Soluble expression of mature Rhizopus chinensis lipase in Escherichia coli and enhancement of its ester synthesis activity

Protein Expression and Purification 163 (2019) 105443

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Soluble expression of mature Rhizopus chinensis lipase in Escherichia coli and enhancement of its ester synthesis activity

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Zhang Zhanga,b, Dong Wanga,b,∗, Yan Xua,b,c,∗∗ a

The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China School of Biotechnology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China c State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Membrane-associated lipase Soluble expression Maltose-binding protein fusion tag Ester synthesis activity

The production of membrane-associated lipase from Rhizopus chinensis (RCL), which has a high ester synthesis activity and important potential applications, is difficult in heterologous expression system such as Escherichia coli and often leads to the formation of inclusion bodies. Here, we describe the soluble expression of mature RCL (mRCL) using maltose-binding protein (MBP) as a solubility-enhancing tag in the E. coli system. Although the MBP-mRCL fusion protein was soluble, mRCL was insoluble after removal of the MBP tag in E. coli BL21 (DE3). Using E. coli BL21 trxB (DE3) as an expression host, soluble mRCL was obtained and expression conditions were optimized. Furthermore, the ester synthesis activity of soluble mRCL was increased by detergent treatment and was found to be 3.5 and 1.5 times higher than those of the untreated enzyme and naturally occurring enzyme, respectively. Overall, this study provides a potential approach for producing active and soluble forms of eukaryotic lipases in a heterologous E. coli expression system.

1. Introduction Lipases (EC 3.1.1.3), triacylglycerol ester hydrolases, are well known for their ability to hydrolyze ester bonds at the interface of the water/non-aqueous phases [1,2]. In contrast to many other enzymes, lipases show remarkable stability and activity in non-aqueous environments, thereby facilitating the catalysis of several special reactions such as esterification and transesterification [3]. Lipase-catalyzed esterification reactions in non-aqueous media are of great interest, owing to their significant applications in several fields [4,5] such as production of flavor esters [6], chiral resolution of racemates [7], production of monoacylglycerols [8], and synthesis of biodiesel [9,10]. Although all lipases exhibit hydrolytic activities, only a few show remarkable esterification capabilities. The strain Rhizopus chinensis (CCTCC M201021) [11] can produce a membrane-associated lipase [12] named R. chinensis lipase (RCL), which has a low hydrolysis activity in aqueous phase and strong ester synthesis activity in non-aqueous and solvent-free phases [11,13]. The production of this membrane-associated protein in the form of a mature lipase from R. chinensis is always low [9]; moreover, the purification of

this enzyme is difficult. A recent study demonstrated the successful heterologous expression of RCL with pro-sequence (r27RCL) by the BL21 trxB (DE3) system, and this r27RCL protein exhibited high hydrolysis activity but low ester synthesis activity [14]. However, the production of active mature lipase, like other Rhizopus lipases, using engineered strains such as Escherichia coli is still challenging [15,16] and often leads to the formation of inclusion bodies (IBs). In general, the heterologous expression of eukaryotic proteins in E. coli often faces problems, such as low expression level, misfolding of protein or formation of inclusion bodies, which usually can be overcame using different vectors or engineering hosts, optimization of culture and induction conditions, fusion with different functional tags, etc. [17]. The widely used expression vectors in the E. coli system include the pET vector series, and the commonly used expression hosts include BL21 (DE3) and some others that possess different functions, such as BL21 trxB (DE3). Thioredoxin reductase (trxB) is an enzyme that can reduce thioredoxin in E. coli cytoplasm [18], and maintains the E. coli cytoplasm in a reduced state which is strongly disadvantageous for the formation of disulfide bonds in proteins [19]. The BL21 trxB (DE3) with the mutation of trxB can assist the formation of disulfide bonds and the

∗ Corresponding author. The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China. ∗∗ Corresponding author. The Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu, China. E-mail addresses: [email protected] (D. Wang), [email protected] (Y. Xu).

https://doi.org/10.1016/j.pep.2019.06.003 Received 12 December 2018; Received in revised form 22 April 2019; Accepted 7 June 2019 Available online 08 June 2019 1046-5928/ © 2019 Elsevier Inc. All rights reserved.

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tagged soluble fusion protein. E. coli JM109 was used for construction and cloning of pMD19-T-mRCL (Nde I), pMD19-T-mRCL (Nco I), pET22b-mRCL, pET-28a-mRCL, and MBP3-mRCL recombinant plasmids and E. coli BL21 (DE3) and BL21 trxB (DE3) were used as hosts for the expression of recombinant lipase. Restriction endonucleases, PrimeSTAR™ HS DNA Polymerase, and T4 DNA ligase were purchased from TaKaRa Biotechnology (Dalian, China), while DNA primers and Plasmid Mini Kit were obtained from Sangon (Shanghai, China). The substrate p-nitrophenyl palmitate (pNPP) used for the determination of lipase hydrolysis activity was purchased from Sigma-Aldrich (Shanghai, China). Heptane, ethanol, and octanoic acid used for synthetic activity assay were obtained from Sinopharm Group Co., Ltd (Shanghai, China). Detergent CHAPS, Triton X-100, DDM, and LPC14, standard protein bovine serum albumin, egg albumin, chymotrypsin, and cytochrome C (bovine heart), and analytical standard ethyl octanoate for ester synthesis activity assay were purchased from Sigma-Aldrich (Shanghai, China). All other chemicals used were of analytical grade and commercially available. The mRCL gene was amplified from genomic DNA of R. chinensis using different forward and reverse primers for different vectors (Table 2). In general, PCR reaction was carried out in a total volume of 25 μL and comprised 2.5 μL of PrimeSTAR buffer, 2.5 μL of dNTP mixture, 0.5 μL of plasmid template (120 ng μL−1), 1 μL of forward primer (20 μM), 1 μL of reverse primer (20 μM), and 0.5 μL of PrimeSTAR™ HS DNA Polymerase. PCR thermocycling conditions comprised a preheating step at 98 °C for 3 min and a denaturation step at 98 °C for 3 min, followed by 30 cycles at 98 °C for 45 s, 59.5 °C for 30 s, and 72 °C for 1 min and a final extension step at 72 °C for 10 min. An approximately 810-bp fragment was amplified. The amplified fragment was cloned into pMD19-T vector, resulting in pMD19-T-mRCL (Nde I) and pMD19-T-mRCL (Nco I). The Nco I/Xho I amplicon was cloned into the Nco I/Xho I digested pET-22b vector to obtain pET-22b-mRCL plasmid under the control of T7 lac promoter. The Nde I/Xho I amplicon was cloned into the Nde I/ Xho I-digested pET-28a and MBP3 vector to yield pET-28a-mRCL and MBP3-mRCL plasmids, respectively, under the control of T7 lac promoter (Fig. 1). MBP tag was fused into N-terminus of target protein to reduce the efficiency of translation, which may eliminate the formation of IBs [36]. The constructed plasmid was transformed into E. coli JM109 by conventional calcium chloride method. Screening for positive colonies was performed using 100 μg mL−1 ampicillin and positive transformants were confirmed by gene sequencing (Sangon, Shanghai, China). The obtained recombinant plasmids were transformed into E.

proper folding of target proteins, especially proteins with several disulfide-bounds that are rarely found naturally folded in the E. coli cytoplasm, under an altered redox environment in E. coli cytoplasm [20–24]. Functional tags, such as histidine (His) tag [25], glutathione Stransferase (GST) tag [26], and maltose-binding protein (MBP) tag [27] are also used for expressing recombinant protein with the aim of achieving easy protein purification or increasing the solubility of target protein in a heterologous expression system [28]. The MBP tag has been used as a fusion partner for over ten years to increase the solubility of proteins that have limited solubility [29]. Through its physical interaction with target protein, a soluble and functionally active product [30,31] can be obtained via proper folding of the fusion protein [30,32]. The MBP tag with signal peptide usually directs fusion proteins to periplasm, and the MBP tag without a signal peptide is highly used for the expression of heterologous proteins in E. coli cytoplasm as it can produce more fusion proteins and increase their solubility [30]. After expression of the fusion protein, the MBP tag is often cleaved using sitespecific proteases to release the target protein, which may cause passenger protein sometimes unable to maintain their solubility or activity. It has been reported that some passenger proteins may undergo precipitation [33,34] and some of them may undergo activities changes when MBP is cleaved by site-specific protease [35], therefore, the solubility and activity regulation of these enzymes is sometimes desirable. Here, we demonstrate the production of soluble mature R. chinensis lipase (mRCL) in E. coli expression system. To prevent the formation of IBs, an MBP-tag was fused to the N-terminus of mRCL. Since three disulfide-bonds are formed in RCL [14], BL21 trxB (DE3) was selected as the expression host to obtain soluble mRCL. After expression conditions were optimized, the ester synthesis activity of soluble mRCL was subsequently recovered using detergents. This expression protocol may serve as a useful method for soluble expression of proteins which are hard to be bio-functionally expressed in E. coli.

2. Materials and methods 2.1. Strains, plasmids, and materials All stains and plasmids used in this study are listed in Table 1. The gene encoding target lipase mRCL was 810 bp in length and cloned using R. chinensis CCTCC (China Center for Type Culture Collection) M201021 genome as template. The expression vector MBP3, with a deletion of signal sequence of malE gene which encoding maltosebinding protein (MBP), was used for the production of N-terminal HisTable 1 Bacterial strains and plasmids used in this study. Strains/plasmids Strains JM109 BL21 (DE3) BL21 trxB (DE3) JM 109/pMD19-T-mRCL BL21 (DE3)/pET-28a-mRCL BL21 (DE3)/pET-22b-mRCL BL21 (DE3)/MBP3-mRCL BL21 trxB (DE3)/MBP3-mRCL Plasmids pMD19-T pET-28a pET-22b MBP3-vector pMD19-T-mRCL (Nde I) pMD19-T-mRCL (Nco I) pET-28a-mRCL pET-22b-mRCL MBP3-mRCL

Genotype or relevant characteristics

Reference or source

e14− (mcrA), endA1, recA1, hsdR17 (rk−, mk−), (lac-proAB) lacIqZM15, relA1 F−, ompT, hsdSB(rB−mB−), gal, dcm, (DE3) F−, ompT, hsdSB(rB−mB−), gal, dcm, trxB15::kan, (DE3) E. coli JM109 harboring pMD19-T-mRCL E. coli BL21 (DE3) harboring pET-28a-mRCL E. coli BL21 (DE3) harboring pET-22b-mRCL E. coli BL21 (DE3) harboring MBP3-mRCL E. coli BL21 trxB (DE3) harboring MBP3-mRCL

Invitrogen Invitrogen Novagen This work This work This work This work This work

Ampr N-Thrombin, N-His, C-His, T7 promoter, lac operator N-pelB, C-His, T7 promoter, lac operator N-8 × His, MBP-tag, TEV protease cleavage site, T7 promoter, lac operator pMD19-T derivative carrying mRCL gene and Nde I and Xho I restriction sites downstream of T7 promoter pMD19-T derivative carrying mRCL gene and Nco I and Xho I restriction sites downstream of T7 promoter pET-28a derivative carrying mRCL gene downstream of T7 promoter pET-22b derivative carrying mRCL gene downstream of T7 promoter MBP3 vector derivative carrying mRCL gene downstream of T7 promoter

TaKaRa Novagen Novagen [14] This work This work This work This work This work

Gene cloning and construction of recombinant expression plasmids. 2

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Table 2 Primers used in this study. The endonucleases Nde I (single solid line), Nco I (double solid line), and Xho I (dash line) are underlined.

Xinchen, Nanjing, China) for 20 min with a 3 s-on/2 s-off program. The cell debris was removed by centrifugation at 10,000×g for 30 min and suspension was filtrated with a 0.22-μm membrane. The supernatant was loaded onto AKTA purifier system and a stepwise elution purification protocol was performed. The impurities were washed from HisTrap HP column (5 mL) using five-column volumes of wash buffer (20 mM Tris-HCl, 150 mM NaCl, 100 mM imidazole, pH 8.0), and MBPmRCL was eluted using five-column volumes of elution buffer (20 mM Tris-HCl, 150 mM NaCl, 300 mM imidazole, pH 8.0). The elution fractions were analyzed by SDS-PAGE and pooled into low-salt buffer (10 mM Tris-HCl and 100 mM NaCl, pH 7.5) and concentrated to 0.5–1 mg mL−1 using Amicon ultrafiltration concentrators (Millipore). MBP-mRCL was overnight incubated with TEV protease at a mass ratio of 50:1 (protein: protease) at 4 °C. The purified mRCL was collected from the flow-through of a Ni-NTA column equilibrated with low-salt buffer.

coli competent cells by calcium chloride method. 2.2. Media and culture conditions For culturing E. coli JM109 and BL21 (DE3) recombinants, LuriaBertani (LB) media (10 g L−1 tryptone, 5 g L−1 yeast extract, and 10 g L−1 NaCl) was supplemented with 100 μg mL−1 ampicillin. For culturing E. coli BL21 trxB (DE3) recombinants, LB media, Terrific broth (TB) media (12 g L−1 tryptone, 24 g L−1 yeast extract, 9.4 g L−1 K2HPO4, 2.2 g L−1 KH2PO4, and 0.4% v/v glycerol), and Autoinduction (AI) media ZYM-5052 [37] were used and supplemented with required antibiotic (50 μg mL−1 kanamycin or 100 μg mL−1 ampicillin). For protein expression in LB and TB media, a single colony carrying recombinant plasmid was picked from a freshly transformed plate and inoculated into 5 mL media, followed by incubation at 37 °C and 200 rpm for 4–6 h to obtain a pre-cultured bacterial suspension. The pre-culture was transferred into 50 mL induction media and incubated under the same condition for 1 h, and protein expression was induced with the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) and by changing the incubation temperature. Protein expression in AI media was performed using procedures similar to those described above. The 5 mL pre-culture was transferred into 50 mL of ZYM-5052 media and incubated at 37 °C and 200 rpm until OD600 reached 0.6–0.8. The incubation temperature was then decreased to 20 °C and protein expression was induced for 72 h.

2.4. R. chinensis culture and lipase extraction R. chinensis CCTCC M201021 was cultured as previously reported [38]. The fermentation process was performed in 20 mL of media after inoculation with spore suspension. The initial spore concentration in fermentation broth was 107 spores·L−1. The fermentation broth contained olive oil (37.5 mL L−1), MgSO4·7H2O (0.5 g L−1), K2HPO4 (2 g L−1), maltose (10 g L−1), and peptone (40 g L−1). After fermentation for 72 h at 30 °C and 200 rpm, fungal biomass was harvested and washed with ultrapure water and 25 mM phosphate buffer (pH 6.2). Lipase was extracted from R. chinensis as previously described method [9]. One gram of mycelium was treated with 15 mL of acetone for 30 min at 4 °C. The mixture was filtered by vacuum filtration to remove solvents and subjected to drying for several hours. The dried cell mass was washed twice with 20 mL of 2.5 mM phosphate buffer (pH 6.2) and once with 20 mL of 2.5 mM phosphate buffer contained 0.1% Triton X-100 (pH 6.2). After centrifugation at 10,000×g for 10 min, the precipitate was shaken in 20 mL of 2.5 mM phosphate buffer containing 1.5% Triton X-100 (pH 6.2) for 4 h. The obtained supernatant was

2.3. Expression analysis, purification, and protease cleavage Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) analysis and protein purification were performed to evaluate the expression of target protein in E. coli. After protein expression, the cultured broth was centrifuged (10,000×g) at 4 °C for 10 min, and pellets were washed twice with 0.9% physiological saline. The washed pellets were resuspended in 25 mL of a binding buffer (20 mM Tris-HCl and 150 mM NaCl, pH 8.0) and sonicated in an ice water bath using a Φ6 probe (XC92-11DN,

Fig. 1. Schematic representation of expression vectors. The target gene was inserted into pET-28a (a), pET-22b (b), and MBP3 (c) vectors following the restriction enzyme digestion and ligation. MBP: the gene sequence encoding maltose-binding protein. TEV: the gene sequence encoding TEV protease recognition site. The restriction sites used during construction are shown in sketch map. 3

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were lyophilized.

collected by centrifugation at 10,000×g for 20 min and stored as the extracted mRCL (eRCL).

2.8. Size-exclusion chromatography 2.5. Lipase lyophilization The samples were centrifuged at 10,000×g for 30 min and suspension was filtrated with a 0.22-μm membrane. The filtered samples were detected by size-exclusion chromatography (SEC) using AKTA purifier system with Superdex 200 increase gel filtration column (GE Healthcare). The column was pre-equilibrated with gel filtration buffer (20 mM Tris-HCl and 150 mM NaCl, pH 8.0). The 0.5 mL of protein sample was injected into AKTA purifier system and eluted by the same buffer described above. The flow rate was set to 0.5 mL·min−−1 at 20 °C. The protein fractions were collected from the column using onecolumn volumes (25 mL) of elution buffer.

Lipase solutions were lyophilized for at least 16 h in a Labconco FreeZone 4.5 system (APS Water Services Corporation, California, USA) after freezing at −80 °C for at least 2 h. The lyophilized lipases were assayed for their ester synthesis activities. 2.6. Activity assay and protein concentration measurements The lipase hydrolysis activity was assayed with some modifications in a previously reported method [39]. One volume of pNPP/2-propanol solution (1 mL of 2-propanol mixed with 3 mg of pNPP powder) was freshly mixed with nine volumes of 50 mM Tris-HCl buffer (pH 8.0) containing 1.16 g L−1 of sodium deoxycholate and 0.56 g L−1 of arabic gum. The standard reaction was started with pre-equilibration of 2.4 mL of above mixture at 40 °C and addition of 0.1 mL of enzyme solution at an appropriate dilution in low-salt buffer. The reaction was maintained for 2 min at 40 °C. The variation in absorbance of the assay reaction at 410 nm wavelength against a blank carrying inactivated enzyme was detected using a UV/Vis spectrophotometer (UNICO UV-3102 PC, China). One enzyme unit was defined as the amount of enzyme releasing 1 μmol of p-nitrophenol per minute under assay conditions. Lipase ester synthesis activity was measured with ester synthesis reaction in heptane with some modifications in the previously described procedure [12,39]. Briefly, 1 mL of substrate solution (final concentration of 0.6 M octanoic acid and 0.6 M ethanol in heptane, with an acid/alcohol molar ratio of 1:1) was added into a 5-mL Eppendorf tube. The reaction was started by the addition of appropriate lyophilized lipase powder and incubation for 30 min at 40 °C and 200 rpm. Insoluble powder was removed by centrifugation at 10,000×g and 4 °C for 3 min. The supernatants (400 μL) were mixed with 100 μL of 2hexanol as an internal standard and analyzed by gas chromatograph (GC). One unit of lipase ester synthesis activity was defined as the amount of enzyme required to produce 1 μmol of ester per minute. The ester products were analyzed by GC according to a previously described protocol [12]. Samples of 1 μL were injected into a gas chromatograph (Agilent 6890) equipped with a DB-WAX capillary column (30 m × 0.25 mm i.d.) and a hydrogen flame-ionization detector. Nitrogen was used as carrier gas, and the injector and detector temperatures were set at 250 °C. Oven temperature was programmed to start at 120 °C and then elevated to 200 °C for 8 min at 10 °C·min−1. Based on mixed standard, a mixture containing 400 μL of 0.2 M pure ethyl heptanoate solution in heptane and 100 μL of 2-hexanol, the ester content in each sample was calculated. The protein concentration was determined by bicinchoninic acid (BCA) protein assay kit (TaKaRa, Dalian, China) using bovine serum albumin (BSA) as a standard.

3. Results and discussion 3.1. Problems with soluble expression of mRCL in E. coli The successful expression of mRCL in BL21 (DE3) was confirmed using SDS-PAGE analysis, wherein a protein band was observed at about 30 kDa on SDS-PAGE gel. However, the protein was expressed in IBs (Fig. 2a and b). Given that heterologous proteins expressed in E. coli cytoplasm are often misfolded or aggregated [17], we used pET-22b with a pelB signal peptide to facilitate periplasm secretion of expressed eukaryotic protein [42]. Nevertheless, mRCL was still obtained in the form of IBs (Fig. 2b) and the aggregated protein showed little activity. Hence, MBP3-mRCL fusion plasmid was constructed, and the target fusion protein of 671 amino acid residues was expressed as predicted by a molecular weight of 73.6 kDa. The results of SDS-PAGE analysis from whole cell protein and soluble protein (Fig. 3a) indicated that the MBP tag facilitated the soluble expression of the fusion protein in E. coli. Purification using a Ni-NTA column yielded the purified fusion protein MBP-mRCL, but the treatment with TEV protease at 4 °C for 16 h to cleave the MBP tag resulted in the formation of inactive precipitate of most mRCL (Fig. 3b). This phenomenon was similar to that seen in other reports [30,34]. MBP can promote the solubility of its fusion protein. However, although the fusion protein is soluble in E. coli, the passenger protein could be in a misfolded or incompletely folded state, possibly owing to the formation of intermolecular micellar structures or MBP acting as a “holdase” [34,43]. Natural mRCL exhibits three disulfide bonds [14], and the misfolding of this protein could occur upon its expression in a system with a reducing environment, such as BL21 (DE3) [44,45], which might result in the formation of an inactive precipitate of mRCL after the MBP tag has been cleaved. Therefore, MBP-mRCL was further expressed in BL21 trxB (DE3), which is known for its ability to alter the redox

2.7. Increase of ester synthesis activity of lipase Lipase was co-lyophilized with detergents to evaluate the increase of ester synthesis activity follow a previously described protocol with some modifications [40]. Four detergents, CHAPS (3-([3-cholamidopropyl] dimthyl-ammonio)-1-propane sulfonate), DDM (n-dodecylbeta-d-maltoside), Triton X-100, and LPC14 (1-myristoyl-2-hydroxy-snglycero-3-phosphocholine) were dissolved in low-salt buffer at 20 mM concentration, and the CMC (critical micelle concentration) values of each detergent are 6.83 mM, 0.22 mM, 0.18 mM and 0.083 mM [41], respectively. Lipase and detergent solutions were mixed at a 1:1 (v/v) ratio to prepare lipase samples. The final concentration of used detergents was 10 mM, equivalent to 55.6 CMC for DDM, 120.5 CMC for LPC14, 1.5 CMC for CHAPS, and 45.5 CMC for Triton X-100, respectively. The ester synthesis activity assay was performed after samples

Fig. 2. Expression of mRCL using pET-28a (a) and pET-22b (b) vector in BL21 (DE3). The expressed mRCL is indicated with arrows. The molecular weight of mRCL was predicted as 29.6 KDa which was consistent with that as seen in the gel. T, total protein; S, soluble protein; P, pellet. 4

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Fig. 3. Expression and purification of MBP-mRCL expressed in BL21 (DE3) (a) and TEV protease treatment of the protein (b). MBP-mRCL was purified using a Ni-NTA column. After purification and buffer exchange, the fusion protein was digested with TEV protease. All segments after cleavage are indicated with arrows. MBP-mRCL and mRCL indicated with arrows were predicted to be 73.6 and 29.6 kDa, respectively. T, total protein; S, soluble protein; FT, flow through from the column; E, eluted protein from the column; P, precipitate.

the cytoplasm in the cells [44,45], which led to protein precipitation after cleavage by TEV protease. However, in BL21 trxB (DE3), although the altered redox environment cannot make mRCL soluble expressed, this environment did facilitate mRCL to correctly fold with the assistance of its fusion partner MBP; therefore, a soluble individual target protein was obtained after cleavage. However, the expression level of soluble fusion protein was quite low using BL21 trxB (DE3) as a host with only about 0.65 mg proteins per liter of culture medium can be obtained. After purification and cleavage, the amount of soluble mRCL protein produced was only 0.075 mg per liter of culture medium. Hence, the increase of soluble expression of mRCL in BL21 trxB (DE3) was desired.

environment in its cytoplasm and to help the folding of proteins that contained disulfide bonds [20], which would result in the correct folding and expression of mRCL. 3.2. Soluble expression of mRCL The soluble expression of MBP-mRCL was also achieved using BL21 trxB (DE3) as host under the similar culture and induction conditions as those used for BL21 (DE3); however, the soluble mRCL was not obtained when expressed without an MBP tag when using BL21 trxB (DE3) as a host (Fig. 4a). After purification of MBP-mRCL and incubation with TEV protease at 4 °C for 16 h, soluble mRCL was successfully collected in the flow-through from a Ni-NTA column, as shown in Fig. 4b. This result indicated that the altered cytoplasmic environment in BL21 trxB (DE3) was important for the proper folding of the protein. However, BL21 trxB (DE3) cannot produce soluble mRCL without the assistance of its fusion partner MBP (Fig. 4a). Therefore, the MBP fusion partner was still required and the combination of host and fusion partner played a critical role in the soluble expression of mRCL. We proposed an explanation about this phenomenon in Fig. 5, which was similar with the model described by Raran-Kurussi [34]. In BL21 (DE3), MBP made fusion protein allowed soluble expression, but mRCL could still be misfolded or incompletely folded because of the absent or mis-formed disulfide bonds in the reducing environment of

3.3. Optimization of expression condition To improve the production of soluble active mRCL, the expression conditions of MBP-mRCL in BL21 trxB (DE3), including culture media, induction temperatures, inducer concentrations, and induction times, were optimized. The expression using three commonly used culture media, LB, TB, and AI, was compared. As shown in Fig. 6a, MBP-mRCL showed maximum expression in LB media. Furthermore, the expression was evaluated at five different induction temperatures (17, 20, 25, 30, and 37 °C) (Fig. 6b). The lower temperatures of 17 °C and 20 °C reduced the cell growth rate and yield, whereas the higher temperature of 37 °C Fig. 4. The expression of MBP-mRCL and mRCL and the cleavage of MBP-mRCL. (a) Expression of MBP-mRCL and mRCL in BL21 trxB (DE3). Ten microliters of each sample were loaded onto the gel and identified with a low molecular weight protein marker; (b) Cleavage of the purified fusion protein MBP-mRCL. The digested proteins were purified using a Ni-NTA column, and mRCL was detected in the flow-through of the column. Twenty microliters of each sample were loaded onto the gel and the samples were identified with a low molecular weight protein marker. T, total protein; S, soluble protein; P, pellet; FT, flow through from the column; E, protein eluted from the column.

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Fig. 5. Schematic diagram about the expression condition of mRCL in different hosts. The 3D structure of mRCL was presented by PyMOL [46]. The red bar, the -SH group of cysteine; the green solid line, the incorrectly formed disulfide bound; the red solid line, the correctly formed disulfide bound; the grey dashed line, the possible interactions between MBP and mRCL.

are known to be toxic to E. coli [47] and may cause metabolic burden on cells, resulting in reduced cell growth. Hence, 0.4 mM of IPTG was selected for expression. In addition, the effects of different induction times on MBP-mRCL expression were studied (Fig. 6d). Cells were induced at 30 °C for 3 h, 5 h, and overnight (O/N). The ratio of target protein to total protein was lowest when induced O/N. No increase in expression level was observed after induction for 5 h and the ratio of target protein to whole cell protein slightly decreased. So, a 3-h-long induction time was chosen for achieving optimum MBP-mRCL expression. Under optimized expression conditions, compared to the expression level before optimization, the yield of MBP-mRCL increased over 11 times and the yield of mRCL increased 10.6 times after treatment

was unsuitable for protein expression because SDS-PAGE showed that the target protein was expressed less at this temperature. As shown by the results of SDS-PAGE analysis, 25 °C and 30 °C showed similar expression levels. However, the induction at 30 °C showed a slightly higher OD600 value than that observed at 25 °C; hence, 30 °C was selected for expressing the fusion protein MBP-mRCL. As shown in Fig. 6c, the effect of the commonly used inducer (IPTG) was evaluated at five different concentrations (0.2, 0.4, 0.6, 0.8, and 1.0 mM), with 0 mM IPTG used as a blank control. The results demonstrated the differences between expression levels at different IPTG concentrations, and expression levels achieved with 0.4 and 0.6 mM IPTG were better than those achieved with other concentrations. High concentrations of IPTG

Fig. 6. Optimization of MBP-mRCL expression condition in BL21 trxB (DE3). Different culture media (a), induction temperatures (b), inducer concentrations (c), and induction times (d) were studied. The details of induction conditions are described in Materials and methods. The target fusion protein MBP-mRCL is indicated with an arrow. Ten microliters of each sample were loaded onto the gel and identified with a low molecular weight protein marker. (a) The cells incubated in LB media without induction were used as blank control. LB: LB media; TB, TB media; AI: Auto-induction media ZYM-5052. (b) Five different induction temperatures (17, 20, 25, 30, and 37 °C) were tested for the expression of MBPmRCL. (c) Five different concentrations of the inducer (0.2, 0.4, 0.6, 0.8, and 1.0 mM) were tested for the expression of MBP-mRCL, with 0 mM IPTG used as a blank control. (d) Three induction times (3 h, 5 h, and overnight) were evaluated.

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Table 3 Protein yield, hydrolysis specific activity, and ester synthesis specific activity of MBP-mRCL and mRCL before and after optimization of expression conditions.

Before optimization After optimization

eRCL MBP-mRCL mRCL MBP-mRCL mRCL

Yield* (mg·L−1)

Hydrolysis specific activity (U·mg−1)

Ester synthesis specific activity (U·mg−1)

3.86 ± 0.08 0.65 ± 0.05 0.075 ± 0.004 7.5 ± 0.1 0.8 ± 0.07

11.4 29.8 59.4 33.6 64.8

10.6 0.83 4.30 0.98 4.55

± ± ± ± ±

0.6 0.8 0.4 0.7 0.5

± ± ± ± ±

0.6 0.09 0.3 0.1 0.4

*The yield indicated the amount of soluble protein (mg) obtained per liter of culture medium.

with TEV protease (Table 3). Although the soluble expression of mRCL was achieved in E. coli, its expression yield was low, which suggested that this lipase could be hard to express in E. coli. After optimization, 7.5 mg of MBP-mRCL per liter culture broth could be obtained, and only about 10% mRCL was recovered after cleavage. There are reports that the MBP fusion expression cannot always produce a high yield of target protein [35]. Using other highly expressed fusion partners and improving the recovery of soluble mRCL will be the further work. The activities of both MBP-mRCL and mRCL were also detected (Table 3). The hydrolysis and ester synthesis specific activities of mRCL were enhanced after removal of MBP tag. It has been reported that the MBP tag may somehow block the active site accessibility of the target protein [35] and lead to a lower activity of the fusion protein when the MBP tag fuses with a smaller molecular weight target protein by a very short linker. This could be the reason why higher activities of mRCL occurred after removal of the MBP tag.

Fig. 7. The effects of detergents on hydrolysis and ester synthesis specific activities of heterogeneously expressed mRCL. The final protein concentrations were 0.01 mg mL−1. Detergents were added at 10 mM final concentrations for the hydrolysis activity assay, and enzymes for the ester synthesis activity assay were prepared by lyophilization at enzyme and detergent concentrations similar to those used in hydrolysis assays. The hydrolysis and ester synthesis assays were performed as described in Materials and methods. The specific activity of untreated mRCL was set to 100%. Size-exclusion chromatography of lipases with or without detergent.

3.4. Increase of ester synthesis activity of mRCL In comparison with natural RCL extracted from the fungus R. chinensis (eRCL), which has higher ester synthesis specific activity, the expressed mRCL showed 6 times higher hydrolysis specific activity but 50% lower ester synthesis specific activity, respectively (Table 3). These results indicate that the activity characteristics of heterogeneously expressed mRCL were quite different from those of the original R. chinensis lipase. The reason underlying these characteristic differences Fig. 8. Size-exclusion chromatography assay of lipase samples with or without detergent. (a) Size-exclusion elution of standard proteins, mRCL, and mRCL with LPC14. The concentrations of samples were 2.5 mg ml−1 of bovine serum albumin (66 kDa, fraction 1) and egg albumin (45 kDa, fraction 2), 4.5 mg ml−1 of chymotrypsin (25 kDa, fraction 3) and cytochrome C (13 kDa, fraction 4), and 0.25 mg ml−1 of mRCL and mRCL with LPC14; (b) Standard curve for calculating protein molecular weight. The molecular weight of fraction A to D were calculated from the standard curve (y = −8.0524x +52.434, R2 = 0.9556); (c) SDS-PAGE of each fractions obtained through size-exclusion elution of different samples.

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Table 4 The ester synthesis activity assay of different fractions of size-exclusion elution.

Protein molecular weight (kDa)* Ester synthesis specific activity (U mg−1) Protein concentration** (μg mL−1)

Fraction A

Fraction B

Fraction C

Fraction D

60.2 (59.2) 3.89 ± 0.12 19.79 ± 0.53

30.9 (29.6) 4.32 ± 0.17 89.72 ± 2.76

61.3 (59.2) 9.92 ± 0.65 30.36 ± 0.69

30.7 (29.6) 15.41 ± 1.20 71.03 ± 3.70

*Protein molecular weight was calculated using the standard curve and the theoretical molecular weight was presented in bracket. **Protein concentration presents the concentration of protein collected for each peak; proteins were diluted to the same concentration (0.02 mg mL−1) for the ester synthesis activity assay.

times higher than that of naturally obtained eRCL. The results of sizeexclusion chromatography indicated that the increase in ester synthesis activity of lipase induced by detergent was not caused by the change in the aggregation of lipase, and further work is necessary. Overall, the protein expression and activity increasing protocols described in this paper may provide a useful approach for the production of membraneassociated eukaryotic proteins in their active soluble forms.

may be related to the membrane-associated expression of eRCL in R. chinensis [12]. As the naturally occurring RCL is a membrane-associated protein and detergents are often used to increase the activity and stability of lipases [48,49], we used four common detergents (CHAPS, Triton X100, DDM, and LPC14) to treat the heterogeneously expressed mRCL for the increase of its ester synthesis activity. The results (Fig. 7) showed that these detergents had no obvious influence on hydrolysis activity of the protein. However, the zwitterionic detergent LPC14 strongly stimulated the ester synthesis activity of mRCL by 3.5 times, which was even 1.5 times higher than that of eRCL. It is possible that the addition of LPC14 influenced the enzyme particle size or the protein aggregation of soluble mRCL. Thus, sizeexclusion chromatography was introduced and the protein sizes of mRCL and mRCL with LPC14 was compared. As shown in Fig. 8a and Table 4, the soluble mRCL obtained in the absence of detergent was presented mainly in monomeric form (fraction B), just as we had proposed. A few dimers of mRCL with molecular weight around 60 KDa (fraction A) were also found, which presented a similar activity of monomeric lipase (Table 4). It seemed that the presence of detergent LPC14 slightly increased the formation of dimers (Fig. 8a, fraction C). However, the activity assay described in Table 4 showed that the ester synthesis specific activity of dimers (fraction C) with detergent was not increased and was 35% lower than that of the monomeric lipase (fraction D). Moreover, the ester synthesis specific activities of both the mRCL dimers and the monomeric lipase were significantly higher than the activities of lipases without detergent. This result indicated that the change of enzyme particle size could not be the reason for the improvement of ester synthesis activity of mRCL by detergent. In the reaction process, the detergent LPC14 may protect lipase proteins in organic solvents and increase their stability. Moreover, proteins always tend to aggregate when reacting in organic solvents. Detergents could improve the dispersion of the enzyme in organic solvents, which could facilitate reaction catalysis by lipase. Alternatively, detergents could interact with the enzyme and altered the micro-environment or conformation of the protein, which may lead to an increase in enzyme activity [50,51]. However, the three other detergents tested did not increase the ester synthesis activity of the lipase. Further research is warranted to understand the intrinsic mechanism of this action, which could be important to promote the application of lipase in non-aqueous phase catalysis.

Conflicts of interest The authors declare that they have no conflict of interest. Acknowledgements This work was supported by the National Natural Science Foundation of China (grant numbers 31271920); the National FirstClass Discipline Program of Light Industry Technology and Engineering (LITE2018-09); and the Top-notch Academic Programs Project of Jiangsu Higher Education Institutions (TAPP). The authors also thanks Prof. John F. Hunt (Columbia University), Prof. Thomas Szyperski (State University of New York at Buffalo), and Prof. Gaetano T. Montelione (Rutgers University) for extensive and useful discussions. The authors also thank Editage (www.editage.cn) for English language editing. References [1] A.M. Brzozowski, U. Derewenda, Z.S. Derewenda, G.G. Dodson, D.M. Lawson, J.P. Turkenburg, F. Bjorkling, B. Huge-Jensen, S.A. Patkar, L. Thim, A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex, Nature 351 (1991) 491–494. [2] X.W. Yu, Y. Xu, R. Xiao, Lipases from the genus Rhizopus: characteristics, expression, protein engineering and application, Prog. Lipid Res. 64 (2016) 57–68. [3] K.E. Jaeger, T. Eggert, Lipases for biotechnology, Curr. Opin. Biotechnol. 13 (2002) 390–397. [4] P.Y. Stergiou, A. Foukis, M. Filippou, M. Koukouritaki, M. Parapouli, L.G. Theodorou, E. Hatziloukas, A. Afendra, A. Pandey, E.M. Papamichael, Advances in lipase-catalyzed esterification reactions, Biotechnol. Adv. 31 (2013) 1846–1859. [5] F. Hasan, A.A. Shah, A. Hameed, Industrial applications of microbial lipases, Enzym. Microb. Technol. 39 (2006) 235–251. [6] A. Larios, H.S. García, R.M. Oliart, G. Valerio-Alfaro, Synthesis of flavor and fragrance esters using Candida antarctica lipase, Appl. Microbiol. Biotechnol. 65 (2004) 373–376. [7] A. Ghanem, H.Y. Aboul-Enein, Lipase-mediated chiral resolution of racemates in organic solvents, Tetrahedron: Asymmetry 15 (2004) 3331–3351. [8] J.B. Monteiro, M.G. Nascimento, J.L. Ninow, Lipase-catalyzed synthesis of monoacylglycerol in a homogeneous system, Biotechnol. Lett. 25 (2003) 641–644. [9] Y. Teng, Y. Xu, D. Wang, Production and regulation of different lipase activities from Rhizopus chinensis in submerged fermentation by lipids, J. Mol. Catal. B Enzym. 57 (2009) 292–298. [10] S. Shah, M.N. Gupta, Lipase catalyzed preparation of biodiesel from Jatropha oil in a solvent free system, Process Biochem. 42 (2007) 409–414. [11] Y. Xu, D. Wang, X.Q. Mu, G.A. Zhao, K.C. Zhang, Biosynthesis of ethyl esters of short-chain fatty acids using whole-cell lipase from Rhizopus chinesis CCTCC M201021 in non-aqueous phase, J. Mol. Catal. B Enzym. 18 (2002) 29–37. [12] D. Wang, Y. Xu, Y. Teng, Synthetic activity enhancement of membrane-bound lipase from Rhizopus chinensis by pretreatment with isooctane, Bioproc. Biosyst. Eng. 30 (2007) 147–155. [13] Q. He, Y. Xu, Y. Teng, D. Wang, Biodiesel production catalyzed by whole-cell lipase from Rhizopus chinensis, Chin. J. Catal. 29 (2008) 41–46. [14] M. Zhang, X. Yu, S. Gvt, R. Xiao, H. Zheng, C. Sha, Y. Xu, G.T. Montelione, Efficient production of 2H, 13C, 15N-enriched industrial enzyme Rhizopus chinensis lipase

4. Conclusion Herein, we have described the production of solubilized mRCL using an MBP tag in an E. coli expression system as well as the increase of its ester synthesis activity. Although the protein obtained using the MBP fusion method in E. coli BL21 (DE3) was insoluble after removing the MBP tag using TEV protease, soluble expression of mRCL was achieved by employing the same expression protocol in E. coli BL21 trxB (DE3). Under optimized expression conditions, the expression level of MBPmRCL increased over 11 times, and the yield of soluble mRCL improved 10.6 times. Furthermore, the zwitterionic detergent LPC14 improved ester synthesis activity of expressed mRCL by 3.5 times, which was 1.5 8

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