JPS58170476A - Preparation of amylase g 4, 5 - Google Patents

Abstract

PURPOSE:To prepare a conjugated enzyme consisting of amylase G 4, 5 or amylase G 4, 5 and pullulanase, by cultivating a bacterium belonging to the genus Bacillus. CONSTITUTION:A bacterium such as Bacillus circulans G 4, 5 (FERM-P 6237) belonging to the genus Bacillus, capable of producing amylase G 4, 5 is cultivated with aeration in 6-9pH at 25-55 deg.C, and amylase G 4, 5 is collected from the culture solution.

Description

[Detailed Description of the Invention] The present invention combines starch with Mar>i>Force! Ryomirror (Q vehicle I) that decomposes lutobentaose into glycated products as its main component
This method corresponds to the Ii method.

As conventional amylase, -17 Mirai was made.

Recently, more molecular weight products such as maltotriose (GJ) and maltotriose (Gel) have been introduced.
Lutopentaose (Gj), maltotriose (Gp)
There is a demand for the development of oligosaccharide manufacturing techniques such as It is believed that these sugars can be widely used in foods, industrial products, and other industrial products as sweeteners, production enhancers, excipients, and clathrating agents, but no method has yet been established. Tina ψ.

The name of this invention is based on the microorganisms of Yushu and Bacillus, which have been widely searched for in soil in search of microorganisms with strong oligosaccharide-producing ability. Lutotetoforce (G
We have focused on producing large amounts of acera (which decomposes into axons whose main components are G) and maltopentaose (G) outside the bacterial cells.

! The enzyme that produces lucitetraose is Ps@m
It is known that amyl 4 produced by domenms 5ttli@ri O generates maltotetraose from the non-reducing end of amylyse and amiupetatin (Arekive @f B1** and @m1try
s+adB1*phys1cm i+zia, 101~
//41屓(197/)) However, this enzyme does not produce maltopentaose.

On the other hand, amylase that produces maltopentaose is found in Bacillus l.
It has been reported that O produces heat-resistant mono-amylase (ζ
The ability of the O# element to generate mal)te)laose O is much smaller than that of maltopentaose, and is on the order of a small amount. Also,
This # element also generates each component of G/-G/J in the same position r#
It is elementary (Ar@h1ma... fBiochemistr
y and Blophys1gs Volumes 1 to 5, λ9O
-291 (1971) I. In addition, 8a @ 1-11ua
mubtlli@'s taste - Amylase also produces a mixture of sugars containing maltotetraose and maltopentaose from starch, but since maltotetraose and maltopentaose are not the main components, these axes are Not suitable for Ichizo.

However, the sugar composition of the starch decomposition product produced by the amylase of the present invention also depends on the DE of the starch used and the reaction conditions during saccharification (starch concentration, pH% during reaction,
# It is slightly activated depending on the amount of leaves, and as the reaction time becomes longer, maltopentaose gradually becomes maltose! decomposes into lutotriose), but usually mal)tesilaose is 1jNJjl and maltopentaose is /jN70
Accounting for these axes will be added to JO-40%.

That is, the amylase produced by the present invention is amylose,
From apropectin, starch, and more! It is divided into units of lucitetraose and maltopentaose to produce a glycated product whose main components are maltotetraose and maltopentaose.
Carp irises with such characteristics are
It is a completely new enzyme that has not been previously known. The inventor named this bulk amylase Gda.1. The enzymatic fermentation of this enzyme is described below. The basic properties of this enzyme are as shown below.

(11) Starch for Sakugawa, amylose, at-petatine, dexF-phosphoric acid, and other dalkanes are decomposed into units of lucidesilaose and maltopentaose to produce saccharified products containing these minuta as the main components.

(2) Action temperature gt range and optimum action temperature; works up to about 70°C, I11 melting temperature is !0.!!!”C (1% starch concentration, reaction at IIk-action pH for 30 minutes, Fig. 1 (
−)).

(31 maximum jlk p HIl, enclosure and optimal action p) IS
p) It acts on i4A3~//, and the most effective pH is 6j~7j ((Soul/Figure (b)).

(Kenthermal stability IQOjM Tris mild solution (p) I 7.
0) Intrinsic activity was measured after release. As a result, it was stable in the pH range of about 6 to about io (Fig. 7(d)).

(6) Stabilization 1 An increase in thermal stability was observed due to the presence/absence of calcium ions.

(7) lll'1m g This enzyme 4! j X / 0
-”MOHgC15%ZIllSOaCu804,
FmBO4t Cyclm, MugNOs etc.
jS is more than disappointed.

(8) The method and molecule h1 enzyme is liquid jIIIli
From the 0-liquid, ammonium sulfate fraction, DEAIA-Sephap-Skatsumta-Ko-Matodatsufi (gradient elution with WC1d-Q4M), Sephadex GJo.

Karamtalo! Togelafie 7-ni 7 Aditutus 010
By 0 katemuri a matodarahui etc., coo! It is often used up to a single word. The molecular lid is about sbh about 110,000 by Se7 Adettas G CoOO Karamuku Tomato Darahui
Rich 0 iso! −Separated, but small ψ of the molecular lid
component occupies the principal component and φ. And, there is a huge difference in the enzymatic properties of the two.

(9) Titer - Traction method 1 dissolved in 101M phosphate buffer
Add an appropriate amount of enzyme to a soluble starch solution (pH 7.0) Qj-, make the total m/- with water, and react at 0°C. Under these conditions, the amount of enzyme that would produce Zhao Yuanli, which corresponds to 1 of glucose per 1#, was defined as the unit of 1.

The literal characteristics are as follows, and the heather #i strain is Huikoken-Yori #
As No. 46237, Industrial Technology Center Hui Biological Industry Technology Center tF
It is discharged by tI.

(Gensugi O; Rod-1 size, width 07~0tμ x length 2j~
5μ, 2~31i! Many things are connected. Uncoupled, Durham negative.

(Z+ fid 1,000 H spores Self-cells swell and form rod-shaped to oval spores. (sl zetetin recruitment.

(4) Good growth on meat juice agar, pale yellowish brown (6)
Dulcose gravy agar 1 Poor growth, pale yellow (6) Dulcose nitrate agar slightly growing, semi-violent ()) Gravy agar slightly tI! , plow, self-deprecation, sedimentation (8) food pot gravy $/N
10Grows even at low nutrient concentrations, promotes growth at 1-j% (9) R-ruta recruitment decomposition is strong, is solidified, then becomes pepsinized, lidomas reduction (toward body) l growth normal, pigment production None 0υTyrisin agar Growth is good, light yellow, tyrosinase negative (b) Glucose-asparagine agar almost no growth (total) No indole production (2) Acetyl methyl carbinol 1 no production ) sulfide water bulking - produced (埒 & sI!lI salt), low positive C nourease, no 94 catalase; positive till starch hydrolysis, positive V4 carbohydrate utilization, glucose, 7 lactose, inose, Galactose, D-xylose, L-7 rabinose, sucrose, maltose, Shinrubose,
Mannitol and starch are produced, but no gas is produced. Lactose, raffinose, sorbitol, and inulin cause weak or almost no raw intoxication ■Methylene blue 1k1 enoic acid I Lifetime stability without use; Suitable growth temperature for eel is approximately JO'C1 Maximum growth temperature is JONj7 ℃ - death 1 degree It does not die even if heated at 1100℃ for 10 minutes.The most uniform growth pH is 7.5-4j.For the above mycological properties, B@rg@y's Mam
nualof D@t@rminatlv* Baet
@riology C1 7th edition and 1st edition (ThIwi
lliams & Wllklms C@mpany/
qj7 and /97), this eubiotic is Bacillus eireu (Ba@1llus eireu).
It was identified as a microorganism closely related to 1aam).

This strain has the ability to produce pullulanase at N time, and this enzyme breaks down the branched bond of amylopetatin, 1-l≦-darcoside bond. l
Act in conjunction with t1! With lutotetraose! It plays an important role in producing lutopentaose in good yield.

The enzymatic properties of the pullulanase produced by this method are as shown below.

(υ action number splits the 1-lz-glucoside bond present in pullulan to produce maltotriose.

It also breaks down the 1-/4-glucosidic bonds of starch, 71m pectin, glycogen or its derivatives.

(2) Action IIA degree range and optimum action temperature: It works up to about 70°C, and the maximum action is so Nss°C (1 stroke pullulan, GOjM) in Liss buffer for 30 minutes).

(3) Acts in the range of main and optimal action pH + pH #Ij to approximately 9, and the optimal action pH is around γ (1%
Pullulan, (103; reacted with DaO″C under M Tris buffer).

(4) Thermoplastic recruitment (10jM) Squirrel buffer* (pH 70)
After heating for 10 minutes at each humidity, the residual activity was determined as 1. As a result, heating at 10"C for 10 minutes resulted in approximately 73% inactivation, and heating at !1"C for 110 minutes resulted in approximately 73% inactivation.

(5) pH stability 1 Stable at pH about 4 to about 9
Measure the residual activity after leaving it for a while).

(6) One inhibitor enzyme cs", zl", h, +, H
/ +, k's'' etc.

(7) Stabilizing oligocalcium ions increase the thermostability of this enzyme.

(8) Purification method: The #element is ammonium sulfate-1DE from the culture IF solution.
AE-Seventh Fa-Four-Scaramtaro! Todarafui (KQI)
02~Q! Due to the kernels eluting with dateient in M, the teeth were separated from Acera 40#j, and then 77 Adetsutus G-
It can be purified to chromatographic homogeneity by co-OO column termadography.

(The molecular weight by the phantom molecule kk+Sephadex G-KoOoge/INF fan method was about 70,000.

01 titer method + 1% dissolved in Q/M phosphate buffer
An appropriate amount of enzyme is added to Pullulan 1lf (pH 7, o) azgLt, the total volume is diluted with water, and the mixture is reacted at 0°C. Under these conditions, the # bulk amount that produces a reducing power equivalent to 1⁻ maltotriose in 1 hour was defined as the unit of ℓ.

For the cultivation of enzyme raw koji according to the invention, a commonly used body medium or a liquid medium is used, and sources for 1 g of the medium for liquid culture include PepFun, meat extract, yeast extract, casein. % and as a carbon source, such as corn staple liquor.

Starch, dextrin,! Lutose, dulcose-shootalose, etc. are used, and a medium containing an inorganic nitrogen source, phosphorus salt, magnesium salt, and metal salt is used as a supplementary nutrient source.

@ Dinner should be at pH 4-9 and temperature! ~rs''c by aerated culture and cultivation.

Amylase G#ji! Since the bulk of the ill is produced externally, after the cultivation is completed, it is evacuated by FA or centrifugation, and the supernatant liquid is collected. Then, as required, concentrate, ammonium sulfate,
By salting out with sodium sulfate, etc., or by salting out with sodium sulfate, etc., or
A 111 solvent is added to obtain the fermented ligation as a precipitate, which is then dried and stored.

The saccharification of starch using this # element is carried out by adding it to liquefied starch at pH 4-9, if it is -60″C.
It will be held in

Next, the present invention will be explained in detail with reference to Examples.

Example / 200-Yong triangle 7? To Sco, Ks HPO4Q J arc,
Mg5Oa l7)bo Q/%, =y->7
．． fE19 was inoculated and cultured at 30°C for 2 days.

After culturing, the culture was sterilized using a Shinshinbun #machine, and the resulting supernatant was measured for live amylase Gd in units of 1Qj per medium. Add 10% ammonium sulfate to culture Pf&
The enzyme was added to saturation to precipitate the entire enzyme, which was collected in a centrifugal R11 machine and stored by transfer.

DE≠ 20 units of the M-nesting agent (76 units of pullulanase)
In addition to the liquefied starch liquid #3 (inner size), calcium chloride was added in an amount of J X / O-''M to make the total volume lO-, and the reaction was carried out at sooC. A portion of the obtained saccharified product was printed on paper After development using the graph method, each sugar segment was cut out, eluted, and fixed using the phenol-sulfuric acid method. As a result, glucose 1.
/%,! Lutose lj.

≦、! Lutotriose/JJ≦, Marsitetraose 3
62≦, maltohexaose 07%, maltohexaose/1%, so@1aus O Example In Example 1, the culture medium was polypeptone S (soybean), K3HPO4Q 7%, Mg5On.・7)
b.

Q/IG, 1% malleable starch, 1000 nium Q to ik#
/% and Coba chloride k) 1 j X / 0-' Mk
@'b114 ground was used. When the fruit was cooked raw, the amount of klGI was 100 ml per 111 ml of culture. In addition, Pullulana-4, which was produced at the same time, was aj earlier than @m/-.

[Brief explanation of drawings]

FIG. 7(a), (b), (c), and (d) are the optimum degrees of starch fractionation reaction by Amylaya a41s, respectively;
Optimal pH 1 thermal stability and pH stability are shown.

Claims (2)

[Claims] (1) Cultivate a microorganism that produces amylase G4 and G5 belonging to Pasillus, and make 7 dishes from the culture. G. Pasyrus amylase G4, which is characterized by collecting ginseng.
5 manufacturing method. (2) A novel method based on Bacillus sp. that is characterized by culturing microorganisms that produce amylase G4 and G5 belonging to Parsis, and collecting a complex enzyme consisting of amylase G, j and pullulanase from the culture.
II of a complex enzyme consisting of ZeGda, S and Pultenar Jkl-b-! fke