JPH0655140B2 - Alkaline cellulase - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application] The present invention relates to a novel alkaline cellulase K-425.

[Conventional technology]

The development of celluloses, which are fibrinolytic enzymes, has hitherto been promoted with the aim of effectively utilizing biomass resources, particularly cellulose resources. The strains that have been isolated as cellulase-producing bacteria are of many types, mainly Aspergillus sp., Penicillium sp., Trichoderma sp., Fusarium sp., Humicola sp., Filamentous fungi such as Acremonium sp., Siudomonas sp. It has also been reported in bacteria such as Bacillus and actinomycetes such as Streptomyces and Thermoactinomyces. However, at present, the use of biomass cellulase on an industrial scale is not large.

On the other hand, as a novel industrial use of cellulase, its use as a blending component of a detergent for clothing has been studied and attracted attention (Japanese Patent Publication No. 59-49279 and Japanese Patent Publication No. 60-23).
No. 158, Japanese Patent Publication No. 60-36240). However, in the natural world, most cellulases produced by microorganisms are classified as so-called neutral or acid cellulases that show the maximum and stable enzyme activity in the neutral to acidic region, and are used for clothing. The presence of cellulase having conditions for incorporation into a detergent composition, that is, the presence of so-called alkali cellulase and alkali-resistant cellulase, which shows maximum activity in the alkaline region or has alkali resistance, is actually very small. . Here, the alkaline cellulase means that the optimum pH is in the alkaline region, and the alkaline-resistant cellulase, the optimum pH is in the neutral to acidic region, but even in the alkaline region at the optimum pH. It refers to a substance which has sufficient activity as compared with the activity and retains stability. Further, the neutral means a pH range of 6 to 8, and the alkaline means a higher pH range.

That is, conventionally, as a method for producing an alkali cellulase and an alkali resistant cellulase which can be used in a detergent composition for clothes, a method of collecting cellulase A by culturing an alkalophilic Bacillus bacterium (Japanese Patent Publication No. 50-285).
No. 15), a method of culturing an alkalophilic bacterium belonging to the genus Cellulomonas to produce alkaline cellulase 301-A (JP-A-58-224686), and culturing alkalophilic Bacillus No. 1139 to produce carboxymethylcellulase. Production method (Fukumori, F., Kudo, T. and Hor
ikoshi, K., J. Gen. Microbiol., 131, 3339, (1985)) and a method for producing an alkaline cellulase using a Streptomyces genus (Japanese Patent Laid-Open No. 61-19483). However, none of them was suitable for industrial fermentative production.

However, recently, the present inventors have found that Bacillus sp. KSM-635 (Bacillus sp.
KSM-635) (FERM P-8872) produces alkaline cellulase K, which is suitable as a detergent cleaning ingredient for clothing, in a high yield, and by further selecting the culture conditions, the productivity is further increased, and the alkaline cellulase industry is improved. It has been found that the fermentative production can be achieved.

[Problems to be solved by the invention]

However, the above Bacillus Espi-KSM-635
The culturing conditions described above are not necessarily industrially advantageous. That is, the alkalophilic strain needs to keep the pH alkaline during the culture, but so far, the so-called alkaline fermentation method using the alkalophilic strain has a short history, and compared with ordinary neutral microorganisms The knowledge about the physiology and biochemistry of microorganisms has not been sufficiently accumulated, and the medium preparation and culture method for industrial fermentation production have been difficult to operate.

Furthermore, among the reported examples described above, Bacillus N is an original alkaline cellulase having an optimum pH in the alkaline region.
1 strain, N2 strain, N3 strain (Japanese Patent Publication No. 50-28515)
The optimum pH values produced by each of the publications are 8-9, 9, 8 respectively.
~ 9 enzyme, Bacillus No. 1139 produced optimum pH
No. 9 and Bacillus Espi-KSM-635 produced an alkaline cellulase K having an optimum pH of 10 (Japanese Patent Application No. 61-
No. 257776), there has been a demand for the provision of an alkaline cellulase which has an optimum pH on the alkaline side and can be used in a detergent composition and has a wide working pH range.

[Means for solving problems]

Under such circumstances, the present inventors have conducted various studies in order to obtain a strain capable of producing alkaline cellulase which grows in a neutral medium and has an excellent action.

In order to solve such a problem, it is possible to use a strain growing in a neutral region as a host and cloning a corresponding cellulase gene, that is, a so-called gene recombination method can be used. It is more effective to search for a neutral microorganism that produces the alkaline cellulase in the natural world and separate it. Then, as a result of seeking the above microorganisms in the natural world, the present inventors have found that the microorganisms isolated from the soil in Haga-gun, Tochigi Prefecture satisfy the above-mentioned demand, and have completed the present invention.

The strain producing the alkaline cellulase of the present invention exhibits the mycological properties as shown below. For the classification of strains, the following media 1 to 25 were used. (Indicated by weight%) Medium 1. Meat extract, 1.0; Bacto peptone, 1.0; NaCl, 0.5; Bacto agar, 1.5 (pH 7.2) Medium 2. Meat extract, 1.0; Bacto peptone, 1.0; NaCl, 0 .5; (pH 7.2) Medium 3. Meat extract, 1.0; Bactopeptone, 1.0; NaCl, 0.5; Gelatin, 1.0 (pH 7.2) Medium 4. Bactrytomas milk, 10.0 Medium 5. Bactopeptone, 1.0; KNO ₃ , 0.1 medium 6. Bactopeptone, 1.0; NaNO ₃ , 1.0 medium 7. Bactopeptone, 0.7; NaCl, 0.5; glucose, 0.5 (pH 7.0) medium 8. Bactopeptone, 1.0 medium 9. TSI agar (Eiken Chemical Co., Ltd. ): Indicated amount Medium 10. Meat extract, 1.0; Bactopeptone, 1.0; NaCl, 0.5; Soluble starch, 0.2; Agar, 1.5 Medium 11. NaNH ₄ HPO ₄・ 4H ₂ O, 0.15; KH ₂ PO _{4 , 0.1; MgSO 4 · 7H 2} O, 0.02: sodium citrate, 0.25 (pH 6.8) medium 12. Christensen (Christensen) medium (manufactured by Eiken Chemical Co.) indicated amounts medium 13. glucose, 1.0; KH ₂ PO _4, 0.1; MgSO ₄ · 7H ₂ O, 0.05; KCl, 0.02; Nitrogen source, 0.1 (pH 7.2) As the nitrogen source, sodium nitrate and ammonium sulfate were used.

Medium 14. King A medium "Eiken" (manufactured by Eiken Chemical): indicated amount Medium 15. King B medium "Eiken" (manufactured by Eiken Chemical): indicated amount Medium 16. Urea medium "Eiken" (Eiken) Chemical): indicated amount medium 17. Cytochrome oxidase test filter paper (manufactured by Nissui Pharmaceutical) medium 18.3% hydrogen peroxide solution medium 19. OF basal medium (manufactured by Difco): indicated amount medium 20. (NH ₄ ) ₂ HPO ₄ , 0.1; KCl, 0.02; MgSO ₄ .7H ₂ O, 0.02; yeast extract, 0.02; Bacto agar, 2.0; BCP (0.2% solution), 0.4 medium 21. Bacto Sabouraud dextrose agar medium (D ifco): indicated amount medium 22. meat extract, 0.3; bactopeptone, 0.5; yeast extract, 1.0; glycerin, 2.0 medium 23. phenylalanine malonate medium (manufactured by Nissui Pharmaceutical Co., Ltd.): indicated amount medium 24. Skim milk, 5.0; Bacto agar, 1.5 Medium 25. Meat extract, 0.3; Bacto peptone, 0.5; L-tyrosine, 0.5; Bacto cold , 1.5 (Mycological properties) (a) Microscopic observation results The size of the cells is 0.4-0.8 μm × 1.5-4.0 μm, and the cells are elliptical at one end. Endospores (0.8-1.2
μm × 1.2-1.5 μm). It has periflagellates and is motile. Gram stain is indeterminate. There is no acid resistance.

(b) Growth condition in various media Meat agar plate culture (medium 1) Growth condition is weak. The shape of the settlement is circular, the surface is smooth and the periphery is also smooth. In addition, the color tone of the settlement is white, semi-transparent and glossy.

Meat broth agar slant culture (medium 1) Growth is weak, the state is spread-like, glossy, and white and translucent.

Broth liquid culture (medium 2) Grows and becomes cloudy.

Meat broth gelatin stab culture (medium 3) Grows, but the condition is weak, and liquefaction of gelatin is not observed.

Litmus milk medium (medium 4) No coagulation or liquefaction of milk is observed. Also, no discoloration of litmus was observed.

(c) Physiological properties Nitrate reduction and denitrification reactions (medium 5, 6) are both negative.

MR test (medium 7) Negative and positive are not attached (pH 5.2).

VP test (medium 7) negative (pH 5.2).

Formation of indole (medium 8) Negative.

Production of hydrogen sulfide (Medium 9) Negative.

Hydrolysis of starch (medium 10) Positive.

Utilization of citric acid (mediums 11 and 12) Negative in both Cosa medium and Christensen medium.

Use of inorganic nitrogen source (medium 13) Negative for both nitrate and ammonium

Pigment formation (medium 14, 15) Negative.

Urease (medium 16) negative.

Oxidase (medium 17) positive.

Catalase (medium 18) positive.

Growth range (medium 2) The growth temperature range is 15 to 37 ° C, and the optimum growth temperature range is 2
5-30 ° C.

The growth pH range is pH 5-10, and the optimum growth pH range is pH 6-9.
It was.

Attitude toward oxygen Facultative anaerobic.

OF test (medium 19) Grows, but grows badly under both aerobic and anaerobic conditions.

Utilization of sugar (+: used, −: not used) 1. L-arabinose + 2. D-xylose + 3. D-glucose + 4. D-mannose + 5. fructose + 6. D-galactose + 7 .Maltose + 8. sucrose + 9. lactose + 10. trehalose + 11. D-sorbit ± 12. D-mannitol + 13. inositto-14. Glycerin ± 15. pH 5.2 Growth in medium containing salt (Modified medium 1) No growth at 5%.

No growth at 7%.

It does not grow at 10%.

Growth at pH 5.7 (medium 21) No growth.

Generation of dihydroxyacetone (medium 22) Negative and positive are not observed.

Deamination of phenylalanine (medium 23) Negative.

Degradation of casein (medium 24) Negative.

Degradation of tyrosine (medium 25) Negative.

Regarding the above-mentioned mycological properties, Vergi's Manual
Of Date Bacteriology (Berge
y's Mannual of Determinative Bacteriology, 8th Edition and The Genus Bacill
us ”, Ruth, E. Gordon, Agriculture Handbook No.42
7. Agricultural Research Service, USDepartment o
As a result of comparison and search with reference to Agriculture, Washington DC (1973), it is recognized that the strain of the present invention is one of the genus Bacillus which is a spore bacillus. And recently, this strain has been found in Hikoshi and Akiba (“Alkalophilic Mic
roorganism ”, Japan Scientific Society Press (Toky
o), 1982), the so-called alkalophilicity (Alk
alophilic microorganism grows in an alkaline medium of pH 8 or higher and cannot grow in a neutral pH range lower than this, whereas it can grow in a weakly acidic range to an alkaline range (pH 5 to 10). Therefore, it is clearly different from the alkalophilic microorganism and can be judged to be a general neutral Bacillus microorganism.

Furthermore, when this strain was compared with other known strains of the genus Bacillus, Bacillus circulans (Ba
cillus circulans). However, a comparison between the known strain belonging to Sirkyurus and this strain shows a difference in the hydrolyzing ability of gelatin. Furthermore, since the above-mentioned known strain does not produce at least alkaline cellulase, the present inventors determined that this strain is a new strain, named Bacillus sp. KSM-425, and deposited it at the Institute for Microbial Technology, Institute of Industrial Science (FERM P -90
07).

Using this strain, the alkaline cellulase K-4 of the present invention
To obtain 25, the strain may be inoculated into a medium and cultured according to a conventional method. It is preferable that an appropriate amount of assimilable carbon source and nitrogen source be contained in the medium. The carbon source and the nitrogen source are not particularly limited, but examples thereof include corn gluten meal as a nitrogen source, soybean flour,
Corn steep liquor, casamino acid, yeast extract, pharmamedia, sardine meal, meat extract, peptone, high pro, ajpower, corn soybean meal, coffee meal, cottonseed oil meal, cultivator, amifrex and adipron, zest, azix, etc. . or,
As a carbon source, in addition to rice husk, malt, filter paper, general paper, plant fiber such as sawdust, molasses, invert sugar, carboxymethyl cellulose (CMC), avicel, cellulose cotton, xylan, pectin, and a carbon source that can be assimilated Examples thereof include arabinose, xylose, glucose, galactose, mannose, fructose, maltose, sucrose, lactose, trehalose, mannitol, sorbit, glycerin, soluble starch, and assimilable organic acids such as acetic acid. In addition, phosphoric acid, Mg ²⁺ , Ca ²⁺ , Mn ²⁺ , Zn ²⁺ ,
Inorganic salts such as Co ²⁺ , Na ⁺ , and K ⁺ , and if necessary, inorganic and organic micronutrient sources can be appropriately added to the medium.

Collection and purification of the target substance, cellulase K-425, from the thus obtained culture can be carried out according to a general enzyme collection and purification means. That is, the crude enzyme solution can be obtained by removing the cells from the culture solution by a usual solid-liquid separation means such as centrifugation or filtration. This crude enzyme solution can be used as it is, but if necessary, a crude enzyme is obtained by a separation means such as a salting-out method, a precipitation method, an ultrafiltration method, and further purified and crystallized by a known method, It is also possible to use it as a purified enzyme.

Thus obtained alkaline cellulase K-4 of the present invention
25 has the following enzymatic properties. The enzyme activity is measured according to the following method, and the buffer solution used is as follows.

pH 3 to 8 McClubein buffer pH 8 to 11 Glycine-sodium hydroxide buffer pH 12 to 13 Potassium chloride-sodium hydroxide buffer Enzyme activity measurement method: (1) CMCase activity 10 mg CMC (A-01L, Sanyo Kokusaku Pulp Co., Ltd.), 10
0.1 ml of the enzyme solution was added to 0.9 ml of a substrate solution containing 0 μmol of various buffer solutions (Mcclebein, phosphoric acid, glycine-NaOH, etc.) and reacted at 30 ° C. for 20 minutes. After the reaction, 3,5-dinitro-salicylic acid (3,5-dinitro-salicylic acid (D
NS)) method was used to quantify reducing sugars. That is, the reaction solution
To 1.0 ml, 1.0 ml of DNS reagent was added, and color was developed by heating at 100 ° C. for 5 minutes, and after cooling, 4.0 ml of deionized water was added to dilute. This was colorimetrically determined at a wavelength of 535 nm. The enzyme titer was defined as 1 unit of the amount of enzyme that produced a reducing sugar corresponding to 1 μmol of glucose per minute under the above conditions.

(2) P-nitrophenyl cellobioside decomposition activity 0.1 μmol
p-nitrophenyl cellobioside (Sigma), 100
An appropriate amount of CMCase was added to 1.0 ml of a reaction solution containing μmol phosphate buffer (pH 7.0) at 30 ° C.
₂ CO ₃ (0.3 ml) and deionized water (1.7 ml) were sequentially added, and liberated p-nitrophenol was colorimetrically determined at 400 nm. The enzyme titer was defined as 1 unit of the amount of enzyme that liberates 1 μmol of p-nitrophenol in 1 minute under the above conditions.

(3) Avicel, cellulose powder Paper degrading activity 20 mg Avicel (Merck) and an appropriate amount of enzyme solution were added to 2.0 ml of a reaction solution containing 200 μmol phosphate buffer (pH 7.0) and shaken at 30 ° C. and 250 rpm. I let it work. After the reaction, cooling centrifugation (5 ° C, 3000 rpm, 20
Min) and 1.0 ml of the supernatant was quantified for reducing sugars by the 3,5-dinitro-salicylic acid (DNS) method. Cellulose powder decomposition activity was measured using cellulose powder (Toyo Roshi Kaisha, Ltd.), and filter paper decomposition activity was measured using filter paper (cellulase activity assay filter paper, Toyo No. 51-special) in the same manner as for avicelase activity. The enzyme titer was defined as 1 unit of the amount of enzyme that produced a reducing sugar corresponding to 1 μmol of glucose per minute under the above conditions.

(4) Cellobiase activity 10 ml of cellobiose (Kanto Chemical Co., Inc.) and 100 μmol of a phosphate buffer (pH 7.0) in a reaction solution of 1.0 ml were reacted with an appropriate amount of enzyme solution at 30 ° C., and then at 100 ° C. After deactivating the enzyme by treating for 2 minutes, the amount of glucose produced is determined by mrotase.
It was measured by the GOD method (Glucose C-Test, Wako Pure Chemical Industries, Ltd.). The enzyme titer was defined as 1 unit of the amount of enzyme that produced 2 μmol of glucose per minute under the above conditions.

(Enzymatic properties) (1) Action It acts well on fibrin such as CMC, cellulose, filter paper, and Avicel, and dissolves them to form reducing sugar such as glucose.

(2) Substrate specificity The enzyme had an activity against cellulose powder, Avicel, filter paper, p-nitrophenyl cellobioside, and cellobiose, in addition to CMC. The cellobiose degrading activity was about 4% of the CMCase activity.

(3) Working pH and optimum pH The working pH range is extremely wide, from 3.5 to 12.5.
The optimum pH is as wide as 8 to 10, and even in the range of 5.5 to 10.5, it has a relative activity of 50% or more of the activity at the optimum pH, and thus has been studied in the past. It can be said that among the alkaline cellulases, the enzyme that exerts the most activity on the alkaline side (Fig. 1).

(4) pH stability The pH stability was examined by measuring the residual activity after each pH was maintained at 30 ° C for 1 hour. As a result, it was extremely stable at pH 5 to 11 and was not inactivated, and even at pH 3 to 12, the activity was maintained at about 50% or more. The enzyme is sufficiently stable even in the high alkaline region (Fig. 2).

(5) Optimum temperature The working temperature was in a wide range of 15 to 75 ° C, and the optimum temperature was 50 ° C. Further, even in the range of 35 to 55 ° C, it had 50% or more of the activity at the optimum temperature (Fig. 3).

(6) Temperature stability After being treated at each temperature for 30 minutes at the optimum pH, the residual activity was measured, and as a result, it was stable at 30 ° C and showed about 50% residual activity even at 50 ° C. I had it (Fig. 4).

(7) Molecular weight This enzyme was used as Sephadex G-1 (Sephadex G-1).
The molecular weight was measured by the gel filtration method according to (00) and was found to be about 35,000.

(8) Effect of metal ions For this enzyme, various metal ions (Al ³⁺ , Fe ³⁺ , Ba ²⁺ , Ca
²⁺ , Cd ²⁺ , Co ²⁺ , Cr ²⁺ , Cu ²⁺ , Fe ²⁺ , Hg ²⁺ , Mn ²⁺ , Mo ²⁺ , Ni ²⁺ , Pb
²⁺ , Zn ²⁺ , Li ⁺ , K ⁺ , Na ⁺ ) were made to coexist during the activity measurement, and the effect was examined (concentration of K ⁺ , Na ⁺ was 50 mM,
For other ions, 1 mM). As a result, H
Inhibition was observed with g ²⁺ and Ba ²⁺ , and activation was observed with Co ²⁺ .

(9) Effect of surfactants Various surfactants (eg LAS, AS, ES, AO
The effects of S, α-SFE, SAS, soap, and polyoxyethylene secondary alkyl ether on the enzyme activity were examined. This enzyme was added to a 0.05% solution of detergent at 30 ° C for 1 hour.
After the treatment for 5 minutes, the activity was measured. As a result, it was not inhibited by any of the surfactants. No inhibition of the activity was observed even with the strong detergent Sodium dodecyl sulphate.

(10) Protease resistance Detergent proteases such as API-21 (Showa Denko), Maxatase (Gist) and Alcalase (Novo) were allowed to coexist (0.1 mg / ml) at the time of activity measurement, and the effect was examined. It was found that they have strong resistance to any protease.

(11) Effect of chelating agent Chelating agents EDTA, EGTA, sodium tripolyphosphate, zeolite, and citric acid were allowed to coexist during the activity measurement, and their effects were examined, but almost no inhibition was observed.

〔The invention's effect〕

The alkaline cellulase K-425 of the present invention has an optimum pH on the high alkaline side (pH 10) as compared with conventional alkaline cellulases. In addition, it has 50% or more of the activity at the optimum pH over a wide range of pH 5.5 to 10.5, and is extremely stable even at pH 5 to 11.

Further, it is hardly affected even by detergent-containing components such as surfactants, proteases and chelating agents. Therefore, the present enzyme can be advantageously used as a blending component of a detergent composition.

〔Example〕

 Hereinafter, the present invention will be described in more detail with reference to examples.

Reference Example 1 Soil of Kaigai-cho, Haga-gun, Tochigi Prefecture was suspended in a spoonful (about 0.5 g) of sterile physiological saline and heat-treated at 80 ° C. for 10 minutes.
The supernatant of this heat treatment solution was appropriately diluted and applied to a separation agar medium (medium 1). Then, this was cultured at 30 ° C. for 3 days to form a colony. A CMCase-producing bacterium was obtained by selecting those that form a transparent zone based on the dissolution of CMC around the settlement. Further, the obtained microorganism was inoculated into the liquid medium of the medium 2 and shake-cultured at 30 ° C. for 3 days. After culturing, the CMCase activity of the supernatant obtained by centrifugation was adjusted to pH 3-1.
3 and screened for alkaline cellulase-producing bacteria.

According to the above method, the Bacillus sp. KSM of the present invention
-425 strain (FERM P-9007) was able to be acquired.

0.05% medium 1.CMC 2% polypeptone 0.5% yeast extract _{KH 2 PO 4 0.1% Na 2} HPO 4 · 12H 2 O 0.25% MgSO 4 · 7H 2 O 0.02% agar 0.75% pH 6.8 medium 2.CMC 1% polypeptone 1 % the reference example Bacillus sp. KSM-425 strain obtained in yeast extract _{0.5% KH 2 PO 4 0.1%} Na 2 HPO 4 · 12H 2 O 0.25% MgSO 4 · 7H 2 O 0.02% pH 6.8 example 1 reference example 1 Was inoculated into the liquid medium 2 of the above and cultured at 30 ° C. for 3 days with shaking. After culturing, the bacterial cells were removed by centrifugation to obtain a crude enzyme solution. Ethanol (3) was added to this crude enzyme solution 1 in dry ice-ethanol, and the resulting precipitate was centrifuged and freeze-dried to obtain a dry powder of alkaline cellulase K-425 (specific activity ^* 10 units. / G)
10 g were obtained.

* Enzyme activity is measured at pH 9.

Example 2 A Bacillus sp. KSM-425 strain was inoculated into a medium having the same composition as the liquid medium 2 of Reference Example 1 except that sucrose was used instead of CMC and 7% CSL was used instead of polypeptone, and the medium was inoculated with Bacillus sp.
Culture was carried out with shaking for one day. This culture was centrifuged, and the CMCase activity of the resulting supernatant was measured to be 160 units /
It was.

[Brief description of drawings]

FIG. 1 is a drawing showing the relationship between enzyme reaction pH and relative activity. FIG. 2 is a drawing showing the relationship between enzyme-treated pH and relative activity. FIG. 3 is a drawing showing the relationship between enzyme reaction temperature and relative activity. FIG. 4 is a drawing showing the relationship between enzyme treatment temperature and relative activity.

Claims (1)

[Claims] 1. An alkaline cellulase K-425 having the following enzymatic properties. (1) Action It works well on fibrin such as carboxymethyl cellulose, cellulose, filter paper, and Avicel, and dissolves them to produce reducing sugar such as glucose. (2) Substrate specificity In addition to carboxymethyl cellulose, it has activity against cellulose powder, Avicel, filter paper, p-nitrophenyl cellobioside, and cellobiose. (3) Working pH and optimum pH The working pH range is 3.5 to 12.5. The optimum pH is 8 to 10, and even in the range of 5.5 to 10.5, the relative activity is 50% or more of the activity at the optimum pH. (4) pH stability It is extremely stable at pH 5 to 11 and does not inactivate, and maintains an activity of about 50% or more even at pH 3 to 12. (5) Optimum temperature The working temperature is in a wide range from 15 to 75 ℃, and the optimum temperature is 50 ℃. Further, it has 50% or more of the activity at the optimum temperature even in the range of 35 to 55 ° C. (6) Molecular weight of about 35,000 (by gel filtration method using Sephadex G100) (7) Effect of metal ion It is inhibited by Hg ²⁺ and Ba ²⁺ and activated by Co ²⁺ . (8) Effect of surfactant LAS, AS, ES, AOS, α-SFE, SAS, soap, and polyoxyethylene secondary alkyl ether hardly inhibit the activity. (9) Protease resistance It has resistance to protease. (10) Effect of chelating agent EDTA, EGTA, citric acid, sodium tripolyphosphate, and zeolite do not inhibit the activity.