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Cloning of the thermostable phytase gene (phy) from Bacillus sp. DS11 and its overexpression in Escherichia coli

Young-Ok Kim, Jung-Kee Lee, Hyung-Kwoun Kim, Ju-Hyun Yu, Tae-Kwang Oh
DOI: http://dx.doi.org/10.1111/j.1574-6968.1998.tb12997.x 185-191 First published online: 1 May 1998


Phytase hydrolyzes phytate to release inorganic phosphate, which would decrease the addition of phosphorus to feedstuffs for monogastric animals and thus reduce environmental pollution. The gene encoding phytase from Bacillus sp. DS11 was cloned in Escherichia coli and its sequence determined. A 560-bp DNA fragment was used as a probe to screen the genomic library. It was obtained through PCR of Bacillus sp. DS11 chromosomal DNA and two oligonucleotide primers based on N-terminal amino acid sequences of the purified protein and the cyanogen bromide-cleaved 21-kDa fragment. The phy cloned was encoded by a 2.2-kb fragment. This gene comprises 1152 nucleotides and encodes a polypeptide of 383 amino acids with a deduced molecular mass of 41 808 Da. Phytase was produced to 20% content of total soluble proteins in E. coli BL21 (DE3) using the pET22b(+) vector with the inducible T7 promoter. This is the first nucleic sequence report on phytase from a bacterial strain.

Key words
  • Bacillus
  • Phytase
  • Phytate
  • Overexpression

1 Introduction

Phytate (myo-inositol hexakisphosphate) is the major phosphate storage compound in seeds of higher plants, mostly cereals and legumes [1]. Phytase (myo-inositol hexakisphosphate phosphohydrolase) hydrolyzes phytate to myo-inositol and inorganic phosphate. Since the digestive tract of monogastric animals including pigs and poultry lacks phytase, phytate-phosphate in the feed cannot be utilized by these animals and thus inorganic phosphate has to be added to secure a sufficient phosphate supply. As a result, phytate and phosphate are excreted in manure, causing environmental problems in areas of intensive livestock production. In addition, phytate forms complexes with multivalent metal ions such as iron, zinc, calcium and proteins, thereby showing anti-nutritional effects [2, 3]. So, the addition of phytase to feed is needed to enhance phosphate utilization from phytate, thereby reducing inorganic phosphate excretion and increasing the feed value.

Phytase activity in microorganisms has been found most frequently in Aspergillus species including A. terreus[4], A. ficuum[5], and A. niger[6]. It also occurs in bacteria such as Aerobacter aerogenes[7], Pseudomonas sp. [8], Escherichia coli[9], Bacillus subtilis[10, 11]. However, the cloning and sequencing of the phytase gene have been only reported for fungal phytases such as A. niger[1214], A. fumigatus[15], A. terrus, and Myceliophthora thermophila[16] and there have been no reports about bacterial phytase genes until now.

In our previous paper [17], we reported a thermostable phytase from Bacillus sp. DS11 (DS11 phytase), which was purified and biochemically characterized. This enzyme is particularly useful in animal nutrition because the thermal stability is valuable for withstanding inactivation during the feed-pelleting and/or expansion processes. In this report, we cloned and sequenced the phytase gene of Bacillus sp. DS11 to elucidate its primary structure. In addition, we overexpressed phytase in E. coli to increase the production level to cost-effective levels.

2 Materials and methods

2.1 Bacterial strains, plasmids and media

E. coli JM83 and BL21 (DE3) were used as hosts for transformation. Plasmid pUC19 was used as a vector for cloning and nucleotide sequencing, and plasmid pET22b(+) was used for overexpression. E. coli was cultured in LB broth (1% tryptone, 0.5% yeast extract, 0.5% NaCl) or on LB agar plate. When needed, ampicillin was added at a concentration of 100 μg ml−1.

2.2 DNA manipulation and sequencing

Total genomic DNA of Bacillus sp. DS11 was isolated according to Marmur et al. [18], and general recombinant DNA manipulation was carried out according to a published protocol [19]. DNA sequencing was performed with the dideoxy chain termination method [20], using the Sequenase version 2.0 DNA sequencing kit from US Biochemical. The DNA clone was sequenced from both strands by universal and internal synthetic primers. The nucleotide sequence of phy has been submitted to GenBank under accession number U85968.

2.3 Internal amino acid sequence analysis

The purification of phytase from Bacillus sp. DS11 was performed as described previously [17]. To determine the internal amino acid sequence of DS11 phytase, the purified enzyme was fragmented by CNBr in 70% formic acid. The peptide mixture was lyophilized and subjected to SDS-polyacrylamide gel (15%). The CNBr-cleaved fragments on the gel were electrophoretically transferred to a sheet of polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The protein bands were cut and their amino acid sequences were analyzed by the Edman degradation method with an Applied Biosystems model 476A protein/peptide sequencer (Applied Biosystems, CA, USA).

2.4 PCR amplification of partial phy sequence

Degenerate oligonucleotides were synthesized on a Model Expedite 8905 DNA synthesizer (Perseptive Biosystems, USA). A set of primers (1 and 2 in Fig. 1) were used as upstream and downstream primers and the total genomic DNA of Bacillus sp. DS11 was used as the template in a PCR reaction. PCR was carried out using the PCR master containing 2.5 U Taq DNA polymerase (Boehringer Mannheim, Germany) in a PCR machine (Perkin-Elmer Cetus, USA). Amplification was performed in 35 cycles of 1 min at 94°C, 1 min at 50°C, and 1 min at 72°C. The PCR product was purified by gel electrophoresis using the Geneclean II kit (Bio-101), cloned into T vector (Novagen), and sequenced.

Figure 1

Amino acid sequence of the purified phytase and primer design. The amino acid sequences of both N-terminal and internal region of the purified phytase are shown with a peptide map. A set of PCR primers was designed based on the underlined amino acid sequences.

2.5 Southern and colony hybridization

Probe labeling and hybridization were carried out at 42°C using DIG Easy Hyb solution according to the manual supplied with the DIG DNA Labeling and Detection Kit (Boehringer Mannheim, Germany). For Southern hybridization, genomic DNA was digested with appropriate restriction enzymes, separated on a 0.8% agarose gel, and transferred to Nylon membrane, positively charged (Boehringer Mannheim, Germany) by the capillary transfer procedure. The genomic library was screened by colony hybridization. E. coli transformants were transferred to Nylon membrane and lysed with 0.5 M NaOH. The denatured DNA was then immobilized, followed by a protease K treatment. Digoxigenin-labeled PCR fragment probe was used for hybridization.

2.6 Construction of phytase overexpression plasmid

The DNA corresponding to the coding region excluding the putative signal sequence was amplified by PCR. The primer (5′-GAACATATGTCTGATCCTTATCAT-3′) was synthesized based on the sequence near the N-terminus of the mature protein (nt 370–384) and the antisense primer (5′-GCAAAGCTTTTATTTTCCGCTTCT-3′) was designed based on the sequence in the vicinity of the termination codon (nt 1417–1431). The former contained a NdeI site and the latter contained a HindIII site at the 5′ end. The amplified product was double digested with HindIII and NdeI, and cloned into the expression vector pET22b(+) (Novagen) previously digested with the same enzymes. The resulting plasmid was introduced into E. coli BL21 (DE3). And the transformant cells were grown at 30°C in LB medium, at the mid-exponential phase isopropyl β-d-thiogalactopyranoside or d-lactose was added to the culture to a final concentration of 1 mM. After further cultivation, the cells were harvested and sonicated. The phytase produced was analyzed by phytase activity as well as SDS-PAGE. To quantify the amount of phytase in total cell protein, each sample was subjected to electrophoresis and scanned by SLR-2D/1D scanning densitometer (Biometer Instruments, USA) after the procedure of staining, destaining, and drying in cellophane foil. The percentage of phytase protein was expressed in relation to the total extinction of protein in a lane (100%).

3 Results and discussion

3.1 Amplification of phy-specific DNA fragment

The N-terminal amino acid sequence of the purified DS11 phytase was reported previously [17]. To design a set of PCR primers to amplify DNA fragments corresponding to the coding region of the phy sequence, an internal amino acid sequence of DS11 phytase was investigated after CNBr fragmentation of the purified enzyme. Between the resulting two CNBr fragments (14 and 21 kDa), the N-terminal sequence of a 14-kDa peptide was identical to that of the intact protein. Subsequently, it was thought that the N-terminal sequence of 21 kDa is the internal amino acid sequence. Based on the determined amino acid sequences, two degenerate oligonucleotide primers were synthesized (Fig. 1). Amplification of Bacillus sp. DS11 DNA fragment by PCR with primer 1 and 2 gave a single PCR product of 600 bp in size, which corresponds to the size of the product expected from the peptide map (Fig. 1). This 600-bp product was ligated into T vector and its nucleotide sequence was analyzed. The amino acid sequence deduced from the nucleotide sequence of the resulting plasmid matches with N-terminal sequences of the purified enzyme and the 21-kDa fragment. So, this 600-bp DNA fragment was used as a hybridization probe for genomic library screening.

3.2 Cloning of the phy gene

Bacillus sp. DS11 chromosomal DNA was digested with BamHI, ClaI, EcoRI, HindIII and PstI, separated on a 0.8% agarose gel, and transferred to Nylon membrane, positively charged. Hybridization was carried out using DIG-labeled PCR product as a probe. As shown in Fig. 2, positive signals were shown in HindIII-, EcoRI- or PstI-digested fragments. A HindIII fragment of 2.2 kb was eluted from the agarose gel, ligated with plasmid pUC19 digested with the same enzyme. The ligation mixture was used to transform E. coli JM83 and the resulting library was screened by colony hybridization using PCR-amplified DNA as a probe. As a result, two positive clones were obtained and those clones contained the same 2.2-kb insert at the HindIII site in pUC19 (Fig. 3). This coincided with the result of Southern hybridization, which showed a 2.2-kb DNA signal and the recombinant plasmid was designated pKP1. To determine the existence of phy in the 2.2-kb fragment, the phytase activity of the clone carrying pKP1 was measured after sonication in 0.1 M Tris-HCl, pH 7.0 buffer containing 1 mM CaCl2. The specific activity was 0.36 units mg−1. This result indicated that the 2.2-kb fragment encodes phytase.

Figure 2

Southern blot analysis of the Bacillus sp. DS11 chromosomal DNA. A 600-bp PCR fragment was used as a probe. Lane 1, λ-DNA digested with HindIII as a molecular weight marker; lanes 2–6, the chromosomal DNA (10 μg) was digested with BamHI, ClaI, EcoRI, HindIII and PstI.

Figure 3

The restriction enzyme maps of the Bacillus sp. DS11 DNA insert having phy gene in pUC19 and that of a HindIII-NdeI derivative subclone. The location and transcription direction of the phy gene are indicated by a solid arrow. Small arrows indicate the orientation of the lac promoter. B, BamHI; E, EcoRI; EV, EcoRV; Hc, HincII; N, NdeI.

3.3 Nucleotide sequence of phy

Deletion analysis of the recombinant plasmid (pKP1) showed phy to be within a 1.8-kb NdeI-HindIII fragment (Fig. 3). Therefore, this fragment was designated pKP2 and sequenced. Computer analysis revealed one major open reading frame (ORF), starting with an ATG initiation codon at base 280 and extending to base 1431 (Fig. 4). The sequence deduced from this ORF includes both the N-terminal and the internal amino acid sequences determined from the purified protein. The ORF encoded a polypeptide of 383 amino acid residues with a calculated molecular mass of 41 808 Da. A 30-amino acid signal sequence and a cleavage site between Leu-30 and Ser-31 were suggested by N-terminal sequencing of the purified DS11 phytase. The most probable Shine-Dalgarno sequence (AGGAGG) was located upstream of the start codon. As a rho-dependent transcription terminator [21], an inverted repeat sequence was observed downstream of the translational termination codon (TAA) of the gene. The theoretical free energy (−24 kcal mol−1) for the formation of the stem and loop structure was quite high.

Figure 4

Nucleotide sequence of the phy gene and its deduced amino acid sequence. The N-terminal and CNBr peptide fragment amino acid of the purified DS11 phytase are underlined. The putative ribosome binding site (RBS) sequence and stop codon (*) are shown. The position of a hairpin-like structure downstream of the coding sequence is indicated by arrows.

The primary structure of phytase has been reported only for fungi such as A. niger[1214] and A. fumigatus[15]. However, bacterial phytase genes have not been cloned until now even though two Bacillus phytases have already been purified and characterized [10, 11]. So this is the first report about the primary structure of bacterial phytase. Interestingly, this DS11 phytase turned out to be quite different from most fungal phytases in molecular size as well as amino acid sequence homology. Even a putative active site motif (RHGXRXP) found in histidine phosphatase including fungal phytases is absent in DS11 phytase. Comparison of the DS11 phytase sequence with protein sequences present in GenBank and related databases showed no significant similarity with other proteins except a B. subtilis hypothetical protein encoded by a hypothetical gene (orf181) located near the 3′ region of cgeAB (a cluster of GerE-controlled genes) [22], which showed 75% identity with the C-terminal region of DS11 phytase. However, in spite of the high similarity between the 3′ regions of the two proteins, the complete sequence and function of the hypothetical protein are not known. Therefore, more studies are needed to reveal the structural and functional relationships between DS11 phytase and the hypothetical protein.

3.4 Overexpression of phy in E. coli

DS11 phytase was thermostable as previously reported [17]. This property would be very useful for the enzyme to withstand inactivation during feed-pelleting or the expansion process and decrease costly formulations to limit activity loss in industrial applications. In order to increase the production level, we constructed an expression plasmid, pEPK1, as described in Section 2. The resulting construct was predicted to encode a polypeptide that is identical to the mature phytase except for one additional methionine residue at the terminus. Full IPTG-dependent induction was achieved after 3 h and phytase was produced to a level as high as 20% of the total cellular protein (Fig. 5). It was 50-fold higher than that of Bacillus sp. DS11, and has similar characteristics as that of native one in thermostability. That is, the optimum temperature for phytase activity was 70°C and about 50% of its original activity remained after incubation at 90°C for 10 min in the presence of 5 mM CaCl2. The expressed enzyme has a molecular mass of 44 kDa, which was larger than the 39 kDa calculated from the deduced amino acid sequence. This discrepancy is probably due to its hydrophobic nature, since the original DS11 phytase had the same molecular mass (44 kDa) as a mature phytase expressed in E. coli. To enhance the economic feasibility for industrial production, lactose was used as an inducer instead of IPTG. When culture was induced by lactose, the product was delayed about 1 h, and maximum phytase content was reached 4 h after induction (data not shown). Neubauer et al. [23] also demonstrated that lactose can be used as an equally efficient inducer as IPTG in E. coli BL21 harboring a recombinant pET3 plasmid provided that the addition of lactose was timed with respect to the glucose level in the growth medium. Therefore, the results show that lactose induces overexpression of phytase in our system with the same strength as IPTG and thus can be used as a suitable inducer in large-scale fermentation for phytase production.

Figure 5

SDS-polyacrylamide gel electrophoresis analysis of the recombinant phytase in E. coli BL21 (DE3)/pEPK1. Lane 1, cell extract from E. coli BL21 (DE3); lane 2, molecular mass markers; lanes 3–8, cell extracts from E. coli BL21 (DE3)/pEPK1 cultured for 0, 1, 2, 3, 4, and 5 h after 1 mM IPTG induction.


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