Characterization of a Mutant from Lactobacillus amylovorus JCM 1126T with Improved Utilization of...
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Characterization of a Mutant from Lactobacillus amylovorus JCM 1126 T with Improved Utilization of Sucrose Yuji Oda, 1 Miyuki Ito 2 1 Upland Agriculture Research Center, Hokkaido National Agricultural Experiment Station, Memuro, Kasai, Hokkaido, 082-0071, Japan 2 Department of Applied Biological Science, Fukuyama University, Fukuyama, Hiroshima 729-0292, Japan Received: 23 May 2000 / Accepted: 26 June 2000 Abstract. A strain YF43, which can grow on sucrose as rapidly as glucose, was isolated by mutation from Lactobacillus amylovorus JCM 1126, the type strain defective in sucrose utilization. Exogenous sucrose stimulated the production of invertase by strains YF43 and JCM 1126 simultaneously. In a medium containing fructooligosaccharide as the sole carbon source, the cells of strain YF43 showed high invertase activity in spite of poor growth. The two invertases produced in the cells grown on sucrose and fructooligosaccharide were an identical b-fructofuranosidase, as judged from properties of partially purified enzymes. These observations indicated that strain YF43 is a mutant improved for permeation of sucrose and not derepressed for the synthesis of invertase. The amount of food wastes has increased as life styles have become convenient, but its disposal brings about serious social problems in cities and surrounding districts [7]. Ensiling for animal feeds may be one way to solve these problems [8], as well as composting [3]. We pre- viously compared production of lactic acid from starch by ten strains classified as Lactobacillus amylovorus to select the best strain for lactic acid fermentation of food waste [12]. Although strain JCM 1126 showed excellent productivity of lactic acid from raw starch, its growth on sucrose was too limited to produce sufficient amounts of lactic acid. This strain is unsuitable for the fermentation of not only food wastes containing sucrose such as sweet bread residue [11], but also by-products derived from confectionaries. Molasses, which includes sucrose as a principal sugar, is sometimes added to ensile raw mate- rials lacking in fermentable sugars [14]. Because strain JCM 1126 can grow well on either glucose or fructose, hydrolysis of sucrose may be a rate-limiting step for its utilization [12]. Thus, we have isolated a mutant from strain JCM 1126 with improved utilization of sucrose. The present paper reports production of invertase by the mutant and characterization of the partially purified en- zyme. Materials and Methods Organism and culture. Lactobacillus amylovorus JCM 1126, the type strain, was obtained from the Institute of Physical Research (Wako-shi, Saitama, Japan). The bacterial cells were grown in 10 ml of MRS medium [5], and 1.0 ml of this culture was inoculated to 100 ml of MRS medium in which glucose had been substituted by 2.0% sucrose. All of the cultures were conducted at 37°C for 2 days under static incubation. Cell weight was estimated from absorbance of medium; 1.0 of A 660 per ml corresponded approximately to 0.22 mg as dry basis. Lactic acid and sucrose in the medium were monitored with a high perfor- mance liquid chromatography (HPLC) [10, 12]. After cultivation, the cells were harvested, washed twice, and resuspended in 0.1 volume of 20 mM Tris-HCl (pH 7.2). A 0.9 ml of the cell suspension mixed with 0.1 ml of lysozyme solution (10 mg/ml) was incubated at 37°C for 1 h and immediately used as crude enzyme for invertase assay. Invertase assay. The reaction mixture contained 50 mM acetate buffer (pH 5.5), 150 mM sucrose, and the crude enzyme in a total volume of 0.25 ml. After incubation of the mixture at 37°C for 30 min, the reaction was stopped by the addition of 3,5-dinitrosalicylic acid reagent [1], and the reducing sugars produced were determined. One unit was defined as the amount of enzyme that released 1 mmol of reducing sugar equivalent to glucose per h under the above conditions. For testing substrate specificity, fructose formed in the reaction mixture was determined with HPLC, as described elsewhere [10]. Enzyme purification. The crude enzyme obtained from 1.0 liter of culture broth was sonicated and centrifuged at 10,000 g for 20 min. The supernatant was purified by the columns of DEAE-Toyopeal 650M and hydroxyapatite, and dialyzed against 20 mM phosphate buffer (pH 6.8). Correspondence to: Y. Oda; E-mail: [email protected] CURRENT MICROBIOLOGY Vol. 41 (2000), pp. 392–395 DOI: 10.1007/s002840010156 Current Microbiology An International Journal © Springer-Verlag New York Inc. 2000
Transcript of Characterization of a Mutant from Lactobacillus amylovorus JCM 1126T with Improved Utilization of...
Characterization of a Mutant fromLactobacillus amylovorusJCM1126T with Improved Utilization of Sucrose
Yuji Oda,1 Miyuki Ito2
1Upland Agriculture Research Center, Hokkaido National Agricultural Experiment Station, Memuro, Kasai, Hokkaido, 082-0071, Japan2Department of Applied Biological Science, Fukuyama University, Fukuyama, Hiroshima 729-0292, Japan
Received: 23 May 2000 / Accepted: 26 June 2000
Abstract. A strain YF43, which can grow on sucrose as rapidly as glucose, was isolated by mutationfrom Lactobacillus amylovorusJCM 1126, the type strain defective in sucrose utilization. Exogenoussucrose stimulated the production of invertase by strains YF43 and JCM 1126 simultaneously. In amedium containing fructooligosaccharide as the sole carbon source, the cells of strain YF43 showed highinvertase activity in spite of poor growth. The two invertases produced in the cells grown on sucrose andfructooligosaccharide were an identicalb-fructofuranosidase, as judged from properties of partiallypurified enzymes. These observations indicated that strain YF43 is a mutant improved for permeation ofsucrose and not derepressed for the synthesis of invertase.
The amount of food wastes has increased as life styleshave become convenient, but its disposal brings aboutserious social problems in cities and surrounding districts[7]. Ensiling for animal feeds may be one way to solvethese problems [8], as well as composting [3]. We pre-viously compared production of lactic acid from starchby ten strains classified asLactobacillus amylovorustoselect the best strain for lactic acid fermentation of foodwaste [12]. Although strain JCM 1126 showed excellentproductivity of lactic acid from raw starch, its growth onsucrose was too limited to produce sufficient amounts oflactic acid. This strain is unsuitable for the fermentationof not only food wastes containing sucrose such as sweetbread residue [11], but also by-products derived fromconfectionaries. Molasses, which includes sucrose as aprincipal sugar, is sometimes added to ensile raw mate-rials lacking in fermentable sugars [14]. Because strainJCM 1126 can grow well on either glucose or fructose,hydrolysis of sucrose may be a rate-limiting step for itsutilization [12]. Thus, we have isolated a mutant fromstrain JCM 1126 with improved utilization of sucrose.The present paper reports production of invertase by themutant and characterization of the partially purified en-zyme.
Materials and Methods
Organism and culture. Lactobacillus amylovorusJCM 1126, the typestrain, was obtained from the Institute of Physical Research (Wako-shi,Saitama, Japan). The bacterial cells were grown in 10 ml of MRSmedium [5], and 1.0 ml of this culture was inoculated to 100 ml ofMRS medium in which glucose had been substituted by 2.0% sucrose.All of the cultures were conducted at 37°C for 2 days under staticincubation.
Cell weight was estimated from absorbance of medium; 1.0 ofA660per ml corresponded approximately to 0.22 mg as dry basis. Lacticacid and sucrose in the medium were monitored with a high perfor-mance liquid chromatography (HPLC) [10, 12]. After cultivation, thecells were harvested, washed twice, and resuspended in 0.1 volume of20 mM Tris-HCl (pH 7.2). A 0.9 ml of the cell suspension mixed with0.1 ml of lysozyme solution (10 mg/ml) was incubated at 37°C for 1 hand immediately used as crude enzyme for invertase assay.
Invertase assay.The reaction mixture contained 50 mM acetate buffer(pH 5.5), 150 mM sucrose, and the crude enzyme in a total volume of0.25 ml. After incubation of the mixture at 37°C for 30 min, thereaction was stopped by the addition of 3,5-dinitrosalicylic acid reagent[1], and the reducing sugars produced were determined. One unit wasdefined as the amount of enzyme that released 1mmol of reducingsugar equivalent to glucose per h under the above conditions. Fortesting substrate specificity, fructose formed in the reaction mixturewas determined with HPLC, as described elsewhere [10].
Enzyme purification. The crude enzyme obtained from 1.0 liter ofculture broth was sonicated and centrifuged at 10,000g for 20 min. Thesupernatant was purified by the columns of DEAE-Toyopeal 650M andhydroxyapatite, and dialyzed against 20 mM phosphate buffer (pH 6.8).Correspondence to:Y. Oda; E-mail: [email protected]
CURRENT MICROBIOLOGY Vol. 41 (2000), pp. 392–395DOI: 10.1007/s002840010156 Current
MicrobiologyAn International Journal© Springer-Verlag New York Inc. 2000
Protein was determined by the method of Bradford [2] with bovineserum albumin as a standard.
Reagent. Fructooligosaccharide (FOS), which contained 1-kestose,nystose, and 1F-b-fructofuranosyl nystose, was obtained from WakoPure Chemical Co. (Osaka, Japan). FOS is commercially produced bytransferring one to three molecules of fructose to the fructose residue insucrose by the action of fungalb-fructofuranosidase.
Reproducibility. Most of the data are shown as the average valuesfrom at least two independent experiments, unless otherwise stated.
Results
Isolation of a mutant. Growing cells of strain JCM1126 were mutated by ethylmethane sulfonate [13]and inoculated to 10 ml of MRS medium containingsucrose as the sole carbon source. After incubation,until the appearance of the medium became cloudy for4 to 7 days, 0.1 ml of the culture was transferred to anew medium and further incubated. The culture brothwas successively diluted and spread on agar plates ofthe same medium. Among isolates forming large col-onies, one strain was arbitrarily selected and namedYF43. Figure 1 compares strains YF43 and JCM 1126with respect to growth, production of lactic acid, andconsumption of sucrose. The concentration of lacticacid produced by strain YF43 increased with growthfor 2 days and was almost constant up to 4 days.Sucrose in the medium was undetectable after 2 days.Strain JCM 1126 grew slowly, but it did not attain alevel comparable to strain YF43.
Production of invertase. Table 1 shows production ofinvertase when strains YF43 and JCM 1126 were grownon various sugars. Glucose had little effect on enzymesynthesis, but it was utilized as the carbon source for cellgrowth. Other sugars more or less induced the productionof invertase, and sucrose was most effective in bothstrains. Little growth was observed in the medium con-taining FOS and raffinose, while the former efficientlystimulated enzyme production by strain YF43. FOScaused higher activity than sucrose for strain YF43, butit can also be assumed that sucrose and FOS induceddifferent enzymes.
Properties of invertase. The enzymes were purifiedfrom the cells of strain YF43 grown on sucrose with190-fold and FOS with 23-fold. Both enzyme prepara-tions still contained many proteins but showed singlecorresponding bands with invertase activity when sub-jected to polyacrylamide gel electrophoresis (Fig. 2).Both enzymes gave maximal activities near pH 4.5–5.0and 60°C, respectively, and degraded sucrose, FOS, raf-finose, and stachyose, but not maltose or melezitose.Relative activities of the enzymes for FOS, raffinose, and
stachyose to sucrose were 13.4, 21.4 and,3. Theseproperties mean the two enzymes produced in the cellsgrown on sucrose and FOS are an identical protein clas-sified tob-fructofuranosidase, as shown in other speciesof Lactobacillus[4].
Discussion
We have first assumed that strain YF43 is a mutantderepressed for the synthesis of invertase, which de-termines the consumption of exogenous sucrose instrain JCM 1126. However, comparable activities ofinvertase produced by strains YF43 and JCM 1126(Table 1) cannot explain the distinct difference ingrowth on sucrose. A rate-limiting step for utilizationof sucrose seems to be its transport into the cells andnot hydrolysis by invertase. Permeation of sucrosemay be improved in strain YF43 by mutation, result-
Fig. 1. Growth of strains YF 43 and JCM 1126 in the mediumcontaining sucrose as the sole carbon source. Open and closed symbolsindicate strains YF43 and JCM 1126, respectively. A, growth; B, lacticacid produced; C, sucrose remained.
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ing in rapid growth on sucrose. It is interesting thatFOS cannot support the growth of strains YF43 andJCM 1126 and stimulated the production of invertaseby strain YF43. FOS composed of 1-kestose, nystose,and 1F-b-fructofuranosyl nystose may be transportedinto the cells by the permeation system specific forsucrose, because those chemical structures are analo-gous to sucrose [9]. Permeation efficiency for each ofFOS must be low as compared with sucrose, and even
in strain YF43, the transported amount may be limitedfor growth but enough to elicit high production ofinvertase. Although the mechanism of sucrose perme-ation is still unknown in detail,L. amylovorusstrainYF43 is applicable for lactic acid fermentation ofmaterials containing both raw starch and sucrose.
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Fig. 2. Locations of protein and invertase activity on the elecro-phoresed gel. Partially purified enzymes from the cells grown onsucrose (A, C) and FOS (B, D) were loaded on the polyacrylamidegel electrophoresis, and protein (A, B) and invertase activity (C, D)were stained [6]. Protein content: A, 45mg; B, 4.5mg; activity: C,32 U; D, 34U.
Table 1. Effect of sugars on the production of invertase in strains YF43 and JCM 1126
Sugars
YF43 JCM 1126
Invertase activity(U/mg cells)
Cell weight(mg/ml)
Invertase activity(U/mg cells)
Cell weight(mg/ml)
None 0.00 0.06 0.00 0.06Glucose 0.00 1.77 0.00 1.84Galactose 0.24 1.09 0.18 1.24Sucrose 2.13 1.75 1.65 0.31Maltose 0.13 1.88 0.07 1.90Soluble starch 0.20 1.63 0.18 1.69FOS 6.05 0.20 0.45 0.18Raffinose 0.54 0.06 0.21 0.06
The cells were grown for 2 days in MRS medium in which glucose had been substituted by each one of 2.0% sugars.
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