Characterization of starch-hydrolyzing lactic acid bacteria isolated from a fermented fish and rice food, “burong isda”, and its amylolytic enzyme

JOURNALOF FERMENTATIONAND BIOENGINEERING Vol. 80, No. 2, 124-130. 1995

Characterization of Starch-Hydrolyzing Lactic Acid Bacteria Isolated from a Fermented Fish and Rice Food, “Burong Isda”, and Its Amylolytic Enzyme MINERVA

OLYMPIA,’

HAJIME

FUKUDA,2 HISAYO ON0,2* YOSHINOBU MITSUO TAKAN02

KANEK0,2

AND

Institute of Fish Processing Technology, College of Fisheries, University of the Philippines in the Visayas Miagao, IIoilo 5023, the Philippines’ and Department of Biotechnology, Faculty of Engineering, Osaka University, Yamadaoka, Suita, Osaka 565, Japan? Received 27 March 1995/Accepted 19 May 1995

Nine strains of lactic acid bacteria that hydrolyze starch were isolated from burong isda, an indigenous fermented food made from fish and rice in the Philippines. Conventional taxonomic and DNA-DNA reassociation studies indicated that all these isolates belong to Lactobacillusplantarum. Each of these isolates harbored more than ten plasmid species with molecular sizes of 2 to 60 kb. The amylolytic activity of L137, one of the isolates, was lost by treatment with novobiocin at 43% frequency, concomitant with curing of a 33.kb plasmid, pLTKl3; this suggested that pLTK13 carries a gene necessary for synthesis of amylolytic enzyme. An acidophilic starch-hydrolyzing enzyme secreted from L137 cells was purified 46.fold with specific activity of 44 units per mg protein. The enzyme was shown to have a molecular mass of about 230 kDa and the optimum temperature and pH for the enzyme reaction with soluble starch were 35°C and 3.8-4.0, respectively. The enzyme hydrolyzed soluble starch, amylopectin, glycogen, and pullulan, and to a small extent amylose, while it exerted no activity on dextran and cyclodextrins. The major reaction products from soluble starch were maltotriose, maltotetraose and maltopentaose, but no panose was detected, and maltotriose was the sole product from pullulan. The K, values for soluble starch, pullulan, and amylose were 4.0, 5.1, and 33 g per liter, respectively. These observations suggest that this enzyme hydrolyzes both a-1,6- and a-1,4-glucosidic linkages. amylolytic

[Key words:

enzyme, amylopullulanase,

burong isda, fermented

rice-fishery

product,

Lactobacil-

ius &antarum, p&mid] strains

newly isolated from several samples of burong collected from markets in the Philippines. An Escherichia coli strain, V517, harboring eight species of plasmids having molecular sizes of 56.4, 7.5, 5.8, 5.3, 4.0, 3.1, 2.8, and 2.2kb (13, 14), was used as reference for estimation of molecular size of plasmids. Type strain of L. plantarum (JCM 1149) was obtained from Japan Collection of Microorganisms (RIKEN, Wako, Saitama). Media For general cultivation of the isolates, MRS agar and broth media (15) purchased from Difco (Detroit, MI, USA) were used. GYP, consisting of 1’; glucose and 0.5% each of yeast extract (Difco) and Bactopeptone (Difco), and GYP + CaC03 (GYP containing O.S.?,’ CaC03) media were prepared as described by Sakai et al. (9) with minor modification and 1.5%’ agar was added to the agar media. The isolates were cultivated at 37°C and kept in MRS agar stabs or in GYP+ CaC03 agar stabs at 4°C by transferring every 2 weeks. For detection of starch hydrolyzing activity of bacterial colonies, a BS plate (blue starch agar plate) was prepared by adding 1% corn starch and 0.2% Starch Azure (Sigma Chemical, St. Louis, MO, USA) to the MRS agar. SYP agar contains log of soluble starch (Wako Pure Chemical, Osaka), 5 g of yeast extract, 5 g of Polypepton (Wako), 5 g of CaC03, 5 ml of mineral stock solution, and 15 g of agar per liter. The mineral stock solution was composed of 110 g of MgS04.7H20, 2g of FeS04. 7H20, and 24 g each of MnS04 and NaCl per liter. YCL broth was composed of 5 g each of casamino acids and yeast extract, log of lactose, 20 g of CaC03, and 5 ml of mineral stock solution per liter. Lactose was used

Several strains of lactic acid bacteria were shown to hydrolyze starch such as those of Streptococcus equinus (l), Streptococcus bovis (2), Lactobacillus amylovorus (3), Lactobacillus sp. (4), Lactobacillus cellobiosus (5, 6), Lactobacillus plantarum (7), and a ruminal lactic acid bacterium (8). Such starch-hydrolyzing lactic acid bacteria have never been reported in fermented foods (9, 10). Tanasupawat et al. (ll), however, suggested a contribution of starch-hydrolyzing lactic acid bacteria to maturation of fermented foods in Thailand. We also found a starch-hydrolyzing lactic acid bacterium (strain L137) in burong bangus, a kind of fermented food of the Philippines made from fish and rice, which is collectively called burong isda (12). This paper is a follow-up to the above study and reports similar starch-hydrolyzing lactic acid bacteria isolated from several samples of burong isda collected from markets in the Philippines. These isolates, along with strain L137, were identified as L. plantarum. The starchhydrolyzing property of L137 was suggested to be associated with a plasmid. The amylolytic enzyme of L137, L137-amylase, hydrolyzes CU-1,4- and a-1,6-glucosidic linkages and yields maltotriose, maltotetraose and maltopentaose as the major products from soluble starch. MATERIALS Bacterial

strains

strain L137 isolated

AND

isda

METHODS

The microorganisms used were in the previous study (12) and eight

* Corresponding author. 124

VOL. 80, 1995

AMYLOLYTIC

in YCL medium because higher amylolytic activity was obtained using this sugar than that obtained using other carbon sources so far tested as in Bacillus arnyloliquefaciens (16). Morphology and physiological characteristics of isolates Taxonomic identification of the isolates was done according to Kandler and Weiss (17) using the conventional method (18). Diaminopimelic acid in cell wall was analyzed according to the method of Hasegawa et al. (19) with minor modifications. One ml of an overnight culture DNA preparation of bacterial strain in MRS broth was inoculated into 3 ml fresh MRS broth and incubated at 30°C for 2 h. Then plasmid DNA was extracted from the cells according to the method of Anderson and McKay (20) and electrophoresed on 0.7% agarose gels in TAE buffer (40mM Tris-acetate, 1 mM EDTA, pH 8.0) at 19 V for 16 to 18 h (21). For study of DNA relatedness, total DNA was prepared from cells cultivated similarly as above. Cell lysis was achieved by treatment with 50 pg of N-acetylmuramidase SC (Seikagaku Corporation, Tokyo) per ml and 0.83% SDS according to the method recommended by the supplier of LA Plate (Snow Brand Milk Prod., Tokyo) described below. Proteins were eliminated by phenol extraction and nucleic acids were recovered by isopropanol precipitation. The precipitate was dissolved in 0.2 ml of TE buffer (10mM Tris-HCl, 1 mM EDTA, pH 8.0) containing 1 c(g of RNase A (Sigma) per ml and incubated at 37°C for 1 h. After cetyltrimethylammoniurn bromide treatment (22) for removal of polysaccharides, phenol extraction was performed again. DNA was precipitated and rinsed with ethanol, then dissolved in 100 ~1 of distilled water and stored at 4°C. DNA concentration of the solution was determined from the absorbance at 260nm. Electrophoresis confirmed that the purified DNA was free of RNA contamination. Determination of DNA relatedness Preliminary experiments were done using LA Plate coated with DNAs of type strains of various Lactobacillus spp. For more detailed examination of the DNA relatedness in the lactic acid bacteria, the microplate hybridization method (23) was used with minor modifications. Probe DNA was prepared by labeling with photobiotin (Bresatec, Adelaide, Australia) in accordance with the manufacturer’s recommendation. Before hybridization with the probe DNAs, 200 ~1 of prehybridization solution consisting of 50% formamide in 2 x SSC (1 X SSC is 0.15 M of NaCl and 0.015 M of trisodium citrate; pH7.0), 2jd blocking reagent (Boehringer), 0.1% N-Iaurylsarcosine, and 0.02% SDS was added to the wells and incubated at 42°C for 30 min. Hybridization was carried out by adding of 100 ~1 of hybridization solution (prehybridization solution containing 1 pg heat-denatured and photobiotin-labeled probe DNA per ml) to the wells and incubating at 42°C overnight. After washing with 0.2x SSC, 100 /11 of PBS (8 mM Na2HP04, 1.5 mM KH2P04, 137mM NaCl, 2.7mM KCl) containing 0.1% streptavidin-horseradish peroxidase conjugate (Gibco BRL, Gaithersburg, MD, USA) was added and incubated at 37°C for 30min. The wells were then washed three times with PBS. The horseradish peroxidase assay was done according to the method recommended by the LA Plate’s supplier. To terminate the reaction, 100 ;ll of 1 N H2S04 was added into each well. Absorbance of the reaction mixture was measured at 450nm (A& with a Microplate Reader photometer Model 450 (Bio-Rad,

LACTIC

ACID BACTERIA

IN A FERMENTED

Richmond, CA, USA). DNA relatedness using the following equation: DNA relatedness

FOOD

125

was calculated

(%) = 100(X-N)/(P-N)

where X is Ab5,, with the DNA solution of isolates, P is &50 of JCM 1149 DNA and N is Ae50 of calf thymus DNA. Purification of Ll37-amylase Cells of a 1.8-1 culture of the organism left standing at 30°C for 48 h in YCL medium were centrifuged at 5,800 X g for 20 min at 4°C. The supernatant obtained was used as enzyme solution. All the following operations were carried out at 4°C. The pH of the supernatant was adjusted to 4.5 by addition of 1 M acetate buffer and phenylmethylsulfonyl fluoride (40mM; Sigma) to give 100mM and 1 mM fmal concentrations, respectively. Pre-filtration was carried out using a Glass Microfibre filter (GF/B; Whatman, Maidstone, England) to remove mucous substances. The filtrate was concentrated to about 20 ml through a 50,000-Da cut-off membrane (Advantec Toyo, Tokyo) under nitrogen atmosphere. The concentrated filtrate was applied to an ion-exchange column (2 x 19 cm) of DEAE-Sepharose CL-6B (Pharmacia) equilibrated with buffer A (10mM acetate buffer, pH 4.0, containing 5 mM CaC12). The column was washed with a 2-fold gel volume of buffer A and eluted with a 4-fold gel volume of buffer A with a linear gradient of NaCl (0 to 0.5 M) at a flow rate of 1 ml per min. Fractions with starchhydrolyzing activity were pooled, desalted and concentrated by ultrafiltration through the 50,OOODa cut-off membrane. The concentrated solution was applied to a Sepharose CL-4B (Pharmacia) column (1.1 x 65 cm) connected to a Toyopearl HW-65 (Toyo Soda, Tokyo) column (0.9~79cm) equilibrated with buffer A. The column was eluted with buffer A at a flow rate of 1 ml per min. The final enzyme solution was kept at -20°C. Protein was determined by measuring absorbance at 280 nm or by the Lowry method (24) using bovine serum albumin as the standard. Enzyme assay Unless otherwise stated, amylolytic activity was assayed at 40°C for 30 min in a mixture of 50 ~1 of 1% soluble starch (biochemical research grade, Nacalai Tesque, Kyoto), glycogen (Merck, Darmstadt, Germany), amylose (average molecular mass 16,000, Nacalai Tesque), amylopectin (Nacalai Tesque), pullulan (Nacalai Tesque), dextran (average molecular mass, 2,000,OOO; Pharmacia), raw starch (from corn; Wako), or (Y-, g- and y-cyclodextrin (Wako), 75 ~1 of 100 mM acetate buffer (pH 4.5), and 25 ~1 of enzyme solution. The reaction was terminated by addition of 50 /ll of 1 N NaOH. Amount of reducing sugars formed was determined by the method of Somogyi-Nelson (25) using glucose as the standard. One unit of the activity was defined as amount of enzyme required to produce 1 pmol reducing sugar per min under the above conditions. To study pH effect on an amylase sample with soluble starch as substrate, 100mM glycine-HCI (pH 2.0-4.0), acetate (pH 4.0-5.3) and phosphate (pH 5.0-6.0) buffers were used. Hydrolyzed products from various carbohydrates by the amylase sample were examined with high-performance liquid chromatography (HPLC) using an Asahipak NH2P-50 (Asahi Kasei, Tokyo) column (4.6 x 250 mm), connected to a Shodex RI-61 detector (Showa Denko, Tokyo). The products were eluted with a mixture of acetonitrile : water (60 : 40) at a flow rate of 1 ml per

126

OLYMPIA

J. FERMENT.BIOENG.,

ET AL.

min and a column temperature of 20°C. Molecular mas? determination Molecular mass of the enzyme was determined by gel filtration column chromatography using Toyopearl HW-65 (0.9 x 79 cm) with 10mM phosphate buffer (pH7.0) at flow rate of 10ml per hour. The standard proteins used for calibration of molecular mass were as follows: ferritin (450 kDa); catalase (240 kDa); aldolase (158 kDa); and bovine serum albumin (dimer, 136 kDa; monomer, 68 kDa) (all these proteins were obtained from Boehringer). RESULTS AND DISCUSSION Survey of burong isda making Results of a survey of local factories of burong isda in Central Luzon, the Philippines, revealed that the rice and freshwater fish were the raw materials. Although there were minor variations among factories in terms of production method, most of them, which were also fish vendors, followed the same basic procedure described in the previous communication (12). Minor variations were as follows. Some factories cooked rice until it became porridge-like for fast maturation of burong isda. Other factories used boiled-dry rice which was believed to produce a better taste than the porridge-like cooked rice. The cooked rice was cooled to lukewarm temperature prior to mixing with fish. The ratio of the amount of rice to fish was not yet standardized. In some factories, rice was inserted into split fish and the whole fish was then covered with cooked rice. In other factories using descaled and eviscerate fish, cooked rice was mixed with filleted fish cut into finger size pieces. The mixed materials were then left to stand for 7 to 10d at room temperature for maturation. Characterization and identification of isolated bacteria All the nine isolates, L131 to L139 including L137 isolated previously (12), formed colonies with a clear halo on GYP +CaC03 and BS plates, indicative of their acidproducing and starch-hydrolyzing activities, respectively, while the authentic strains of L. plantarum, JCM 1149 and IF0 3070, formed colonies without a clear halo on BS plates, indicating a lack of starch-hydrolyzing activity (Fig. 1). All the isolates were slender, straight non-motile rods occurring singly or in short chains and were gram-positive. Colonies on SYP agar were circular and convex. Deep colonies were mostly bi-convex discs while some were tri-convex, and the surface of the colonies was grayish-white and buttery. Young cultures in MRS broth were cloudy, with neither surface growth nor sediments, while the cultures incubated for one week or longer deposited aggregates at the bottom. TABLE Strain L131 L132 L133 L134 L135 L136 L137 L138 L139

Ado ~ ~ ~ ~-

1. Ara _ _ + _ _ +

Utilization Dul _

of various Esc _ _

_ _ _ _ _

+ + _ + +

carbohydrates Glu + + + + + + + + +

Mnl _

_

_

_

_

L133 L136 L139 3070

FIG. 1. Halo formation of isolated bacteria on GYP+CaCO, and BS plates. Overnight cultures of 9 isolates in MRS agar at 30°C were streaked on GYP + CaCOj (A) and BS (B) plates and the plates were incubated at 30°C for 3 d.

All the nine isolates were catalase-negative and grew in the presence of 4% salt but not in the presence of 10% salt. Three isolates (L137, L138, and L139) grew at 15”C, while the remaining six strains showed very slow or no growth at this temperature. All the nine isolates produced acid from glucose, and D and L forms of lactic acid were found to be the major products. These results indicate that the isolates are homolactic fermentors. They could not grow on media containing adonitol, dulcitol, lactose, mannitol or rhamnose as the sole source of carbon and showed severally different behavior of growth on arabinose, esculin, mannose, melibiose, salicin, sorbitol, sucrose, and xylose (Table 1). All of the isolates utilized glucose and starch. All these observations agree with the descriptions for the genus Lactobacillus (17). Cell wall peptidoglycans of the isolates contained meso-diaminopimelic acid. This fact strongly suggests that the isolates are L. plantarum, because most of the other lactobacilli have Lys-D-Asp-type cross-linkages (17). These observations indicate that amylolytic strains of L. plantarum are widely distributed in burong isda and must be essential for hydrolysis of starch in its maturation. Identification of isolates by DNA-DNA hybridization DNA homologies between the nine isolates and 22 type strains of L. acidophilus, L. bifermentans, L. brevis, L. buchneri, L. casei, L. confusus, L. corymformis, L. crispatus, L. curvatus, L. delbrueckii, L. fermentum, L.

by starch-hydrolyzing

Lac _ _ _ _

_ _

L131 L132 L134 L135 L137 L138 JCM 1149 IF0

Mno _ _ f * + + + +

lactic acid bacteria Mel _ _ _ + + +

t +

isolated

Rha _ _ _ _ _

Sal

from burong isda

Sor

Sta i

_

t

_

_

_

_

* _ _

+ + +

_

+

_

_

+

_

sue _

f +

+

_ t +

+

t

t +

XYl +

f

* _ * *

Abbreviations: Ado, adonitol; Ara, arabinose; Dul, dulcitol; Esc, esculin; Glu, glucose; Lac, lactose; Mnl, mannitol; Mno, mannose; Mel, melibiose; Rha, rhamnose; Sal, salicin; Sor, sorbitol; Sta, starch; Sue, sucrose; Xyl, xylose; + , positive reaction; k, weak reaction; ~, negative reaction.

AMYLOLYTIC

VOL. 80, 1995

TABLE

2. DNA relatedness of starch-hydrolyzing lactic acid bacteria with the type strain of L. plantarum

ACID

BACTERIA

IN A FERMENTED

127

FOOD

TABLE 3. Number and size of plasmid species in the starchhydrolyzing lactic acid bacteria isolated from burong isda

DNA relatedness (%)” with JCM 1149T

Strain

84i 3 105&24 78i 7 65* 5 6X* 6 51*11 97* 9 81* 7 98+14 100

L131 L132 L133 L134 L135 L136 L137 L138 L139 JCM 1149T T Type strain. a Average and standard

LACTIC

deviation

in triplicate

A L137

15

B L131 L132 L134 L135 L139 c L133 L136 L138

11 11 11 11 11 11 11 11

1.8, 30, 7.1, 7.1, 7.1, 7.1, 7.1, 7.1, 7.1, 7.1,

2.3, 4.8, 5.4, 33, 45, 60 8.1, 10, 16.5, 8.1, 10, 16.5, 8.1, 10, 16.5, 8.1, 10, 16.5, 8.1, 10, 16.5, 8.1, 10, 16.5, 8.1, 10, 16.5, 8.1, 10, 16.5,

5.9, 6.7, 7.2, 11, 12, 15, 28,

. 19, 19, 19, 19, 19, 19, 19, 19,

25, 28, 25, 28, 25, 28, 25, 28, 25, 28, 25, 28, 25,28, 25, 28,

34, 40, 34, 40, 34, 40, 34, 40, 34, 40, 37, 40, 37,40, 37, 40,

45, 45, 45, 45, 45, 45, 45, 45,

58 58 58 58 58 58 58 58

determinations.

gasseri, L. gallinarum, L. helveticus, L. johnsonii, L. maltaromicus, L. parabuchneri, L. paracasei, L. pentoSW, L. plantarum, L. rhamnosus, L. viridescens, and E. coli (control) were examined using LA Plate. The DNAs of the nine isolates showed strong hybridization signals only with the DNA of L. plantarum (data not shown). Results of the DNA-DNA reassociation experiments with the labeled DNA from the type strain of L. plantarum (JCM 1149) showed hybridization index of 78% or higher for the six isolates (Table 2), while the other three strains, L134, L135, and L136, showed moderate values (51 to 68%). These data are consistent with the fact that L. plantarum strains consist of two different groups showing 50 to 60% DNA homology (17). Thus, all the nine isolates should belong to a single species, L. plantarum. The G+C content of L137 (45.2%) as reported previously (12) was also found to be similar to that (4446%) of L. plantarum (17). It was Plasmids and starch-hydrolyzing activity reported that L. plantarum harbors a variety of plasmids

FIG. 2. Agarose gel electrophoresis of plasmid DNAs prepared from the starch-hydrolyzing bacteria. Plasmid DNAs were from strain L131 (lane 1), L132 (lane 2), L133 (lane 3), L134 (lane 4), L135 (lane 5), L136 (lane 6). L138 (lane 7), L139 (lane 8), and L137 (lane 9). Arrowheads indicate the positions of the marker plasmids of E. coli V517 and their corresponding molecular sizes (13, 14). Conditions for electrophoresis and staining plasmid bands were described in the text.

(14, 26, 27). When total DNAs of the isolates were prepared for DNA-DNA hybridization experiments, all of the isolates showed plasmid bands in agarose gel electrophoresis. To investigate the plasmids in detail, plasmid DNAs were extracted from the isolates. We found that each of the isolates harbored 11 to 15 plasmid species (Fig. 2) and that the isolates were divided into three different classes according to their plasmid profiles; strain L137 (type A) exhibited a distinctive plasmid band profile in comparison with the other eight strains and contained 15 plasmid species (Table 3). The remaining eight isolates also harbored various different species of plasmids; five strains (L131, L132, L134, L135, and L139) showed a common profile (type B) and the other three strains (L133, L136, and L138) shared another profile (type C). Then we investigated whether the plasmid play a role in the amylolytic activity of the isolates by plasmid curing. Cells were cultivated in MRS medium supplemented with 0.5 pg of novobiocin per ml at 30°C for 24 h as described by Ruiz-Barba et al. (14). The cultures were diluted serially by one tenth with 0.85% saline and 100 ~1 each of the diluted cultures was spread on BS plates. The plates were incubated at 30°C for 2 d and amylolytic activity of the colonies was examined by observing halo formation. No colonies showing the halo-phenotype (i.e., unable to form a halo) were found in strains L131, L132, L133, L135, and L136. However, strain L137 segregated halo- colonies (Fig. 3) with 43,?4 frequency (215 colonies showing the halo- phenotype of 501 colonies examined) and strains L133, L138, and

FIG. 3. Appearance of colonies showing the halo- phenotype on MRS plate containing corn starch by spreading cells of strain L137. Cells were cultivated in MRS medium containing 0.5 pg of novobiocin per ml at 30°C overnight. The cultures were diluted appropriately and spread on MRS-starch plates. The plates were incubated at 30°C for 2 d and exposed to I2 vapor in a desiccator.

128

OLYMPIA

.I. FERMENT.BIOENG.,

ET AL.

80

90

100

110

120

Elution volume (ml)

FIG. 4. Agarose gel electrophoresis of plasmid fractions of the halo- clones of L137. Samples were the plasmid DNA from original strain of L137 (lane l), halo+ clone of L137 treated with novobiocin (lane 2), and 8 independent haloclones derived from L137 by novobiocin treatment (lanes 3 to 10). The arrowhead indicates the band of a 33-kb plasmid which is missing in the halo- clones.

L139 showed much lower frequencies of halo- segregation (data not shown) compared with strain L137. Several halo+ and halo clones of strain L137 were isolated and it was found that all of the 43 halo clones were examined lacked the 33-kb plasmid (only data for 8 haloclones are shown in Fig. 4). Thus, the halo- phenotype was, most probably, due to the loss of the 33-kb plasmid, suggesting that the 33-kb plasmid, pLTK13, carries a gene necessary for synthesis of amylolytic enzyme. The faint band observed at the same migration site as that of pLTK13 in the halo- clones was, most probably, due to contaminating chromosomal DNA. We attempted to Purification of amylolytic enzyme purify and characterize the amylolytic enzyme of strain L137, because this strain showed the highest activity among the isolates, and amylolytic enzyme synthesis might be associated with the pLTK13 plasmid. After cultivation of strain L137 in YCL medium, approximately 130 units per liter of amylolytic activity was detected in the culture filtrate. Results of a typical purification procedure of the enzyme, L137-amylase, are summarized in Table 4. At the final purification step of L137-amylase, we obtained 46-fold purification with a 19% overall yield and specific activity of 44 units per mg protein. The final purified product of L137-amylase showed a single absorbance peak at 280 nm and the peak of hydrolyzing activities on starch and pullulan coincided completely with the peak of the UV absorbance (data not shown). TABLE Purification

4.

step

Culture filtratea Ultrafiltration (50 kDa cut-off) DEAE-Sepharose Sepharose CL-4B & Toyopearl

Purification

of amylolytic

Total protein (mg) _b

HW-65

a A 1.8-l culture was used for purification b Not shown because of high background

184 8 1 of the enzyme. protein content

FIG. 5. Determination of molecular mass of L137-amylase by gel filtration. Standard proteins used were a, ferritin (450 kDa); b, catalase (240 kDa); c, aldolase (158 kDa); d, bovine serum albumin (dimer, 136 kDa); and e, bovine serum albumin (monomer, 68 kDa). The open circle indicates Ll37-amylase.

Molecular mass of L137-amylase Molecular mass of the enzyme was estimated to be about 230 kDa by gel filtration column chromatography (Fig. 5). Most of the known amylases have molecular sizes ranging from 22.5 to 76 kDa (5, 28-30). Examples of extremely large molecular size enzymes are pullulanase-amylase complex enzyme from B. subtilis (450 kDa) (31) or amylase-pullulanase enzyme from B. circulans (218 kDa) (32), in the cases of a kind of complex enzyme or an enzyme with two active sites for amylase and pullulan, respectively. The largest extracellular amylase so far identified in prokaryotes, with gene cloning, is A-180 amylase of approximately 180 kDa in an alkalophilic gram-positive bacterium (33). Thus, L137-amylase is considered as one of the high-molecular-size amylases. A similar large-molecular size of 150 kDa was reported for an cu-amylase secreted by L. amylovorus (34). Staining of L137-amylase on polyacrylamide gel was unsuccessful in spite of use of several stains; e.g., Coomassie brilliant blue staining, silver staining, or activity staining. Only copper negative staining (35) showed a very faint broad band on the gel after SDS-PAGE or native PAGE with 7.5% acrylamide gel (data not shown). The position of this band was found at a position close to the top of the separating gel which was higher position than that of myosin having a molecular mass of 200 kDa. The above results suggest that L137-amylase is a monomeric protein. The high molecular mass and the difficulty in staining of this enzyme might be due to the presence of carbohydrate moieties, although glycoproteins are not commonly present in bacterial enzymes . Some characteristics of Ll37-amylase The optienzyme

from L. plantarum strain

Total activity (units) 230 175 159 44 owing to YCL medium.

Specific activity (units/mg protein) _b

0.96 19.9 44.0

L137 Purification (fold) _b

Yield 00) 100

I 21 46

77 69 19

AMYLOLYTIC

VOL. 80, 1995

LACTIC

^lOO *

5

‘2

3

4 PH

5

6

ACID

BACTERIA

: 40 g a 20 B ; H 80 0 IIIILI 10 20 30 40 50 60 Temperature ( ’ C)

pH of L137-amylase for amylolytic activity was at pH 3.8-4.0 (Fig. 6A) and the enzyme was stable from pH 3.5 to 5.3 (data not shown). These results indicate that L137-amylase is more acidophilic than the reported amylases in lactic acid bacteria (4, 6, 34, 36). The optimum temperature of L137-amylase under the conditions of pH4.5 and 30-min reaction time was 35°C and the activity decreased quickly above 45°C (Fig. 6B). Thus, the enzyme seems thermolabile and the residual activity showed an almost linear decrease at temperatures above 30°C (Fig. 6C). The time course of hydrolysis of soluble starch with the final purified product of L137-amylase (Fig. 7) indicated that it is an endo-type amylase. In the early stages of substrate hydrolysis, maltotriose, maltotetraose, and maltopentaose were produced predominantly, and these three maltooligosaccharides comprised 75 mol% of the total products of hydrolysis after prolonged incubation of up to 24 h (data not shown). No panose or isopanose was detected in the reaction mixture. L137-amylase hydrolyzed amylose to maltotriose, maltotetraose, and maltopentaose. It also hydrolyzed maltohexaose to maltotriose, but not maltopentaose, maltotetraose, maltotriose, or maltose, as observed with the crude enzyme preparation (37). These results indicate that L137-amylase differs from most of the n-amylases whose major products of starch hydrolysis are glucose and maltose, and also from a-amylases with pullulanase activity (31,

70

60

(min)

FIG. 7. Products of starch hydrolysis with L137-amylase. Reaction mixture contained soluble starch (1%; Nacalai Tesque) and L137-amylase preparation (1 unit per 150 ,nl of the reaction mixture) in 50 mM acetate buffer (pH 4.5) and the mixture was incubated at 30°C. Products were glucose ( q), maltose ( n ), maltotriose ( l), maltotetraose (0), maltopentaose (A), and maltohexaose (A). The amount of oligosaccharides formed was indicated by the mole percentage of the total amount of products of hydrolysis at a given time.

20

30 40 50 - 60 Temperature (OC)

Buffers used in A were; glycine-HCl temperature (B) and thermostability

buffer (0 ; (C) were

32, 38-40). Substrate specificity of L137-amylase was studied with various polysaccharides and cyclodextrins (Table 5). Under the conditions of pH 4.5 and 4O”C, the enzyme effectively hydrolyzed amylopectin, soluble starch, and glycogen. It also showed significant activity toward pullulan and yielded maltotriose (data not shown), but low activity toward amylose. Raw starch, dextran and a-, p- and y-cyclodextrins were not hydrolyzed at all. These results suggest that the enzyme prefers to digest polysaccharides containing both a-1,4 and a-1,6-glucosidic linkages as substrates than the linear amylose molecule containing only the n-1,4 linkage. From the Lineweaver-Burk plots, the K, values of the enzyme for soluble starch, pullulan, and amylose were determined to be 4.0, 5.1, and 33 g per liter, respectively (data not shown). These results indicate that L137-amylase has similar affinities to soluble starch and pullulan as an amylopullulanase (38). Such enzymes which hydrolyze both the (x-1,4- and n-1,6glucosidic linkages have been reported in thermophilic bacteria, e.g., Clostridium thermosulforogenes (36) and Bacillus sp. (38), or in thermophilic archaea, Pyrococcus furiosus and Thermococcus litoralis (40). However, L137-amylase is produced from L. plantarum, a mesophilic bacterium. The dual activities of amylopullulanase in B. subtilis and B. circulans, both aerobic mesophiles, are interpreted to be due to two active sites (31, 32), whereas those of thermophiles, e.g., B. stearothermophilus (41) and Thermoanaerobium (42), are due to a single active site for those two substrates. Dual activities at a single active site are also found in anaerobic bacteria growing on starch (39, 40) and it was thought that the enzymes function as an efficient energy-yielding systems for the anaerobes (39). These possibilities of L137amylase remain to be elucidated.

TABLE

40

129

60

mum

Time

FOOD

B

FIG. 6. Effects of pH and temperature on the activity and thermostability of L137-amylase. pH 2.0-4.0); acetate buffer ( n ; pH 4.0-5.3); and phosphate buffer (0 ; pH 5.0-6.0). Optimum determined in 50 mM acetate buffer (pH 4.5) and incubated for 30 min.

20

IN A FERMENTED

5.

Relative

activity

of purified substrates

Substrate

Relative

Soluble starch Glycogen Amylose Amylopectin Pullulan Dextran Raw starch Cyclodextrin (n, 9, 7) a Conditions

L137-amylase

for the assay for activity

on different activity=

100% 96 39 116 50 0 0 0 were described

in the text.

130

OLYMPIA

J. FERMENT. BIOENG.,

ET AL

ACKNOWLEDGMENTS We thank Yasuji Oshima, Osaka University, for his valuable discussions, and also Takanori Koso, of Osaka University, for his technical assistance. This study was supported in part by the Komori Memorial Foundation.

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