Crystallization and preliminary X-ray analysis of a novel type of lipolytic hydrolase from Bacillus licheniformis

The hydrolase BL28 from B. licheniformis was crystallized in the tetragonal space group P4₁2₁2 and diffraction data were collected to 1.67 Å resolution.

Abstract

With increasing demand in biotechnological applications, the identification and characterization of novel lipolytic enzymes are of great importance. The crystallization and preliminary X-ray crystallographic study of a novel type of hydrolase from Bacillus licheniformis (BL28) are described here. Recombinant BL28 protein containing a C-terminal His tag was overproduced in Escherichia coli and purified to homogeneity. BL28 was crystallized using 0.2 M ammonium acetate, 0.1 M sodium citrate tribasic dihydrate pH 5.6, 30%(w/v) PEG 4000 as a crystallizing solution. X-ray diffraction data were collected to a resolution of 1.67 Å with an R _merge of 5.8%. The BL28 crystals belonged to the tetragonal space group P4₁2₁2, with unit-cell parameters a = b = 57.89, c = 167.25 Å. A molecular-replacement solution was obtained and structure refinement of BL28 is in progress.

1. Introduction  

Lipolytic enzymes such as esterases, lipases and phospholipases are generally involved in the hydrolysis of esters, transesterifications and condensations of small metabolites or nutrients (Casas-Godoy et al., 2012 ▶; Hasan et al., 2009 ▶). Most lipolytic enzymes have been classified into eight families (families I–VIII) on the basis of conserved motifs, sequence identity, evolutionary relationships and functional properties (Arpigny & Jaeger, 1999 ▶). These enzymes generally show hydrolytic activity against a broad range of ester-bond-containing substrates with high regiospecificities and stereospecificities and share catalytic mechanisms without the need for any cofactors (Tan et al., 2010 ▶; Schmidt et al., 2009 ▶). Lipolytic enzymes are currently used for a number of industrial purposes, including paper and textile preparation; processing of oils, lipids, foods and beverages; synthesis of high-grade chemicals and pharmaceutical intermediates; and biodiesel production (Serdakowski & Dordick, 2008 ▶; Panda & Gowrishankar, 2005 ▶).

Owing to their significant roles and increasing importance in industrial applications, there is an ongoing need to identify novel lipolytic enzymes with improved enzymatic properties. Enzyme discovery using a metagenomic technique provides an opportunity to identify a large number of new enzymes for biotechnological applications (Nagarajan, 2012 ▶; Reyes-Duarte et al., 2012 ▶). Interestingly, a number of enzymes that could not be categorized into any of the eight conventional families have been identified, including EstOF4 from Bacillus pseudofirmus (Rao et al., 2013 ▶), EstA3 from Thermoanaerobacter tengcongensis (Rao et al., 2011 ▶), EstGtA2 from Geobacillus thermodenitrificans (Charbonneau et al., 2010 ▶), CE_Gk from G. kaustophilus (Montoro-García et al., 2009 ▶), LipEH166 and EML1 from several metagenomic libraries (Kim et al., 2009 ▶; Jeon et al., 2009 ▶), EstD from Thermotoga maritima (Levisson et al., 2007 ▶) and Est30 from G. stearothermophilus (Ewis et al., 2004 ▶). These enzymes are considered to be noncanonical lipolytic hydrolases.

A previous report identified BL28, a lipolytic hydrolase, from the genome of B. licheniformis and characterized it for potential biotechnological applications (Ju et al., 2013 ▶). Primary sequence analysis clearly showed that this enzyme has no similarity to the eight conventional lipolytic hydrolase families (families I–VIII). However, it has significant (66%) sequence identity to Est30 from G. stearo­thermophilus, which was recently shown to encode a noncanonical lipolytic hydrolase (Rao et al., 2013 ▶; Liu et al., 2004 ▶). After cloning and expression of the gene in Escherichia coli, the catalytic properties of BL28 were investigated (Ju et al., 2013 ▶). BL28 hydrolyzed p-nitrophenyl esters with acyl chains of up to 12 C atoms (p-nitrophenyl dodecanoate), with the highest activity for short-chain esters, while no significant enzyme activity was observed for p-nitrophenyl phosphate (p-NP). Cross-linked enzyme aggregates (CLEAs) of BL28 retained 32% of their initial activity after a 2 h incubation at 80°C, while free soluble BL28 only showed 8% of its initial activity.

Herein, we report the crystallization and preliminary crystallographic analysis of BL28. Structural analysis of BL28 is essential for understanding the structure–function relationship of this novel lipolytic hydrolase and for providing a molecular platform to be used in numerous industrial applications.

2. Experimental procedures  

2.1. Overproduction and purification of BL28  

A novel type of lipolytic hydrolase (BL28; GenBank accession No. YP_080734) from B. licheniformis has previously been identified and characterized (Ju et al., 2013 ▶). Briefly, the BL28 gene was amplified from the chromosomal DNA of B. licheniformis (KCTC 1026, Korean Collection for Type Cultures) by polymerase chain reaction (PCR) using two primers (forward, 5′-GCGGATCCATGAAAATTGTCAAA-3′, BamHI site in bold; reverse, 5′-GCCTCGAGTGTCTGCCAATCCAA-3′, XhoI site in bold). Both primers were purchased from Bionix Technologies (Seoul, Republic of Korea) and used without further purification. The PCR was carried out with 30 cycles of denaturation at 92°C for 60 s, annealing at 62°C for 30 s and amplification at 72°C for 60 s, followed by a final extension step at 72°C for 10 min in a buffer consisting of 200 µM dNTP, 1.0 mM MgCl₂, 10 pmol of primers, 5 U of DNA polymerase (Roche, USA). The amplified product (∼1.2 kb) was digested with BamHI and XhoI, and then cloned into the pET-21a vector to produce the recombinant plasmid (pET-21a-BL28). The C-terminus of the recombinant BL28 protein was fused to a tag of six histidines (hexa-His tag), resulting in eight additional residues (LEHHHHHH) at the C-terminus.

After confirming the full-length DNA sequence of the BL28 gene, the recombinant plasmid (pET-21a-BL28) was transformed into E. coli BL21 (DE3) cells and positive clones were selected on a Luria–Bertani (LB) agar plate with 100 µg ml⁻¹ ampicillin. For protein overproduction, a single positive colony was inoculated into 5 ml of LB medium supplemented with 100 µg ml⁻¹ ampicillin and grown overnight at 37°C. The overnight cultures were then diluted (1:50) in 250 ml fresh LB broth containing 100 µg ml⁻¹ ampicillin at 37°C, and were allowed to grow with a shaking speed of 200 rev min⁻¹ until mid-log phase (OD₆₀₀ = 0.4–0.5). The overproduction of BL28 was induced by the addition of isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 1.0 mM at 37°C with constant shaking. After 4 h, the cells were harvested by centrifugation (7000 rev min⁻¹, 20 min) and washed with PBS buffer three times. Cell pellets (∼4.7 g of wet mass) were then completely resuspended in 40 ml lysis buffer (20 mM Tris pH 8.0, 150 mM NaCl, 10 mM imidazole, 1 mM EDTA) and lysed on ice by five sonication steps [1 min each at 30% amplitude using a Vibracell VCX 750 (Sonics & Materials, Connecticut, USA)]. The cell lysate was then ultracentrifuged at 36 000 rev min⁻¹ in a Ti70 rotor (Beckman, USA) at 4°C for 1 h to obtain soluble fractions for the purification of BL28.

The final purification step was executed on a HisTrap High Performance column (5 ml, GE Healthcare) connected to an ÄKTA Fast Protein Liquid chromatography (FPLC) system under the control of UNICORN 4.12 software (GE Healthcare; Kim et al., 2012 ▶). The supernatant was loaded onto a column pre-equilibrated with lysis buffer at a flow rate of 0.5 ml min⁻¹. The unbound protein was then washed extensively with washing buffer (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1 mM EDTA) containing 20 mM imidazole until the absorbance at 280 nm (A ₂₈₀) was insignificant. After extensive washing, the protein was directly eluted with elution buffer (20 mM Tris pH 8.0, 150 mM NaCl, 200 mM imidazole, 1 mM EDTA) and desalted on a PD-10 column with 20 mM Tris–HCl pH 8.0. The fractions were pooled and concentrated using a Vivaspin 20 (3 kDa molecular-mass cutoff, Vivascience). Recombinant BL28 concentration was determined by bicinchoninic acid (BCA) protein assay (Pierce, Rockford, USA) using bovine serum albumin as a standard. The purity (∼92%) of BL28 was analyzed on SDS–PAGE. The yield of purified protein was typically ∼8 mg per litre of culture. The purified proteins (10 mg ml⁻¹) were stored at −20°C without further modifications.

2.2. Crystallization  

Crystallization trials on purified BL28 were carried out via a microbatch method (Chayen et al., 1990 ▶) under Al’s Oil using Wizard I and II (Emerald Bio, Washington, USA), Crystal Screen and PEG/Ion screen solutions (Hampton Research, California, USA) at 22°C (Ngo et al., 2013 ▶; Ryu et al., 2012 ▶). A drop consisting of 1 µl screening solution and 1 µl protein solution (10 mg ml⁻¹) was placed into each well of a Nunc 96-well Mini Tray (Nalge Nunc International, USA).

2.3. X-ray data collection and data processing  

Crystals were transferred to a cryosolution consisting of screening solution supplemented with 20% glycerol and flash-cooled in a cold nitrogen-gas stream at −173°C prior to data collection. Diffraction data were collected on an ADSC Quantum 315 CCD detector on the PAL 7A beamline, Pohang Accelerator Laboratory, Republic of Korea at −173°C. The wavelength of synchrotron X-rays and the crystal-to-detector distance were 1.000 Å and 200 mm, respectively. The exposure time was 1 s per frame. Crystals were rotated through a total of 90° with 1° oscillation range per frame. The data were processed using the HKL-2000 package (Otwinowski & Minor, 1997 ▶) and the structure solution was obtained by the molecular-replacement (MR) method with the Phaser program (McCoy et al., 2007 ▶) included in the CCP4 suite (Winn et al., 2011 ▶).

3. Results and discussion  

A novel type of lipolytic enzyme (BL28) from B. licheniformis was overproduced in E. coli BL21 (DE3) cells and purified to homogeneity for crystallization. The crystals were obtained from four different conditions during initial crystallization screening (Nos. 9, 15, 17 and 18 of Crystal Screen). Among them, condition No. 9 [0.2 M ammonium acetate, 0.1 M sodium citrate tribasic dihydrate pH 5.6, 30%(w/v) polyethylene glycol 4000] was selected for further optimization. Rectangular-shaped crystals appeared 3 d after setting up the crystallization condition. Diffraction-quality crystals were obtained on increasing the volume of protein and reservoir solutions to 1.5 µl. The crystals grew to final dimensions of 0.15 × 0.05 × 0.05 mm (Fig. 1 ▶). The crystals belonged to the tetragonal space group P4₁2₁2, as indicated by systematic absences, with unit-cell parameters of a = b = 57.89, c = 167.25 Å. The diffraction data set was processed to 1.67 Å resolution with 95.8% completeness and an R _merge of 5.8%. The data-collection statistics are given in Table 1 ▶. Assuming the presence of one molecule per asymmetric unit, the Matthews coefficient (V _M) and solvent content were calculated to be 2.47 Å Da⁻¹ and 50.2%, respectively, which are within the ranges commonly observed for protein crystals (Matthews, 1968 ▶). The crystallographic structural solution of BL28 was obtained by MR using the crystal structure of Est30 (PDB entry 1r1d; Midwest Center for Structural Genomics, unpublished work) as a search template. The best results were obtained in the tetragonal space group P4₁2₁2, with an initial R factor of 47.0% (R _free = 47.9%) and good crystal packing without any clashes. Structure refinement of BL28 and a functional study of ligand binding are currently in progress. The crystal structure of BL28 will hopefully reveal molecular details of this novel lipolytic hydrolase family that could be used as an information platform for the development of biotechnological applications.

Figure 1.

BL28 crystals were obtained using PEG 4000 as a crystallizing agent by the microbatch method. Crystal dimensions are 0.12 × 0.05 × 0.05 mm.

Table 1. X-ray data-collection and processing statistics.

Values in parentheses are for the highest resolution shell.

Space group	P41212
Unit-cell parameters (Å)	a = b = 57.89, c = 167.25
Wavelength (Å)	1.000
Resolution (Å)	25.0–1.67 (1.73–1.67)
Total reflections	218834
Unique reflections	32658
Completeness (%)	95.8 (63.5)
Multiplicity	6.7 (5.2)
R merge † (%)	5.8 (31.7)
Mean I/σ(I)	37.6 (2.4)

^† R _merge = .