JPS63240777A - Microorganism capable of producing alkaline cellulase - Google Patents

Abstract

NEW MATERIAL:Bacillus sp. KSM-580 (FERM P-9013), belonging to the genus Bacillus and capable of producing alkaline cellulase K-580 having the following propertries. Action; Dissolving cellulose, such as carboxymethyl cellulose (CMC), cellulose or Avicel(R), to form reducing sugar, such as glucose. Action pH; 3-12.5 (optimum pH; 7-10). Action temperature; 15-80 deg.C (optimum temperature; 65 deg.C). Molecular weight; having peaks of molecular weight at 18,000 and 50,000 (measured by a gel filtration method). Effects of metallic ions; Inhibited by Hg<2+> and activated by Ba<2+>, Ca<2+>, Cd<2+> and Co<2+> with hardly affected by surfactants, such as LAS, chelating agents, such as EDTA. USE:A microorganism capable of producing alkaline cellulase which is a blending agent for detergent compositions, growing in a neutral culture medium and industrially producing the alkaline cellulase having excellent action. PREPARATION:For example, an extract solution obtained from soil of ICHIKAI Town, HAGA District in TOCHIGI Prefecture is treated at 80 deg.C for 10min and the resultant treated solution is cultivated at 30 deg.C for about 3 days to collect carboxymethyl cellulase (CMCase)-producing microorganisms, which are then searched for.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a novel microorganism of the genus Bacillus that produces -580 alkaline cellulase and grows in a neutral medium.

[Conventional technology]

The development of cellulase, a fibrinolytic enzyme, has traditionally been carried out with the single goal of effectively utilizing biomass resources, particularly cellulose resources. There are many types of bacterial strains that have been isolated as cellulase-producing bacteria, mainly filamentous fungi such as Aspergillus, Penicillium, Trichoderma, Hexathalium, Humicola, and Acremonium, as well as Shutomonas and Cellulomonas. It has also been reported in bacteria such as , Ruminococcus, and Bacillus, as well as actinobacteria such as Streftomyces and Thermoactinomyces.

However, at present, there are not many uses for biomass cellulases on an industrial scale.On the other hand, as a new industrial use of cellulases, the use as a compounding ingredient in laundry detergents is being considered and is attracting attention. (% Publication No. 59-49279, Special Publication No. 60-23
1158, Japanese Patent Publication No. 60-36240).

However, in the natural world, most cellulases produced by microorganisms are classified as so-called neutral or acidic cellulases, which exhibit maximum and stable enzymatic activity in neutral to acidic regions, and are suitable for use in clothing. Cellulases that have the conditions for inclusion in detergent compositions, ie, exhibit maximum activity in the alkaline region, or have alkaline resistance. The reality is that the existence of so-called alkaline cellulases and alkaline-resistant cellulases is extremely rare. Here, alkaline cellulase refers to a cellulase whose optimum pH is in the alkaline region, and alkaline-tolerant cellulase refers to a cellulase whose optimum pH is in the neutral to acidic region, but which has an optimum pH in the alkaline region as well. It refers to something that has sufficient activity and maintains stability compared to the activity. Also, neutral means pH 6~
8, and alkaline refers to a higher pH range.

That is, conventional methods for producing alkaline cellulases and alkali-resistant cellulases that can be used in laundry detergent compositions include a method of culturing alkalophilic bacteria and collecting cellulase A using Co2+ (Japanese Patent Publication No. 1973- No. 28515), alkaline cellulase 301-
Method of producing people (%Kaisei No. 58-224886)
, A method for producing carboxymethyl cellulase by culturing alkalophilic Bacillus Nn1139 (Fukun+o
rl.

F., Kudo, T. and Horikoshi, in, J.'Gen.

Mlcrobiol, 1.31, 3339,
(1985)) and a method for producing alkaline cellulase using a species of Stretmyces (Japanese Unexamined Patent Publication No. 1981-1999)
No. 19483) have been reported, and none of them are suitable for industrial fermentation production.

However, recently, the present inventors have investigated Bacillus sp. KSM-635, a type of alkalophilic bacterium.
USSP, KSM-635) (FEBMP-
8872) can produce alkaline cellulase K, which is suitable as a component in laundry detergents, in good yields, and by selecting culture conditions, productivity can be increased and industrial fermentation production of alkaline cellulase is possible. I found that.

[Problem that the invention seeks to solve]

However, the above Bacillus sp. KPM-635
The culture conditions are not necessarily industrially advantageous. In other words, it is necessary for alkaline-loving bacterial strains to keep the % pl (% pl) alkaline during cultivation, but to date, the so-called alkaline fermentation method using alkaline-loving strains has only a short history, and compared to normal neutral microorganisms, these preferable Knowledge about the physiology and biochemistry of alkaline microorganisms has not been sufficiently accumulated.

Medium preparation and culture methods have been operationally difficult for industrial fermentation production.

Furthermore, among the reported examples mentioned above, the original alkaline cellulases whose optimal p)I is in the alkaline region include Bacillus N1 strain, N2 strain, and N3 strain (Japanese Patent Publication No. 50-285).
No. 15), the optimum pH is 8 to 9.
9.8-9 enzyme, produced by Bacillus Nn 1139. Optimum pH 9 and Bacillus sp. KSM-
Optimal polo alkaline cellulase K produced by 635
However, the optimum pH that can be incorporated into cleaning compositions is on the alkaline side, and its action pH is on the alkaline side.
There was a need to provide alkaline cellulases with a wide range of coverage.

[Means for solving problems]

Under these circumstances, the present inventors conducted various studies in order to obtain a strain that can grow in a neutral medium and produce alkaline cellulase with excellent activity.

To solve this problem, it is possible to use a so-called genetic recombination technique in which the relevant cellulase gene is cloned using a bacterial strain that grows in a neutral region as a host; It is most effective to search for neutral microorganisms in nature that produce alkaline cellulase-producing microorganisms and isolate them. As a result of searching for the above-mentioned microorganism in nature, the present inventors discovered that a microorganism isolated from the soil of Haga District, Tochigi Prefecture satisfies the above-mentioned requirements, and completed the present invention.

The strain of the present invention exhibits the mycological properties shown below. Incidentally, for the classification of bacterial strains, the following five mediums 1 to 25 were used. (Displayed as 1% by weight) Medium 1. Meat extract, 1.0; bactopeptone.

1, O; NaCl, 0,5; Bacto agar, L5 (p
H72) Medium 2 Meat Extract, 1.0; Bact-(f ton, 1.0;
NaCl, C5; (pH'7:2) medium a meat extract, 1.0; bactopezotone.

LO; NaC1, C5: Gelatin, 10 (pH 72) Medium 4 Bactolitmus milk, 1α0 Medium a Bactopepty, 1.0; KNO, 0
, 1 medium G Bactopezot 7 , L Oy NaNO3,
LO medium 7 bactopezoton r Q 7 ; NaCZ
, Q 5; Glucose, C5 (pH 70) Medium & Bactopezotone, 1.0 Medium 9. TSI agar (manufactured by Eiken Chemical): indicated amount medium IQ meat extract, LO; bactopezotone.

1, O; NaCl, Q 5; Soluble starch.

C2; Agar, 1.5 Medium 11. NaNH4HPO4・4H, O, C15;
KH, P.O.

C1; Mg5O, 7HtO+ Q O2s Sodium citrate, α25 (pHa8) Medium 12 Christensen (Chrlat@n+sen
) Medium (manufactured by Eiken Chemical): indicated amount medium 1a glucose, 1. O; IGI, PO,,Q
1; MgSO4・7I(, o, a o rs
;KCl.

o, 02; Nitrogen source 0.1 (PH'72) The nitrogen source is
Sodium nitrate and ammonium sulfate were used.

Medium 14 King human medium “Eiken” (manufactured by Eiken Chemical): Indicated amount medium 1a King Bi soil “Eiken Pa (manufactured by Eiken Chemical): Indicated amount medium 1a Urea medium “Eiken’ (manufactured by Eiken Chemical) : Indication amount medium 17 Filter paper for cytochrome oxidase test (manufactured by Hinaga Pharmaceutical Co., Ltd.) Medium 183% hydrogen peroxide aqueous medium 19. OF basal medium (Difco #): Indicated amount medium 2 Q (NH,), HPO4,0,1; KCl
, 0.02;MgSO4-7H,O,aO2;
Yeast extract.

002; Bacto agar, 20; BCP (02% solution), 04 medium 21. Bacto-Sabouraud dextrose agar medium (manufactured by Difeo): indicated amount medium 22 meat extract, O, a; bactopetetone.

05: Yeast extract, 1.0: Glycerin filtration 0 Medium 2a Phenylalanine malonate medium (manufactured by Hinaga Pharmaceutical Co., Ltd.): Indicated amount medium 24 Skim milk, aO: Pact agar.

1.5 Medium 2a meat extract, Q3: Pactopezotone.

α5: L-tyrosine, 05: Pact agar, 1.5 (Mycological properties) (,) Microscopic observation results The size of the bacterial body is % Q4 ~ α8 μm XL5 ~ aO μm rods, at one end of the bacterial body Cylindrical or oval endospores (
04-08 μmXα8-1.2 μm). It has periflagella and is motile. Durham staining was positive. It has no anti-acid properties.

(b) Growth status in various media■ Broth agar plate culture (Medium 1) Growth status is weak. The shape of the colony is circular or irregular, the surface is smooth, and the periphery is smooth or leaf-like. The color of the village is pale yellow, translucent and shiny.

■ Broth agar slant culture (Medium 1) Growth is weak, spread-like, shiny, pale yellow and translucent.

■ Gravy liquid culture medium (medium 2) Grow. Especially the upper layer becomes cloudy.

■ Meat juice gelatin puncture culture (medium 3) Growth occurs on the surface layer, and liquefaction of gelatin is observed.

■ Lidomus Milk Medium (Medium 4) Liquefaction of milk is observed. No discoloration was observed in my father, Lidmus.

(c) Physiological properties ■ Nitrate reduction and denitrification reactions (Medium 5 and 6)
negative.

■ MR test (Medium 7) Negative and positive are not clear (pH 62) ■ VP test (medium 7) Negative and positive are unclear (pH 5, 2).

■ Production of indole (medium 8) negative.

■ Production of hydrogen sulfide (medium 9) negative.

■ Starch hydrolysis (medium 10) Positive.

■ Utilization of citric acid (Medium 11.12) Negative in Cosa medium, positive in Christensen medium.

■ Utilization of inorganic nitrogen sources (medium 13) Both nitrates and ammonium salts were positive.

■ Pigment production (medium 14.15) negative.

[Phase] Urease (medium 16) negative.

0 Oxidase (medium 17) Positive.

O catalase (medium 18) Positive.

O growth range (medium 2) The growth temperature range is 15-50℃, the optimal growth temperature range is 2
5-40℃.

The pH range for growth is pH 5 to 11, and the optimum pH range for growth is p.
It was H8-10.

Attitude towards oxygen: Facultatively anaerobic T131 0-F test (Medium 19) Grows, but both aerobic and aerobic sister plants grow poorly.

■ Sugar utilization (+: used, - not used) 1, L-arabinose +2 D-
xylose +aD-glucose
+4 D-mannose
+a fructose +aD
-galactose +7 maltose
10a sucrose 19, lactose 10IQ trehalose -11,
D-Sorvit +12 D-Mannit +1a Inosit
+14 Grise, Rin
+15 Starch +○ pH in vp medium (medium 7) Ha2 0 Growth in salt-containing medium (modified medium 1) 5%
Grow in

It grows at 7%.

No growth at 10%.

Growth in @pHa7 (Medium 21) Grows.

■ Production of zohydroxyacetone (medium 22) negative.

[Phase] Deamination of phenylalanine (medium 23)
Negative■ Casein degradation (medium 24) Positive.

○ Degradation of Tyroshi/ (Medium 25) negative.

Regarding the above mycological properties, please refer to the Bersey's Manual
Of DetamiNative Bacteriolozo (Ber)
gey+g Manual of Determina
tive Bacteriology), 8th edition and The Genus Ba
cillus” by Ruth, E. Gordon, Agr.
icultureHandbook Nn 427,
Agricultural Re5earchSsr
vlce, U, S, Department of
Agriculture.

As a result of comparison and searching with reference to Washington D.C. (1973), the strain of the present invention is recognized as a type of genus Baaillua, which is a sporobacillus. And this strain has recently been used in digging and Akiha (
” Alkalophllie Mlcroorgan
Japan 5cientific 5e
ct・ty Press (Tokyo), 1982) claims that the so-called Alkalop
microorganisms grow in an alkaline medium with a pH of 8 or higher, and cannot grow in a neutral pH range below this, but in a slightly acidic to alkaline range (pH 5 to 11).
), it is clearly different from this alkaliphilic microorganism.

It can be determined that it is a common Bacillus microorganism that grows in neutral conditions.

Furthermore, when this strain is compared with other known strains of the genus Bacillus, Bacillus licheniformis (Ba.
cillus 1ieh@nlformis). However, when comparing known strains and this strain, they differ in their ability to reduce nitrate. Furthermore, since the above-mentioned known strains do not produce at least alkaline cellulase, the present inventors determined that this strain is a new strain, and
It was named Varnish ff-KSM-580 and deposited with the Institute of Microbial Technology, Agency of Industrial Science and Technology (FERMP-9013).
.

In order to obtain -580 alkaline cellulase using the strain of the present invention, the strain may be inoculated into a medium and cultured according to a conventional method. It is preferable that the medium contains appropriate amounts of assimilable carbon sources and nitrogen sources. There are no particular restrictions on the carbon and nitrogen sources, but examples include corn gluten meal, soybean flour, corn stew sol liquor, casamino acids, yeast extract, Pharmamedia, sardine meal, meat extract, and bezoton. , No\izoro, Azono Q-war, corn soybean meal, coffee grounds, cottonseed oil cake, Cartebeta, Amiflex and Azozolone, Zest, Flex, and the like. In addition, carbon sources include rice husk, wheat flour, filter paper, patterned paper, vegetable fibers such as sawdust, blackstrap molasses, invert sugar, carboxymethylcellulose (CMC), avicel, cellulose cotton, xylan, pectin, and assimilated Carbon sources to be obtained, such as arabinose, xylose, glucose, galactose,
Examples include mannose, 7-lactose, maltose, sucrose, milk sugar, mannitol, inosit, sorbitol, glycerin, soluble starch, and assimilated organic acids such as acetic acid and citric acid. Also.

Others: phosphoric acid, Mg”, Ca %Mn, Z
n.

Inorganic salts such as Co'', Na'', and K+, and, if necessary, inorganic and organic trace nutrients can also be appropriately added to the medium.

Collection and purification of cellulase K-580, the target substance, from the culture thus obtained can be carried out in accordance with general enzyme collection and purification methods. That is, the crude enzyme solution can be obtained by removing the bacterial cells from the culture solution using conventional solid-liquid separation means such as centrifugation or filtration. This crude enzyme solution can be used as it is, but if necessary, the crude enzyme can be obtained by separation methods such as salting-out method, precipitation method, ultra-permeability, etc., and then purified and crystallized by a known method. It is also possible to use it as a purified enzyme.

The alkaline cellulase K-580 thus obtained is
It has the enzymatic properties shown below. The enzyme activity was measured according to the following method, and the buffers used were as follows: O pH 3-8 McLuvain buffer p) I8-11 Glycine-sodium hydroxide buffer pH 12-13 Potassium chloride-water Sodium oxide buffer enzyme activity measurement method: (110MCase activity I CIIpCMC (A-, 01L, Iduga Kokusaku)).

100μItIol various buffer solutions (Mac/I/
The enzyme solution of 01 was added to the substrate solution of 09 at containing (hein, phosphoric acid, glycine-NaOH, etc.).

The reaction was carried out at 30°C for 0 minutes. After the reaction, 3,5-dinitro-esteric acid (3,5-dlnLtro-8&mcyll)
Reducing sugars were quantified by the c acid (DNS) method. That is, 10 DNS reagents were added to 10 R1 of the reaction solution.
0 rat f was added, the color was developed by heating at 100"C for 5 minutes, and after cooling, the mixture was diluted with 40H1 deionized water and subjected to colorimetric determination at a wavelength of 535 nm. The enzyme titer was as follows:
The amount of enzyme that produced reducing sugar equivalent to 1 μrnol of glucose per minute under the above conditions was defined as 1 unit.

(2) p-nitrophenyl cellobioside decomposition activity 01μnool p-nitrophenyl cellobioside (
Sigma), 100 μmol phosphate buffer (ptt 7
0) containing the reaction solution 1. After allowing an appropriate amount of CMCase to act in Od at 30°C, IMNa 2 COl was added to 03
G, deionized water i 1.7 d sequentially added, t, free p-2□trophenol' (z quantified colorimetrically at 400 nm. Enzyme titer was 1 μmol p-trophenol per minute under the above conditions). The amount of enzyme that releases nitrophenol was defined as 1 unit.

(3) Add an appropriate amount of C to a reaction solution containing 2Od of Avicel, cellulose powder, and filter paper decomposition activity, 20 mfl Avicel (Merck), 00 μmol phosphate buffer (pH 70).
MCase was added and allowed to act at 30° C. and 25 Orpm while photographing. After the reaction, refrigerated centrifugation (5°C @ 3
000 rpm.

20 minutes) and the supernatant 1.0! /'i3,5-dinitro-salicylic acid (3,5-dinitro-5al
Reducing sugars were determined by the icylic acid (DNS) method. Cellulose powder decomposition activity was measured using cellulose powder (Toyo Roshi Co., Ltd.), and filter paper decomposition activity was performed using filter paper (filter paper for cellulase activity assay, Toyo Koji 51-Toku) in the same manner as for avicelase activity. The enzyme titer is 0 (4) where 1 unit is the amount of enzyme that produces reducing sugar equivalent to 1 μrIIO1 glucose per minute under the above conditions.Cellopiase activity 10 mg cellobiose (Kanto Kagaku Co., Ltd.), 100 μrr+
OL') Reaction solution containing acid buffer (TIH70) LO
After allowing an appropriate amount of CMCase to act at 30°C during d,
After inactivating CMCase by filtration at 100°C, the amount of glucose produced was measured using the mulotase/GOD method (Glucose GOD method).
C-Te5t (Wako Tsumugi Kogyo Co., Ltd.). For the enzyme titer, one unit was the amount of enzyme that produced 2 μmol of glucose per minute under the above conditions.

(Enzymatic properties) (1) Action CM C* Cellulose% Acts well on cellulose such as filter paper and Avicel, and dissolves them.

Produces reducing sugars such as glucose.

(2) Substrate specificity In addition to CMC, this enzyme had activity against cellulose powder, Avicel, and filter paper.

(3) Working pH and optimal pH The working pH range is extremely wide, from 3 to 125.

The optimum pH is wide ranging from 7 to 10, and even in the range of 45 to 10.5, the relative activity is more than 50% of the activity at the optimum pi (pi). Among them, it can be said that it is the enzyme that is most fully active in the alkaline side (Figure 1).

L4) pH stability We measured the residual activity after holding each pH at 30°C for 1 hour and investigated the pH stability.As a result, the pH was 415.
It was extremely stable and did not lose its activity at pH 35 to 125, and maintained about 50% or more activity even at pH 35 to 125. This enzyme is sufficiently stable even in this highly alkaline region (Fig. 8g2).

(5) Optimal temperature The working temperature ranged over a wide range of 15 to 80°C9, and the optimum temperature was 65°C. father.

Even in the range of 50-75℃, the activity at the optimum temperature is 5%.
0% or more (Figure 8g3).

Furthermore, even at 15°C, the activity is 40% or more of the activity at 30°C.

(6) Temperature stability After treatment at each temperature for 30 minutes at the optimum pH, residual activity was measured, and the results showed that it was stable at 55°C and 65°C.
It had a residual activity of approximately 50% even in the following conditions (Figure 4)
.

(7) Molecular weight The molecular weight of this enzyme was measured based on the glue filtration method using Sephadex G-100 (5ephadex G-100), and it was approximately L80,000 and 500,000.

(8) Effect of metal ions Regarding this enzyme, various metal ions (At”, Fe”+
, Ba2+, ca2+, Cd"+, Co", Cr"
,Cu”.

Fe”, Hg”, Mn”, Mo”, Nl”,
Pb”, Zn”±, LL+, K”, Na+) were allowed to coexist during the activity measurement, and their influence l# was investigated (K, Na
The concentration 'k was set to 50 mM for ions, and 1 mM for other ions). As a result, inhibition in ug, Ha%Ca”, Co”, Cd”, Co
Activation was observed by 2+.

(9) Effect of surfactant Each surfactant (for example, LAS, AS, ES).

AO8, α-8FE, SAS, soap,? The effect of lyoxyethylene secondary alkyl ether) on enzyme activity was investigated. The enzyme was treated with a 005% surfactant solution at 30°C for 115 minutes, and then the activity was measured. As a result, no inhibition of the activity was observed even by sodium dodecyl sulfate, which is a strong detersoent that is not inhibited by any surfactant.

(10) f-Lothiase resistant grotease for detergents, e.g. API-21 (Showa Denko)
, Maxatase (Gist) and Alcalase (Novo) coexisted during activity measurement ((10) tQ/al
) and investigated its effects, it was found that it has strong resistance to all proteases.

Uυ Influence of chelating agent Chelating agent EDTA% EiGTA, )su, p
IJ sodium phosphate, zeolite, and citric acid were allowed to coexist during activity measurement, and their effects were examined, but no inhibition was observed.

〔Effect of the invention〕

The N strain of the present invention, Bacillus sp. K3M-580, is a strain that grows in neutral conditions.

Alkaline cellulase can be produced industrially more easily than with alkaliphilic strains.

The alkaline cellulase K-580 produced by the strain of the present invention, Co2+, has an optimal pHt on the highly alkaline side (pH 10) compared to conventional alkaline cellulases. Moreover, in a wide range of pH 45 to IQ5, it has an activity that is 50% or more of the activity at the optimal pH, and is extremely stable even at pH 45 to 12. It is hardly inhibited by detergent ingredients such as detergents. Therefore, this enzyme can be advantageously used as a component of detergent compositions.

〔Example〕

Hereinafter, the present invention will be explained in more detail with reference to Examples and Reference Examples.

Example 1 A spoonful of soil from Ichikai-cho, Haga-gun, Tochigi Prefecture (approximately 0.5 y
) It was suspended in sterile physiological saline and heat-treated at 80°C for 10 minutes. The supernatant of this heat-treated solution was diluted to an appropriate level and applied to a 1-separation agar medium (medium 1). Next, add this to 30
The bacteria were cultured for 3 days in C and allowed to form colonies. Those that formed a zona pellucida around the colony due to dissolution of CMC were selected to obtain CMCase producing bacteria.

The obtained bacteria were inoculated into a liquid medium of medium 2, and cultured with shaking at 30°C for 3 days. After culturing, the CMCase activity of the centrifuged supernatant was measured at pH 3 to 13 to screen for alkaline cellulase producing bacteria.

Bacillus sp. KSM of the present invention by the method described above.
-580 shares (FEBM P-9013) were able to be obtained.

Medium 1-CMC2%? Lipezot 7 0.5% Yeast Extract 0.05% K) I2PO, 01% Na2HPO4H12H, OO, 25% MgSO4.7
H, OQO2% l1as medium 2 CMC1%? Ribezoton 1% Yeast extract 0.5% KH, PO40
1% ・Na 2HPO, ・12H, 00, 25%Mg5O
, 7HtOO, 02% pI (a8 Reference Example 1 Bacillus sp. KSM-580 strain obtained in Example 1 was inoculated into liquid medium 2 of the same example, and cultured with shaking at 30°C for 3 days. After culturing, the bacterial cells was removed by centrifugation to obtain a crude enzyme solution.To 1 ton of this crude enzyme solution, 3 t of ethanol was added in dry ice-ethanol, the resulting precipitate was centrifuged, and further freeze-dried to dry. As a powder, alkaline cellulase K-580 (specific activity*33 units/P)
IIF was obtained.

This enzyme activity is a value measured at pH 9.

Reference example 2: Replace CMC with sucrose? Bacillus sp. KSM-580 strain was inoculated into a medium having the same composition as liquid medium 2 of Example 1, except that lipezoton was replaced with 7% C8L, and
The cells were cultured with shaking for days. This culture was centrifuged, and the CMCase activity of the resulting supernatant was measured at 6 O units/l.
Met.

[Brief explanation of drawings]

FIG. 1 is a drawing showing the relationship between enzyme reaction pH and relative activity. FIG. 2 is a diagram showing the relationship between enzyme treatment pH and relative activity. FIG. 3 is a diagram showing the relationship between enzyme reaction temperature and relative activity. Figure 4 shows the activity at 30°C versus 20°C and 15°C.
FIG. 5, which may be a diagram showing the activity at °C, is a diagram showing the relationship between enzyme treatment temperature and relative activity. that's all

Claims (1)

[Scope of Claims] 1. A microorganism that belongs to the genus Bacillus and has the following enzymatic properties and is capable of producing alkaline cellulase K-580. (1) Action It acts well on cellulose such as carboxymethyl cellulose, cellulose filter paper, and Avicel, dissolving them and producing reducing sugars such as glucose. (2) Substrate specificity In addition to carboxymethyl cellulose, it has activity against cellulose powder, Avicel, and filter paper. (3) Working pH and optimum pH The working pH range is 3 to 12.5. The optimum pH is 7
~10, and even in the range of 4.5 to 10.5, it has a relative activity that is 50% or more of the activity at the optimum pH. (4) pH stability Extremely stable at pH 4.5 to 12 without deactivation, pH 3.5
~12.5, the activity is maintained at about 50% or more. (5) Optimal temperature The working temperature ranges over a wide range from 15 to 80°C, and the optimum temperature is 65°C. Moreover, even in the range of 50 to 75°C, it has 50% or more of the activity at the optimum temperature. (6) It has molecular weight peaks at about 18,000 and about 50,000 (by gel filtration using Sephadex G100). (7) Influence of metal ions: inhibited by Hg^2^+, Ba^2^+, Ca^2
It is activated by ^+, Cd^2^+, and Co^2^+. (8) Effect of surfactants LAS, AS, ES, AOS, α-SFE, SAS, soap, and polyoxyethylene secondary alkyl ether hardly inhibit the activity. (9) Protease resistance Resistant to proteases. (10) Effect of chelating agents EDTA, EGTA, citric acid, sodium tripolyphosphate, and zeolite do not inhibit the activity.