TECHNICAL FIELD

The present invention relates to novel micro¬ organisms, novel enzymes obtainable herefrom, and to a method of producing the novel enzymes. More specifically, the inven¬ tion relates to novel enzymes obtainable from strains of the novel alkalophilic species Bacillus sp. AC13.

Moreover, the invention relates to a method for producing the enzymes of the invention, and to the use of the enzymes, particularly in detergents or in the paper pulp industry.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide novel microorganisms capable of expressing valuable novel enzymes for industrial applications, as well as novel enzymes of different kinds obtainable from these organisms.

Accordingly, the invention provides isolated bio¬ logically pure cultures of strains of the alkalophilic species Bacillus sp. AC13. In another aspect, the invention provides enzymes obtainable from strains of Bacillus sp. AC13. and having immunochemical properties identical or partially identical to those of an enzyme derived from Bacillus sp. AC13 _P NCIMB No. 40482. In a more specific aspect, the invention provides proteases obtainable from strains of Bacillus sp. AC13. and having immunochemical properties identical or partially identical to those of a protease derived from Bacillus sp. AC13. NCIMB No. 40482. In another specific aspect, the invention provides xylanases obtainable from strains of Bacillus sp. AC13. an having immunochemical properties identical or partiall identical to those of a xylanase derived from Bacillus sp. AC13. NCIMB No. 40482.

In a third specific aspect, the invention provide cellulases obtainable from strains of Bacillus sp. AC13. an having immunochemical properties identical or partiall identical to those of a cellulase derived from Bacillus sp AC13, NCIMB No. 40482.

In a third aspect, the invention provides a proces for the preparation of an enzyme of the invention, the proces comprising cultivation of a strain of Bacillus sp. AC13 preferably the strain Bacillus sp. AC13. NCIMB No. 40482, or mutant or a variant thereof, in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts followed by recovery of the desired enzyme.

In further aspects, the invention provides detergen additives and detergent compositions comprising an enzyme o the invention.

Moreover, the invention relates to the use of xylanase of the invention in processes for treatment o lignocellulosic pulp.

BRIEF DESCRIPTION OF DRAWINGS

The present invention is further illustrated b reference to the accompanying drawings, in which:

Fig. 1 shows the relative activity (% rel.) of protease of the invention at various pH, determined at 25° with casein as substrate; and Fig. 2 shows the relative activity (% rel.) of protease of the invention at various temperatures (I Buffer p 9.5; D Buffer pH 9.5 containing 0.1% STPP) , determined at p 9.5 with casein as substrate.

DETAILED DISCLOSURE OF THE INVENTION

The Microorganism

The present invention relates to microorganisms o the novel alkalophilic species Bacillus sp. AC13. represente by the type culture Bacillus sp. AC13. NCIMB 40482.

The strain Bacillus sp. AC13. NCIMB 40482, has been deposited on 3 March 1992 according to the Budapest Treaty on the International Recognition of the Deposits of Microorganisms for the Purpose of Patent Procedures, at National Collections of Industrial and Marine Bacteria Ltd. , 23 St. Machar Drive, Aberdeen AB2 1RY, Scotland, UK, under Accession No. NCIMB 40482.

The microorganisms of this invention are aerobic, rod shaped, spore forming bacteria, and therefore belonging to the genus Bacillus.

Morphologically they can be described as rods having a diameter of 0.6-0.9 μm and a length of 4-8 μm. The spores are ellipsoid to round, approximately 1.2 x 0.8 μm, terminal swelling the sporangia, giving the sporangium a characteristic racket or drumstick like shape.

The microorganisms of Bacillus sp. AC13 are obligate- ly alkalophilic, requiring carbonate buffer pH 9 to 10 in the agar media for growth. Optimal growth is observed at 37°C, at pH 9.5-10. No growth at 50°C and no growth at pH 7. Colonies on potato dextrose agar (Difco™) added 0.1

M sodium sesquicarbonate are white with characteristic dendroid to hairy edges.

The microorganisms can be further described by the following characteristics.

NaCl tolerance 0-10% weak growth at 12% Growth temperature < 45°C no growth at 50°C Hydrolysis of casein positive gelatine positive pullulan negative starch positive cellulose positive xylan positive

Catalase reaction positive Aminopeptidase test negative Deamination of phenylalanine negative

Reduction of nitrate positive Major fatty acids C 15:0 ISO (« 45%) C 15:0 ANTEISO (« 25%) ISO 17:1 wlOc (« 10%) Unsaturated: 20% Branched approx. 50%

Cultivation of the Microorganism

The microorganism of the invention can be cultivate under aerobic conditions in a nutrient medium containin assimilable carbon and nitrogen together with other essentia nutrients, the medium being composed in accordance with th principles of the known art.

Suitable carbon sources are carbohydrates such a sucrose, glucose and starch, or carbohydrate containin materials such as cereal grain, malt, rice and sorghum. Th carbohydrate concentration incorporated in the medium may var widely, e.g. up to 25% and down to 1 - 5%, but usually 8 - 10 will be suitable, the percentages being calculated as equi valents of glucose. The nitrogen source in the nutrient medium may be o inorganic and/or organic nature. Suitable inorganic nitroge sources are nitrates and ammonium salts. Among the organi nitrogen sources quite a number are used regularly in fermenta tion processes involving the cultivation of bacteria. Illustra tive examples are soybean meal, cotton seed meal, peanut meal casein, corn, corn steep liquor, yeast extract, urea an albumin. In addition, the nutrient medium should also contai usual trace substances.

Since the novel Bacillus species of this inventio are alkalophilic and unable to grow at pH below 7, the cultiva tion is preferably conducted at alkaline pH values, which ca be obtained by addition of suitable buffers such as sodiu carbonate or mixtures of sodium carbonate and sodium bicarbon ate, after sterilization of the growth medium. For cultivatio in tank fermentors it is necessary to use artificial aeration The rate of aeration is similar to that used in conventiona tank fermentation.

After fermentation, liquid enzyme concentrates may be produced by removal of coarse material from the broth or, if desired, concentration of the broth by evaporation at lo temperature or by reverse osmosis. Finally, preservatives may be added to the concentrate.

Solid enzyme preparations may be prepared from the purified and/or concentrated broth by precipitation with salts, such as Na ₂ S0 ₄ or water- iscible solvents, such as ethanol o acetone. Removal of the water in the broth by suitable dryin methods such as spray-drying may also be employed.

The microorganisms of the invention have been foun to be able to produce valuable novel enzymes, in particula proteases, xylanases and cellulases.

The Enzymes The enzymes of the invention are obtainable b cultivation of a microorganism of the invention, preferabl Bacillus sp. AC13, NCIMB No. 40482, or a mutant or a varian thereof, in a suitable nutrient medium, containing carbon an nitrogen sources and inorganic salts. The enzymes may also b obtained by recombinant DNA-technology.

In more specific aspects, the enzymes of the presen invention can be further described by the following physical chemical characteristics.

The Proteases In a specific embodiment of the invention, a proteas can be further characterized by having an apparent molecula weight of approximately 30 kD as determined by SDS-PAGE, and pi of approximately 9.3 as determined by isoelectric focusin on LKB Ampholine PAG plates. The protease can also be characterized by havin proteolytic activity at pH values of from below pH 6 to abov pH 11, having optimum above pH 10, around pH 11, when determ ined at 25°C with casein as substrate.

Moreover, the protease can be characterized by havin proteolytic activity at temperatures of from approximately 15° to above 70°C, having activity optimum at temperatures in th

range 45-55°C, around 50°C, when determined at pH 9.5 with casein as substrate. This activity optimum can be detected with or without sodium tripolyphosphate, which is a common in¬ gredient in many commercial detergents.

The Xylanases

In a specific embodiment of the invention, a xylanase can be further characterized by having an apparent molecular weight of approximately 25 kD when determined by SDS-PAGE, and a pi of approximately 9 when determined by isoelectric focusing on LKB Ampholine PAG plates.

The Cellulases

In a specific embodiment of the invention, two cel¬ lulases can be further characterized, one by having an apparent molecular weight of approximately 45 kD when determined by SDS- PAGE, and a pi of approximately 4.3 when determined by iso¬ electric focusing on LKB Ampholine PAG plates, and one by having an apparent molecular weight of approximately 55 kD when determined by SDS-PAGE, and a pi of approximately 4.5 when determined by isoelectric focusing on LKB Ampholine PAG plates.

Immunochemical Properties

The enzymes of the invention have immunochemical properties identical or partially identical (i.e. at least partially identical) to those of an enzyme derived from the strain Bacillus sp. AC13. NCIMB No. 40482. The immunochemical properties can be determined immunologically by cross-reaction identity tests. The identity tests can be performed by the well-known Ouchterlony double immunodiffusion procedure or by tandem crossed immunoelectro- phoresis according to Axelsen N.H. ; Handbook of Immunopre- cipitation-in-Gel Techniques; Blackwell Scientific Publications (1983), chapters 5 and 14. The terms "antigenic identity" an "partial antigenic identity" are described in the same book, chapters 5, 19 and 20.

Monospecific antisera are generated according to the above mentioned method by immunizing rabbits with the purified enzymes of the invention. The immunogens are mixed with Freund\'s adjuvant and injected subcutaneously into rabbits 5 every second week. Antisera are obtained after a total im¬ munization period of 8 weeks, and immunoglobulins are prepared therefrom as described by Axelsen N.H.. supra.

Assay for Proteolytic Activity

The proteolytic activity is determined with casein as 10 substrate.

One Casein Protease Unit (CPU) is defined as the amount of enzyme liberating 1 mM of primary amino groups

(determined by comparison with a serine standard) per minute under standard conditions, i.e. incubation for 30 minutes at

1525"C and pH 9.5.

A folder F 228/1 describing the analytical method is available upon request to Novo Nordisk A/S, Denmark, which folder is hereby included by reference.

Assay for Xylanolytic Activity 20 The xylanolytic activity is measured in endo-xylanase units (EXU) , determined at pH 9.0 with remazol-xylan as substrate.

A xylanase sample is incubated with remazol-xylan substrate. The background of non-degraded dyed substrate is 25 precipitated by ethanol. The remaining blue colour in the supernatant is proportional to the xylanase activity, and the xylanase units are then determined relatively to an enzym standard at standard reaction conditions, i.e. at 50.0 +/-

0.1°C, pH 9.0, and 30 minutes ¹ reaction time. 30 A folder AF 293.9/1 describing the analytical metho is available upon request to Novo Nordisk A/S, Denmark, whic folder is hereby included by reference.

Assay for Cellulytic Activity

The cellulytic activity is measured in cellulas viscosity units (CEVU) , determined at pH 9.0 with carboxymethy cellulose (CMC) as substrate. Cellulase viscosity units are determined relativel to an enzyme standard ( < 1% water, kept in N ₂ atmosphere at -20°C; arch standard at -80°C). The standard used, 17-1187, i 4400 CEVU/g under standard incubation conditions, i.e. pH 9.0 Tris Buffer 0.1 M, CMC 7 LFD substrate 33.3 g/1, 40.0°C for 3 minutes.

A folder AF 253/1 describing the analytical method i available upon request to Novo Nordisk A/S, Denmark, whic folder is hereby included by reference.

Industrial Applications The enzymes of this invention possess valuabl properties allowing for various industrial applications. I particular the proteases and cellulases of the invention, i being alkaline, find potential application in e.g. detergen compositions. The cellulases may find potential application i the textile industry, e.g. for Bio-Polishing. The xylanases ma find application in e.g. the paper pulp industry.

The following examples further illustrate the presen invention, and they are not intended to be in any way limitin to the scope of the invention as claimed.

EXAMPLE 1

Cultivation Example

The strain Bacillus sp. AC13. NCIMB 40482, wa cultivated at 30°C on a rotary shaking table (300 r.p.m.) i 500 ml baffled Erlenmeyer flasks containing 100 ml of medium o the following composition (per litre) :

Potato starch 100 g

Ground barley 50 g

Soybean flour 20 g

Na ₂ HP0 ₄ x 12 H ₂ 0 9 g Pluronic ^® 0.1 g

Sodium caseinate 10 g

The starch in the medium is liquified with α-amylase and the medium is sterilized by heating at 120°C for 45 minutes. After sterilization the pH of the medium is adjusted to 9.7 by addition of 10 ml 1 M sodium sesquicarbonate to each flask.

After 3 days of incubation the following activities were observed: Proteolytic activity 20 CPU/1

Xylanolytic activity 10 EXU/g Cellulytic activity 4 CEVU/g

Isoelectric focusing on gels overlayered with different substrates at least 2 different proteases, at least 4 different xylanases, and at least 2 different cellulases were detected.

The major proteolytic band has a pi of approximately 9.3 and a molecular weight of approximately 30 kD.

The major xylanolytic band has a pi of approximately 9 and a molecular weight of approximately 25 kD.

The major cellulytic band has a pi of approximately 4.3 and a molecular weight of approximately 45 kD.

EXAMPLE 2

Purification of the Proteolytic Compounds After cultivation, the proteolytic activity of the fermentation broth of Ex. 1 was found to be 20 CPU/1. In this fermentation broth at least two proteolytic enzymes have been identified by isoelectric focusing on LKB Ampholine PAG plates.

After separation of the solid material, the majo proteolytic component was purified by a conventional chroma tographic method. From 1 litre of culture broth yield was 50 m of protease preparation with a proteolytic activity of 23 CPU/1 (60%) .

The purified protease has an apparent pi value of 9. when determined by isoelectric focusing on LKB Ampholine PA plates. By SDS-PAGE the apparent molecular weight of th protease is found to be 30 kD. Purity was more than 90% a judged by both SDS-PAGE and isoelectric focusing.

The activity was determined using the assay fo proteolytic activity described above. The results of thes experiments are presented on the appended Figs. 1-2.

EXAMPLE 3

Purification of the Xylanolytic Compounds

In the fermentation broth, as obtained according t Ex. 1, at least four xylanolytic enzymes have been identifie by isoelectric focusing combined with an overlayer of xylan. The xylanolytic enzymes cover the pi range of from 5 to 9.5. From the fermentation broth of Ex. 1, a xylanase wit an alkaline pi (the major xylanolytic component) was purifie to homogeneity by conventional chromatographic technique involving cation exchange chromatography on S-Sepharose Hig Load™ and Mono S™, hydrophobic adsorption chromatography o Phenyl-Sepharose, as well as affinity chromatography t specific removal of proteinases.

The purified xylanase has an apparent pi value of in a 3.5 to 9.5 isoelectric focusing gel. By SDS-PAGE th apparent molecular weight of the xylanase is found to be 25 kD

EXAMPLE 4

Purification of the Cellulytic Compounds

In the fermentation broth obtained according to Ex 1 at least two cellulytic enzymes have been identified b isoelectric focusing.

The fermentation broth of Ex. 1 was filtrated and applied on a cellulase affinity column. After wash at pH 8.5 in

Tris buffer, the column was eluted at high pH 11.8 with triethylamine. The pH in the eluate was adjusted to 7.5 and UF- concentrated and washed out with Tris buffer.

The concentrate was applied on a Mono Q™ column (Pharmacia) and eluted with a linear gradient with 15 column volumes in Tris buffer pH 9.0 with 0.5 M NaCl.

The cellulase containing fractions were subjected to isoelectric focusing and SDS-PAGE, and two cellulytic com¬ ponents were found, one having a MW of approx. 45 kD and a pi of approx. 4.3, and one having a MW of approx. 55 kD and a pi of approx. 4.5.

EXAMPLE 5

N-Terminal A ino-Acid Analysis

The N-terminal amino-acid sequence of the cellulase, having a MW of approx. 45 kD, obtained according to Ex. 4, was determined using standard methods for obtaining and sequencing peptides [Findlay & Geisow (Eds.), Protein sequencing - a practical approach, 1989, IRL Press].

The N-terminal amino-acid sequence was found to be (SEQ ID No. 1 of the attached sequence listing) :

Asp-Xaa-Asp-Xaa-Val-Val-Glu-Glu-His-Gly-Gln-Leu-Arg-Ile- Xaa-Asn-Gly-Xaa-Leu.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: NOVO NORDISK Α/S (B) STREET: Novo Alle

(C) CITY: DK-2880 Bagsvaerd

(E) COUNTRY: Denmark

(F) POSTAL CODE (ZIP) : DK-2880

(G) TETEFHONE: 45 44 44 88 88 (H) TELEFAX: 44 49 32 56

(I) TELEX: 37304

(ϋ) TITLE OF INVENTION: NOVEL MICROORGANISMS (iii) NUMBER OF SEQUENCES: 1 (iv) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) CX.MEUTER: IBM PC cortpatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFIWARE: Patentin Release #1.0, Version #1.25 (EPO) (v) CURRENT APPLICATION DATA: APPLICATION NUMBER:

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTEEISTICS: (A) LENGTH: 19 amino acids (B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOIOGY: linear

(ii) MDLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (v) FRAGMENT TYPE: N-terminal

(Vi) ORIGINAL SOURCE:

(A) ORGANISM: Bacillus sp.

(B) STRAIN: AC 13 NCIMB 40482

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Asp Xaa Asp Xaa Val Val Glu Glu His Gly Gin Leu Arg lie Xaa Asn 1 5 10 15

Gly Xaa Leu

International Application No: PCT/

Form PCT/ftO/lM (January 1M1)