Characterization of Alkaline Phosphatase Producing Bacteria Isolated from Thai Fermented Fish Products

Seventeen isolates of alkaline phosphatase (ALP) producing bacteria were screened and systematically studied. They were divided into 10 groups on the basis of their phenotypic characteristics and 16S rRNA gene sequence analyses. The Gram-positive coccal isolates in Group I (3 isolates), Group II (3 isolates) and Group III (1 isolate) showed 99.2%, 99.5-99.7% and 99.8% sequences similarity to Staphylococcus saprophyticus, S. napalensis and S. sciuri, respectively. Each Gram-positive rod-shaped isolates in Group IV and V belonged to the genus Bacillus and was closely related to B. vietnamensis and B. safensis with 99.2% and 99.4% sequences similarity, respectively. The Gram-positive, moderately halophilic rod-shaped bacteria in Group VI (3 isolates), Group VII (1 isolate), Group VIII (1 isolate) and Group IX (2 isolates) were closely related to Virgibacillus halodenitrificans (98.5-99.1%), Oceanobacillus iheyensis (99.3%), Halobacillus mangrove (97.9%) and H. dabanensis (98.5-98.6%), respectively. One Gram-negative isolate of moderately halophilic bacterium (Group X) was closely related to Idiomarina zobelli (98.0%). They possessed phosphatase activities ranged from 10.08-70.96 U ml-. The isolate NSW 13-2 (Group VI) showed the highest ALP.


Introduction
Alkaline phosphatase (ALP) is involved in removing of 5'-and 3'-phosphate groups from DNA, RNA and nucleotides.It can also remove phosphate groups from proteins.This enzyme can be widely found in human tissues, calf, shrimp as well as bacteria.Besides its effect on human health problem, ALP are usefully applied in various fields such as molecular research tool as well as enzyme immunoassays especially bacterial alkaline phosphatase (Dong & Zeikus, 1997;Sun et al., 2007).),milk industry for validation of milk pasteurization (Rankin et al., 2010), cosmetics as a substance for the regeneration and metabolism of cells, environment for monitoring herbicides or pesticides (Muginova et al., 2007), clinical diagnosis as a marker and medicine such as calf intestinal alkaline phosphates, a therapeutic drug for lipopolysaccharide-mediated diseases (Beumer et al., 2003).Despite its wide application, ALP with greater activity or novel properties suitable for commercial applications is still required such as ALP which is active under the high salt concentration and high thermal stability or storage stability.
Halophilic bacteria play important role in Thai traditional fermented fish (pla-ra) (Chamroensaksri et al., 2008).In the course of our study on biodiversity of halophilic bacteria in fermented foods, these bacteria are isolated and should be the promising source of bacterial ALP with interesting properties.This work deals with the screening and characterization the halophillic bacteria isolated from Thai fermented fish and salted fish samples that able to produce ALP.

Screening of Alkaline Phosphatase (ALP) Producing Bacteria
The isolates were screened by cultivating on Heart Infusion agar (Difco) containing 0.01 % phenolphthalein bisphosphate tetrasodium salt (Sigma) and 10% NaCl (w/v) as the method descdribed by Barber & Kuper (1951).After incubation at 37 °C for 1-2 days, all pink colonies were selected as potential ALP-producing strains.All selected isolates were separately confirmed for their abilities to produce ALP with the following procedure; a loopfull of the selected ALP-producing strain was inoculated into 5 ml of JCM no.377 broth and incubated on a rotary shaker at 37 o C (150 rpm) for 24 h for the seed culture.The seed culture broth (0.5 ml) was transferred into 50 ml of modified JCM No. 377 broth in 250 ml Erlenmeyer flask (duplicate) and incubated as above conditions.Supernatant obtained after centrifugation of the cultures at 10,000 rpm (13,300 g), 4 o C for 10 min was used as crude enzyme for ALP activity detection.
ALP activity assay was done by method described by Helianti et al. (2007).Reaction mixture composing of 1.0 ml of 10 mM p-nitrophenylphosphate (pNPP) (Sigma) in 0.2 M Tris-HCl buffer pH 10.0 with 5 mM MgCl 2 , and 0.1 ml of the crude enzyme was incubated at 37 o C for 15 min.The reaction was stopped with 1 ml of 1M NaOH and its absorbance was measured at 405 nm.One unit of alkaline phosphatase (ALP) was defined as the amount of the enzyme yielding 1 micromole of p-nitrophenol within 1 minute per milligram protein under the assay conditions.The protein concentration was estimated by Lowry method (Lowry et al., 1951) using bovine serum albumin as the standard.

16S rRNA Gene Sequence and Phylogenetic Analysis
The 16S rDNA fragments were amplified with 357R, 802R, BF1 and BR1 primers.After purification the PCR products were sequenced using BigDye Terminator Cycle Sequencing Ready Reaction kit (ver.3.0: Applied Biosystems) in the ABI PRISM 310 Genetic analyzer (Applied Biosystems, USA) The 16S rRNA gene sequence were subsequently aligned along with the selected sequences obtained from the GenBank/EMBL/DDBJ databases by using program CLUSTAL X (version 1.81) (Thompson et al., 1997).Gaps and ambiguous bases were eliminated from the calculations.The phylogenetic tree was constructed by using the neighbor-joining method (Saitou & Nei, 1987) in the MEGA program version 4 (Tamura et al., 2007).The confidence values of branches of the phylogenetic tree were determined using bootstrap analyses (Felsenstein, 1985) based on 1,000 re-samplings.

Identification and Characterization of ALP-producing Isolates
Seventeen isolates of ALP-producing bacteria were divided into 10 groups based on their 16S rRNA gene sequence analyses and phenotypic characteristics.Seven isolates (Group I, II and III) belonged to the genus Staphylococcus were Gram-positive non-motile and non-spore forming cocci.They were catalase-positive but oxidase-negative.Two isolates in Group IV and V belonged to the genus Bacillus were Gram-positive spore forming rods.The moderately halophilic bacteria in Group VI (3 isolates belonged to the genus Virgibacillus), Group VII (1 isolate belonged to the genus Oceanobacillus), Group VIII (1isolate) and Group IX (2 isolates) belonged to the genus Halobacillus, were Gram-positive, spore forming rods.They produced catalase and most of them were oxidase negative.All isolates contained meso-diaminopimelic acid in the cell wall.They showed the difference in the colonial pigmentation and the growth in high concentration of NaCl (w/v).One isolate of moderately halophilic bacterium in Group X belonged to genus Idiomarina was a Gram-negative non-spore forming rod shaped bacterium.All the isolates in each group were negative reaction for citrate utilization, indole and tyrosine hydrolysis.The details of their characterization are described below.
Group III contained one isolate, KL6-2.Colonies were circular, low convex, glistening surface and gray white with yellowish (less than 3 mm in diameter).Growth occurred aerobically and anaerobically in the presence of 0-15% NaCl (w/v), at 25-45 o C (optimum at 30-37 o C), at optimum pH 7-8.The isolate was positive for MR, nitrate reduction and hydrolysis of aesculin and gelatin but negative for VP and starch hydrolysis.Acid was produced from D-mannose, D-raffinose, D-ribose, salicin, sucrose and trehalose, but was not from many sugars.The phenotypic characteristics were different from S. sciuri subsp.scuiri DSM 20345 T (Kloss et al., 1976) as shown in Table 2.The isolate KL6-2 showed 99.8% sequence (1457 bp) similarity to S. sciuri subsp.scuiri DSM 20345 T based on 16S rRNA gene (Figure 1).Therefore, it was identified as S. sciuri (Kloss et al., 1976).

Bv
Group VI contained three isolates KL1-1, KL2-4 and NSW 13-2.Colonies were circular to slightly irregular, raised, opaque cream yellow for KL1-1 and NSW 13-2 and cream yellow colour for KL 2-4, (1-2 mm diameter).They grew under aerobic and anaerobic conditions, in NaCl up to 20% (w/v), at 25-45 o C, at pH 7-8 (KL1-1, NSW13-2) and pH 6-8 for KL2-4.They were positive for nitrate reduction and hydrolysis of gelatin but negative for MR-VP, hydrolysis of aesculin and starch.The isolate KL2-4 hydrolyzed Tween 80 but KL1-1 and NSW13-2 did not.Acid was produced from D-glucose, D-ribose, maltose and sucrose.No acid was produced from glycerol, inositol, inulin, D-mannose, D-melezitose, D-melibiose, raffinose, rhamnose, salicin and D-xylose.The variable of acid production from sugars between these three isolates and the closely type strain was shown in Table 3.The isolates KL1-1(1468 bp), KL2-4 (1477 bp) and NSW13-2 (1474 bp) were closely related to V. halodenitrificans DSM 10037 T with 99.1, 98.6 and 99.1% sequence similarity, respectively (Figure 3).On the basis of 16S rRNA gene sequence analyses, these three isolates were placed in the genus Virgibacillus.Their phenotypic characteristics were closed to V. halodenitrificans DSM 10037 T as shown in Table 3.Therefore, they were identified as V. halodenitrificans (Yoon et al., 2004).On the basis of 16S rRNA gene sequence analyses, this isolate was placed in the genus Oceanobacillus.The isolate NSW13-4 (1467 bp) was closely related to O. iheyensis JCM11309 T with 99.3% sequence similarity (Figure 3).The ability to produce acid from sugars of isolate NSW13-4 was variable and was different from Oceanobacillus iheyensis JCM 11309 T (Lu et al., 2001) as shown in Table 3.However, this isolate was identified as O. iheyensis (Lu et al., 2001).
Group VIII contained one isolate, NSW13-3.Colonies of isolate NSW13-3 were entire, circular, low convex and pale yellow colour.Growth occurred aerobically in the presence of 5-20% (w/v) NaCl, at 25-45 o C (optimum at 30-37 o C) and at pH 7-8.It was positive for MR, hydrolysis of gelatin, starch and Tween 80 but negative for VP, nitrate reduction and hydrolysis of aesculin and arginine.No acid was produced from L-arabinose, inulin, D-melezitose, D-melibiose, rhamnose and D-xylose.On the basis of 16S rRNA gene sequence analyses, this isolate was placed in the genus Halobacillus.The isolate NSW13-3 (1472 bp) was closely related to H. mangrovi MS10 T with 97.9% sequence similarity (Figure 3) (Soto-Ramírez et al., 2008).The isolate showed different phenotypic characteristics from Halobacillus mangrovi MS10 T (Table 3) and contained meso-diaminopimelic acid in the cell wall which was absent in Halobacillus mangrovi MS10 T (Soto-Ramírez et al., 2008).Therefore, this isolate was kept unidentified.3. On the basis of 16S rRNA gene sequence analyses, both isolates were placed in the genus Halobacillus, the isolate NSW13-5 (1467 bp) and SMK24-1 (1468 bp) were closely related to H. dabanensis JCM 12772 T with 98.6 and 98.5 % similarity respectively (Figure 3) (Liu et al., 2005).They showed different phenotypic characteristics from H. dabanensis JCM 12772 T (Table 3) and contained meso-diaminopimelic acid which was absent in H. dabanensis JCM 12772 T (Liu et al., 2005).Therefore, these isolates were kept unidentified.
Three isolates, KL1-1, KL2-4 and NSW 13-2 (Group VI) from pla-ra identified as V. halodenitrificans was isolated as mentioned above (Tanasupawat et al., 2010).One isolate, NSW13-4 (Group VII) from pla-ra identified as O. iheyensis was found.Three novel species of the isolates in Group VIII (NSW13-3 from pla-ra) and Group IX (NSW13-5 from pla-ra and SMK24-1 from hoi-dong, fermented mollusces) were closely related to H. mangrovi MS10 T and H. dabanensis JCM12772 T , respectively.One isolate, MK10-1 (Group X) from pla-ra that related to I. zobellii KMM231 T was kept unidentified.The ALP producing bacterial isolates in this study were successfully identified as the genera level, however, the isolates in Group VIII, IX and X are required for further study on their DNA-DNA hybridization to confirm their taxonomic position and to propose them as the novel species.

Conclusion
From 32 fermented fish samples, only 17 isolates were shown to be able to produce extracellular ALP.They were identified as the isolates in the genera Bacillus, Halobacillus, Idiomarina, Oceanobacillus, Staphylococcus and Virgibacillus based on their phenotypic characteristics and 16S rRNA gene sequence analysis.The isolate NSW13-2 identified as V. halodenitrificans isolated from Nakornsawan fermented fish, was provided as a potential ALP-producing strain.A high level of extracellular alkaline phosphatase production was detected at 70.9 U ml -1 .Optimization of ALP production as well as purification and characterization of ALPs from strain NSW13-2 are under study.

Figure 2 .
Figure 2. Phylogenetic relationships of isolates KL2-3, MK10-2 and related taxa based on 16S rRNA gene sequence analysis.The branching pattern was generated by the neighbour-joining method.Bootstrap values (expressed as percentages of 1000 replications) >50% are shown at nodes.Bar, 0.01 substitutions per 100 nt

Figure 4 .
Figure 4. Phylogenetic relationships of isolate MK10-1 and related taxa based on 16S rRNA gene sequence analysis.The branching pattern was generated by the neighbour-joining method.Bootstrap values (expressed as percentages of 1000 replications) >50% are shown at nodes.Bar, 0.01 substitutions per 100 nt

Table 1 .
Isolate number, source, location, nearest relatives, sequence identity (%) and ALP activity of isolates

Table 2 .
Differential phenotypic characteristics of Staphylococcus isolates and related type strains

Table 3 .
Differential phenotypic characteristics of Bacillus, Oceanobacillus, Virgibacillus, Halobacillus and Idiomarina isolates and related type strains