ARS/USDA, Peoria, IL 61604, USA. 2Biopolymer Research Unit .... function of time in a Brookfield LVTDV-1 (Brookfield, Illinois) with a small sample adapter ...
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RRENT MICROBIOLOGY
Vol. 32 (1996), pp. 343-348
Current Microbiology An International Journal © Springer-Verlag New York Inc. 1996
Isolation and Characterization of Microorganisms with Alternan Hydrolytic Activity H.A. Wyckoff,l G.L. Cote,2 P. Biely3 lFermentation Biochemistry Research Unit, ational Center for Agricultural Utilization Research, 1815 North University Street, ARS/USDA, Peoria, IL 61604, USA 2Biopolymer Research Unit, ational Center for Agricultural Utilization Research, 1815 orth University Street ARS/USDA, Peoria, IL 61604, USA 3Institute of Chemistry, Slovak Academy of Sciences, Bratislava, Slovakia
Abstract. Alternan is an unusual a-o-glucan containing alternating (1 ~ 3), (1 ~ 6) linkages that exhibits remarkable resistance to enzymatic hydrolysis. The commercial potential of the polysaccharide may be enhanced by the ability to economically modify the native form into fractions of varying molecular weight. By employing isolation procedures with covalently dyed alternan as the substrate, several bacterial isolates that produced endohydrolytic activity were obtained in pure culture. The activity was confirmed by decreases in viscosity and by direct examination of the hydrolysis products with thin layer chromatography. Analysis of the hydrolysis products established that all isolates produced enzymes with identical alternan depolymerizing activity, producing a cyclic tetrasaccharide as a major product. All alternanase activity was shown to be extracellularly located. A single strain exhibited constitutive production of alternanase, while all other isolates required the presence of alternan in the growth media for enzyme production. All isolates were phenotypically similar, produced heat-resistant spores, and were tentatively identified as members of the genus Bacillus.
Alternan is a microbial glucan structurally related to dextrans [9]. Whereas dextrans consist of o-glucose units linked a-(1 ~ 6) to one another, with various amounts of secondary branch linkages, alternan consists of alternating a-(1 ~ 3)- and a-(1 ~ 6)-linked o-glucose residues [5]. The unique characteristics of alternan make it potentially useful as a replacement for gum arabic in applications requiring a lowviscosity, highly soluble gum [2]. Dextran is produced by numerous strains of Leuconostoc and Streptococcus, but alternan is only known to be produced by three strains of Leuconostoc mesenteroides [9]. This relative scarcity of alternan in nature would suggest enzymes capable of degrading alternan are equally rare. In fact, the only enzymes known to degrade ames are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.
Correspondence to: H.A. Wyckoff
alternan to a significant degree are isomaltodextranases, which hydrolyze isomaltose units from the nonreducing ends of the polysaccharide [23, 24]. 0 enzymes capable of endolytic cleavage of alternan have been previously reported. In our work on possible practical applications of alternan, it has become apparent that enzymes capable of hydrolyzing alternan to reduce its molecular weight and viscosity would be very useful. Recently, we have reported on the production of a new type of bacterial endoglucanase that depolymerizes alternan [1]. The natural habitat of Leuconostoc is commonly believed to be vegetable matter [7]; therefore, environmental samples consisting primarily of plant material and soil samples were screened for microorganisms having the ability to degrade alternan. Dyed substrates have frequently been used as indicators of polysaccharide hydrolysis [21, 22] and for isolation of organisms possessing endohydrolytic activities [6]. Thus, our screening procedure used formation of zones of hydrolysis around colonies on media contain-
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ing covalently dyed altern an. In this work, we describe the isolation and purification of the bacterial strains and their tentative identification, and a comparison of the enzymes produced by individual strains. Materials and Methods Strain isolation and characterization. Approximately O.5-cc samples of soil or plant material were inoculated into 20 mlliquid medium in 50-ml culture flasks and incubated 48 h at 30°C with agitation (250 rpm). Each sample was inoculated into four different media, LB [17], YEPD [16], Dex-lO [6] without blue dextran, or PMN [18] with glucose substituted for lactose. After incubation, aliquots of the liquid cultures were streaked or spread on the same medium solidified with 1.5% agar and containing 0.5% Remazol brilliant blue dyed alternan (RBBA), prepared by the method described below, and incubated at 30°C. Colonies showing zones of hydrolysis after 72-96 h were restreaked for purity and to confirm activity on the dyed substrate. For routine maintenance and for determination of enzymatic activity, cultures were grown at 30°C, shaken at 250 rpm in tryptic soy broth [17 g L -I Bacto tryptone, 3.0 g L -I Bacto soytone (Difco, Detroit, Michigan), 5.0 g L -I NaCl, 2.5 g L -I K2HP0 4 ] supplemented with 0.25% glucose or alternan as the major carbon source. Cultures were routinely transferred with a 1% inoculum, and 96-h cultures were used for enzyme determinations. Isolates were screened with the Biolog GP microplate system and identified with a Gram-positive data base (Biolog, Hayward, California) following manufacturer's instructions. Phenotypic characterization of isolates was carried out with standard protocols as outlined by Smibert and Krieg [26]. Spores were harvested from cultures grown in Schaeffer sporulation media [25], and suspensions in water were tested for heat resistance by being heated to 80°C for 10 min in a water bath. Spore viability was determined by plating on tryptic soy broth agar containing 0.5% Remazol brilliant blue dyed alternan. Synthesis of dyed substrate. Alternan was synthesized enzymatically, with alternansucrase from Leuconostoc mesenteroides RRL B-1355 [2, 5], and a soluble dye-labeled derivative of alternan was synthesized by linkage to Remazol brilliant blue (Sigma Chemical Co., St. Louis, Missouri) according to the procedure of Rinderknecht et al. [21]. Following the reaction, the mixture was precipitated twice with equal volumes of ethanol and exhaustively dialyzed against water to remove excess dye and low-molecularweight carbohydrates. Analytical procedures. Enzymatic activity in cell-free supernatants was determined with a procedure based on the release of alcoholsoluble fragments from the dyed substrate [1]. For quantitation of enzymatic activity, the procedure was calibrated with a reducing sugar assay [1]. Confirmation of endohydrolytic cleavage of alternan was achieved by thin-layer chromatography of the products with previously described procedures [1, 4] and by viscosity reduction. Alternan digests were conducted by incubating 2 J.l.1 of culture supernatant concentrated 50-100 times (Centriprep 10, Amicon, Beverly, Massachusetts) with 20 J.l.1 of a 2% alternan solution in distilled water at 30°C. Viscosity reduction was measured by mixing 1 ml of NRRL B-21195 alternanase which had been purified through the gel-filtration step [1] with 7 ml 9% (wt/vol) alternan in distilled water. Viscosity was monitored as a function of time in a Brookfield LVTDV-1 (Brookfield, Illinois) with a small sample adapter 13R/18 at 30°C.
CURRENT MICROBIOLOGY Vol. 32 (1996)
Electrophoresis and enzyme detection. Alternanase-containing, cell-free culture fluids were desalted and concentrated (Centriprep 10, Amicon, Beverly, Massachusetts) approximately 50 times and analyzed for alternanase forms by polyacrylamide gel electrophoresis (PAGE) under native conditions following the Laemmli protocol [11]. After resolution of the proteins, the separation gel was washed twice with 0.1 M phosphate buffer (pH 7.0) for 10 min and brought into contact with a 1% agarose detection gel containing 0.5% dyed alternan in 0.025 M phosphate buffer (pH 7.0). The sandwich was incubated at 40°C in a wet chamber until slight changes in the blue background were observed in the detection gel. The detection gel was then separated from the separation gel and gently shaken in 2:1 solution of ethanol: 0.05 M acetate buffer (pH 5.0) to clear zones of hydrolyzed substrate. Phylogenie analysis and base composition. Genomic DNA was isolated from cells harvested from 48-h tryptic soy broth cultures by the method of Pitcher et al. [19]. Small subunit ribosomal RNA was amplified by the procedure of Weisburg et al. [27] and sequenced with the primers described by Lane [12], with a Taq Dye Deoxy@ Terminator cycle sequencing kit (Applied Biosystems, Foster City, California) following manufacturer's instructions. Reactions were resolved on an ABI 373 automated sequencer (Applied Biosysterns), and sequences were analyzed with the Ribosomal Database [13]. DNA base composition was determined by thermal denaturation by the method of Hayward and Sly [8] by use of E. coli B genomic DNA with a base composition of 50% G + C (Sigma Chemical Co.) as an internal standard. Moles percentage guanine plus cytosine values were calculated with a modified [10] Marmur and Doty [14] equation.
Results and Discussion Seven strains designated NRRL B-21189, NRRL B-21190, NRRL B-21191, NRRL B-21192, NRRL B-21193, NRRL B-21194, and NRRL B-21195 were isolated, on the basis of their ability to form clearing zones on agar media supplemented with RBBA (Fig. 1). Isolates were obtained from only two media, LB and modified Dex-l0. No isolates with alternanase activity were recovered from cultures derived in YEPD or PMN media. All strains required between 72 and 96 h of growth before zones of activity appeared around the colonies. Cells of all isolates were 0.6-0.8 x 1.2-2.0 f.Lm in size and contained centrally located, ellipsoidal refractile bodies which swelled the sporangia and were visible in stained cells or under phase contrast microscopy. Spore suspensions remained viable after 10 min at 80°C and were able to germinate and form colonies with enzymatic activity upon plating and incubation on media containing RBBA. Of particular interest was the propensity of all the isolates to stain Gramnegative at all times. Gram-negative staining isolates identified as Bacillus have been described by Marshall and Ohye [15]. They concluded that low concentrations of amino sugars in the cell wall were responsible for the atypical staining reaction of their isolates. This
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H.A. Wyckoff et al.: Alternan Hydrolytic Activity
Fig. 1. Colonies of isolates displaying typical zones of activity on media containing Remazol brilliant blue-dyed alternan. Table 1. Characteristics of strains Obligatelyaerobic Catalase positive Oxidase negative V-P negative Production of heat-resistant spores Acid or gas not produced from glucose
may also explain the unusual staining characteristics of our isolates. Comparison of the metabolic activities of the isolates against the Biolog database established their identity as members of the genus Bacillus; however, speciation of the isolates from the database was not possible. The general characteristics of the isolated strains are listed in Table 1. With the exception of staining Gram-negative during all phases of growth, these characteristics are consistent with members of the genus Bacillus. To confirm the classification of the isolates, partial16s rRNA sequences spanning the VI and V2 regions were determined. Similarity ranking of sequences against the small subunit ribosomal sequences in the Ribosomal Database confirm that the most similar sequences are of members of the
Table 2. D A base composition of alternanase-producing strains Strain RRL B 21189 NRRL B 21190 RRL B 21191 NRRLB21192 RRL B 21193 NRRLB21194 NRRL B 21195
Mole %G
+C
43.7 42.6 41.5 41.3 40.7 41.6 44.0
genus Bacillus. However the low SAB values (0.6-0.65) indicate the isolates may not be members of recognized species. The DNA base composition of the genomic DNA of the strains ranged from 41 to 44 mol% G + C (Table 2), which is in good agreement with the values of many Bacillus species [20]. Attempts to use a defined medium to determine if the isolates could use alternan as a sole carbon and energy source were unsuccessful. No defined medium that would support growth was identified. However, growth in tryptic soy broth was greatly retarded without the addition of a carbon source. With all strains, a higher cell density was observed in media
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Vol. 32 (1996)
Table 3. Alternanase production by bacterial strains degrading Remazol brilliant blue-dyed alternan Relative enzyme activity in tryptic soy broth-grown cultures Strain RRL B-21189 NRRL B-21190 NRRL B-21191 NRRL B-21192 NRRL B-21193 NRRLB-21194 NRRL B-21195
Glucose
Alternan
0.6 0.8 100
50 ]
o
,L----------,------------r--------,----
o
120
60
180
Minutes
Fig. 3. Viscosity reduction of 8% alteman solution by partially purified altemanase from strain NRRL B-21195.
G
II
m N Fig. 4. Thin layer chromatogram of 2-1J.1 samples of alteman digestion products from enzymes produced by all seven isolates. 1 = 24-h incubation; 2 = 48-h incubation. Lane A, strain NRRL B-21189; lane B, strain NRRL B-21190; lane C, strain NRRL B-21191; lane 0, strain NRRL B-21192; lane E, strain NRRL B-21193; lane F, strain RRL B-21194; lane G, strain RRL B-21195. G, o-glucose; II, isomaltose; III-VIII, novel oligosaccharides produced by altemanase hydrolysis of alteman.
nan for enzyme production. The alternanase of strain NRRL 8-21195 has been purified and characterized [1], and the novel non reducing digestion products of alternan have been identified [4]. Seven isolates identified as belonging to the genus Bacillus produced an extracellular, endo-acting enzyme. Thus, with
genetic systems and vectors for well-known species such as B. subtilis and B. megatarium, genetic engineering of these isolates to overproduce alternanase and/or clone the gene is possible. In addition to the production of low-molecular-weight derivatives of alternan, alternanase may prove useful in the study of
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alternan structure and in the purification of alternansucrase, which is known to be difficult to separate from alternan during purification of the enzyme [3]. ACKNOWLEDGMENTS
The authors acknowledge the excellent technical support of Ms Mary Kinney and thank Dr. Robert Hespell for critical review of the manuscript.
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Su~plled bY U.S. Dept. of Agricultur NatlOna ljenter for Agricultural Utilizat on Research, Peoria, fmnOlS
