WO1986001832A1

WO1986001832A1 - THERMOSTABLE beta-AMYLASE

Info

Publication number: WO1986001832A1
Application number: PCT/US1985/001774
Authority: WO
Inventors: Joseph Gregory Zeikus; Hyung-Hwan Hyun
Original assignee: Michigan Biotechnology Institute
Priority date: 1984-09-18
Filing date: 1985-09-13
Publication date: 1986-03-27
Also published as: DK158522B; JPS62500422A; US4647538A; FI861960A0; JPH0570428B2; EP0195802B1; EP0195802A4; DK158522C; EP0195802A1; DK226486D0; DE3582729D1; FI861960A; DK226486A

Abstract

A thermostable and thermoactive, extracellular $g(b)-amylase is produced by $i(Clostridium thermosulfurogenes). Methods of producing the $g(b)-amylase and using it to convert starch to maltose are also disclosed.

Description

THERMOSTABLE β-AMYLASE

Technical Field The present invention relates to enzymes. More particularly, it relates to a thermostable β-Amylase.

Background Art

The food and beverage industries principally^'employ the enzyme, β-amylase (EC 3.2.1.41); to convert starch into maltose solutions. β-Amylase hydrolyzes the α-1,4-glucosidic linkages in an exo-fashion from the non-reducing end of starch-type subtrates, and pro¬ duces both maltose in the β-anomeric configuration and β-limit dextrins from starch. The β-amylase employed in most industrial applications is obtained from plants. Attempts have been made to obtain a more active, thermostable and extracellular enzyme from microorgan¬ isms. However, the known microbial β-amylases are not active or thermostable enough to substitute for the plant enzyme. Therefore, the plant β-amylases con¬ tinue to be used for maltose production although they are expensive and relatively unstable.

Only a few enzymes of current industrial interest have been isolated and characterized from thermoanaerobes. The thermostable and active enzymes characterized to date from thermoanaerobes, include an endoglucanase of Clo- stridium thermoce1lum, an alcohol dehydrogenase of

Thermoanaerobium brockii and C. thermohydrosulfuricum and a polygalacturonate hydrolyase of ^_ thermosulfurogenes. It would obviously be desirable to have a more thermo¬ stable β-amylase than those currently available.

Disclosure of the Invention The primary objects of the present invention are to disclose a thermostable β-amylase and a method of producing that enzyme.

We have discovered that the organism Clostridium thermosulfurogenes produces a unique thermostable and thermoactive β-amylase. The enzyme is extracellular and has an optimum temperature for activity at 75°C. It is stable up to 70°C in the absence of substrate and up to 80°C in the presence of 5% starch. The optimum activ¬ ity of the enzyme is at about pH 5.5 to about 6.0. The β-amylase of the present invention appears to be particularly suited for use in industrial applications. - The β-amylase is produced by culturing C. thermosulfurogenes anaerobically on a substrate of starch in a nutrient medium containing essential vitamins, minerals and growth factors until substantial enzymatic activity is detectable and then isolating the β-amylase. The β-amylase is produced extracellularly and there¬ fore is easily separated from the cells and isolated in purified form substantially free of interfering enzymatic activity. In the preferred method of the invention, the β-amylase is produced by growing a pure culture of C. thermosulfurogenes anaerobically on a substrate of starch in a medium containing essential vitamins, minerals and growth factors. The β-amylase is obtained from the culture supernatant substantially free of interfering enzymatic activity.

A detailed description of the preparation and charac¬ terization of the enzyme follows: Organism and cultivation. C. thermosulfurogenes strain 4B was isolated from Octopus Spring algal-bacterial mat in Yellowstone National Park and it was deposited in both the Deutsche Sammlung von Mikroorganismen (DSM 2229) and the American Type Culture Collection, Rockville, MD (ATCC 33743). Stringent anaerobic culture techniques were employed for medium preparation and cultivation. The organism was routinely grown at 60°C in 26 ml anaero¬ bic pressure tubes containing 10 ml of TYE medium (24) with 0.5% glucose or soluble starch and ₂-C0₂ (95:5) gas headspace. Culture medium was autoclaved for 45 min to ensure destruction of the extremely heat resistant spores of thermoanaerobes.

For determination of starch hydrolysis reaction on petri dishes, the organism was streaked in an anaerobic chamber onto plates of TYE medium that contained 1.0% soluble starch and 3.0% purified agar. The plates were placed into an anoxic paint can under nitrogen and incu¬ bated at 60°C for 4 days, The plates were removed from the paint can and flooded with iodine solution (1% I₂, and 2% KI in H_?0) and hydrolysis zones were visually observed.

Preparation of enzyme. Washed cells and culture supernatant as the amylase sources were prepared by cultivating the strain in serum vials that contained 50 ml of TYE medium with 0.5% soluble starch. Stationary phase cultures were then centrifuged at 12,000 x g for 10 min. Cell suspensions were prepared by washing twice with 20 mM sodium acetate buffer (pH 6.0) and then suspending the cells in buffer. Large-scale growth for preparation of concentrated extra-cellular amylase was performed in a 14-liter fermentor that contained 10 liters of LPBB medium with 1.0% maltose and 0.02% yeast extract under N -CO (95:5). Culture supernatant was obtained from cultures grown at 60°C until the late exponential phase, using a continuous-flow centrifuge system. The superna¬ tant (10 liters) was concentrated first by precipitating proteins with 2 volumes of cold ethanol (20 liters), then filtering through charcoal powder to collect the precipi¬ tate, then the precipitate was suspended in 300 ml of 20 mM sodium acetate butter (pH 6.0), and ammonium sulfate was added to 70%. The precipitated proteins were centri¬ fuged at 15,000 x g and then suspended in 100 ml of the same buffer as above. This suspension was centrifuged again to remove insoluble materials and then precipitated with 2 volumes of cold acetone. This last precipitate was suspended into 100 ml of the same buffer and, then 100 ml of the enzyme solution was dialyzed against 4 liters of double distilled water for 24 h.

The specific activity of the concentrated supernatant amylase solution was 60 units per mg-protein. Cells were mass-cultured and harvested for prepara¬ tion of cell extracts by the same procedures as described above, except TYE medium supplemented with 0.5% soluble starch was employed. Cell extracts were prepared aerobi- cally by suspending 3 g of wet cell paste in 12.5 ml of 20 mM sodium acetate buffer (pH 6.0). This suspension was treated at 37°C for 1 hr with 5 mg of lysozyme from chicken egg white and disrupted by passage through a

2

French pressure cell at 20,000 lb/in . The supernatant was collected by centrifugation at 30,000 x g for 30 min at 4°C. Protein concentration was determined.

Enzyme assays. Amylase or β-amylase activity was assayed by measurement of reducing sugar released from reaction on starch. The reaction mixtures (1 ml) contained 10% soluble starch, 1 ml of 0.5 M sodium acetate buffer (pH 6.0); and 3 ml enzyme solution which was appropriately diluted with water. The reaction was stopped by cooling on ice after 30 min incubation at 60°C.

Reducing power was measured by the dinitrosalicylic acid method. For assay of pullulanase activity, the reaction mixture consisted of 0.5 ml of 2% pullulan in 0.2 M sodium acetate buffer (pH 6.0) and 0.5 ml enzyme solution. After incubation at 60°C for 30 min, the reaction was stopped by cooling on ice and by adding 4 ml of 3,5-dinitrosalicylic acid.

One unit of amylase, β-amylase or pullulanase is defined as the amount of enzyme that produced 1 ymol of reducing sugar as a glucose standard per min under the above conditions.

Glucoamylase activity was assayed by incubating 1 ml of reaction mixture that contained 1% soluble starch, 0.1 M sodium acetate buffer (pH 4.8) and the appropriately diluted enzyme solution at 60°C for 30 min. The reaction was stopped immediately by cooling on ice and then by boiling in a steam bath for 10 min. These reaction mixtures were centrifuged to remove the insoluble precipi tates, and then released glucose was analyzed by the hexokinase and glucose-6-phosphate dehydrogenase method. A unit of glucoamylase is defined as the amount of enzyme that produced one μmol of glucose per min under the assay conditions. Qualitative and quantitative analysis of starch- hydrolysis products. Enzyme (12 units) was placed into 20 ml of 0.1 M sodium acetate butter (pH 6) containing 1%o soluble starch and incubated at 65°C. Samples were with¬ drawn at intervals and reducing sugar and glucose content were analyzed as described above,

Maltose was identified in the starch hydrolysates by high pressure liquid chromatography (HPLC). The separa¬ tion system consisted of a liquid chromatograph equipped with a data station, a refractive index detector and a oligosaccharide analysis column (300 x 7.8 mm) fitted with a microguard pre-column (40 x 4.6 mm). Starch hydrolysate samples were centrifuged at 5,000 x g and loaded onto the column (50 1).

Determination of reducing value and blue value curve A reaction mixture used, enzyme and a 0.25% a ylose solu¬ tion in 0.1 M sodium acetate buffer (pH 6.0) and were incubated at 65°C. Samples (0.5 ml) were withdrawn at 5 min intervals and were placed in tubes that contained 2 ml of 0.1 M HC1 for determination of blue value. After mixing, 0.5 ml samples were withdrawn and added to 5 ml of iodine solution (0.05 g of iodine and 0.5 g of potas- sium iodide per liter). Then, absorbance was measured at 620 nm in a spectrophotometer blanked against water- iodine. The blue value was calculated by dividing the absorbance of the digest with that of the substrate-iodine blank and multiplying by 100. Another 0.5 ml sample of the reaction mixture was withdrawn for determinations of reducing value. The reducing power was analyzed by the Nelson colorimetric copper method. Total carbohydrate was assayed by the phenol-sulfuric acid method.

Optical rotation study. A reaction mixture (5 ml) consisting of 1% starch solution in 0.1 M sodium acetate buffer (pH 6.0) and enzyme was added to a 10 cm cell. Optical rotation was measured at room temperature in a polarimeter using the sodium line. When the optimal rotation became approximately constant, about 20 mg of solid sodium carbonate was added and the mutarotation of the mixture was measured.

Enzyme characterization. The enzyme inhibition and kinetic studies were performed under the standard assay conditions described above. The stability studies em- ployed enzyme solution (about 40 units/ml) in 0.1 M sodium acetate buffer (pH 6.0) under the described condi¬ tions.

Location and type of amylase activities. In prelimi¬ nary experiments, large reddish color zones against a blue background were detected around colonies on starch agar plates that were stained with iodine solution. This result implied the C__;_ thermosulfurogenes produces amylases that are not capable of completely hydrolyzing starch.

Table 1 summarizes the results of experiments demon- strating the cellular location and types of different amylase activities in C^_ thermosulfurogenes. The organism produced extracellular amylase, whose activity will be classifed below as β-amylase, but not extracellular pullulanase or glucoamylase during growth. In addition, cell bound glucoamylase and amylase but not pullulanase activity was detected. Neither amylase nor glucoamylase activities were inhibited by aerobic assay conditions.

Table 1. Amylolytic Enzyme Activities and Cellular Location in C _ thermosulfurogenes

Location -- Activity

Cell-Free

Super¬ Washed Extracts

Assay natant Cells (U/mg

Enzyme Condition (U/ml) (U/ml) Protein)

Amylase Aerobic 5.5 0.07 0.26

Anaerobic 5.5 0.07 0.26

Pullulanase Aerobic ^'o.oo 0.00 0.00

Anaerobic 0.00 0.00 0.00

Glucoamylase Aerobic 0.00 0.02 0.07

Anaerobic 0.00 0.02 0.07

C. thermosulfurogenes was grown to the late exponential phase on TYE medium that contained 0.5% soluble starch as carbon source. Anaerobic assay conditions were estab¬ lished via addition of dithiothreitol (2 mM) and gassing with _? gas in preparation of enzyme solution and all reactants.

HPLC analysis of starch hydrolysates formed by extracellular amylase revealed that only maltose was produced as a detectable product in addition to high molecular weight oligosaccharides (i.e., limit-dextrins) . In spite of prolonged incubation, only reducing sugars but not glucose were detected by quantatitive analysis of starch hydrolysates. Enzymatic hydrolysis of maltotriose and maltotetraose hydrolysis were also studied. Only maltose (8 mg/ml was) produced as a result of incubating 1% maltotetroase in 0.1 M sodium acetate buffer with 12 units of the enzyme at 65°C for 3 hrs. Under these same conditions, enzymatic hydrolysis of maltotriose (1%) yielded both glucose (0.4 mg/ml) and maltose.

The release of reducing power and reduction of iodine staining 'capacity by action of the enzyme on amylase was determined. The slow decrease of blue value against reducing value in comparison with that of the α-amylase control indicates that the enzyme is an exo-acting amylase. From the mutarotation study, which employed α-amylase as control, the upward shift of optical rotation upon the addition of base to the starch hydrolysate formed by the C_^ thermosulfurogenes enzyme indicates that the hydrolysis products have a β-anomeric configuration. The data above provided evidence that the extracellular amylase activity isolated from C_^ thermo¬ sulfurogenes was a β-amylase.

Physicochemical properties of β-amylase. The effect of temperature on the activity of β-amylase was determined. The optimum temperature for the enzyme activity was 75°C, and 85% of maximal activity was mea¬ sured at 80°C. When Arhennious plots of specific activ¬ ity versus 1/temperatures were determined (data not shown), Q-,₀. values of 1.7 were calculated for β-amylase. The effect of temperature on the stability of the β-amylase in the absence of substrate also was deter¬ mined. The enzyme was completely stable up to 70°C and 83% of the original activity was retained after treatment at 75°C for 1 hr. The enzyme was almost completely. destroyed within 20 min at 80°C in the absence of starch, but addition of starch to the enzyme solution greatly enhanced the heat-stability in proportion to the concen¬ tration of starch. The enzyme was entirely stable at 80°C in the presence of 5% starch.

The effect of pH on β-amylase activity and stability was determined. The optimum activity was o served at pH 5.5 to 6.0, and the activity dropped gradually at more alkaline or acid pH values. Higher than 43% of maximal activity was detected between pH values of 2.5 and 9.0. The enzyme was stable in the pH range of 3.5 to 6.5, but unstable below pH 3.0 and above pH 7.5.

The effects of sulfhydryl reagents, metal ions and Schardinger dextrins (α and β-cyclodextrin) on β-amylase activity were examined. β-Amylase

++ ++ activity was strongly inhibited by Cu , Hg and p-chloromercuribenzoate (PCMB), but weakly inhibited by hydrogen peroxide, 5,5'- dithiobis-2-nitrobenzoic acid, N-ethylmaleimide (NEM), iodocetic acid and Zn . The inhibitions could be prevented and restored by the addi- tion of dithiothreitol (10 mM) . The Schardinger dex¬ trins, unexpectedly, did not inhibit enzyme activity and were riot hydrolyzed by the β-amylase. Under the assay condition used, the enzyme activity was not affect¬ ed by the addition of 1 to 30 mM of calcium ion.

Table 2. Effects of Sulfhydryl Reagents, Metal Ions and

Schardinger Dextrins on -Amylase Activity

Concentration of Reagent Added % Activity

(mM) Remaining

None 100

Hydrogen peroxidase - 1 98

5 86 5,5'-difhiobis-2-nitrobenzoic acid (DTNB) - 0.2 96 N-ethylmaleimide (NEM) - 1 93

5 54

Iodocaetate (IA) - 1 100

5 52 p-chloromercuribenzoic acid - 0.02 0.2

(pCMB) - 0.1 0.0

CuCl₂ - 1 1.8

HgCl₂ - 1 0.0 2nCl₂ - 1 93

-10 73

CaCl₂ - 5 100

-30 100 α-Cylcodextrin - 10 100 β-Cyclodextrin - 10 100

el 100% activity corresponds to 24.4 units/mg protein under the standard assay conditions.

The effects of ethanol on the enzyme activity and stability were also performed, β-Amylase activity was not notably affected in the presence of 3% (v/v) ethanol, but it dropped slowly in proportion to the concentration of ethanol at higher than 3% (v/v) . In the presence of 10% (v/v) ethanol, 65% of maximal activity was observed. This effect is attributed to the precipitation of starch substrate by ethanol because β-amalyse was completely stable at 65°C for 1 hr in presence of 10% (v/v) ethanol prior to activity analysis.

Kinetic properties of β-amylase. The apparent kinetic constants were determined for β-amylase on starch versus amylose by determination of production formation rate versus time curves. It was demonstrated that the dependence of the rate of enzymatic hydrolysis on the substrate concentration followed Michaelis-Menten kinetics and linear relationships of 1/V versus 1/[S] were obtained. The apparent [S]_{0 ςv} and Vmax as determined from the double reciprocal for starch versus amylose were: 1 mg/ml and 60 U/mg protein verus 1.67 mg/ml and 30 U/mg protein, respectively. *

In general, the foregoing data establish that C. thermosulfurogenes produces both cell bound glucoamylase and extracellular β-amylase activities. The β-amylase is produced in high yield as a primary metabolite during grown on starch. The β-amylase possessed a high thermal stability which may make it of industrial utility in brewing and starch processing. The extracellular amylase was classifed as a β-amylase based on certain criteria. The action of the enzyme on starch yielded only maltose as a detectable product besides high molecular weight limit dextrin, but not glucose or other low molecular maltosaccharides. Maltotetraose hydrolysis yielded only maltose, and malto- triose yielded glucose and maltose. The enzyme was inactive against pullulan, sucrose, cellobiose, trehalose, Schardinger dextrins and maltose. A large^* release of reducing power with relatively little reduction of the iodine-staining capacity on hydrolysis of amylose and the upward mutarotation of starch digest upon the addition of alkali indicated that the enzyme acts in an exo-fashion and produced maltose with the β-anomeric configuration. In these respects, the extracellular amylase of C__;_ thermo- sulfurogenes was similar in the action patterns of plant and other microbial β-amylases.

The β-amylase has industrial applications in the production of various sugar solutions or alcohols from starch because of its novel features. The enzyme is extracellular, a primary metabolite and it is active and stable at high temperature. The enzyme displays a high maximum temperature (75°C) for enzyme activity and is stable up to 80°C in the presence of substrate. The β-amylase is also active and stable at a wide range of pH. It has an optimal activity (pH 5.5-6.0), and stability (pH 3.5-6.5) in the acidic range, which distin¬ guishes it from most other β-amylases that display optimal activity and stability around a neutral pH.- The β-amylase also may have novel applications for produc¬ tion of maltose syrups in conjunction with a pullulanase which is active at nearly the same pH and temperature range. β-Amylases from higher plant and microorganisms are sulfhydryl enzymes and are inactivated by sulfhydryl reagents or oxidation. The present data indicates that C. thermosulfurogenes β-amylase is also inactivated by sulfhydryl reagents (i.e., PCMB, NEM). However, this inhibition could be prevented and reversed by the addition of reducing agent (dithiothreitol). Also, metal ions (e.g., Cu , Hg- and Fe ) inhibited the β-amylase. However, Schardinger dextrins which notably are competi- tive inhibitors of other β-amylases (5, 6), did not inhibit the. β-amylase of C^ thermosulfurogenes.

The β-amylase activity and stability was little affected by ethanol although growth of this species was very sensitive (i.e., no growth in the presence of 2% ethanol).

It will be appreciated by those skilled in the art that β-amylase productivity may be enhanced through mutation of the organism or by genetic recombination techniques. Therefore, the scope of the invention should not be limited to the specific strain of the organism described above because any organism capable of producing a thermostable β-amylase similar to that produced by C. thermosulfurogenes having the identifying characteris¬ tics of ATCC 33743 can be used. In view of the foregoing, it is to be understood that the invention is not to be limited by any of the specific embodiments described but only by the claims which follow:

Claims

The embodiments of the invention in which an exclu¬ sive property or privilege is claimed are defined as follows:

1. A thermostable β-amylase, substantially free of interfering saccharidase enzymatic activity, which is obtain from the the microorganism Clostridium thermosulfurogenes.

2. The use of Clostridium thermosulfurogenes for the production of a thermostable β-amylase.

3. The use of the β-amylase of claim 1 to prepare maltose from starch.

4. The use of the β-amylase of claim 1 in the preparation of alcohol from starch.

5. A thermostable β-amlyase having an optimum temperature for activity of about 75°C; said β-amylase being stable up to 70°C in the absence of substrate and up to 80°C in the presence of substrate; said enzyme having optimum activity at about pH 5.5 to about 6.0 and being stable from about pH 3.5 to about 6.5.

6. A method for producing a thermostable β-amylase which comprise anaerobically culturing a β-amylase producing Clostridium thermosulfurogenes in a nutrient medium until substantial enzymatic activity is detectable and thereafter isolatin the thermostable β-amylase.

7. A thermostable β-amylase having the follow¬ ing physiochemical properties:

(1) Reactivity: It produces only maltose and limit dextrins when transforming starch hydrolysates; (2) Substrate specificity: It produces only maltose from starch or maltotetraose and glucose and maltose from maltotriose;

(3) Optimum pH value: About 5.5 to about 6.0;

(4) pH stability: Stable from about pH 3.5 to about 6.5;

(5) Optimum temperature: 75°C, 85% of maximal activity at 80°C;

(6) Temperature stability: It is stable up to 70°C in the absence of substrate and up to 80°C in the presence of 5% starch; and

(7) Influence of inhibitors: β-amylase activity inhibited by metal ions (Cu . and Hg . ) and p-chlo_.omer- curibenzoate but not by calcium Schandingder dextrins or ethanol.

TFK27:B