Supplementary Information

A novel phospholipase D constitutively secreted by Ochrobactrum sp. DZQ2a capable of enzymatic synthesis of phosphatidylserine

Fei Hu · Huai Wang · Zhang-Qun Duan · Ri-Sheng Yao

F. Hu · H. Wang · R.-S. Yao

Hefei University of Technology, Hefei 230009, People’s Republic of China

Z.-Q. Duan

Academy of State Administration of Grain, Beijing 100037, People’s Republic of China

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Screening of high phospholipase D (PLD)-producing microorganism

Oily soil samples collected from Beijing, which have received the accumulation of industry oil sediments or kitchen ventilator grease stain for several years, were selected as the initial inocula for PLD enrichments. The sample was inoculated in 100 ml flasks containing 25 ml of sterile minimal medium. And PC supplied as the sole carbon and energy source at a concentration of 0.15 mM. The cultures were incubated in an orbital shaker at 30 °C and 120 rpm, and repeated for three times. The strains in the degrading culture were isolated by repetitive streaking onto PC-agar medium. These pure cultures were then re-inoculated into sterile minimal medium containing 0.15 mM of PC as the carbon source. The strain was monitored by measuring the optical density (OD) at 530 nm and the activity of produced PLD was detected under assay conditions. All the isolated strains produced PLD to various extents. Among them, strain DZQ2a produced the highest quantity of PLD (data not shown) and therefore was selected as the target strain for the further study.

Strain identification

Genomic DNA of strain DZQ2a was extracted with UNIQ-10 genome DNA isolation kit (Sangon Ltd., China). 16S rDNA was amplified in PCR using the template and the microorganism primers were F7 (5'-CAGAGTTTGATC CTGGCT-3') and R1540 (5'-AGGAGGTGATCCAGCCGCA-3'). Amplification procedures were as follows: initial denaturation at 98 °C for 5 min followed by 35 cycles of denaturation at 95 °C for 35 s, annealing at 55 °C for 35 s and elongation at 72 °C for 30 s, and final elongation at 72 °C for 8 min. The PCR product was purified with a UNIQ-10 PCR DNA extraction kit (Sangon Ltd., China), and the sequence reaction mixtures were electrophoresed by using a model 3730 automatic DNA sequencer (Applied Biosystem Ltd., USA). The resulting sequence was submitted to GenBank. Multiple alignments of sequences and construction of neighbor-joining phylogenetic trees were performed with the MEGA5 software. The stability of relationships was assessed by a bootstrap analysis of 1000 trials. The strain identification result was presented in Fig. 1s. Obviously, BLAST analysis showed that 16S rDNA sequence of the strain had more than 99% identity with that of Ochrobactrum intermedium TA13 (Accession No. AM490628) and the accession number of this strain was KC146415. The results suggested that it was an Ochrobactrum strain and therefore we designated it as Ochrobactrum sp. DZQ2a. As far as we know, this is the first report that PLD can be produced by an Ochrobactrum strain.

Fig. 1s Neighbor-joining phylogenetic tree based on 16S rDNA gene sequences showing the relationships between Ochrobactrum sp. DZQ2a and related species. The number at each branch point represents the bootstrap percentages. Bar 0.05 substitutions per nucleotide position.

Effects of various metal ions and detergents

The effects of 1 mM of various metal ions (the corresponding salts were NaCl, KCl, CuCl2, MnCl2, CaCl2, MgCl2, ZnCl2, FeCl2, CoCl2, BaCl2 and AlCl3, respectively) and detergents (including Tris, SDS, Urea and EDTA) on the PLD activity were examined using the standard assay described earlier. The enzyme activity without additives was taken as the control and defined as 100%. The relative activity was defined as the ratio of the enzyme activity in the presence of the tested ions or reagents to the control. As pointed out in Table 1s, although the PLD behavior in the presence of different reagents was various, none of them was particularly effective in either increasing or decreasing the activity of the enzyme.

Table 1s Effect of various reagents on the PLD activity*

Reagent / Relative activity (%) / Reagent / Relative activity (%)
None / 100 / FeCl2 / 88±2
NaCl / 111±2 / CoCl2 / 85±3
KCl / 93±3 / BaCl2 / 89±2
CuCl2 / 74±5 / AlCl3 / 91±3
MnCl2 / 86±4 / Tris / 88±4
CaCl2 / 79±3 / SDS / 78±5
MgCl2 / 85±4 / Urea / 98±1
ZnCl2 / 71±5 / EDTA / 49±6

* Various reagents (1 mM) were added to L-serine solution (0.15 mM) containing the purified enzyme and then incubated at room temperature for 60 min. The reaction was initiated by addition of PC solution (0.05mM) and the residual activity was measured under the standard conditions. The values were means of three independent experiments.

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