DK153799B - Maltogenic amylase, process for its preparation, and a plasmid used for obtaining it - Google Patents

Description

1 DK 153799 B

The present invention relates to a novel maltogenic amylase, methods for its preparation and plasmid for use in its preparation.

Beta-amylases are maltogenic exo-amylases which hydrolyze alpha-1,4-glycosidic bonds from the non-reducing end of amylose, amylopectin or glycogen to give the beta form of maltose. The beta form of maltose will spontaneously isomerize in aqueous solutions to a mixture of the alpha and beta forms.

Beta-amylases can be used to prepare maltose-containing syrups for use in the confectionery, bakery and brewery industries.

Beta-amylases have been isolated from various 15 plants and microorganisms (W.M. Fogarty and C.T. Kelly,

Progress in Industrial Microbiology, Vol. 15, pp. 112 - 115, 1979). Beta-amylases known from plants (barley, sweet potatoes and soybeans) and from the Bacillus species B. mycoides, B. megatrium and B. polymyxa are all enzymes whose activity is inhibited by sulfydryl reagents, e.g. PCMB (parachlormercuribene zoat), indicating that the active site involves an SH group.

So far, only a beta-amylase which is not inhibited by PCMB, namely a beta-amylase prepared from Bacillus circulans (U.S. Pat.

4,001,136). This B. circulans beta amylase is more thermostable than the above beta amylases. However, it is rapidly inactivated at and above 65 ° C.

However, in a process for producing maltose, wherein starch in an aqueous medium is hydrolyzed by a beta-amylase, it is advantageous to use a process temperature of approx. 60 - 70 ° C to prevent retrogradation and to prevent microbial infections.

The above-mentioned Bacillus circulans beta-amylase is therefore less suitable for commercial use at ca. 65 - 70 ° C

p.g.a. one for quick deactivation.

2 DK 153799B

IN USA. U.S. Patent No. 3,804,715 discloses a heat-resistant beta-amylase extracted from wheat bran as described in British Patent No. 1,130,398. However, this beta-amylase is less attractive in a commercial process compared to an enzyme produced from a bacterial source, because the latter can be produced on a larger scale at relatively low cost compared to the cost of a plant-origin beta-amylase.

Therefore, there is a need for an effective microbial maltogenic amylase preparation sufficiently thermostable to be used at 65-70 ° C for a sufficient period of time to allow starch hydrolysis in an economical manner.

The object of the invention is to provide a novel microbial maltogenic amylase that is not inactivated by sulphydryl reagents such as PCMB, and additionally has a higher thermostability compared to previously known microbial beta-amylases.

The present invention is based on the surprising realization that a novel extracellular maltogenic enzyme (C599 amylase) with such good properties can be prepared from a novel microorganism belonging to the Bacillus stearothermophilus complex.

For a better understanding of the present invention and the following description, reference is made to the drawing, in which: 1 graphically illustrates the relative activity of the maltogenic amylase enzyme against the temperature; FIG. 2 illustrates graphically the relative activity against pH, and FIG. Figure 3 shows the restriction endo-nuclease cleavage map of the novel plasmid pDN452.

In its first aspect, the present invention relates to a thermostable maltogenic amylase which is property 35

3 DK 153799B

in that it has a temperature optimum (measured at 30 minutes reaction time in acetate buffer (0.1 M)) at pH 5.5 of approx. 60 ° C, a pH optimum at approx. 60 ° C in the range of 4.5 to 6 (determined in a MC Ilvaine buffer at 30 minutes reaction time), a residual activity after 60 minutes at 70 ° C (in acetate buffer (0.1 M) at pH 5.5) of at least 75%, maltotriose quantitatively cleaves in equimolar amounts of maltose and glucose and can be prepared by growing Bacillus NCIB 11837 in a suitable nutrient medium containing carbon and 10 nitrogen sources and inorganic salts, and subsequent isolation of the amylase formed with the aforementioned properties of culture fluid.

The maltogenic amylase product may be in solid or liquid form. In solid form, it usually has an activity 15 of from 500 to 25,000 E (defined below) per gram.

According to its second aspect, the present invention relates to a process for preparing the above-mentioned maltogenic, thermostable amylase, and this process is characterized in that a Bacillus strain NCIB 11837 20 or variants or mutants thereof having substantially the same properties as it is grown in a suitable nutrient medium. containing carbon and nitrogen sources and inorganic salts, after which the maltogenic amylase is recovered from the culture liquid.

In addition, by DNA recombinant technique, the gene encoding the novel thermostable maltogenic amylase has been transferred into another microorganism which under appropriate culture conditions produces the maltogenic amylase in substantially greater amounts compared to what can be obtained. by culturing the donor microorganism NCIB 11837.

According to its third aspect, the present invention relates to a process for preparing the above-mentioned maltogenic amylase, which is characterized in that a transformed host strain from the genus Bacillus to which the gene encoding the thermostable maltogenic amylase is transferred Bacillus strain NCIB 11837 is grown in a suitable nutrient medium containing carbon and nitro

4 DK 153799B

gene sources and inorganic salts, followed by recovery of the maltogenic amylase from the culture fluid.

A recombinant plasmid containing the gene encoding the thermostable maltogenic amylase can be produced by cleavage of chromosomal DNA from Bacillus NCIB 11837, with a suitable restriction enzyme to obtain a linear DNA sequence containing the amylase coding gene. cleavage of an appropriate vector to obtain a further linear DNA sequence and joining of the two linear 10 DNA sequences to obtain a recombinant plasmid containing the amylase gene.

The vector, preferably an E. coli plasmid, is cleaved with an appropriate restriction enzyme which yields linear DNA with ends that can be joined to the ends of the fragments of donor DNA.

The joining of the two linear sources of the DNA sequences is performed by a ligase using well known techniques.

According to a preferred embodiment of the method of the present invention, the host strain is preferably selected from Bacillus subtilis.

The transformation of the host strain is performed in a well-known manner by transforming the recombinant plasmid into E. coli cells, identifying starch-degrading transformants, and subcloning the recombinant plasmid from such Amy + transformants into the selected host strain.

The host strain (which preferably does not exhibit amylase activity), upon acquisition of the recombinant plasmid, can produce the novel maltogenic amylase when the strain is grown in a suitable culture medium. The amylase is secreted into the growth medium, providing a simple recovery step.

The present invention also relates to a plasmid for use in a Bacillus strain in producing the maltogenic amylase of the present invention and this plasmid is characterized in that it is a recombinant plasmid containing the gene transferred from Bacillus strain NCIB 11837.

5 DK 153799 B

encoding the maltogenic amylase as well as a nucleotide sequence that ensures replication in a Bacillus subtilis host. This sequence is preferably according to the invention plasmid pBD64 or pUB110 or derivatives thereof having substantially the same properties. Such a plasmid can be prepared by cleaving chromosomal DNA from Bacillus NCIB 11837 with the restriction enzyme Mbol, isolating DNA fragments in the range of 4 - 12 kb (kilobase pairs), joining with E. coli plasmid pACYC184 that has been cleaved with the restriction enzyme BamH1 , transformation into E. coli cells, identification of starch degrading transformants containing plasmids with the amylase gene, cleavage of these plasmids with restriction enzyme Sau3Al, joining the DNA fragment expressing amylase activity with plasmid 15 pBD64 that has been cleaved with the restriction enzyme B , and transformation of the new recombinant plasmid into B. subtilis.

According to the invention, the plasmid is preferably pDN452 with the restriction endonuclease cleavage map shown in Figure 3 of the drawing. Plasmid pDN452 is a 7.6 kb hybrid plasmid containing the nucleotide sequence of pBD64 and the DNA sequence of Bacillus NCIB 11837 encoding the subject amylase inserted at the BamH1 site of this plasmid.

The maltogenic amylase of the invention is well suited for preparing maltose-rich syrups in which the starch is treated with the maltogenic amylase in an aqueous medium.

Tests have shown that the maltogenic amylase is suitable for the preparation of maltose and maltose-rich syrups. Such 30 products have considerable interest in the manufacture of certain confectionery products because poor hygroscopicity of maltose, low viscosity in solution, good heat stability and mild, not too sweet taste.

The industrial production process for maltose syrups involves the liquefaction of starch, sugars with a maltose-forming enzyme, optionally supplemented with an enzyme which

6 DK 153799B

gaps.1,6-branching points of amylopectin, e.g. and alpha-1,6-amyloglucosidase.

Although the amylase in question responds in many respects, like the known beta-amylases 5, it differs from these in several essential points, as will be apparent from the following detailed description, and consequently the new enzyme has been characterized as a maltogenic amylase, and not a beta-amylase.

The new enzyme migrates by SDS-polyacrylamide gel-10 electrophoresis as a single band corresponding to a molecular weight of approx. 70,000 Dalton. The isoelectric point is found by 8.5 isoelectric focusing in plate gel.

The enzyme hydrolyzes amylopectin, glycogen and amylose, with maltose being the major part of the reaction products. Glucose is formed in small amounts corresponding to 1 - 10% of the maltose formed.

From branched polysaccharides such as amylopectin and glycogen, the enzyme forms boundary dextrins which can be hydrolyzed with glycoamylase.

Sulphydryl reagents such as para-chloro-mercuribenzoate and chelate-binding reagents such as EDTA have no influence on enzyme activity.

The maltogenic amylase of the invention differs from the known beta-amylases as follows: 1. It quantitatively hydrolyzes Schardinger cyclodextrins. Schardinger beta-cyclodextrin is hydrolyzed to maltose + glucose at a molar ratio of 3: 1, while alpha-cyclodextrin is hydrolyzed to maltose + glucose at a molar ratio of 10: 1. HNMR spectral analysis of alpha-cyclodextrins incubated with the maltogenic amylase shows an initial formation of alpha-maltose as the first major product.

2. Maltotriose is quantitatively cleaved in equimolar amounts of maltose and glucose. HNMR spectral analysis of maltotriose incubated with the maltogenic amylase shows that the hydrolysis product is alpha-maltose + glucose.

3. It is stable in buffer at 70 ° C.

7 DK 153799B

4. The boundary dextrins of the maltogenic amylase do not form colored complexes with I2 ~ KI reagents. Thus, the maltogenic amylase of the invention is an exoenzyme which attacks alpha-1,4-glycosidic bonds, the main hydrolysis product being alpha-maltose.

Since there has been an increased interest in maltose-rich syrups containing more than 80% maltose in recent years, the ability of the maltogenic amylase of the present invention to cleave maltotriose found in 10 known maltose syrups is quantitative in maltose and glucose. extremely advantageous because the maltose yield is thereby increased.

A microorganism capable of producing the maltogenic amylase of the present invention was selected based on its ability to grow on an agar substrate prepared as follows:

Tryptone (Difco) (10 g), amylopectin (CPC snowflake 10 g), Bactoagar (agar-agar) (Difco) (40 g) and ion-exchanged water (1000 ml) were mixed aseptically at 55 ° C with a corresponding amount of saline solution having the following composition: (.NH 2 SO 4: 0.04% by weight

MgSO 4, 7H 2 O: 0.1 -

CaCl 2: 0.04 - KH 2 PO 4: 0.6 - 25 The pH of the saline solution was adjusted to 3.0 with 10 N sulfuric acid.

Soil samples collected at Krisuvik, an area of hot springs in Iceland, were dispersed on the aforementioned agar substrate and incubated at 65 ° C.

After 48 hours, the agar surface was stained with iodine vapors and a colony, C599, which was surrounded by a zone of unstained amylopectin was isolated.

The isolated colony of C599 with underlying agar was incubated overnight with starch at pH 4.5 at 35 ° C and 70 ° C. A thin-layer chromatographic study revealed that maltose was the main product of the hydrolysis.

8 DK 153799B

The isolated microorganism C599 was deposited with The National Collection of Industrial Bacteria, Torry Research Station, Aberdeen, Scotland, on March 15, 1983 and was given reference number NCIB 11837.

5

taxonomy

The newly discovered microorganism is an aerobic, rod-shaped and spore-forming bacterium. It thus belongs to the genus Bacillus.

10 On agar substrates where the sporulation is poor, the culture autolyses rapidly and dies. Therefore, it has been impossible to carry out the normal taxonomic program for the classification of Bacillus species.

The morphology and growth temperature between 50 and 70 ° C indicate that the new microorganism belongs to the Bacillus stearothermophilus complex.

morphology

The spores are oval approx. 1 x 1.6 µ and located 20 terminally to subterminally.

The sporangia are strongly swollen and resemble ketche or drumsticks.

Determination of Enzyme Activity 25 A maltogenic amylase unit (E) is defined as the amount of enzyme which, under standard conditions (temperature 60 ° C, pH 5.5 and reaction time 30 minutes) produces reducing sugars corresponding to 1 μιηοΐ maltose per minute.

A 0.5% soluble starch (from Merck) in 0.1 M acetate buffer or 0.05 M phosphate buffer (pH 5) is incubated with 1 ml of the enzyme dissolved in ion-exchanged water containing 0.1 -0.2 U per ml. The reaction is quenched after 30 minutes of reaction time by the addition of 4 ml of 0.5 N NaOH.

The content of reducing sugar is determined according to the Somogyi method (Somogyi: J. Biol. Chem. 153, p. 375-80 (1944)).

9 DK 153799 B

An alternative way of determining enzyme activity is based on the ability of the maltogenic amylase to quantitatively cleave maltotriose in equimolar amounts of maltose and glucose.

5 A maltogenic amylase NOVO unit (MANE) is defined as the amount of enzyme that, under standard conditions, cleaves a μιηοΐ maltotriose per minute. The enzyme reaction is stopped by changing the pH to approx. 11. The glucose formed is converted by glucose dehydrogenase (Merck, GlucDH) to 10 gluconolactone to form NADH. The amount of NADH formed is measured colorimetrically at 340 nm.

Standard conditions: Temperature 37 ° C ± 0.05 ° C

pH 5.0

Incubation time 30 min.

15

Reagents 1. Dissolve 0.1 M citrate buffer, pH 5.0 5.25 g citric acid (CgHgO7, H2 O) in ca. 200 ml of demimerized water and the pH is adjusted to 5.0 with 4.0 / 1.0 N NaOH. Make up to 250 ml with demineralized water and check the pH. The buffer solution can be stored for one week in the refrigerator (the pH should be checked before use), but preferably prepared 25 every test day.

2. Maltotriose substrate 20 mg / ml

To 500 (1000) mg of maltotriose (Sigma M 8378) citrate buffer (reagent 1) is added to 25 (50) ml. Must 30 be made every day.

3. GlucDH reagent

Enzyme mixture, Merck No. 14055 Bottle "1" and "2", is filled with buffer solution, Merck No. 14051. After 15 35 minutes, the contents of the bottles are transferred to a 500 ml graduated flask containing approx. 200 ml buffer (Merck no.

10 DK 153799B

14051), and additional buffer is added up to 500 ml.

Stable 14 days in refrigerator.

4. Stop Reagent 5 0.06 N NaOH.

Glucose standard curve Dissolve 1.6 g glucose in 1000 ml demineralized water and dilute samples of 1.0, 2.0, 4.0, 6.0 and 10.0 up to 100 ml with demineralized water. The 5 standard solutions obtained have a glueose concentration of 88.8, 177.6, 355.2, 532.9 and 888.1 μιηοΐ / liter, respectively.

The glucose standard curve is prepared by mixing 2.0 ml of the above-mentioned glucose glucose solutions with 3.0 ml of GlucDH reagent and incubating for 30 minutes at room temperature, whereupon OD-g is measured. As a blank, a sample of demineralized water is used instead of glucose.

20 Enzyme test sample

The test samples are diluted with demineralized water so that the final dilution is within the range covered by the standard curve.

Assay

To 500 μΐ enzyme (preheated to 37 ° C), add 500 μΐ maltotriose substrate (preheated to 37 ° C) and place the mixture after careful mixing on a water bath (37 ° C).

After 30 minutes of reaction time, the test tube is removed from the 30 bath and 1000 μΐ stop reagent is added. Then 3.0 ml of GlucDH reagent is added and OD34g is measured after 30 minutes at room temperature. As a blank, a sample containing enzyme, stop reagent, and maltotriose substrate is used. The maltotriose substrate is not added until immediately after the stop reagent.

11 DK 153799 B

enzyme Preparation

A Bacillus strain capable of producing the maltogenic amylase of the invention is usually propagated on a solid substrate before being grown under aerobic conditions in a suitable culture medium. Both media contain assimilable carbon and nitrogen sources in addition to inorganic salts optionally together with growth-promoting nutrients, e.g. yeast extract. Cultivation is typically carried out at 50-55 ° C and at pH 6.5, which is preferably kept constant by automatic aids. The enzyme is secreted into the medium.

The resulting fermentation liquid can be liberated from bacterial cells and bacterial residues together with other solids by e.g. filtration. The supernatant containing the enzyme can be further clarified, e.g. by filtration or centrifugation and then optionally concentrated by, for example, ultrafiltration or in an evaporator under reduced pressure. The resulting concentrate can, if desired, be converted to dryness, e.g. by lyophilization or spray drying. The resulting crude enzyme product typically exhibits an activity in the range of from ca. 500 - 25,000 E per gram.

Purification of the enzyme

The maltogenic amylase of the invention can be purified from a culture liquid from a batch fermentation as follows: 250 liters of the culture liquid with an enzyme activity of 4 U per ml is filtered and the filtrate is ultrafiltered, seed filtered and lyophilized. 193 g of freeze-dried powder is obtained with an activity of 2400 E per g, corresponding to 47% of the original activity.

The powder is dissolved in 15 mM acetate buffer, pH 5.0 and dialyzed against 15 mM acetate buffer pH 5.0 until the conductivity is approx. 1 mS. The dialysate is then transferred to a cation-exchange CM-Sepharose C1-6B® (Pharmacia Fine Chemicals) equilibrated with the same buffer. The amylase passes

12 DK 153799 B

through the column, while 60% of the remaining proteins are retained by the ion exchanger.

The pH of the passage from this column is adjusted to 4.0 with acetic acid and then the eluate is added 5 to a CM-Sepharose C1-6B® column equilibrated with 15 mM acetate buffer pH 4.0. Under these circumstances, the amylase is adsorbed by the ion exchanger. The enzyme is then eluted with pH 4.0 acetate buffer with increasing ionic strength. The enzyme activity in the eluate follows the protein content of a symmetrical peak.

The top material exhibits a single sharp protein band by SDS-polyacrylamide gel electrophoresis. The molecular weight is approx.

70,000 Dalton, pi is approx. 8.5 determined by isoelectric focusing in plate gel. The specific activity is 325 MANE / mg protein of the crystallized, redissolved and freeze-dried product.

Immunological properties

Monospecific antiserum was prepared by immunizing rabbits with purified maltogenic amylase according to the method described by N.H. Axelsen et al., A Manual and Quantitative Immunoelectrophoresis (Oslo 1973), Chapter 23.

Crossed immune electrophoresis according to the same author of crude C599 amylase against this serum gave a single peak of immunoprecipitate, confirming the monospecificity of the antisera.

Enzyme chemical properties

The relationship between the activity of the maltogenic amylase of the invention and the pH and temperature was determined by the method described above using a reaction mixture in which the pH and temperature were adjusted to predetermined values.

BRIEF DESCRIPTION OF THE DRAWINGS Referring again to the drawing, in which FIG. 1 graphically illustrates the relative activity set against temperature (substrate 4% soluble starch, pH 5.5 (0.1 M acetate), 30 minutes reaction time), and

13, DK 153799B

Figure 2 graphically illustrates the relative activity set against pH (temperature 60 ° C, substrate 2% soluble starch, 30 minutes reaction time, MC Ilvaine buffer).

It can be seen from the drawing that the maltogenic amylase 5 has an activity optimum at pH 5.5 of approx. 60 ° C and its pH optimum is in the range of 4.5 - 6.0. More than 50% of the maximum activity is still found at 80 ° C.

To determine the thermostability of the maltogenic enzyme, the enzyme preparation, 1500 U / g, is mixed with 0.1 M acetate-10 buffer (150 mg / ml) at pH 5.5 at a temperature of 50 ° C, 60 ° C and 70, respectively. ° C. The residual amylase activity was determined in the manner described above. The results are shown in the following table.

Table I

v

14 DK 153799B

Temperature Time, min. Percentage residual activity 5 0 100 15 100 50 30 100 60 100 10 -. 15 '15 100 60 30 100 60 100 15 0 100 15 90 70 30 80 60 75 20

After 60 minutes at 70 ° C, 75% of the enzyme activity is preserved. None of the known beta-amylases so far exhibit such good thermostability.

The influence of various reagents on the activity of the maltogenic amylase of the invention is shown in the following Table II.

Table II

15 DK 153799 B

Inhibition of the amylase of the invention 5

Inhibitors Residual activity after 60 min.

at room temperature,% ______ _ 100 '~ 10 PCMB, ImM 92 EDTA, ImM 104

Schardinger alpha-cyclodextrin, 1% 109

Schardinger beta-cyclodextrin, 1% 107

CaCl- ImM 85 lOmM 73 KC1 ImM 95 lOmM 94 20 MgCl- ImM 95 lOmM 93

CoCl9 ImM 91 lOmM 44

FeCl-, ImM 96 25 oMM 74

MnCl ~ ImM 78 lOmM 52

NaCl ImM 98 lOmM 96 30 CuCl ~ ImM 10 lOmM 1

ZnCl-ImM 51 µmM 15

BaCl 'ImM 98 35 1 lOmM 92 A1C1- ImM 98 J lOmM 84

HgCl ~ 0, ImM 3

ImM 0 40 ___________

16 DK 153799 B

Heavy metal ions such as Hg ++ and Ca ++ inhibit the activity of the amylase, whereas neither PCMB, EDTA nor Schardinger cyclodextrins have any effect on the activity.

5

Cloning of the maltogenic amylase gene from Bacillus NCIB 11837

As described in further detail below, the maltogenic amylase gene can be cloned and expressed in B. subtilis as follows.

Chromosomal DNA from Bacillus NCIB 11837 is partially degraded by the restriction enzyme Mbol (Biolabs Cat. No. 147). Fragments ranging from 4 - 12 kb are isolated and joined (with DNA ligase (Biolabs Cat. No. 202)) with E. coli plasmid PACYC184 (Chang et al., J. Bacteriol. 134, 1141-56, 1978), 15 that has been cleaved with restriction enzyme BamH1 (Biolabs Cat. No. 136).

Plasmid pACYCl84 exhibits resistance to both tetracycline and chloramphenicol and contains a single restriction site for the restriction enzyme BamH1. Insertion of DNA into the 20 BamH1 site destroys the ability to exhibit resistance to tetracycline but not to chloramphenicol.

The ligated mixture is transformed into competent E. coli cells. The transformed cells are spread on a suitable selection medium containing soluble starch 25 and chloramphenicol. Chloramphenicol-resistant transformants containing a cloned fragment in the BamH1 site are identified by being tetracycline sensitive. Among these transformants, transformants exhibiting amyla activity are identified by the bright zone around such colonies when the starch-containing agar is exposed to iodine vapor. Such a transformant contains a first recombinant plasmid containing a gene encoding the novel maltogenic amylase.

Plasmid pBD64 (a derivative of Staphylococcus aureus 35 plasmid pUB110 (Gryczan et al., J. Bacteriol. 141, 246-53, 1980)) is used for subcloning in B. subtilis. pBD64 is in

17 DK 153799B

able to replicate in B. subtilis and exhibit resistance to kanamycin and chloramphenicol.

pBD64 is digested with BamH1 and joined to the above-described first recombinant plasmid partially digested with Sau3Al (Biolabs Cat. No. 169). This subcloning is preferably performed by the so-called rescue technique (Gryczan et al., Mol.Gen.Genet. 177, 459-67, 1980) using an amylase negative B. subtilis strain containing plasmid pUB110, a plasmid from Staphylococcus 10 aureus which can be transformed into B. subtilis.

Transformed cells of the pUB110-containing B. subtilis strain are then spread on a suitable selection medium containing soluble starch and chloramphenicol.

An Amy + transformant containing a second recombinant plasmid is identified as above, and the plasmid from this transformant is isolated and transformed into an amylase negative B. subtilis containing no pUB110.

Transformed cells are then spread on a suitable cell. . + txone medium and Amy transformants containing the above-mentioned second recombinant plasmid are identified. This recombinant plasmid can then be transformed into a suitable host, e.g. and B. subtilis.

The invention will be explained in more detail by means of the following examples.

25

Example 1

Preparation of Maltogenic Amylase from Bacillus Strain NCIB 11837 The NCIB 11837 culture was grown at 60 ° C for 1-2 days on the following agar:

Bactodextrose (glucose) (Difco) 2 g Bactoagar (agar-agar) (Difco) 25-Ammonium sulphate 0.5- Trace metals * 3 ml per liter of agar (NH4) 2S04 0.5 g

Water 1000 ml

18 DK 153799 B

* Trace metals: H ^ BO ^ 500 mg

CuS04 40 -

At 100 -

FeCl3 200 - 5 MnS04 400 -

ZnS04 400 -

Na molybdate 200-ion exchanged water 3000 ml 10 Pre-culture

A freeze-dried culture from the above growth medium was propagated in 500 ml shake flasks in 100 ml of the following substrate: 15 Trypton (Difco) 10 g

Glucose 10 -

Potassium Hydrogen Phosphate 3 -

Water 1000 ml The incubation was performed for 1-2 days at 50-55 ° C.

a) Batch fermentation 500 ml shake flasks were supplied with 100 ml of a substrate of the following composition:

Pressure Intravenous Casein (Sheffield) 10 g Yeast Extract (Bacto) 5 -

Potassium Hydrogen Phosphate 3 - 30 Maltodextrin 10 g

Water 1000 ml

The shake flasks were seeded with 1 - 5 ml of the above culture. It was incubated at 50 ° C for 2-3 days.

The resulting culture fluid contained approx. 10 - 20 U / ml.

Propagation from shake flasks to 550 liters of steel tanks can be carried out step by step by increasing the volume of

W DK 153799B

by a factor of 3 - 5 in each step. On a 550 liter scale, the yield was approx. 5 E per ml.

b) Continuous fermentation Continuous fermentation was performed with TMP and SMP substrates of the following composition: TMP substrate: Trypton (Difco) 30 g

Maltodextrin (MC03L, amylase hydrolyzed corn starch) 20 -

Potassium Hydrogen Phosphate 3 -

Pluronic® 0.5-

Water 1000 ml 15 SMP Substrate: Hydrolyzed Soy Protein (Difco) 20 g

Maltodextrin 10 -

Potassium Hydrogen Phosphate 3 -

Pluronic® 0.5-20 Water 1000 ml The fermentation was carried out in an Eschweiler fermentation tank of type S 10 with a working volume of 1 liter.

Ferments were started with 100 ml of the above culture and the substrate dosing was started after 24 hours at 55 ° C. The pH was adjusted to 6.5 with 3% sulfuric acid and the temperature was maintained at 55 ° C.

Aeration: 1 liter / liter substrate / minute 30 Stirring: 1000 rpm

Dilution rate: D = 0.05 hr ^

Under the above conditions, the activity yield was 50 - 75 U per ml on TMP and 40 - 50 E per ml on 35 SMP.

Example 2

20 DK 153799 B

Cloning of the maltogenic amylase gene from Bacillus NCIB 11837 to B. subtilis 5 A. Microorganisms and plasmids

All Bacillus subtilis strains were derivatives of B. subtilis 168, (Spizizen, Proc.Natl.Acad.Sci. 44, 1072 -78, 1958).

DN304: aroI906 sacA321 amy is a Met "1" transferant of QB1133 (Steinmetz et al., Mol. Gen. Genet 148, 281-85, 1976) and has been deposited with the National Collection of Industrial Bacteria, Torry Research Station Aberdeen .

Scotland, March 7, 1984 with reference number NCIB 11957.

DN314 is DN304 pUBD10 and DN311 is DN304 pBD64. The plasmids pUBHO and pBD64 (Gryczan et al., J. Bacteriol. 134, 318 -329, 1978, and Gryczan et al., J. Bacteriol. 141, 246-53, 1980) were isolated from B. subtilis strains BD366 and BD624, respectively. . pUB110 and pBD64 both exhibit resistance to kanamycin and pBD64 also to chloramphenicol. B. subtilis strains 168, QB1133, BD366 and BD624 can be obtained from the Bacillus Genetic Stock Center, Columbus, Ohio, USA (strain filing number, BGSC1A1, 1A289, 1E6, and 1E22, respectively).

All Escherichia coli strains were derivatives of E.

25 coli K-12. E. coli No. 802 with r- m + gal lac (Wood, J. Mol. Biol. 16, 118-33, 1966) and E. coli MC1000: (Arab) 7697 ara D139 lac x 74 gal K gal U StrA (Casadaban et al., J. Mol. Biol., 138, 179-207, 1980) containing plasmid pACYCl84 (Chang et al., J. Bacteriol. 134, 1141-56, 1958) have both been deposited d. March 7, 1984 and received the deposit numbers NCIB 11958 and NCIB 11956 respectively.

B. Transformation of E. coli

A culture of E. coli K-12 strain # 802, stored overnight in LB (10 g Bactotrypton, 5 g Bacto yeast extract and 10 g NaCl per liter of water, pH 7.0) was diluted 100 times in 500 ml of LB and grown at 37 ° C to OD

2i DK 153799 B

= 0.4. The culture was cooled, placed 15 minutes on ice, centrifuged for 15 minutes at 3000 rpm (in a Sorvall GS3 rotor), resuspended in 200 ml cold 0.1 M CaCl2, placed on ice for 20 minutes, centrifuged for 10 minutes at 3000 rpm. , Resuspended in 5 ml of cold 0.1 M CaCl2 and placed on ice for 20 hours. Then, cold glycerol was added to a content of 10% and samples were frozen in liquid nitrogen and stored at -70 ° C. Frozen cells were thawed on ice, DNA was added, the mixture was incubated for 45 minutes on ice, for 2 minutes 10 at 37 ° C and then spread on a suitable selection medium.

C. Transformation of Bacillus subtilis

Competent Bacillus subtilis cells were prepared according to Yasbin et al. (J. Bacteriol. 121, 296-304, 1975). The cells were harvested by centrifugation (7000 rpm, 3 minutes), resuspended in a tenth volume of the supernatant comprising 20% glycerol, frozen in liquid nitrogen and stored at -70 ° C. For transformation, cells 20 were thawed at 42 ° C and mixed with a volume of buffer (Spizizen's minimal medium (Spizizen, Proe. Haiti. Acad. Sci. USA 10, 72-78, 1958) with 0.4% glucose, 0.04 M MgCl2 and 0.002 M EGTA). DNA was added and the mixture was incubated with shaking at 37 ° C for 20 minutes. The cells were then spread on a suitable selection medium.

D. Preparation of plasmid pACYCl84 from E. coli E. coli K-12 strain MC1000 containing plasmid PACYC184 was grown overnight in 250 ml LB, 0.4% glucose 30 and 20 μg / ml chloramphenicol. Cells were harvested by centrifugation and resuspended in 4 ml of buffer 1 (0.025 M Tris HCl, pH = 8.0, 0.01 M EDTA, 0.05 M glucose, 2 mg / ml lysozyme). The suspension was incubated at 0 ° C for 15 minutes and then mixed with 8 ml of buffer 2 (0.2 M NaOH, 1% SDS).

Then 6 ml of buffer 3 (3M Naacetate, pH = 4.8) was added, the mixture was allowed to stand at 0 ° C for 60 minutes and then centrifuged for 20 minutes at 19,000 rpm (about 45,000 g in

22 DK 153799B

a Sorvall SS34 rotor). The supernatant was precipitated with 0.6 volumes of cold isopropanol and resuspended in 1.2 ml of 5TE (0.05 M Tris HCl, pH = 8.0, 0.005 M EDTA) and 20 μΐ boiled RNase A (Boehringer) (2 mg / ml ). Thirty minutes later, solution 5 was placed on top of 4.0 ml of buffer 4 (80 g of CsCl plus 56 ml of 5TE) and 0.1 ml of EtBr (ethidium bromide) in a VTi65 glass. The mixture was centrifuged at 45,000 rpm for 20 hours. The plasmid was then recovered from the glass, dialyzed and extracted as described in section G.

E. Preparation of plasmid pBD64 from Bacillus subtilis DN311

The plasmid was prepared as described for E. coli strains (see section D), but with the following modifications. Growth was made in LB containing 0.01 M potassium phosphate, pH = 7.0 and 6 µg / ml chloramphenicol. After harvest, the cells were incubated at 37 ° C with lysozyme. Buffer 2 was replaced by a mixture of 1 volume of buffer 2 and 3 volumes of buffer 5 (0.2 M glycine, 0.2 M NaCl and 1% SDS). The following 20 steps were the same as in D.

F. Production of small-scale B. subtilis plasmids

The plasmid from 5 ml of B. subtilis in LB (containing 0.01 M phosphate pH = 7.0 and appropriate antibiotics) was prepared as in section E except: buffer volume was reduced four times, 0.5 ml of phenol and 0.5 ml of chloroform was added after buffer 3, after centrifugation at 19,000 rpm the supernatant was precipitated with ethanol, resuspended in 400 μΐ buffer 6 (0.05 M Tris HCl pH = 8.0, 0.1 M Naacetate), the plasmid were re-precipitated, resuspended in 400 μΐ buffer 6, precipitated, washed and resuspended in 100 μΐ TE with 1 μg / ml boiled RNase A (Boehringer).

G. Preparation of Bacillus NCIB 11837 Chromosomal DNA A pellet of frozen cells from ca. 50 ml of culture was resuspended in 1.1 ml of buffer (0.05 M Tris HCl, pH = 7.4, 0.1 M NaCl, 25% sucrose). 100 μΐ lyzosym (25 mg / ml) and

23 DK 153799 B

150 μΐ EDTA (0.5 M, pH = 8.0) was added. The mixture was incubated at 37 ° C for 30 minutes, 2 ml of 0.2% SDS was added followed by the incubation for 30 minutes at 37 ° C. Then 1 g of CsCl and 0.05 ml of EtBr (10 mg / ml) per 0.95 5 ml of mixture were added and the mixture was centrifuged at 45,000 rpm at 15 ° C for 20 hours in a VTi65 rotor (Beckman).

DNA was located under a long-wave UV lamp and removed by puncturing the glass with a syringe. EtBr was extracted with isopropanol and the solution was dialyzed for 2 hours against TEE (0.01 M Tris HCl, pH = 8.0, 0.01 M EDTA). The solution was then adjusted to 8 ml with TEE and extracted twice with phenol and once with chloroform.

DNA was precipitated with 0.1 M NaCl and cold ethanol and dissolved in 1 ml TE (0.01 M Tris HCl, pH = 8.0, 0.001 M EDTA).

The chromosomal DNA (chr DNA) solution was stored at 4 ° C.

H. Partial Cleavage and Fractionation of Chr DNA from Bacillus NCIB 11837 20 60 µg of Chr DNA from Bacillus NCIB 11837 (see section G) was digested into 900 µΐ buffer (0.01 M Tris HCl, pH = 7.4, 0.075 M NaCl , 0.01 M MgCl 2, 0.006 M mercaptoethanol, 100 μg / ml gelatin) with 10 units of Mbol (Biolabs Cat. No. 147) at 37 ° C. After 10 minutes and 40 minutes, samples of 25 450 μΐ were extracted with phenol and precipitated with 0.1 M NaCl and cold ethanol. Each sample was dissolved in 100 μΐ TEE. 100 μΐ DNA was applied per 12 ml sucrose gradient (Maniatis T. et al., Cell 15, 687-701, 1978) and centrifuged at 27,000 rpm for 22 hours at 20 ° C in a SW41 rotor (Beckman).

30 0.5 ml fractions were drained after puncturing the bottom of the glass and precipitated with cold ethanol. DNA was dissolved in 50 μΐ TE and the size of DNA in each fraction was determined on a 0.7% agarose gel stained with 0.5 μg / ml EtBr. The fractions containing DNA from ca. 4 - 12 kb, were collected, extracted with phenol, precipitated with ethanol and dissolved in TE.

DK 153799B

24 I. Isolation of recombinant plasmid containing an amylase gene 4 μ9 pACYCl84 (see section D) was digested with 8 U BamHl (Biolabs Cat. No. 136) for 1 hour at 37 ° C in 50 μΐ 5 buffer (0.006 M Tris HCl , pH = 8.0, 0.15 M NaCl, 0.006 M MgCl 2, 0.006 M mercaptoethanol, 100 μg / ml gelatin). The linear DNA was treated with calf intestinal alkaline phosphatase (Sigma P 4502), (Goodman et al. Methods in Enzymology, 68, 75-90, 1979). pACYC184 treated with 0.4 µg BamHl and 10 phosphatase were then joined with ca. 1 µg Bacillus NCIB 11837 chr DNA from sucrose gradients with approx. 100 U DNA ligase (Biolabs Cat. No. 202) for 4 hours at 16 ° C in 100 μΐ buffer (0.066 M Tris HCl, pH = 7.5, 0.01 M MgCC * 25 μg / ml gelatin, 0.001 M ATP, 0.01 M DTT). The joined DNA was transformed into a competent E. coli strain # 802 as described in section B.

The cells were spread on freshly poured agar plates consisting of two layers: The bottom layer was 10 ml LB agar with 0.5% soluble starch and 60 μg / ml chloramphenicol and the top layer 20 was 25 ml of the same agar but without chloramphenicol. After overnight incubation at 37 ° C, ca. 1200 chloramphenicol resistant transformants. About 600 of these were tetracycline sensitive, indicating that plasmid from these transformants contained a cloned fragment at the 25 BamH1 site. Among these 600 colonies (representing a gene bank of Bacillus NCIB 11837), a strain DN400 exhibited amylase activity, as evidenced by the pale zone around this colony following exposure of the starch-containing agar to iodine vapors. Strain DN400 was found to contain a plasmid, called pDN400, of ca. 14,000 base pairs including the amylase coding gene.

J. Subcloning of pDN400 in Bacillus subtilis

Ca. 1 µg of pDN400 (constructed as described in Section I and prepared from E. coli strain DN400 as described for pACYC184 in section D) was digested at 37 ° C in 100 µg buffer (0.006 M Tris HCl, pH = 7, 5, 0.05 M NaCl,

DK 153799 B

0.005 M magnesium chloride, 100 μg / ml gelatin) with 0.06 U Sau3Al (Biolabs Cat no. 169). Samples of 25 μΐ were transferred to phenol after 10, 20, 30 and 40 minutes, and pheonically extracted and precipitated with 0.1 M NaCl and cold ethanol.

Half of the DNA was joined (see section H) with 0.4 μg of plasmid pBD64 (see section E) that had been cleaved with BamH1 (as described for pACYCl84 in section I).

Bacillus subtilis strain DN314 was then transformed (see section C) with the ligation mixture and spread on LB agar plates containing 0.01 M potassium phosphate pH = 7.0, 0.5% soluble starch and 6 μg / ml chloramphenicol. Among the approx. 6000 transformants 1 -f were identified

Amy (starch degrading according to the iodine sample).

Plasmid from this strain was isolated (as described in section F) and transformed into Bacillus subtilis strain DN304 (as described in section C). An Amy + transformant strain DN463 containing plasmid pDN452 was isolated. As can be seen from the following Example 3, culturing DN463 gives increased yields of the novel thermostable amylase of the present invention.

Example 3 Preparation of thermostable maltogenic amylase from Bacillus subtilis strain DN463

A culture of the above cloned microorganism DN463 was seeded in 500 ml shake flasks on 100 ml of the following substrate:

Liquid starch 12.5% w / v (tap water)

Soybean 7.5%

Na2HPO4 1% 35 To the above substrate, 5 µg / ml chloramphenicol was added prior to inoculation. The incubation was performed at 30 ° C for 3 - 4 days. The culture liquid was centrifuged in 20

26 DK 153799 B

minutes at 6000 g. The centrifugate containing ca. 200 U / ml was used directly in the following example.

Example 4

Substrates for saccharification were prepared by redissolving 7DE spray-dried maltodextrin in deionized water to give ca. 30% dry matter. The assay experiments were performed in standard laboratory batch reactors of 500 ml.

Samples of these substrate were heated to 60 ° C and the pH was adjusted to 5.5, to which 50 amylase units / g of dry matter was added. After 72 hours at 60 ° C the content of glucose, maltose and maltotriose in the syrup was as follows: glucose: 5% maltose: 67% maltotriose: 0%

Sugar with 25 beta-amylase units / g dry matter 20 of Biozym Mil (soybean betamylase, 20,000 beta-amylase / g) under the same conditions gave a syrup containing 0.3% glucose, 61% maltose and 7% maltotriose.

Claims (8)

27 DK 153799B 5 1. Maltogenic amylase, characterized in that it has a temperature optimum at pH 5.5 of approx. 60 ° C, a 10 pH optimum at 60 ° C in the range of 4.5 to 6 and a residual activity after 60 minutes at 70 ° C of at least 75%, cleaves maltotriose quantitatively in equimolar amounts of maltose and glucose, and can be prepared by cultivation of Bacillus NCIB 11837 in a suitable nutrient medium containing 15 carbon and nitrogen sources and inorganic salts, and subsequent isolation of the amylase formed with the above-mentioned culture liquid properties. Process for the preparation of the maltogenic amylase according to claim 1, characterized in that a Bacillus strain NCIB 11837 or variants or mutants thereof having substantially the same properties as it is grown in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts, on which the maltogenic amylase is recovered from the culture liquid. Process for producing the maltogenic amylase according to claim 1, characterized in that a transformed host strain of the genus Bacillus to which the gene encoding the thermostable maltogenic amylase according to claim 1 is transferred from Bacillus strain NCIB 11837 is grown. in a suitable nutrient medium containing carbon and nitrogen sources and inorganic salts, upon which the maltogenic amylase is recovered from the culture liquid. The method according to claim 3, characterized in that the host strain belongs to the species Bacillus subtilis. 28 DK 153799B Method according to claim 4, characterized in that the host strain is Bacillus subtilis 168 or a variant or mutant thereof. Plasmid for use in a Bacillus strain in the preparation of the maltogenic amylase according to claim 1, characterized in that it is a recombinant plasmid containing the gene transferred from Bacillus strain NCIB 11837 encoding the thermostable maltogenic amylase according to claim 1 , as well as a nucleotide sequence ensuring replication in a Bacillus subtilis host. Plasmid according to claim 6, characterized in that the nucleotide sequence which ensures replication in a Bacillus subtilis host is plasmid pBD64 or pUB110 or derivatives thereof having essentially the same properties. Plasmid according to claim 6, characterized in that it is plasmid pDN452 with the restriction endonuclease cleavage map shown in FIG. 3.