extract, 0.1% potassium diphosphate, 0.05% magnesium sulfate, pH6.0

Fermentation medium: 3% amphoteric starch (or oxidized starch, soluble starch, corn starch, glucose, sucrose) polypeptone 0.7%, yeast extract 0.3%, dipotassium phosphate 0.3%, magnesium sulfate 0.1%, iron sulfate 0.01 %, Calcium chloride 0.02%, pH6.0

Cultivation method: Dispense 50 ml of the seed medium into 500 ml shake flask, sterilize and cool, inoculate the new strain LSM35 (KFCC-10846) of the present invention, and centrifuge the culture at 37 ℃ for 24 hours as seed culture solution. . 200ml fermentation broth is dispensed into 1L shake triangle fructose, sterilized and cooled at 121 ° C. for 15 minutes, followed by 10% inoculation of the seed culture solution, followed by 170 rpm centrifugation culture at 37 ° C. for 3 days.

After the incubation was completed, the cells were removed by centrifugation at 5,000 rpm for 10 minutes and the supernatant was used as an experimental solution. The enzyme titer is measured by the method of 2). At this time, the main raw material was oxidized starch as 1.35u / ㎖, and the positive starch as the main raw material was 2.92u / ㎖.

Experimental Example 2

Fermentation broth of Example 1 using positive starch was injected 2.5L into a 5L fermentation tank, sterilized and cooled, and then inoculated with 50 ml of pre-prepared seed culture solution, followed by incubation for 3 days under agitation 400rpm, aeration 1vvm, 37 ° C. The enzyme titer of the culture broth was measured by the method of Example 1. The titer of the crude enzyme solution was 6 u / ml. As a result of purifying the culture termination solution by the method of Experimental Example 3, the titer of the concentrated enzyme solution was 60 u / ml.

As described above, Streptomyces sp. KFCC-10846 KSM35, a strain producing maltotetraose-producing amylase, was isolated by the composition of the present invention, and the enzymes produced by these strains are particularly conventional maltotetraose. It is particularly useful for the production of maltotetraose-producing amylase due to its high enzymatic activity in cationic and oxidized starches, which are much cheaper than the soluble starches used for production.

Claims (3)

In the production of maltotetraose-producing amylase using microorganisms, maltotetraose-producing amylase was produced by aerobic culture of Streptomyces sp. KSM35 (KFCC-10846) isolated from compost in a fermentation medium containing starch as the main ingredient. forming amylase one side G4-amylase) is a method for producing maltooligosaccharide-producing amylase, characterized in that the accumulation in cells and culture. The method for producing maltooligosaccharide-producing amylase according to claim 1, wherein the starch is used as a carbon source, in particular, a cationic starch and an oxidized starch at a concentration of 3%. The method for producing maltotetraose producing amylase according to claim 1 or 2, wherein said culture is carried out aerobicly at 37 DEG C while maintaining a culture pH of 6.0.