Expression and Purification of Three Lipases (LipAD1, LipAD2, and LipAD3) and a Lipase Chaperone (LipBD) from Acinetobacter schindleri DYL129

Acinetobacter schindleri DYL129 유래의 3개 lipases와 chaperone의 발현과 정제

Abstract

Previously, three kinds of lipases, lipAD1, lipAD2, and lipAD3, and lipase chaperone, lipBD, of Acinetobacter schindleri DYL129 isolated from soil sample were reported. In this report, three lipase and lipase chaperone were cloned into the pET32a(+) or pGEX-6P-1 vectors for protein expression in Escherichia coli, and named as pETLAD1, pETLAD2, pETLAD3 and pETLBD or pGEXLAD1, pGEXLAD 2, pGEXLAD3 and pGEXLBD, respectively. Protein expression rate was higher in pET system than in pGEX system. Although LipAD1 and LipAD2 were produced as inclusion bodies, their expression levels were high. So LipAD1 and LipAD2 could be solubilized in 8 M urea buffer and purified. LipAD3 and LipBD were overexpressed in soluble form and purified. Those proteins were purified by His-tag affinity chromatography connected in AKTA prime system. The activities of the purified lipases were demonstrated with 1% tributyrin agar plate. After purification, molecular mass was determined with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. LipAD1 showed high activity toward ${\rho}$-nitrophenyl acetate and ${\rho}$-nitrophenyl butyrate, LipAD2 showed high activity toward ${\rho}$-nitrophenyl acetate and ${\rho}$-nitrophenyl myristate, and LipAD3 showed high activity toward ${\rho}$-nitrophenyl acetate, ${\rho}$-nitrophenyl butyrate, and ${\rho}$-nitrophenyl miristate, respectively. Three lipases, LipAD1, LipAD2, and LipAD3, showed optimal reaction at $50^{\circ}C$ using ${\rho}$-nitrophenyl butyrate, as substrate.

기존 연구를 통하여 토양에서 분리한 Acinetobacter schindleri DYL129로부터 3개의 lipase 유전자(lipAD1, lipAD2와 lipAD3)들과 1개의 chaperone (lipBD) 유전자를 보고하였다. 본 연구에서는 각 유전자들의 발현을 위해서 pET32a(+)와 pGEX-6P-1 벡터에 클로닝하여 각각을 pETLAD1-3와 pETLBD 또는 pGEXLAD1-3와 pGEXLB로 명명하였으며, 단백질의 발현량은 pET 시스템을 사용할 때 1.5 배 정도 향상됨을 확인하였다. LipAD1과 LipAD2는 inclusion body 형태로 발현이 되었으며, LipAD3과 LipBD는 soluble type으로도 발현되었다. Inclusion body 형태의 LipAD1과 LipAD2는 고농도의 우레아를 처리하여 refolding 시켰다. LipAD1은 C4와 C2를, LipAD2는 C2와 C14를 그리고 lipAD3은 C2, C4와 C14를 기질로 잘 이용하는 것을 확인하였다. 그리고 모든 효소들은 $50^{\circ}C$에서 최적 활성을 나타내었다.

Fig. 3. The three lipases were purified by affinity chromatography. A) SDS-PAGE analysis of the samples of purified lipases and lipase chaperone. Samples were resolved on 12% polyacrylamide gel and stained with Coomassie blue R-250. Lane 1: molecular size markers (100, 70, 50, 40, 30, 20, 15 kDa); lane 2: N His- LipBD; lane 3: P His- LipBD, 57 kDa; lane 4: native His-LipAD1; lane 5: P His-LipAD1, 56 kDa; lane 6: N His-LipAD2; lane 7: P His-LipAD2, 56 kDa; lane 8: N His-LipAD3; lane 9: P His-LipAD3, 34 kDa (N: native protein, P: purified protein). B) Activity test for the purified and dialyzed lipases. The enzyme activities of fractions 6-9 were tested with 1% tributyrin agar plate containing 0.01% phenol red. The yellow color indicates the released fatty acids from tributyrin.

Fig. 4. Substrate specificity of His-LipAD1, His-LipAD2, and His-LipAD3. Acyl chain length specificity of purified lipases was determined from their activities toward various esters of ρ-NP (0.5 mM). Percentages shown are relative to maximum activity.

Fig. 5. Effect of temperature on the activity of lipases. The enzyme was incubated in 20 mM Tris-HCl buffer (pH 7.4) for 30 min at various temperatures. The activity was determined using ρ-NP butyrate as substrate. The value obtained for His-LipAD2 at 50℃ was considered as 100%.

Fig. 1. Overexpression of LipAD1 and LipAD2. A) M, size marker (100, 70, 50, 40, 30, 20, 15 kDa); 1-4 insoluble proteins (1: NIP pET, 2: IP pET, 3: NIP His-LipAD1, 4: IP His-LipAD1); 5-8 soluble proteins (5: NI pET, 6: I pET, 7: NI His-LipAD1, 8: I LipAD2), B) M, size marker; 1-4 soluble proteins (1: NI pET, 2: I pET, 3: NI His-LipAD2, 4: I His-LipAD2); 5-8 insoluble proteins (5: NIP pET, 6: IP pET, 7: NIP His-LipAD2, 8: IP His-LipAD2). Arrow indicates expressed protein (about 56 kDa) NIP: uninduced protein pellet, IP: induced protein pellet, NI: uninduced protein, I: induced protein.

Fig. 2. Expression of LipAD3 and LipBD. A) The soluble proteins were loaded onto a 12% polyacrylamide gel. M: size marker; 1: NI pET; 2: I pET; 3: NI His-LipAD3; 4, 5: I His-LipAD3; 6: NI LipBD; 7, 8: IP His- LipBD. B) Insoluble proteins. M: size marker; 1: NIP pET; 2: IP pET; 3: NIP His-LipAD3; 4, 5: IP His-LipAD3; 6: NIP His-LipBD; 7, 8: IP His- LipBD. Arrow indicates expressed proteins (about 36 and 57 kDa, respectively). NIP: uninduced protein pellet, IP: induced protein pellet, NI: uninduced protein, I: induced protein.

Table 1. PCR primers used for the expression of three lipases and the lipase chaperone

Table 2. Purification of lipases from Acinetobacter schindleri DYL129