Galactosidase Gene of Thermus thermophilus KNOUC 112 Isolated from Hot Springs of a Volcanic Area in New Zealand : Identification of the Bacteria , Cloning and Expression of the Gene in Escherichia coli *

To isolate the β-galactosidase producing thermophilic bacteria, samples of mud and water were collected from hot springs of avolcanic area near Golden Springs in New Zealand. Among eleven isolated strains, the strain of KNOUC112 produced the highest amounts of β-galactosidase at 40 h incubation time (0.013 unit). This strain was aerobic, asporogenic bacilli, immobile, gram negative, catalase positive, oxidase positive, and pigment producing. Optimum growth was at 70-72°C, pH 7.0-7.2, and it could grow in the presence of 3% NaCl. The main fatty acids of cell components were iso-15:0 (30.26%), and iso-17:0 (31.31%). Based on morphological and biochemical properties and fatty acid composition, the strain could be identified as genus Thermus, and finally as Thermus thermophilus by phylogenetic analysis based on 16S rRNA sequence. So the strain is designated as Thermus thermophilus KNOUC112. A gene from Thermus thermophilus KNOUC112 encoding β-galactosidase was amplified by PCR using redundancy primers prepared based on the structure of β-galactosidase gene of Thermus sp. A4 and Thermus sp. strain T2, cloned and expressed in E. coli JM109 DE3. The gene of Thermus thermophilus KNOUC112 β-galactosidase(KNOUC112β-gal) consisted of a 1,938 bp open reading frame, encoding a protein of 73 kDa that was composed of 645 amino acids. KNOUC112β-gal was expressed as dimer and trimer in E. coli JM109 (DE3) via pET-5b. (Asian-Aust. J. Anim. Sci. 2004. Vol 17, No. 11 : 1591-1598)


INTRODUCTION
Many thermophiles, whose optima temperature for growth 45-80°C, are present in hot springs as well as other thermal environments (Campbell and Pace, 1968).These organisms offer some major advantages for industrial and biotechnological process.Many of which reproduce rapidly and efficiently at high temperatures, their enzymes are capable of catalyzing biochemical reactions at high temperatures and are generally more stable than enzyme from mesophlies, thus self-life of the enzyme can be longer.The enzymes stable at high temperature generally represent high stability not only to heat, but also to other protein denaturants such as detergent and organic solvents.Because of those properties mentioned above, thermostable enzymes are utilized extensively in industry (Thomas and William, 1986) β-Galactosidase (EC 3.2.1.23)catalyzes not only hydrolysis of β-D-galactopyranosides, such as lactose, but also a trans-galactosylation reaction that produces galactooligosaccharides (Onishi and Tanaka, 1995).The enzyme is useful in dairy industry for prevention of lactose crystallization in frozen and condensed milk products, for decreasing water pollution caused by whey from cheese production, and for remedy of lactose intolerance (Kern and Struthers Jr., 1966).Galacto-oligosaccharides, enzymatic trans-galactosylation reaction products from lactose, have become of interest for human health.Galactooligosaccharides are recognized as a growth-promoting factor for intestinal bifido bacteria (Ohtsuka et al., 1989), which are helpful for maintenance of human health (Hughes and Hooner, 1991).Thermostable β-galactosidase can be a good tool for the efficient use of lactose and for the difficulties caused by lactose.Many β-galactosidases from microbial sources have been reported (Dumortier et al., 1994;Brady et al., 1995;Choi et al., 1995;Berger et al., 1997), but studies on the thermostable β-galactosidase is not much.From the late of 1990', gene of highly thermostable β-galactosidase began to be reported, and Ohtsu et al. (1998) and Vian et al. (1998) purified and characterized a thermostable β-galactosidase from Thermus sp.A4 and Thermus sp.strain T2 respectively.Still more research is required to find better thermostable β-galactosidase.
Accordingly, in this study a β-galactosidase producing thermophilic bacteria from hot spring was isolated and identified.And the β-galactosidase gene of the bacteria was

MATERIALS AND METHODS
Isolation of β-galactosidase producing thermophilic bacteria β-Galactosidase producing thermophilic microorganisms were isolated from mud and water collected near by Golden Springs in New Zealand, as described below.To cultivate the thermophilic microorganisms, 1 g of mud sample or 1 ml of water sample from hot spring was added to the medium of ATCC 1598 (Alfredsson et al., 1985) and incubated at 70°C aerobically by shaking (200 rpm) for 48 h.One liter of this medium was composed of Bacto-tryptone, 2.5 g; yeast extract, 2.5 g; nitrilotriacetic acid, 100.0 mg; CaSO 4 To screen β-galactosidase producing bacteria, the cultures were streaked onto ATCC 1598 solid medium containing 1% (v/w) lactose and 0.02% (v/w) 5-bromo-4chloro-3-indolyl-β-D-galactopyranosidase (X-gal).After incubation at 70°C for 48 h, blue colonies were selected, then cultivated in ATCC 1598 liquid medium for identification and testing β-galactosidase activity.

Assay of β-galactosidase activity
Microorganisms were harvested by centrifugation at 8,000×g for 10 min at 4°C, suspended in sodium phosphate buffer (10 mM, pH 6.8), washed 2 times by the same buffer and sonificated 5 times of 30 sec (100 Hz).Cell debris was eliminated by centrifugation (12,000×g) for 20 min at 4°C.
The cell free extract was used for the assay of βgalactosidase activity using o-nitrophenyl β-Dgalactopyranoside (ONPG) as substrate (Craven et al., 1965).β-Galactosidase activity was determined by release of o-nitrophenol from 0.04 M ONPG dissolved in sodium phosphate buffer (100 mM, pH 6.8).An aliquot of cell free extract (0.5 ml) was added to 2.5 ml of ONPG solution and incubated at 70°C for 10 min.The reaction was stopped by addition of 3 ml of Na 2 CO 3 (0.5 M), and the absorbance at 420 nm was measured.One unit of enzyme activity is defined as the activity hydrolyzing 1 µmol of ONPG per min by cell free extract from 1 ml of culture.

Identification of β-galactosidase-producing bacteria
Morphology and physiological characteristics : The bacterial strain showing the highest β-galactosidase activity was selected, then tested for Gram staining, morphological, biochemical, and physiological properties.The morphology of the isolated bacteria was observed under scanning electron microscope (SEM).Catalase reaction, oxidase reaction, NaCl tolerance, casein hydrolysis, starch hydrolysis, gelatin liquefaction, H 2 S production, citrate utilization, urease production, ONPG hydrolysis, indole production, nitrate reduction, and acid production from carbohydrates were tested.Most tests were examined as applied by Santos et al. (1989) and Manaia and da Costa (1991).The pigment producing property was determined by carotenoide production (Brock and Brock, 1967).All biochemical and physiological tests were done at 70°C for 48 h.
Composition of cellular fatty acid : Fatty acid methyl esters were obtained from fresh wet biomass by saponification, methylation and extraction as described by Kuykendall et al. (1988).The fatty acid methly esters were separated by using a model 5890 GC system (Hewlett Packard) equipped with a flame ionization detector fitted with a 5% phenyl methyl silicone capillary column (0.2 mm ×25 m).The carrier gas was H 2 of high-purity, the column head pressure was 60 kPa, the septum purge was 5 ml/min, the column split ratio was 55:1 and the injection port temperature was 300°C.The temperature program was run from 170 to 270°C at the rate of 5°C/min.The numerical analysis of fatty acid methyl esters and fatty acid profiles were performed by using the standard MIS Library Generation Software (Microbial ID Inc.).
16S rRNA sequence determination and phylogenic analysis : Isolation of genomic DNA, PCR amplification of the 16S rRNA gene and sequencing of the purified PCR products were carried out as described by Rainey et al. (1996).Universal primers of fD1 (5'agagtttgatcctggctcag3') and rD1 (5'acggctaccttgttaccactt3') were used to amplify the gene of 16S rRNA.Sequence reaction products were purified by ethanol precipitation and electrophoresis with a model 377 Genetic Analyzer (Perkin-Elmer, USA.).The 16S rRNA sequences obtained in this study were aligned against the previously determined sequences of the genus of Meiothermus/Thermus sequences available from the Ribosomal Database Project (Maidak et al., 1996).The evolutionary tree for the data set was inferred using the neighbor-joining method (Saitou and Nei, 1987).The PHYLIP package (Felsentein, 1993) was used for constructing the tree.

Isolation, cloning and sequence determination of βgalactosidase gene
β-galactosidase gene was amplified by Touch Down PCR in gene cycler (Bio-Rad, Japan).PCR was performed using redundancy primers and chromosomal DNA of the isolated bacteria.Redundancy primers were prepared on the basis of gene structures of β-galactosidase from Thermus sp.A4 (Ohtsu et al., 1998) and Thermus T2 (Vian et al., 1998).
The nucleotide sequence of forward primer was 5'atgYtSggcgtttgYtaYtacc3', and that of reverse primer was 5'tcatgYctcctcccaSacg3'.DNA fragment produed in PCR was isolated by agarose electrophoresis and Geneclean Kit method (Bio101, USA), ligated into pGEM-T Easy (Promega, USA) and cloned in Escherichia coli JM109.Luria-Bertani (LB) medium was used for culturing E. coli JM109.Ampicilline was added at the concentration of 100 µg/ml to screen E. coli JM109 transformed by pGEM-T Easy harboring the DNA fragment.Sequence of the DNA fragment was determined using Big Dye Automatic sequencer ABI377 (Perkin Elmer, USA) and PE9600 Thermocycler (Perkin Elmer, USA).The DNA and amino acids sequence analysis was performed by the DNASIS software system.The homology search was done using the World Wide Web server from BLAST search maintained at National Center for Biotechnology Information (NCBI).

Expression of β-galactosidase gene
Expression plasmid was constructed by inserting the isolated gene into pET-5b (Promega, USA) regulated by LacUV5 promoter.E. coli JM109 (DE3) was transformed with the pET-5b harboring the gene and cultured on LB solid medium containing ampicillin (100 µg/ml) at 37°C.After heating colonies formed on the LB solid medium at 70°C for 3 h, Z-buffer (Ausubel et al., 1998) containing 1.4 mg of X-gal/ml was poured over those colonies and those colonies were incubated 3 h at 70°C.Blue colony was picked and used to confirm the expression of βgalactosidase gene.The confirmation of expression was performed by cell free extracts ONPG hydrolysis at 70°C and by X-gal hydrolysis of native PAGE gel soaked and incubated at 70°C in Z-buffer of X-gal (1.4 mg/ml).

Electrophoresis
Observation of DNA and protein was performed by agarose electrophoresis and polyacrylamide gel electrophoresis (PAGE) respectively.Agarose electrophoresis was done on 1% agarose gel.SDS polyacrylamide gel electrophoresis was performed by the method of Laemmli (1970) on 7.5% polyacrylamide slab gel, and native polyacrylamide gel electrophoresis was done on the same way with that of SDS polyacrylamide gel electrophoresis expect without addition of SDS and without denaturation of sample before electrophoresis.

Isolation of β-galactosidase-producing thermophilic bacteria
β-galactosidase-producing bacterial strains were isolated from 11 samples of mud and water collected from 11 hot springs of 4 regions near by Golden springs in New Zealand (Table 1).The temperatures of those hot springs were 50 to 90°C, where thermophilic organisms may highly predominate.The range of sample pH was from 3.5 to 10. From each of 11 samples, a colony of blue color showing hydrolysis of X-gal on ATCC 1598 solid medium was isolated.Those 11 strains were tested for β-galactosidase activity at 40 h incubation time using ONPG as substrate.Among 11 strain, the strain of KNOUC112 (0.013 unit) isolated from a hot spring of Orakeikorako was selected due to its highest β-galactosidase activity.

Identification of β-galactosidase-producing thermophilic bacteria
Morphological and biochemical characteristics : The morphological and biochemical characteristics of the strain KNOUC112 were summarized in Table 2.The strain KNOUC 112 was found to be a Gram-negative, non-motile, non-spore forming bacteria, and it formed yellowpigmented colonies.As shown in Figure 1, it was rodshaped (0.2×2.0~3.5 µm).The optimum growth temperature was about 70~72°C and growth did not occur below 45°C  Alfredsson et al. (1985) reported that most thermophilic bacteria utilized many kinds of monosaccahide, pyruvate and glutamate, and hydrolyzed casein.All strains of genus Thermus formed yellow-pigmented colonies, were Gram-negative rods and not motile, did not from spores, and their optimum growth temperatures were about 70°C (Brock and Brock, 1967).T. thermophilus could be distinguished from all other species of the genus Thermus by their ability to grow at temperatures above 80°C (Degryse et al., 1978) and in medium containing 2-3% NaCl (Manaia and da Costa, 1991).The strain KNOUC112 showed same properties with those of genus Thermus reported by Brock(1984) for morphology and optimum growth temperature, and it coincides with Thermus thermophilus reported by Mania and da Costa (1991) in growth above 80°C and in the medium containing NaCl of 3%.The carbon-source utilization of KNOUC112 also closely resembles those of other Thermus strains (Degryse et al., 1978) with the only difference of utilizing malate as a single carbon source by this isolate.
Therefore, on the basis of its morphological, biochemical and physiological characteristics, strain KNOUC 112 was identified as Thermus thermophilus, and the strain is named as T. thermophilus KNOUC112.et al., 1998) and Thermus sp.strain T2 (Gene bank accession No. g2765752, Vian et al., 1998) respectively whose genes were referred for the preparation of redundancy primers used to amplify the β-galactosidase gene of KNOUC112 in PCR.KNOUC112β-gal also has high identity of 90% and 74% with those of Thermus sp., Thermus sp.IB-21 (Gene bank accession No. g25989599) and Thermus brockianus (Gene bank accession No. g4928634) respectively.All Thermus spp.above have βgalactosidase of 645 a.a, but the β-galactosidases of Pyrococcus woesei (Dabrowski et al., 2000), Bacillus stearothermophilus (Gene bank accession No. g80200), Thermotoga maritime (Moore et al., 1994) and E. coli (Jacobson, 1994) are composed of 510, 672, 1,037, 1,023 a.a respectively that are much different with those of Thermus thermophilus KNOUC112 and other Thermus spp.

Expression of β-galactosidase gene
Colony of E. coli JM109 (DE3) transformed by pET-5b carrying the KNOUC112β-gal hydrolyzed X-gal in solid LB medium at 70°C, and it's β-galactosidase activity of hydrolyzing ONPG was detected in the cell free extract of E. coli JM109(DE3) showing that the β-galactosidase was produced intracellularily.In the gel of SDS-PAGE, there was a new protein of about 73 kDa (Figure 4-C line) fitting with the deduced molecular weight by the nucleotide sequence of KNOUC112β-gal.In native PAGE, the cell free extract of E. coli having KNOUC112β-gal showed no protein of 73 kDa (Figure 5-C line), but of about 140 kDa (dimmer) and about 210 kDa (trimer).The dimmer and trimer showed activity of X-gal hydrolysis (Figure 5-D line), and the activity of dimer was much stronger than that of trimer.It means that KNOUC112β-gal was expressed as active form of dimer and trimer in E. coli JM109 DE3, and the dimer may be the main product of KNOUC112β-gal.E. coli β-galactosidase (Wickson and Huber, 1970) and Kluyveromyces fragilis β-galactosidase (Kulikova et al., 1972) had activity when they were tetramer.Monomer and dimer of Streptococcus lactis 7962 β-galactosidase were active (Mcfeters et al., 1969).
Thermus thermophilus KNOUC112, in 40 h incubation β-Galactosidase Gene of Thermus thermophilus KNOUC112 Isolated from Hot Springs of a Volcanic Area in New Zealand: Identification of the Bacteria, Cloning and Expression of the Gene in Escherichia coli * cloned and expressed in E. coli.
β-galactosidase gene DNA fragment of about 2 kb was produced by PCR.The fragment was a β-galactosidase gene consisting of a 1,938 bp open reading frame, encoding a protein of 645 amino acids.The deduced molecular weight of the protein is 72,896 dalton.The nucleotide sequence of the DNA fragment named as KNOUC112β-gal and it's deduced amino acid sequence are shown in Figure 3. KNOUC112βgal has high homology of 99% and 83% with those of Thermus sp.A4 (Gene bank accession No. g3157465, Ohtsu

Figure 2 .
Figure 2. Phylogenetic tree based on 16S rRNA sequences showing the positions of strain KNOUC112, the type strains of Thermus species and the representatives of some other related taxa.Scale bar represents 0.01 substitution per nucleotide position.

Table 1 .
Origin of strains isolated near by Golden springs in New Zealand

Table 3 .
Composition of cellular fatty acids of the strain KNOUC112 Fatty acid

Table 2 .
Morphological and physiological characteristics of strain

Table 4 .
Levels of 16S rRNA similarity for strain KNOUC 112, the type strains of Thermus species and representatives of some related