Activity of Two Histidine Decarboxylases from Photobacterium phosphoreum at Different Temperatures, phs, and NaCl Concentrations

Transcription

1 76 Journal of Food Protection, Vol. 67, No. 8, 2004, Pages Copyright, International Association for Food Protection Activity of Two Histidine Decarboxylases from Photobacterium phosphoreum at Different Temperatures, phs, and NaCl Concentrations HIDEAKI MORII* AND KENTARO KASAMA Faculty of Fisheries, Nagasaki University, Bunkyo, Nagasaki , Japan MS 0-555: Received 5 December 200/Accepted 29 March 2004 ABSTRACT The major causative agent of scombroid poisoning is histamine formed by bacterial decarboxylation of histidine. The authors reported previously that histamine was exclusively formed by the psychrotrophic halophilic bacteria Photobacterium phosphoreum in scombroid fish during storage at or below 0 C. Moreover, histamine-forming ability was affected by two histidine decarboxylases: constitutive and inducible s. This article reports the effect of various growth and reaction conditions, such as temperature, ph, and NaCl concentration, on the activity of two histidine decarboxylases that were isolated and separated by gel chromatography from cell-free extracts of P. phosphoreum. The histidine decarboxylase activity of the cell-free extracts was highest in 7 C culture; in 5% NaCl, culture growth was inhibited, and growth was best in the culture grown at ph 6.0. Moreover, percent activity of the constitutive and inducible s was highest for the inducible in cultures grown at 7 C and ph 7.5 and in 5% NaCl. The temperature and ph dependences of histidine decarboxylase differed between the constitutive and inducible s; that is, the activity of histidine decarboxylases was optimum at 0 C and ph 6.5 for the inducible and 40 C and ph 6.0 for the constitutive. The differences in the temperature and ph dependences between the two s extended the activity range of histidine decarboxylase under reaction conditions. On the other hand, histidine decarboxylase activity was optimum in 0% NaCl for the two s. Additionally, the effects of reaction temperature, ph, and NaCl concentration on the constitutive activity of the cell-free extracts were almost the same as those on the whole histidine decarboxylase activity of the cell-free extracts, suggesting that the constitutive activity reflected the whole histidine decarboxylase activity. Scombroid poisoning generally results from eating fish belonging to the families Scomberesocidae and Scombridae, which include tuna, bonito, and mackerel. These fish can give rise to toxic levels of histamine under storage conditions, leading to histidine decarboxylation, because they characteristically contain large amounts of free L-histidine in the muscle. The causative agent of scombroid poisoning is as yet entirely unknown, but histamine is commonly present and therefore widely used as an indicator for this type of food poisoning. In previous reports, histamine was formed in scombroid fish during storage at or below 0 C (6, 4). The histamine was exclusively formed by psychrotrophic halophilic luminous bacteria Photobacterium phosphoreum, which dominated scombroid fish during storage at low temperatures (6, 4, 5). The histamine-forming ability of the washed cell suspension and the cell-free extracts of P. phosphoreum was influenced by various growth and reaction conditions (6). The histamine-forming ability of the cell-free extracts of P. phosphoreum was affected by two histidine decarboxylases, namely constitutive and inducible s (7). Moreover, the two histidine decarboxylase activities varied with oxygen tensions for growth (7). This article reports the effect of growth and reaction temperatures, phs, and NaCl concentrations on the two en- * Author for correspondence. Tel: ; Fax: ; morii@net.nagasaki-u.ac.jp. zyme activities of P. phosphoreum. This study is significant because even though detailed studies of this type have been done with washed cell suspensions, few studies have been conducted with the use of cell-free extracts, especially of constitutive and inducible s. Also, this study is important for predicting histamine toxicity from scombroid fish during storage at low temperatures. MATERIALS AND METHODS Bacteria, media, and growth conditions. One strain of P. phosphoreum, NUFM 262, isolated from mackerel stored in ice was used to investigate the effect of various growth and reaction conditions on the activity of two histidine decarboxylases: constitutive and inducible s (7). Generally, the bacterium was incubated at 20 C with SWYP broth (0.5% Bacto Peptone [Difco, Detroit, Mich.] and 0.% yeast extract powder [Oxoid, Basingstoke, England] in 75% filtered aged seawater [.5% salinity] at ph 6.2) containing 0.2% L-histidine. However, the ph of the medium was adjusted from 5.0 to 8.0 at increments of 0.5 with.0 N HCl or.0 N NaOH to determine the effect of initial ph in the medium;,, and 5% NaCl solutions were used in place of 75% seawater in SWYP broth to examine the effect of NaCl concentrations in the medium; and the cultures were grown at 4, 7, 5, 20, 25, and 27 C to determine the effect of growth temperatures on histidine decarboxylase production. Morganella morganii IFO 848 was used for comparison and to resolve histidine decarboxylase activity. The bacterium was incubated at 7 C with YP broth (.0% polypeptone and 0.2% yeast

2 J. Food Prot., Vol. 67, No. 8 HISTIDINE DECARBOXYLASE FORMATION BY P. PHOSPHOREUM 77 FIGURE. Typical elution profile of Photobacterium phosphoreum histidine decarboxylase from a Sepharose CL-6B column. Cell-free extracts applied to the column were prepared from Photobacterium phosphoreum grown in SWYP broth containing 0.2% L-histidine at 20 C for 24 h under aerobic stationary conditions., histidine decarboxylase activity;, absorbance at 280 nm. The figure presents the result of a single analysis. FIGURE 2. Typical elution profile of Morganella morganii histidine decarboxylase from a Sepharose CL-6B column. Cell-free extracts applied to the column was prepared from M. morganii grown in YP broth containing 0.2% L-histidine at 7 C for 24 h under aerobic stationary conditions., histidine decarboxylase activity;, absorbance at 280 nm. The figure presents the result of a single analysis. extract powder [Difco] in distilled water at ph 6.2) supplemented with 0.2% L-histidine. Preparation of cell-free extracts. The bacterial strain was usually grown under static conditions for 24 h in 200 ml of SWYP or YP broth in a 500-ml Erlenmeyer flask. The cultures at 4 and 7 C were grown for 9 and days, respectively. Cells were harvested from 800 ml (400 ml in the case of M. morganii) of the culture by centrifugation at,000 g at 4 C for 5 min and suspended in 7 ml of 0. M acetate buffer adjusted to ph 6.2. The suspended cells were lysed in an ice bath with an ultrasonic disruptor (model UR-200P, Tomy Seiko, Tokyo, Japan) at an output of 5 (80 W). The cell debris was removed by centrifugation at 7,000 g at 4 C for 0 min. The supernatant fluid (cell-free extract) thus obtained ( 7.5 ml) was used as a source for assay, protein determination, and gel filtration studies. Histidine decarboxylase assay. A mixture of 0. ml of solution in 0.02 M acetate buffer (ph 6.2), 0.5 ml of 0. M acetate buffer (ph 6.2), 0. ml of L-histidine solution (0 mg/ml), and 0. ml of 50 mm dithiothreitol (DTT) or distilled water was incubated at 25 C for h. The reaction was terminated after heating in a boiling water bath for 5 min. A blank assay solution was prepared by adding the histidine solution after boiling. The histamine formed was determined by the method of Taylor et al. (2). One unit was defined as the amount of that formed mol of histamine at 25 C inh. Protein determination. Quantitative determination of protein was performed by the method of Lowry et al. (). The eluates of protein from a Sepharose CL-6B column were monitored by measuring the absorbance at 280 nm. Gel filtration. The cell-free extracts (7 ml) were loaded onto a Sepharose CL-6B column (95 by 2.64 cm) equilibrated with 0.02 M acetate buffer (ph 6.2) and eluted with the acetate buffer. Dithiothreitol to a final concentration of 5 mm was added to the cell-free extracts and the acetate buffer. Eluates of 7.8 ml each were collected at a flow rate of.6 ml/h. To estimate the molecular mass of the, the column was calibrated with the following protein markers: thyroglobulin (669 kda), ferritin (440 kda), catalase (22 kda), bovine serum albumin (67 kda), chymotrypsinogen A (25 kda), and N-2,4-DNP-L-alanine (255.2 Da). Measurement of histidine decarboxylase activity under various growth and reaction conditions. To measure the effect of various growth temperatures, initial medium phs, and medium NaCl concentrations on histidine decarboxylase formation, the constitutive and inducible s that were isolated and fractionated by gel chromatography were pooled and used for this experiment. (Constitutive is regularly formed under any environment in which the organism is placed in contact with the substrate for which the is specific. Inducible is synthesized by the cell in response to the presence of an inducer, which can also serve as the substrate.) To measure the two activities under various reaction temperatures, phs, and NaCl concentrations, the assay mixtures mentioned in the Histidine decarboxylase assay section were incubated from 0 to C at intervals of 0 C, adjusted with McIlvaine buffer (0. M citrate and 0.2 M phosphate) from ph 5.0 to 8.0 at increments of.0, and supplemented with 0 to 5.0% NaCl at increments of.0%, respectively. These experiments usually were not replicated. RESULTS AND DISCUSSION Histidine decarboxylase activity of P. phosphoreum and M. morganii. Typical elution profiles from a Sepharose CL-6B column of the cell-free extracts from P. phosphoreum and M. morganii are shown in Figures and 2, respectively. In P. phosphoreum (Fig. ), the constitutive activity, which showed the same level in the cultures grown either in the presence or absence of histidine (7), was resolved in fractions 7 to 4 (peak I). The inducible activity, which was practically absent in the culture grown without histidine (7), was resolved in fractions 47 to 5 (peak II). Other activity was resolved in fractions 67

3 78 MORII AND KASAMA J. Food Prot., Vol. 67, No. 8 TABLE. The effects of growth temperature on the formation of constitutive and inducible histidine decarboxylases from Photobacterium phosphoreum a Temperature ( C) Incubation period (days) Protein content (mg) Cell-free extract Total (U) Specific activity (U/mg) % activity of Constitutive Inducible recovery a The data are the result of a single analysis., histidine decarboxylase. to 75 (peak III), but it was found not to be an source on the basis of the molecular mass described below. Moreover, the eluates of peak III contained intracellular histamine and other substances (the difference between the intracellular histamine levels determined by the assay and the blank assay). The elution volume corresponded to a molecular mass of about 700 kda in peak I, about 07 kda in peak II, and less than Da in peak III. In M. morganii (Fig. 2), histidine decarboxylase activity was resolved only in fractions 4 to 5, which was almost the same as peak II in P. phosphoreum. The activity was practically absent in the culture grown in the absence of histidine, suggesting that it is inducible activity. On the other hand, other activity was resolved in fractions 69 to 75, which was almost the same as peak III in P. phosphoreum, suggesting that the activity was not an source. Histidine decarboxylase activity of P. phosphoreum grown under various growth conditions. Protein content, total and specific activities of histidine decarboxylase, and percent activity of the constitutive and inducible s of the cell-free extracts from P. phosphoreum grown at various temperatures, initial medium phs, and medium NaCl concentrations are shown in Tables through, respectively. In the cultures grown at various temperatures (Table ), the specific activity of histidine decarboxylase of the cell-free extracts was extremely high for the 7 C culture, medium for 5 to 25 C cultures, and low for 4 and 27 C cultures. The temperature for maximizing growth was 25 C as previously described (6), suggesting that the highest activity of histidine decarboxylase of the cell-free extracts was obtained when the culture was grown at a low temperature rather than the optimum temperature. This suggestion is also supported from the results obtained for the washed cell suspension from other bacteria. Gale (4), for example, reported that amino acid decarboxylases of the washed cell suspension from Escherichia coli were most active at growth temperatures of 20 to 26 C. The maximal activity of histidine decarboxylase of the washed cell suspension from M. morganii (2, 8 0) and Photobacterium strains (P. histaminum and P. phosphoreum) (2) was also obtained at growth temperatures of 20 to 25 C and 5 to 20 C, respectively. In fact, P. phosphoreum was the same bacterial species as that used in this study. However, the results obtained differed from our results. The difference between the two results could be in the histamine formation by the cell-free extracts and the washed cell suspension. On the other hand, percent activity of the constitutive and inducible s (Table ) mostly accounted for the inducible in 7 to 25 C cultures (8 to 9%). Percent activity of the inducible lowered in 4 C (6%) and 27 C cultures (7%). These results did not change even if the low recovery of histidine decarboxylase activity achieved by gel filtration was taken into account, because the activity lost by gel filtration originated in the constitutive activity (7). Besides, growth rate was very low at 4 C and growth was poor at 27 C, considering incubation time and protein content of the culture, respectively. The results from specific and percent activities suggest that histidine decarboxylase of P. phosphoreum is actively induced at growth temperatures of 7 to 25 C, incubation being the highest at 7 C, which is important when considering formation of histamine in scombroid fish stored at low temperatures. In cultures grown in medium at various initial phs (Table 2), the specific activity of histidine decarboxylase in the cell-free extracts was highest for the culture grown at ph 6.0. The activity decreased abruptly with a decrease from ph 6.0 (8% of the maximal activity in ph 5.0 culture) and gradually with an increase from ph 6.0 (29% of the maximal activity in ph 8.0 culture). Allin () concluded that during growth of E. coli and Salmonella, an acid medium stimulates the formation of histidine decarboxylase of the washed cell suspension. Ienistea (5) found, in a study with a washed cell suspension, that the optimal ph for histidine decarboxylase induction in most bacteria species is between 5.0 and 5.5. Very little histidine decarboxylase activity was noted when the culture was grown at ph 8.5. Similar results also have been obtained for the histidine decarboxylase activity of the washed cell suspension from M. morganii (2, 8) and Photobacterium strains (2). Additionally, in the Photobacterium strains, the histidine decarboxylase activity was highest at ph 4.5 (2). These results obtained from the washed cell suspension were similar to those obtained from the cell-free extracts in this study. However, the maximal activity of histidine decarboxylase in the washed cell sus-

4 J. Food Prot., Vol. 67, No. 8 HISTIDINE DECARBOXYLASE FORMATION BY P. PHOSPHOREUM 79 TABLE 2. The effects of initial medium ph level on the formation of constitutive and inducible histidine decarboxylases from Photobacterium phosphoreum a Cell-free extract % activity of ph Protein content (mg) Total (U) Specific activity (U/mg) Constitutive Inducible recovery a The data are the result of a single analysis., histidine decarboxylase. pension was obtained at ph levels in which growth was inhibited, suggesting the protective action theory postulated by Koessler et al. (). This theory deals with the induction of certain s whose end products remedy the low ph microenvironmental condition, which threatens the microorganisms. However, the highest activity of histidine decarboxylase in cell-free extracts (this study) was obtained at the ph level where growth was best. The ph of fresh scombroid fish generally ranges from 5.5 to 6.5 (8 20). The storage of scombroid fish at low temperatures could therefore enhance the production of histamine by P. phosphoreum. For example, the concentration of histamine formed from P. phosphoreum strains in Maller s basal medium (ph 6.0) with % histidine increased with time and reached values ranging from 5. to 46.0 mg/00 ml after 6 days of incubation at 0 C (4). On the other hand, percent activity of the constitutive and inducible s (Table 2) increased for the constitutive in more acidity (below ph 5.5) and for the inducible in more alkalinity (above ph 7.5). However, it is unclear whether these facts correlate with those described below (Fig. 4), in which the relative activity of histidine decarboxylase was higher for the constitutive in more acidity and for the inducible in more alkalinity. In the cultures grown at various NaCl concentrations in the medium (Table ), the specific activity of histidine decarboxylase in the cell-free extracts was higher in 5% NaCl culture than in and % cultures. Growth was poor in 5% NaCl and optimum in % NaCl (6). However, percent activity of the inducible increased rapidly with the increase of NaCl concentration from 6% activity in % NaCl culture to 97% activity in 5% NaCl culture. These facts suggest that the high concentration of NaCl promotes the induction of histidine decarboxylase. The storage of scombroid fish in ice might therefore inhibit the formation of histidine decarboxylase by P. phosphoreum. However, the histamine content was at a maximum value of 0.8 and.9 mg/00 g in the outer muscle of mackerel during 24 days of storage in ice and at the temperature of ice, respectively, although luminous bacterial counts were below 0 2 CFU/cm 2 in the skin of mackerel during 24 days of storage in both ice and at the temperature of ice. In addition, the recovery rate of histidine decarboxylase from the cell-free extracts applied to the column (Table ) was about 85% in all cultures grown in different NaCl concentrations. However, the recovery rate was about % in the cultures grown in the medium with seawater instead of NaCl solution (see Tables and 2), perhaps because the constitutive was stabilized in the culture grown in the NaCl solution, although the reason for this is unclear. Histidine decarboxylase activity of P. phosphoreum under various reaction conditions. The relative activities of the constitutive and inducible s of P. phosphoreum under various reaction temperatures, phs, and NaCl concentrations are shown in Figures through 5, respectively. The histidine decarboxylase activity was optimum at 0 C for the inducible and at 40 C for the constitutive and decreased rapidly on both sides of the TABLE. The effects of medium NaCl concentration on the formation of constitutive and inducible histidine decarboxylases from Photobacterium phosphoreum a Cell-free extract % activity of NaCl Protein content (mg) Total (U) Specific activity (U/mg) Constitutive Inducible recovery a The data are the result of a single analysis., histidine decarboxylase.

5 740 MORII AND KASAMA J. Food Prot., Vol. 67, No. 8 FIGURE. The effects of reaction temperatures on the activity of constitutive and inducible histidine decarboxylases from Photobacterium phosphoreum., constitutive histidine decarboxylase;, inducible histidine decarboxylase. The figure presents the result of a single analysis. FIGURE 4. The effects of reaction ph levels on the activity of constitutive and inducible histidine decarboxylases from Photobacterium phosphoreum., constitutive histidine decarboxylase;, inducible histidine decarboxylase. The figure presents the result of a single analysis. optimum value for the two s (Fig. ); that is, the temperature dependence of the differed between the two s. Growth was very poor at 0 C and did not occur at 40 C (6). The drop in activity was probably the result of histidine decarboxylase thermolability. Gale () demonstrated a general decline in activity for most bacterial amino acid decarboxylases in the washed cell suspension at reaction temperatures above 40 C. Similar results have also been reported for histidine decarboxylase in the washed cell suspensions of M. morganii (), P. histaminum (2), and P. phosphoreum (2, 6). Under different phs in the reaction mixture (Fig. 4), histidine decarboxylase activity was optimum at ph 6.0 for the constitutive and at ph 6.5 for the inducible and decreased suddenly on both sides of the optimum value for the two s; that is, ph dependence differed between the two s. These results in the two activities were similar to those in the histidine decarboxylase activities in the washed cell suspensions of M. morganii (2, 7), Proteus vulgaris (7), P. histaminum (2), and P. phosphoreum (2, 6). Under different NaCl concentrations in the reaction mixture (Fig. 5), the constitutive and inducible activities were highest in 0% NaCl concentration and decreased gradually with the increase in NaCl concentration; that is, the salt dependence of the was almost the same between the two s. Fresh scombroid fish during storage at low temperatures could therefore have enhanced production of histamine by the mutual reaction of the two s after the cells are autolyzed. Additionally, salt dependence of the two s in the cell-free extracts was almost the same as that of the whole histidine decarboxylase in the cell-free extracts (6) but was entirely different from that of the whole in the washed cell suspension (6). The histidine decarboxylase activity was higher in the inducible than in the constitutive at 0 C incubation (Fig. ), suggesting that the inducible, unlike the constitutive, might function at temperatures below zero. Moreover, activity was not detected at 50 C for the inducible but was detected at C for the constitutive. Activity was detected at ph 5.0 for the constitutive but not the inducible and at ph 8.0 for the inducible but not the constitutive (Fig. 4). These results suggest that the sum of these two s extends the range of reaction temperature and ph for histidine decarboxylase activity. In scombroid fish during storage at low temperature, P. phos- FIGURE 5. The effects of reaction NaCl concentrations on the activity of constitutive and inducible histidine decarboxylases from Photobacterium phosphoreum., constitutive histidine decarboxylase;, inducible histidine decarboxylase. The figure presents the result of a single analysis.

6 J. Food Prot., Vol. 67, No. 8 HISTIDINE DECARBOXYLASE FORMATION BY P. PHOSPHOREUM 74 phoreum histidine decarboxylase activity could therefore take effect because of its ability to occur in a wide range of environmental situations after bacterial cells are autolyzed. The effects of various reaction temperatures and phs on the constitutive activities of cell-free extracts (this study) were almost the same as those on the whole activity of the cell-free extracts (6); that is, as far as reaction temperature and ph in the reaction mixture are concerned, constitutive activity reflects the whole activity, although the reason for this is not clear. On the other hand, the effects of various NaCl concentrations in the reaction mixture on the constitutive and inducible activities of the cell-free extracts (this study) were almost the same as those of NaCl concentrations in the reaction mixture on the whole activity of the cell-free extracts (6). These results suggest that, with respect to the effect of NaCl concentration in the reaction mixture, the constitutive activity reflects the whole activity, considering the effects of reaction temperature and ph on the constitutive activity as mentioned before. This study suggests that histidine decarboxylase of P. phosphoreum is maximally induced at a growth temperature of 7 C. Previous articles (4, 5) reported that luminous bacterial counts and histamine content in scombroid fish were higher in storage at 0 C than in ice. However, this study suggests that the high concentration of NaCl in the medium promotes the induction of histidine decarboxylase by P. phosphoreum. In a previous study (9), histamine content was at a maximal value of.8 and 2.4 mg/00 g in the ventral muscle of mackerel during 24 days of storage in ice and at the temperature of ice, respectively. Moreover, luminous bacteria were detected in the muscle of mackerel during 24 days of storage at the temperature of ice, but not in those stored in ice. These results suggest that it is better to store scombroid fish in ice and freshwater as quickly as possible after fishing and throughout distribution in order to keep histamine levels low. The effects of various reaction conditions on histamine formation were different between the washed cell suspension and the cell-free extracts of P. phosphoreum (6). The histamine-forming ability of the washed cell suspension and the cell-free extracts varied with culture age (6, 7) and growth and reaction conditions such as oxygen tension (6, 7), temperature (6, this study), ph (6, this study), and NaCl concentration (6, this study). Histidine decarboxylase of the cell-free extracts was composed of the constitutive and inducible s, and the temperature and ph dependences of the were different between the two s (7, this study). Moreover, percent activity of the constitutive and inducible s varied with various growth conditions. As previously described, the ability and the process of histamine formation by P. phosphoreum varied with various environmental conditions. P. phosphoreum, if given the opportunity to grow and given the conditions to form high levels of histamine, can cause histamine accumulation to toxic levels. Kimata and Kawai (9) have reported that mackerel fillets produced 54 and 50 to 70 mg/00 g of histamine during storage at 7 C for 75 h and at 6 to 7 C for 50 to 200 h, respectively. Therefore, a completely clear understanding of histamine formation by P. phosphoreum is needed. It is hoped that this type of study will be continued in the future and that the results of this study will be used for predicting histamine toxicity from fresh scombroid fish during storage at low temperatures. REFERENCES. Allin, K Bacterial production and destruction of histamine in vitro. 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