Appl Microbiol Biotechnol (1990) 33:340-344
Applied Microbiology Biotechnology © Springer-Verlag 1990
Physiological and enzymatic characterization of a novel pullulan-degrading thermophilic Bacillus strain 3183 Gwo-Jenn Shen*, Kailash C. Srivastava, Badal C. Saha, and J. Gregory Zeikus Michigan BiotechnologyInstitute, 3900 Collins Road, Lansing, MI 48910, USA Received 10 October 1989/Accepted 6 February 1990
Summary. A new thermophilic Bacillus strain 3183 (ATCC 49341) was isolated from hot-spring sediments. The organism grew on pullulan as a carbon source and showed optimum pH and temperature at pH 5.5 and 62 ° C, respectively, for growth. The strain reduced nitrate to nitrite both aerobically and anaerobically. It produced extracellular thermostable pullulanase and saccharidase activities which degraded pullulan and starch into maltotriose, maltose, and glucose. Medium growth conditions for pullulanase production were optimized. The optimum pH and temperature for pullulanase activity were at pH 6.0 and 75 ° C, respectively. The enzyme was stable at pH 5.5-7.0 and temperature up to 70 ° C in the absence of substrate. The K,, for pullulan at pH 6.0 and 75°C was 0.4 mg/ml. The pullulanase activity was stimulated and stabilized by Ca 2+. It was inhibited by ethylenediaminetetraacetate (EDTA), betaand gamma-cyclodextrins but not by alpha-cyclodextrin and reagents that inhibit essential enzyme SHgroups.
Introduction Pullulanase (pullulan 6-glucanohydrolase, EC 3.2.1.41) is an enzyme which is capable of hydrolyzing the alpha-l,6-glucosidic linkages of pullulan and produces maltotriose as the end product (Abdullah and French 1970). As this enzyme can debranch amylopectin, it is used to increase the efficiency of starch saccharification. Pullulanase can be used with glucoamylase for the production of high glucose syrups or with beta-amylase for high maltose syrup production, where it functions to increase yield and decrease reaction times (Norman 1979; Saha and Zeikus 1987). Importance has been placed on the pullulanase property of environmental * Present address: The Research Institute of Scripps Clinic, La Jolla, CA 92037, USA Offprint requests to: B. C. Saha
compatibility with glucoamylase or beta-amylase. Many mesophiles can produce pullulanase (Fogarty 1983) but these pullulanases lack high thermostability. Recently, several thermostable pullulanases have been discovered from extreme thermophiles such as Clostridium thermohydrosulfuricum (Hyun and Zeikus 1985; Melasniemi 1987), Thermoanaerobium brockii (Coleman et al. 1987), Thermoanaerobium Tok6-B1 (Plant et al. 1987), a new Clostridium isolate (Antranikian et al. 1987), Thermus aquaticus YT-1 (Plant et al. 1986) and Thermus sp. (Nakamura et al. 1987). Some novel pullulanase preparations display the unique property of not only degrading alpha-l,6-glucosidic linkages in pullulan but also degrading the alpha-l,4 linkages of soluble starch, amylose and amylopectin (Coleman et al. 1987; Plant et al. 1987; Saha and Zeikus 1989). Although Suzuki and Chishiro (1983) and Imanaka and Kuriki (1989) reported that Bacillus stearothermophilus strains produce pullulan hydrolases, the enzymes were not able to cleave alpha-l,6-1inkages of pullulan and were not classified as true pullulanases. We have isolated a pullulan-degrading thermophilic Bacillus species, strain 3183 (deposited with American Type Culture Collection, Rockville, Md, USA; ATCC 49341), which is able to grow at a pH lower than that of B. stearotherrnophilus and produces an extracellular thermostable pullulanase. In this paper we report on the general physiological properties of this strain, and the general biochemical properties of its pullulanase activity.
Materials and methods Screenin#, isolation and identification of microbial strains. The se-
diment samples for screening were obtained from hot-spring sources in Yellowstone National Park. The organism was enriched in a screening medium (pH 4.0) containing (g/l): NH4C1, 1.0; CaC12-2H20, 0.05; MgSO4.7HzO, 0.5; NaC1, 1.0; NaH2PO4, 1.0; vitamin solution (10 ml); trace mineral solution (10 ml); yeast extract, 0.1; and pullulan, 2.5. The enrichment was performed with shaking (200 rpm) at 60°C in rubber-bung- and crimp-topsealed test tubes containing 10 ml medium and 1.0 ml sediment
341 sample. The enrichments that showed growth were then plated onto agar plates (2.3% agar) in screening medium. Subsequently, single isolated colonies were transferred into test tubes containing 10 ml of the same medium. These procedures were repeated several times to ensure the purity of the culture.
Table 1. Morphological and physiological characteristics of Bacillus strain 3183 (ATCC 49341) Rods (2.7 to 3.0 txM by 0.7 to 1.0 I~M), occurring as single cells, pairs or chains of 3-4 cells, non-motile, Gram positive, bacillary body swollen, terminal, oval spores
Morphology:
Measurement of cel! growth and enzyme activities. All experiments on cell growth and enzyme production were performed in large sealed serum vials (158 ml total volume, Wheaton, Millville, NJ, USA) containing 20 ml screening medium and the vials were incubated at 60° C with shaking. Experiments on the optimum growth pH and optimum enzyme production pH were performed in a 1-1 fermentor (Multigen, New Brunswick, Scientific Co., Edison, N J, USA) with 350-ml working volume at 60° C. The pH was controlled with 2 N NaOH solution. Growth was monitored by measuring optical density at 660 nm. Standard assay of pullulanase activity employed a reaction mixture (1 ml) containing pullulan (1%), acetate buffer (50 raM, pH 6.0, with 5 mM CaCI2) and an enzyme source (as indicated) with incubation at 60° C for 30 rain. The reducing sugars released from pullulan was determined by the dinitrosalicylic acid (DNS) method with glucose as the standard (Bernfeld 1955). Amylasesaccharidase activity was measured by the same procedure, except that starch was used as the substrate. One unit (U) of enzyme activity is defined as the quantity producing 1 ~tmol reducing sugar (as glucose equivalent) per minute under the above conditions.
Prepartation of the enzyme. Enzyme sources used were either cellfree culture broth or partially purified extracellular enzyme preparation. Cultures (1 1) obtained during the late exponential growth phase were centrifuged (15000g, 15 rnin) to remove cells. Solid (NH4)2SO4 was added to the supernatant solution to give 80% saturation. The precipitate formed was collected by centrifugation at 20000 g for 30 rain and dissolved in acetate buffer (50 mM, pH 6.0) with 5 mM CaClz. The enzyme solution was then dialyzed in the same buffer (41) for 48 h at 4° C. This crude enzyme preparation (specific activity, 2.74 U/mg protein) was used for the characterization of pullulanase. Analysis of hydrolysis products. An HPLC system was employed to analyze the enzymatic hydrolyzate products of starch and pullulan. For analysis of oligosaccharides, a multisolvent delivery system (600, Waters Chromatography Division, Millipore Corporation, Milford, Mass, USA) equipped with an autosampler (712, WISP, Waters), a refractive index detector (410 Differential refractometer, Waters), and an oligosaccharide analysis column (Aminex HPX-42A, Bio-Rad Laboratories, Richmond, Calif, USA) with a de-ashing system (Bio-Rad) was used. The column was maintained at 85° C and elution of sugars was performed with distilled water. The distinction between maltotriose and panose or isopanose was made by using a Supelcosil LC-NH2 (Supelgo, Bellefonte, Pa, USA) column, and the acetonitrile and water (75:25) solvent system as described by Nikolov et al. (1985).
Other methods. Protein was estimated according to Lowry et al. (1951) with bovine serum albumin as standard. The Km for pullulan was determined by the Lineweaver-Burk method (Linewaver and Burk 1934). The taxonomic characterization of the isolated strain 3183 was performed according to the methods described in Bergey's manual (Sneath et al. 1986) and other sources (Cowan and Steel 1974).
Growth: Agar Broth Aerobic Anaerobic NaC1 broth pH 5.7 Temperature pH Azide glucose broth Casein Catalase Gelatin Milk Nitrate Starch Tyrosine Production of: Acetylmethyl carbinol Indole Urease Utilization of: Carbohydrates
Citrate Galacturonic acid Glucoronic acid Succinate
Acid without gas from pullulan, cellobiose, cyclodextrin, fructose, glucose, lactose, maltose, mannose, starch, sucrose, and trehalose Not utilized Utilized Utilized Utilized
Identification o f amylolytic enzymes T a b l e 2 shows the e n z y m a t i c hydrolysis p r o d u c t s o f the c u l t u r e s u p e r n a t a n t s o l u t i o n f r o m s o l u b l e starch a n d
Table 2. Analysis of product formation from soluble starch and pullnlan by extracellular amylolytic enzymes of Bacillus strain 3183 Substate
Pullulan Soluble Starch
T h e characteristics o f strain 3183 are s u m m a r i z e d i n T a b l e 1. M a n y o f the characteristics o f strain 3183 are
Not produced Not produced Not produced
s i m i l a r to B. stearothermophilus a n d B. acidocaldarius. H o w e v e r , this strain differs b e c a u s e it denitrifies, grows o n p u l l u l a n , a n d shows a growth p H r a n g e o f 4.0-6.0.
Results
Taxonomic assessment o f strain 3183
Abundant, white colonies with serrated margins Good growth, no sediment Abundant growth Little growth, microaerophilic No growth in 5%, 7% or 10% Grows well Optimum, 62° C; range 45°-65° C Optimum, 6; range 4-6 None Hydrolysed Negative Liquefied Coagulated and peptonized Reduced to nitrite both aerobically and anaerobically Hydrolysed Scant growth
Reaction Degradation products (%) time (h) DP1 DP2 DP3 DP4 6 72 6 72
5.92 24.78 4.20 9.71
11.30 31.46 22.78 40.75
> DP4
70.94 7 . 7 9 4.05 34.58 7.65 1.53 27.08 19:58 26.36 35.56 8.86 5.12
The reaction was performed at 60°C and pH 6.0. The reaction mixture (5 ml) contained 1% substrate, 50 mM acetate buffer, 5 mM Ca 2+ and 1 ml cell-free culture broth
342 A
10
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Fig. 2. ~ cffc~ o~ medium pH on growth a.d cxt~accl]ular pullu]a.asc producfio, by B ~ c i l l ~ strain 318~. Experiments wc~c ;
;
I'0 Minutes
1;
1A, B. HPLC analysisof pullulan hydrolysisproducts of Bacillus strain 3183 crude pullulanase preparation. A, Separation of
Fig.
maltotriose and panose. B, Pullulan hydrolysisproduct pattern pullulan. Initially, more DP3 was formed from pullulan than from soluble starch. However, DP3 was further degraded into DP1 and DP2 during a longer incubation time. Hydrolysis of alpha-l,6-glucosidic linkages of pullulan by this pullulanase preparation was confirmed by analyzing the end-product(s) of the enzyme reaction by HPLC. The column and solvent system used was able to separate maltotriose from panose and isopanose. Both panose and iospanose were co-eluted in this system. The DP3 fraction was confirmed to be maltotriose and not panose or isopanose (Fig. 1). This indicated that the alpha-l,6-glucosidic linkages in pullulan were degraded to maltotriose. However, the degradation of maltotriose to maltose and glucose indicated that alpha-l,4-glucosidic linkages were also hydrolyzed. These results show that the Bacillus strain 3183 produced both extracellular pullulanase activity and additional amylases-saccharidases for production of glucose, maltose and other oligosaccharides.
pc#o~mcd at ~0°C in a 1-1 ~u]t~gc. ~crmcntor with a 3~0-ml wor~.g volume. Scrccni.g medium was supplemented whh maltose (0.~%) aad co~ steep liquor (0.0~%). E.zymc a~ivity w~s mcasurcd usi.g ccll-~rcc culture b~oth and standard assay co.difions: ~, g~owth at pH ~.0; ~, enzyme a~Jvity at pH ~.0; ~ , gro~h ~t pH ~.0; e , c ~ y m c ~cfivity at pH ~.0. O.D.~o.~ =optical dc.sity measured at ~ 0 . m
liquor (0.05%) (data not shown). The addition of P O ~ -3 up to 37 mM in the basic screening medium also enhanced final pullulanase activity levels (data not shown). The optimum temperature and pH for growth of strain 3183 were determined in 1-1 fermentors. Under the pH-controlled conditions (pH 6o0), strain 3183 grew in the range of 40 °-65 ° C with an optimum growth temperature of 62 ° C. No growth occurred at 70 ° C. The fermentation time course for extracellular pullulanase production and growth at pH 5.0 and 6.0 is shown in Fig. 2. Growth was initiated with a much shorter lag phase at pH 6.0 than at 5.0. The final cell density was highest at pH 6.0, whereas the final pullulanase activity was highest at pH 5.0. Pullulanase synthesis was associated with the exponential growth phase at pH 5.0 but not at pH 6.0. The decrease in pullulanase activity at the end of the growth period was associated with the microscopic observation of cell lysis.
Characterization of pullulanase Growth conditions for pullulanase production The effect of carbon sources on growth and extracellular pullulanase production was investigated. Strain 3183 grew on glucose, maltose, maltotriose, maltotetraose, maltohexaose, mannitol, sorbitol, sucrose, cellobiose, starch, amylopectin, beta-limit dextrin, pullulan and wheat flour but not on lactose. Pullulanase activity was highest with wheat flour or pullulan as carbon substrate (data not shown). Nitrogen-based nutritional studies indicated that yeast extract (0.05%), trypticase (0.05%), or casamino acids (0.05%) were better promoters of growth, but lower pullulanase activity was detected in the broth as compared to use of corn steep
General biochemical properties of extracellular pullulanase were determined in order to compare these features with other described microbial pullulanases and Bacillus saccharidases. The pullulanase activity was maximal at 75°C and at pH 6.0; the enzyme was not active below pH 4.0. The pullulanase preparation was stable at pH 5.5-7.0 but was unstable below pH 5.5 or above 8.0. In the absence of substrate, the pullulanase rapidly lost activity at temperatures above 75 ° C. The enzyme displayed a half-life of 96 h when incubated at pH 6.0 and at 70°C without substrate. The calculated apparent K,~ value for this enzyme activity on pullulan was 0.4 mg/ml at pH 6.0 and 75 ° C. The influence of inhibitory or stabilizing reagents
343 Table 3. Comparison of enzymeeffectors on pullulanase activity of Bacillus strain 3183
Reagent
Control CaC12 MgC12 MnC12 COC12 ZnC12 FeC13 EDTA pCMB N-Bromosuccinimide DTT Alpha-cyclodextrin Beta-cyclodextrin Gamma-cyclodextrin
Concentration (raM) -10 10 10 10 10 10 10 0.02 10 10 10 10 10
Relative activity (%) 100 186 80 46 71 1 1 0 99 0 108 109 41 23
The enzyme activity was assayed under standard assay conditions: EDTA=ethylenediaminetetraacetate; pCMB=para-chloromercuribenzoate; DTT- dithiothreitol
on the pullulanase activity was investigated (Table 3). Heavy metal ions such as Fe z+ or Zn 2+ inhibited enzyme activity. Enzyme activity was also inhibited by beta- and gamma-cyclodextrins but not by alpha-cyclodextrin. Calcium ions greatly stimulated the pullulan-degrading activity. Ethylenediaminetetraacetate (EDTA) also completely inhibited the enzyme activity at a concentration of 1 mM. The inhibition by EDTA was relieved by Ca 2+, and pullulanase reactivation was dependent on the concentration of Ca 2+ added (Fig. 3).
Discussion
An acidophilic thermophile, Bacillus strain 3183, was isolated from the hot-spring sediments of Yellowstone National Park. The new strain showed physiological properties similar to B. stearothermophilus and B. acidocaldarius strains although its optimum growth pH and pH range for growth differs from those described for these species (Cowan and Steel 1974; Sneath et al. 1986). The cells of this organism are much smaller (Table 1) than that of B. stearothermophilus KP 1064 (Suzuki and Imai 1985). The Bacillus strain 3183 is also different from B. stearothermophilus, B. acidocaldarius, and B. acidopullulyticus strains (Jensen and Norman 1984; Suzuki and Imai 1985; Sneath et al. 1986) because it can assimilate succinate and denitrify NO~- to NO~- both aerobically and anaerobically. Strain 3183 is different from B. acidopullulyticus because of different growth temperature (60°C for strain 3183 vs. 37°C for B. acidopullulyticus (Jensen and Norman 1985). Unlike strain 3183, B. acidocaldarius does not grow on pullulan (Sneath et al. 1986). At present, further taxonomic studies on DNA homology are needed to compare this newly-isolated strain with previously described Bacillus
2O0
180
~ ~ ' ~ f f i ~ " ~ "
~" 160 _~ 140 •-~ ~ ~0 ~ 10~ ~0 .-~ ~
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~ 4~ ~0
°o
I
i
,
~ ,
CaCI 2
(mM)
Fig. 3. Relationship of CaC12 concentrationto pullulanase activ-
ity of Bacillus strain 3183 in the absence ((3), versus the presence (~) of I mM ethylenediaminetetraacetate(EDTA)
species before proper taxonomic identification can be made. The newly isolated Bacillus strain described here produces an extracellular thermostable pullulanase which exhibited maximum activity at 75 ° C and pH 6.0. Several new sources of thermostable pullulanases have been reported, mainly in thermoanaerobes (Hyun and Zeikus 1985; Antranikian et al. 1987; Coleman et al. 1987; Melasniemi 1987; Nakamura et al. 1987; Plant et al. 1987; Saha and Zeikus 1989). Although mesophilic Bacillus strains can produce pullulanase (Nakamura et al. 1975; Adams and Priest 1977; Jensen and Norman 1984), these activities are not thermostable at temperatures higher than 60 ° C. The crude extracellular enzyme preparation of Bacillus strain 3183 contains pullulanase, amylase and saccharidase activities which can hydrolyze soluble starch into a variety of products that may have potential industrial utility. Starch, maltose or pullulan supported both good growth and pullulanase production. Wheat flour was a practical source of carbon and nitrogen for both growth and enzyme production. Supplementation of starch medium with corn steep liquor enhanced enzyme production and this is an inexpensive source of growth factors. Like other microorganisms grown on starch as the carbon source, Bacillus strain 3183 may produce a multicomponent complex saccharidase-amylase system. The crude enzyme preparation we studied was able to degrade not only pullulan but also amylose, amylopectin and soluble starch. In general, pullulanase debranches amylopectin, beta-limit dextrins and soluble starch, but cannot act on amylose (Abdullah and French 1970). However, it has been shown that pullulanases from thermophiles such as C. therrnohydrosulfuricum (Melasniemi 1987; Saha et al. 1988), Thermoanaerobium brockii (Coleman et al. 1987), Thermoanaerobium TokB1 (Plant et al. 1987) and mesophilic B. subtilis (Takasaki 1987) are different from other described pullulanases. These pullulanases degrade alpha-l,4-glucosidic linkages of starch to produce mixtures of DP2-DP4. The high starch and pullulan degrading activities in the culture broth of Bacillus strain 3183 suggests that it may have a similar pullulanase activity (Coleman et al.
344 1987; P l a n t et al. 1987) a n d / o r a m i x t u r e o f p u l l u l a n a s e , a l p h a - a m y l a s e o r a l p h a - g l u c o s i d a s e activities. I n a separate report, we have demonstrated that an amylop u l l u l a n a s e was p u r i f i e d f r o m t h e c r u d e e n z y m e p r e p a r a t i o n t h a t d e g r a d e s b o t h a l p h a - l , 4 b o n d s in s t a r c h a n d a l p h a - l , 6 b o n d s in p u l l u l a n ( S a h a et al. 1989).
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lulanase activities of Clostridium thermohydrosulfuricum. Biochem J 246:193-197 Nakamura N, Watanabe K, Horikoshi K (1975) Purification and some properties of alkaline pullulanase from a strain of Bacillus no. 202-1, an alkaline microorganism. Biochem Biophys Acta 397:188-193 Nakamura N, Sashihara N, Nagayama H, Horikoshi K (1987) Production of a thermostable pullulanase by a Thermus sp. J Jpn Soc Starch Sci 34:38-44 Nikolov ZL, Meagher MM, Reilly PJ (1985) High-performance liquid chromatography of trisaccharides on amine-bonded silica columns. J Chromatogr 321:393-399 Norman BE (1979) The application of polysacchadde degrading enzymes in the starch industry. In: Berkeley RCW, Gooday GW, Ellwood DC (eds) Microbiol polysaccharides and polysacchaddases. Academic Press, New York, pp 339-376 Plant AR, Morgan HW, Daniel RM (1986) A highly stable pullulanase from Thermus aquaticus YT-1. Enzyme Microb Technol 8:668-672 Plant AR, Clemens RM, Daniel RM, Morgan HW (1987) Purification and preliminary characterization of an extracellular pullulanase from Thermoanaerobium Tok6-B1. Appl Microbiol Biotechnol 26:427-433 Saha BC, Zeikus JG (1987) Biotechnology of maltose syrup production. Process Biochem 22:78-82 Saha BC, Zeikus JG (1989) Novel highly thermostable pullulanase from thermophiles. Trends Biotechnol 7:234-239 Saha BC, Mathupala SP, Zeikus JG (1988) Purification and characterization of a highly thermostable novel pullulanase from Clostridium thermohydrosulfuricum. Biochem J 252;343-348 Saha BC, Shen G-J, Srivastava KC, I_~cureux LW, Zeikus JG (1989) New thermostable alpha-amylase-like pullulanase from thermophilic Bacillus spo 3183. Enzyme Microb Technol 11:760-764 Sneath PHA, Mair NS, Sharpe EM, Holt JG (eds) (1986) Bergey's manual of systematic bacteriology, vol 2. Williams and Wilkins, Baltimore, pp 1130-1139 Suzuki Y, Chishiro M (1983) Production of extracelluiar thermostable pullulanase by an amylolytic obligatory thermophilic soil bacterium Bacillus stearothermophilus KP 1064. Appl Microbiol Biotechnol 17:24-29 Suzuki Y, Imai T (1985) Bacillus stearothermophilus KP 1064 pullulan hydrolase. Its assignment to a unique type of maltogenic alpha-amylase but neither to pullulanase nor isopullulanase. Appl Microbiol Biotechnol 21:20-26 Takasaki Y (1987) Pullulanase-amylase complex enzyme from Bacillus subtilis. Agric Biol Chem 51:9-16 Zeikus JG, Ben-Bassat A, Hegge PW (1980) Microbiology of methanogenesis in thermal, volcanic environments. J Bacteriol 143: 432-440
