JPH01252282A - Enzyme from filamentous fungi - Google Patents

Abstract

NEW MATERIAL: This enzymic preparation comprises a protease derived from the entomopathogenic filamentous fungal genus Metarhizium, Beauvaria, Verticillium and Aschersonia, etc., has no activity for a film protein azure, a cuticle of Locusta migratoria and an elastin and specificity for the formula (Bz is benzoyl; NA is p-nitroanilide) without being inhibited with a soybean trypsin inhibitor.

USE: For production of medicines, agriculture, etc.

PREPARATION: For example, a separated strain of Metarhizium anisopliae of the entomopathogenic filamentous fungal genus is cultured and grown in a culture medium and the resultant culture solution is then centrifuged to wash the obtained solid substance with a 0.1 M potassium phosphate buffer solution. Thereby, an electrostatically bound enzyme is taken out, filtered and centrifuged to dialyze the filtrate. The prepared dialyzate is concentrated and fractionated according to isoelectric focusing to afford the enzyme.

COPYRIGHT: (C)1989,JPO

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a filamentous fungal enzyme, and more specifically to a highly specific protease produced by entomopathogenic filamentous fungi.

Entomopathogenic filamentous fungus Metarhizium, Anisobria (neck d
Side 11...''■ Buttonai'' (MEI strain) was shown to produce a mixture of extracellular proteases (R, J, S
T, leger, A, K, Charnley and RoM, Cooper, Characteriz.
ation of cut+cle-degradin
g proteases produced by t
he entomo-p a t h o gene
fieJa-□□zo+1Lax M E l
-Arclives of Biochemistry
y and Biophysics (1987), 2
2+-232). These proteases were fractionated into two major components. That is, Gimo elastase (Pr
) and trypsin-like protease (referred to as Pr2
), and there is an isoenzyme of Pr2.

Prl exhibits broad substrate specificity, whereas Pr2 exhibits a much narrower substrate range. Pr2 rapidly hydrolyzes casein and synthetic substrates including alkinine or lysine, but not the host insect cuticle, elastin, or synthetic substrates for chymotrypsin and elastase. It shows little or no activity.

Specific active site inhibitors confirmed the similarity between Pr2 and trypsin.

Eight isolates of entomopathogenic strains producing extracellular proteases were identified, namely Metarhizium anisobria (4 strains), Bowvaria bassiana (-right side), and J\Rucillium lecani (-) (2). Ltd.) and Ashelsonia arelobuis (bd residence) 1
Hariga nLLS) was further screened. All isolates produced Prl and Pr2 proteases or
Surprisingly, the proteases of six of these isolates were similar to the aforementioned Pr of Metarhizium anisoplia (MEI).
It shows extremely narrow substrate specificity compared to 2 proteases.

Metarhizium anisoplia isolate MEI, R5549
The Pi·2 protease of R523 exhibits activity against the membrane protein a2ure, but not against the cuticle and elastin of the locust (Table 1). In contrast, P from six other isolates
r2 protease is inactive against all three proteins. Furthermore, proof of the limited substrate specificity of these Pr2 proteases was obtained by experiments with synthetic substrates (nitroanilide derivatives of peptides). The Pr2 enzymes from MEI, R3549 and R523 hydrolyzed a series of substrates Bz-AA-AA-Arg-NA, showing little distinction in their secondary subsite specificities ( (See Table 2).

In contrast, the Pr2 protease from the other six isolates was Bz-P 11 e -V a l -A r g
- Showed specificity for NA. These enzymes thus require highly specific substrate sequences, representing a previously undescribed class of fungal proteases. The only other enzyme with similar limited specificity is thrombocytin
tin) (botlobus atocus (flch L■L
It is a type of thrombin, such as (revised) poison origin). Trompositin is B z-P J> e -V a l -
A r g -N A is immediately cut off, and Bz-P r
It shows lower activity towards o-Ph e-Ar g -NA.

P r 2 from MEI, R3549 and RS 23
Differences between the protease and Pr2bu0 dease from the other six isolates were further revealed by inhibitor experiments (see Table 3).

P r 2 from MEI, RS 549 and R523
Like trompositin, Pr2 proteases from soybean trypsin inhibitors are inhibited by soybean trypsin inhibitor, but Pr2 proteases from six other isolates are not inhibited (with the exception of Asiersonia allelobuis, which was not tested).

These highly specific proteases will be effectively used in pharmaceutical manufacturing, agriculture, or industry.

The present invention thus provides an enzyme preparation consisting of at least one protease derived from an entomopathogenic filamentous fungus of any one of the following fungal genera:

That is, the fungal genera are Metarhizium, Bouvaria, Verticillium, and Acielsonia, and this enzyme preparation is characterized by the following properties.

(a) is substantially inactive against the membrane proteins azul, Tonosamahata cuticle and elastin; (b) Bz-Pb e-Va I-Ar g-N
(C) Not inhibited by soybean trypsin inhibitor.

In a preferred embodiment, the protease is derived from one of the following bacterial species: Namely, Metarhizium anisobria, Bouvaria hasiana, Perticillium lecani, and Asiersonia arelobuis.

In a particularly preferred embodiment, the protease is derived from one of the strains listed below, or one of their derivatives or mutants capable of producing the protease. These strains are Commonwea I thMycologie.
cal 1nstit, ute (CMI), Ferr
y Lane.

Kew, Richmond, 5urrey, TW
93AF, Ll, on October 12, 1987, and has the following strain deposit number:

1 student, 1 person, Jinsha senior ■ Theft No. 1 Metarhizium Anisobria var major R5324319793 var
major R5298'319794 Herticillium lecani l 3+97952, 31
9796 Bouvaria hasiana 319797 Asiersonia arelobuis 31979B The characteristics of the deposited strain described above are basically consistent with the published description of the type strain.

The present invention is illustrated in the following examples.

Bribe 1 B z -A A -A A -A r g-N A
:N-benzoyl-■5-amino acid-L-amino acid-L
-Arginine-p-nitroanilide Bz-Phe -V
a l-Arg-NA: N-pensoyl-■, -phenylalanine-L-valine-L-arginine-p-nitroanilide B z-Val -G I y-A r g-NA: N
-Hensyl-1-valine-glycine-1-arkynine-p-nitroanilide Bz-Pro-Phe-Arg-NA: N-bensoyl-proline-L-phenylalanine-L-arginine-p-nitroanilide CBZ-01y-P r o-Ar g-NA: N-carbobenzoxy-glycine-proline-L-arginine-1)-nitroanilide B z -A r g-N
A: N-benzoyl-L-arginine-p-nitroanilide D-Vat-Leu-Lys-NA: D-valine-■,-leucine=L-lysine-p-nitroanilide 5uc-(Ala)2-Pro-Phe -NA: N-succinyl-L-alanine-l-alanine-L-proline-L-phenylalanine-p-nitroanilide 5uc-Phe-Leu-Phe-NA: N-succinyl-l-phenylalanine-L-leucine-[ , -phenylalanine-p-nitroanilide 5uc-(Ala) 2-Pro-Leu-NA: N-succinyl-shi-alanine-shi-alanine-L-proline-L-leucine-p-nitroanilide 5uc-(Ala) 2-Pro-Abu-NA: N-succinyl-7-lanine-■,-alanine-L-proline-L-amino-n-butyric acid-p-nitroanilide 5uc-(Ala)2-Val-Ala-NA: N-succinyl-L-alanine-L-alanine-L-valine-
L-alanine-p-nitroanilide A c -(AI a) 3-N A: N-acetyl-L-alanine-L-alanine-L-alanine-p-nitroanilide PMSF: Phenylmethylsulfonyl fluoride Tos- Ly
s-CH2Cl: N-1)-)Syl-Syl-Syn-Chloromethyalpha KetoneTos-Pbe-CH2Cl N-)Syl-L-Phenylalanine-Chloromethyl Ketone BOC-Gly-Leu-Phe-CH2C1:N-
t, ert-phthoxycarbonyl-glycine-L-leucine-1-phenylalanine-chloromethylketone Example] 1 Metarhizium anisobria var, mLlo engineering RS
324 Metarhizium anisoplia Vat・-1fl
tLQf R5298 Verticillium lecani 1 Vernacilium lecani 2 Houbaria hacianaacielsonia arelobuis 2) Historically cultivated Metarhizium and Verticillium isolates were grown on Sabouraud dextrose agar at 23°C (Herticillium lecani) or at 27°C (Metarzium・Anisoplia> The Bouvaria hasiana isolate was stored in Cz a
It was stored at 27°C in p e , k -D ox medium.

Asiersonia allelobuis isolates were stored at 23°C on maltose agar.

100n+ containing filamentous fungi in a 250ml Erlenmeyer flask
1 of growth medium. The growth medium was as follows.

1% F/V carbon source 0.1%w/vKH2PO4 0.05% w/vM g S 0.

0.05M4-morpholine ethanesulfonic acid 1l- pH6,0 0, 271g/n+l FeSO4・7H201,0
μg/ml ZnSO4-7H200, 271g/m
l NaMoO4・2H200, 02μg/ml
CuSO4・5H200, 0271g/ml M
Non-inhibitory carbon sources for the nCl 264 H20 polymer are used: locust cuticle, bovine serum albumin, laminarin, cellulose, elastin and collagen. If cellulose is used, the growth medium must be supplemented with 0.2% w/v NaNo3 as a nitrogen source.

Insoluble carbon sources were added to previously sterilized (121°C for 15 minutes) basal medium and autoclaved at 115°C for 5 minutes. Soluble carbon sources were autoclaved in basal medium at 115°C for 20 minutes.

3 x 106 samples taken from agar plates cultured for 7-12 days.
After inoculation with 150 spores, the flasks were placed in a rotating incubator (150 revolutions/min) at 23°C (Herticillium lecani, Asiersonia arelobuis) or 2 spores.
Shake at 7.5°C (Metallidium anisoplia and Bouvaria hasiana).

3) 1% Tonosama grasshopper cuticle basal salt medium (200ml
0 ml conical flask (rotating at 150 ml) for 5 days (for Asiersonia arelobuis, 14 ml)
Cultures grown for 1 day) were clarified by filtration (through Whatman filter paper 1) and centrifugation (8,000 g, 15 min at 3° C.). The solid matter (cuticle and filamentous fungal cells) is washed with 0.1 M potassium phosphate buffer (approximately 3% w/v, wet weight) to remove electrostatically bound enzymes, and filtered. Centrifuged. This 11! The culture filtrate and the original culture filtrate were separately dialyzed (2×300 volumes of distilled water for 12 hours at 4°C, pH 6.0) and concentrated (molecular weight 20.0
00 polyethylene glycol) and combined (final volume 50 ml) before being subjected to isoelectric focusing (IEF) for the preparation described below. The pH of the fraction was determined (the gradient was discontinuous at pIs of 410.0 and above, so pl values of 10.0 and above were approximate values) and the enzyme activity of the fraction was adjusted to 0.0. It was assayed after dialysis against 2M KC,1 (12 hours at 4°C) and then distilled water (12 hours at 4°C).

IEF (pH 3,5-10.0) for preparation, 44
Narrow range IEF was performed on a 110+nl column (LKB8101). The method was as described in the manufacturer's manual. The enzyme activity and protein profile in the eluate were determined. 0.2M
Measurements were made after removal of the ampholytes by complete dialysis at pH 6.0 with KCl (14 hours at 4°C, 300 volumes) and distilled water (12 hours at 4°C, 300 volumes).

4) 1 bottle a) Non-specific protease activity of 14-tα stagnation was measured using the membrane protein Azul. A 20 m3 substrate, 4 ml of a Britton-Robinson buffer solution at a predetermined pH, and 1 ml of an enzyme solution were placed in a 5 ml bottle, the bottle was stoppered, and the bottle was fixed on a rotary disk.

After incubation at 30°C, trichloroacetic acid #(0,
The reaction was terminated by adding 25ml (1.500g/liter). After centrifugation (10 min at 5,000 g), absorbance was measured at 595 nm.
It was measured with Activity is expressed as μg trypsin equivalent 1 jt/ml/hr calculated from a standard curve of trypsin activity against the membrane protein Azur. The results are shown in Table 1.

Elastase activity was determined using 4 ml Tris buffer (0.05 M
, pH 8.0), in a reaction mixture containing 1 ml enzyme solution and Elastin-Congo Red (protein concentration 1 mg/ml).

After an appropriate time interval at 30°C, the mixture was centrifuged (5000g for 10 min) and the absorbance was measured at 450 nm. One elastin solubility unit was defined as the amount of enzyme that solubilizes 1 mg of elastin in 30 minutes.

Activity against purified protein was assayed as follows. Add 0.5 ml of enzyme solution and 2.0 ml of protein. 5
Containing at a concentration of mg/ml1. Inkyuno\-! with 0ml of 0.05M) Lys buffer (pH 8,0)! ·did. After incubation at 30°C for an appropriate time,
The reaction was terminated by the addition of ml of 10% trichloroacetic acid and left for 1 hour. Whatman no.

The absorbance of the filtrate was reduced to 280 by filtration through 50m paper.
Measured in nm. Enzyme activity was measured at μ8 per ml for 1 minute.
Expressed as trypsin equivalent. Enzymatic release of free amino groups from proteins was determined by ninhydrin analysis.

The activity against Tonozamahata cuticle was measured as follows.

30 mg of ground locust cuticle (prepared using 1% potassium tetraborate aqueous solution), 2 ml enzyme, 4 ml
1 Fritonorohinson buffer (pH 5,0) and Shake vigorously at 30° C. with 0.5 ml of toluene. Samples (0.05 ml) were incubated for 24 hours, taking them at regular intervals. Amino acid release due to proteolytic activity was measured with ninhydrin.

The results are shown in Table 1.

The activity towards nitroanilide substrate is 0. 1 ml enzyme solution and 1. 8ml Friton-Robinson buffer (p
The measurement was carried out in a reaction mixture containing H8,Q).

After 3 min at 23°C, 0.1 ml of substrate (dissolved in dimethyl sulfooxide) was added and the reaction was incubated at 410 nm.
I tracked it. Results are expressed as 71 mol nitronitrile liberated per ml in 1 minute.

An extinction coefficient of 8800 at 410 nm was used. The results are shown in Table 2.

All controls contained autoclaved enzyme at 121c for 30 minutes in place of the non-heat treated enzyme solution.

b) Molecular weight is SDS of neutral or alkali-gel system.
Determined by polyacrylamide gel electrophoresis and Kell filtration on Sephadex G-100 fines using 0.15M phosphate buffer. The results are shown in Table 4.

Claims (1)

[Scope of Claims] 1. Entomopathogenic fungi of the genus Metarhizim and Bea
uvaria￥), Verticillium (￥Vertici
llium￥) and Aschersonia (￥Ascher
1. An enzyme preparation comprising at least one protease derived from any one of the following: (a) has substantially no activity against the membrane proteins azul, locust cuticle and elastin; (b) has specificity for Bz-Phe-Val-Arg-NA; and (c) soybean trypsin. Not inhibited by inhibitors. 2. Protease is produced by Metarhizium anisoplia (￥
Metarhizium anisopliae￥),
Beauvaria bassiana (￥Beauvaria bas
siana￥), Verticillium lecani (￥Vert
icillium lecanii) and Aschersonia areirodis (Aschersonia
The enzyme preparation according to claim 1, which is derived from any one species of B. aleyrodis¥. 3. Protease is CMI319793, CMI31
9794, CMI319795, CMI319796,
Enzyme preparation according to claim 1 or 2, derived from any one of the strains CMI319797 and CMI319798, or a derivative or mutant strain thereof capable of producing the protease. 4. The protease has a molecular weight of 25,000-30,000, an optimum pH for activity is about 8.0, an optimum pH for stability is about 6.0-9.0, and is stable at temperatures up to 50°C. 4. An enzyme preparation according to any one of claims 1-3.