GU Xiaoqian, GUI Yuanyuan, LI Jiang, , ZHANG Xuelei, and ZILDA Dewi Seswita

1) MNR Key Laboratory for Science & Technology of Marine Ecosystems, First Institute of Oceanography, Ministry of Natural Resources, Qingdao 266061, China

2) College of Environmental Engineering, Qingdao University, Qingdao 266061, China

3) Research Center for Marine and Fisheries Product Processing and Biotechnology, Agency for Marine and Fisheries Research and Development, Ministry of Marine and Fisheries Affairs, Jakarta 40115, Indonesia

Abstract A novel lipase gene (lip4346) encoding a primary translation product with 176 amino acids was screened from the genome fine mapping of the macroalgae-associated bacterial strain Microbulbifer sp. YNDZ01. Macroalgae were collected from the coast of the Halmahera Island of Indonesia. The lip4346 gene was cloned and heterologously expressed in Escherichia coli. The purified recombinant Lip4346 protein had a molecular mass of 19 kDa, a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and a maximum enzyme activity of 31.2 U mL?1. The optimal temperature and pH for the lipase activity of Lip4346 were 70℃ and 10.0, respectively. Lip4346 was tolerant with a number of organic solvents and detergents, and was active toward triacylglycerols and p-nitrophenyl esters with short- and medium-chain lengths. The unique characteristics of Lip4346 indicate that it is a promising nonaqueous biocatalyst for industrial applications.

Key words Microbulbifer; lipase; alkali-tolerant; high temperature-tolerant

1 Introduction

Lipases (acylglycerol acylhydrolases, EC 3.1.1.3) are enzymes that catalyze the hydrolysis of various fatty esters. They are distributed widely in animals, plants, and microorganisms (Desnuelle, 1972). Microbial lipases have received considerable attention because of their actual and potential applications in the detergent, oil and fat, dairy,and pharmaceutical industries (Gaoaet al., 2000). Most lipases derived from mesophilic microorganisms are active over a wide pH range but are generally unstable above 60℃. The unique biochemical properties of thermostable lipases are advantageous in their potential applications in biotechnological industries (Rajeshet al., 2019). The advantages of using thermophilic enzymes as biocatalysts include increased substrate solubility and diffusion rates, faster mass transfer because of reduced viscosity, and reduced risk of contamination (Abdel-Fattah, 2002; Haki and Rakshit, 2003; Li and Zhang, 2005).

Lipases are unusual soluble enzymes because they exhibit activity at the water-oil interphase, a characteristic called interfacial activation (Verger and DeHaas, 1976).Lipases in aqueous media hydrolyze long-chain triglycerides into diacylglycerides, monoglycerides, free fatty acids,and glycerol, whereas lipases in nonaqueous media synthesize esters from glycerol and long-chain fatty acids(Balanet al., 2012; Masomianet al., 2012). In addition,lipases that are active and stable in organic solvents can enhance substrate solubility and product recovery during enzymatic synthesis (Sharma and Kanwar, 2014). However,the low catalytic efficiency of lipases in organic solvents needs to be overcome (Doukyua and Ogino, 2010).

In general, enzymes from thermophilic microorganisms are more thermostable than enzymes from mesophilic microorganisms (Nawani and Kaur, 2000). Commercial lipases need to be thermostable because high temperatures can increase enzymatic reaction rates, reduce microorganism contamination risks, and boost equilibrium displacement in endothermic reactions (Liet al., 2011; Yuet al.,2012). Thermophilic lipases often have good thermostability and high activity in organic solvents (Sharmaet al.,2017). Lipases are environmentally friendly alternatives for many industrial processes because they can reduce the amounts of chemicals. Lipases have been used as biodegradable and non-toxic additives in detergents, resulting in harmless residues (Sharmaet al., 2017). However, it is still a challenge to find lipases with distinct properties,including tolerance to organic solvents, tolerance to high salt and alkaline conditions, and high enzymatic activity at high temperatures (Sharmaet al., 2017).

In this paper, a novel lipase gene (lip4346) was cloned from the identifiedMicrobulbifersp. YNDZ01 and expressed inEscherichia coli. The purified lipase showed high stability at high temperatures, good stability in alkaline conditions, and tolerance to organic solvents. These properties indicate that Lip4346 can serve as a nonaqueous biocatalyst and food additive in biotechnological processes including the manufacture of drugs, detergents, and cosmetics.

2 Materials and Methods

2.1 Bacterial Strains

TheMicrobulbifersp. YNDZ01 strain was isolated from the surfaces of macroalgae collected on the coast of Halmahera Island, Indonesia (127?52?E, 0?36?N).

2.2 Plasmids, Vectors, and Culture Media

The vector pET-30a (+) and host strainE. coliBL21 (DE3)cells were purchased from Novagen (Merck, Darmstadt,Germany). The cloning vector pUC118 and host strainE.coliDH5α cells were purchased from Takara (Dalian, China), and a His-tag-specific purification kit was obtained from GE Healthcare Co., Ltd. (Pittsburgh, PA, USA). TheE. coliDH5α andE. coliBL21 (DE3) cells were cultured at 37℃ in Luria-Bertani (LB) medium containing 100 μg mL?1ampicillin and 50 μg mL?1kanamycin, respectively.

2.3 Preparation of DNA Samples of Microbulbifer sp. YNDZ01

The collected macroalgae samples were flushed using sterile seawater to remove debris and loose bacteria from their surfaces. The flushed samples were immediately placed into sterile tubes and stored at 4℃ until used.

Prior to swabbing, the thalli were flushed with sterile seawater. Then they were swabbed using sterile swab cotton, streaked on screening plates (tryptone 0.5%, yeast powder 0.1%, agar 1.5%, and tributyrin 0.1%), and cultured at 30℃. After purification and identification, the total genomic DNA ofMicrobulbifersp. YNDZ01 was extracted using a PowerSoil DNA Isolation kit (MP BIO Laboratories, Inc., CA., USA) in accordance with the manufacturer’s protocol. Fine mapping and gene analysis ofMicrobulbifersp. YNDZ01 were performed by Novogene Biological Co., Ltd. (Beijing, China).

2.4 Cloning and Sequence Analysis of lip4346

The presumptive lipase genelip4363was obtained from the fine mapping of the YNDZ01 genome. For cloninglip4346, specific primers were designed and synthesized as follows: Primer F: 5’-ACGCGTCGAC CGACACCCA TTACATTCACC-3’ (underlined bases are the addedSalI site) and Primer R: 5’-CCGCTCGAG TTTATTCCATT CCGCTGATA-3’ (underlined bases are the addedXhoI site). The PCR amplification conditions were as follows:35 cycles of denaturation at 95℃ for 30 s and annealing at 56℃ for 30 s, followed by an extension at 72℃ for 2 min.The nucleotide and amino acid sequences were identified by database searches using BLASTn and BLASTp (http://blast.ncbi.nlm.nih.gov/Blast.cgi), respectively, and the DNAMAN software package (http://www.lynnon.com/) was used for multiple sequence alignment.

2.5 Expression of lip4346 and Purification of the Recombinant Protein

Thelip4346gene was ligated into a pET-30a expression vector and transformed intoE. coliBL21 cells. The positive clones were identified on a tributyrin plate. Then the cells harboring the pET30a–lip4346recombinant plasmid were cultured in LB medium containing 50 μg mL?1kanamycin with shaking (150 r min?1) at 37℃. When the OD600of the culture medium reached 0.6–0.8, the expression of the fusion protein Lip4363 was induced by adding 0.5 mmol L?1isopropyl-β-D-thiogalactopyranoside (IPTG).After induction at 15℃ for 16 h, the cells were harvested(6000 ×gfor 10 min at 4℃) and disrupted using a highpressure cell cracker (Constant Systems, Co., Ltd., UK) (Li and Liu, 2017).

The recombinant Lip4346 protein was purified using a His Bind Purification kit (GE Healthcare Co, Ltd.). After washing with binding buffer (20 mmol L?1sodium phosphate, 0.5 mol L?1NaCl, 20 mmol L?1imidazole, pH 7.4),the recombinant Lip4346 was eluted using elution buffer(20 mmol L?1sodium phosphate, 500 mmol L?1NaCl, pH 7.4) with a linear gradient to 250 mmol L?1imidazole (Rieraet al., 2003). The eluent containing the target protein was assessed by SDS-PAGE followed by staining with Coomassie brilliant blue R-250 (Blakesley and Boezi, 1977).The activity of the purified recombinant Lip4346 was determined using a standard method.

2.6 Lipase Activity and Substrate Specificity Assays

Lipase activity was measured using thep-nitrophenylpalmitate (p-NPP) method (Pencreach and Baratti, 1996)with minor modifications. In brief, the assay was carried out in a 1 mL reaction mixture containing 100 μL ofp-nitrophenyl caproate (10 mmol L?1) in a 1.5 mL tube containing 890 μL of glycine-NaOH buffer (pH 10.0). Then 10 μL of diluted lipase elution was added and mixed. The reaction mixture was incubated at the optimal temperature for 10 min, then the reaction was stopped by adding 10%SDS. The amount ofp-nitrophenol produced was quantified using a spectrophotometer at 410 nm. One unit (U) of lipase activity was defined as the amount of enzyme that caused the liberation of 1 μmolp-NP per min from thep-NPP substrate under standard assay conditions (Ameriet al., 2019).

p-Nitrophenyl esters with different acyl chain lengths,includingp-nitrophenyl caprylate (C2),p-nitrophenyl butyrate (C4),p-nitrophenyl caproate (C6),p-nitrophenyl caprylate (C8),p-nitrophenyl caprate (C10),p-nitrophenyl laurate (C12),p-nitrophenyl myristate (C14),p-nitrophenol palmitate (C16), andp-nitrophenyl stearate (C18:0),were used as substrates under standard assay conditions to determine the substrate specificity of Lip4346. The hydrolytic activities of Lip4346 toward the triacylglyceride substrates triacetin (C2), tributyrin (C4), tricaproin (C6),tricaprylin (C8), tricaprin (C10), trilaurin (C12), trimyristin (C14), tripalmitin (C16), and tristearin (C18) were also determined. All assays were performed in triplicate.

2.7 Effects of Temperature and pH on Lip4346 Activity and Stability

Lip4346 was pre-incubated at 30–90℃ in intervals of 10℃ for 6 h to examine the effect of temperature on Lip4346 activity. After 6 h, the residual lipase activity was determined under standard assay conditions. As the control, the group with the highest enzyme activity was defined as 100%.

The effects of pH on Lip4346 activity and stability were measured at pH 3.0 – 11.0 in intervals of 1.0 usingp-nitrophenyl caproate as the substrate. The reaction mixtures were incubated at 70℃ for 15 min in different buffer systems, including Na2HPO4-citrate (pH 3.0 – 6.0), NaH2PO4-Na2HPO4(pH 6.0 – 8.0), Tris-HCl (pH 8.0 – 9.0), and glycine-NaOH (pH 9.0 – 11.0). Lipase activity was determined using the standard method. As the control, the group with highes enzyme activity was defined as 100%.

2.8 Effects of Metal Ions and Inhibitors on Lip4346 Activity

The effects of metal cations (Mn2+, Fe3+, Ni2+, Ca2+, Fe2+,Sr2+, Mg2+, Cd2+, and Gu2+) on Lip4346 activity were evaluated by measuring the residual lipase activity after preincubating Lip4346 with the different metal ions (2 mmol L?1) for 30 min at 70℃ and pH 10.0. As the control, the lipase activity without metal ions was set as 100%.

The effects of different inhibitors (detergents, reducing/oxidizing agents, and additives) on Lip4346 stability were evaluated by measuring the residual lipase activity after Lip4346 was incubated with each reagent for 30 min at 70℃. As the control, the lipase activity without the inhibitors was set as 100%. All assays were performed in triplicate.

2.9 Tolerance of Lip4346 to Organic Solvents

The effects of organic solvents (50% v/v) on Lip4346 activity were investigated to examine the tolerance of Lip4346 to organic solvents. Different organic solvents were preincubated with Lip4346 for 30 min at 70℃ and pH 10.0 with shaking at 120 r min?1. Then the lipase activity was measured under standard assay conditions relative to the control (without organic solvent). All assays were performed in triplicate.

3 Results and Discussion

3.1 Cloning and Sequence Analysis of Lip4346 from Microbulbifer sp. YNDZ01

We detected thelip4346gene with primers designed on the basis of theMicrobulbifersp. YNDZ01 genome sequence. The predicted open reading frame consisted of 528 bp, which encoded a protein with 175 amino acids. The deduced Lip4346 protein had a putative molecular mass of 19 kDa and an isoelectric point of 5.8. Comparative sequence analyses using BLAST showed that thelip4346nucleotide sequence had no overall nucleotide sequence similarity to any of characterized lipases in the NCBI nucleotide sequence database. However, the Lip4364 amino acid sequence shared 66.67%, 65.14%, 60.59%, 56.57%,55.17%, 53.71%, 45.78%, 42.48%, 37.82%, and 35.23%similarity with lipases fromMicrobulbiferthermotolerans(GenPept: WP_067156140.1),Microbulbifer marinus(Gen-Pept: WP_091387438.1),Microbulbifer yueqingensis(Gen-Pept: WP_091515587.1),Microbulbifer agarilyticus(Gen-Pept: WP_050901684.1),Microbulbifer aggregans(Gen-Pept: WP_083261088.1),Microbulbifersp. Q7 (GenPept:WP_082859526.1),Methylobactersp. (GenPept: PPC90 620.1),Planktothricoidessp. SR001 (GenPept: WP_0544 65766.1),Crinalium epipsammum(GenPept: WP_015203 750.1), andCyanothecesp. PCC 7822 (GenPept: WP_013324487.1), respectively. The multiple sequence alignment (Fig.1) indicated that the active serine and oxyanion-forming residues of Lip4346 were at the center of the typical penta-peptide motif (GNSMG) and a catalytic triad was formed by Ser53-Asp129-His153, which confirmed Lip4346 as a new member of lipase family IV.

The surface of macroalgae is a protective and nutrientrich habitat that harbors a dense, complex, and highly dynamic microbial community that plays important roles in the physiology, ecology, and evolution of the host (Eganet al., 2008). However, microbial communities on algae remain largely underexplored despite their huge biodiversity and marked differences from microbial communities that live freely in seawater. These microorganisms interact with algae in multiple and complex ways, making them a rich source of novel bioactive compounds with biotechnological potential, such as dehalogenases, antimicrobials,alga-specific polysaccharidases, and other enzymes. In this study, we investigated the lipolytic activity of a putative lipase encoded bylip4346fromMicrobulbifersp. YNDZ01,which was isolated from the surface of macroalgae collected from the coast of the Halmahera Island (127?52?E,0?36?N). The novellip4346gene was detected by constructing a complete genome map of the lipolytic activity ofMicrobulbifersp. YNDZ01.

3.2 Expression, Purification, and Identification of Lip4346

Fig.1 Multiple sequence alignment of the deduced Lip4346 amino acid sequence and the amino acid sequences of known lipases in GenPept. The putative penta-peptide motif (GFSMG) is indicated by a red box. Conserved residues are highlighted in blue. The conserved amino acid residues that form the putative catalytic triad of Lip4346 are marked by asterisks. PPC90620.1, lipase (Methylobacter sp.); WP_013324487.1, triacylglycerol lipase (Cyanothece sp. PCC 7822); WP_015203750.1, triacylglycerol lipase (Crinalium epipsammum); WP_050901684.1, lipase (Microbulbifer agarilyticus); WP_054465766.1, triacylglycerol lipase (Planktothricoides sp. SR001); WP_067156140.1, lipase (Microbulbifer thermotolerans); WP_082859526.1, lipase (Microbulbifer sp. Q7), WP_083261088.1, lipase (Microbulbifer aggregans); WP_091387438.1, lipase (Microbulbifer marinus); and WP_091515587.1, lipase (Microbulbifer yueqingensis).

Fig.2 Three positive clones of recombinant Lip4346 on a tributyrin plate.

Thelip4346gene was expressed inE. coliBL21 (DE3)cells by induction with IPTG at a final concentration of 0.5 mmol L?1, and the positive clones showed high activity on a tributyrin plate (Fig.2). Fig.3 shows that the recombinant lipase Lip4346 was mainly expressed in soluble form. After purification by Ni-NTA affinity chromatography, a single band was obtained by SDS-PAGE, which corresponded to the calculated mass (approximately 19 kDa) of Lip4346. Then, the enzyme activity of purified recombinant Lip4346 was determined at an optimal condition, and the average activity was 31.2 U mL?1. The enzyme activity of Lip4346 was much higher than those of previously reported commercial strains that efficiently produce lipase, such asProteussp. NH 2-2 (1.662 U mL?1)(Shaoet al., 2019),Penicilliumsp. (1.62 U mL?1) (Turatiet al., 2019), andBurkholderiasp. C20 (3.9 U mL?1) (Liuet al., 2006), but lower than those ofSerratia rubidaeastrain Nehal-mou (41.13 U mL?1) (Nehalet al., 2019) andPseudomonassp. (87.5 U mL?1) (Gaoaet al., 2000).

Fig.3 SDS-PAGE analysis shows purified Lip4346. M,Protein marker; Lane 1, Precipitate of recombinant E. coli BL21(DE3) induced by IPTG; Lane 2, Supernatant of recombinant E. coli BL21(DE3) induced by IPTG; Lane 3,Purified protein Lip4346.

3.3 Effects of Temperature and pH on Lip4346 Activity

The purified recombinant Lip4346 exhibited stable activity between 50℃ and 80℃. The highest lipase activity was achieved at 70℃, and 30% of the maximum activity was retained at 90℃ (Fig.4). This result indicates that Lip4346 is high temperature-tolerant, which is an important characteristic of enzymes used for industrial biotransformation. The lipase isolated fromCohnellasp. A01 (Cardenaset al., 2001) showed maximum activity at 70℃,whereas all other previously reported bacterial lipases showed optimal activity when the temperature is below 70℃ (Zhenget al., 2011). These results suggest that the high optimum temperature for Lip4346 activity and stability make it a favorable lipase in the leather, medical,cosmetic, textile, and food industries (Houdeet al., 2004).

Fig.4 Effect of temperature on the activity of Lip4346 under standard assay conditions.

Fig.5 Effects of different pH values on the activity of Lip4346 at 70℃ under standard assay conditions. The pH value of Na2HPO4-citric acid buffer is 3.0–6.0. The pH value of NaH2PO4-Na2HPO4 buffer is 6.0–8.0. The pH value of Tris-HCl buffer is 8.0–9.0. The pH value of glycine-NaOH buffer is 9.0–11.0.

The optimal pH for Lip4346 was 10.0; however, it retained more than 50% of its maximum activity at pH 7.0–10.0, which indicated it had high stability across a broad pH range (Fig.5). Our results revealed that Lip4346 is an alkaline lipase with optimal activity at pH 10.0, which is another important characteristic of enzymes used for industrial biotransformations. Lipases that are stable under alkaline conditions are promising candidates for detergent formulations capable of fat stain removal (Emtenaniet al.,2013) and for treating waste from the dairy industry (Lotrakul and Dharmsthiti, 1997). Most lipases have pH optima of 4.0–8.0 (Sharmaet al., 2011; Singh and Mukhopadhyay, 2012), but the lipases produced byHalobacillussp. AP-MSU 8 (Houdeet al., 2004),Xanthomonas oryzaepv.oryzaeYB103 (Emtenaniet al., 2013), andTrichoderma lentiformeACCC30425 (Wanget al., 2018) show maximum activity and stability at pH 9.0. Alkaline lipases are now popularly exploited for a large variety of industrial purposes.

3.4 Effects of Metal Ions and Inhibitors on Lip4346 Activity

Recombinant Lip4346 was activated by Mn2+, Fe3+, Cd2+,and especially Ca2+, which increased the lipase activity by 40%. Conversely, it was significantly inhibited by Ni2+,Fe2+, Sr+, Mg2+, and especially Cu2+, which sharply reduced lipase activity by 70% (Fig.6).

Fig.6 Effects of metal ions on the activity of Lip4346.

It is well known that metal ions can affect enzyme activity. The significant improvement effect of Ca2+on Lip 4346 activity is consistent with the results of a previous report (Shuet al., 2012). The activity of many enzymes from marine microorganisms is enhanced by Ca2+probably by forming metal–carbonyl coordination bonds with amino acids in the active centers of enzymes, thereby stabling their 3D structures. However, Cu2+can inhibit Ca2+funtion by competitively binding to the same active centers of enzymes, which is similar to the results obtained in our study. Despite these findings, the exact catalytic mechanism of Lip4346 remains unknown and requires further elucidation.

Most of the inhibitors and detergents tested exerted obvious effects on Lip4346 activity. The exceptions were acetonitrile and ethylene-diaminetetraacetic acid (EDTA),which slightly reduced the lipase activity by only 7.34%and 8.76%, respectively. Lip4346 activity obviously decreased after incubation with Tween 80, Tween 20, Tween 100, SDS, dimethylsulfoxide (DMSO), and phenylmethanesulfonyl fluoride (PMSF) (Table 1).

Table 1 Effects of inhibitors and detergents on the lipase activity of Lip4346

3.5 Tolerance of Lip4346 to Organic Solvents

Lip4346 showed high activity in the presence of organic solvents, such as butyl alcohol and glycerol up to a concentration of 50% (v/v). Indeed, Lip4346 showed 220%and 145% activity after incubation for 6 h in 50% (v/v)butyl alcohol and glycerol, which are widely used solvents in lipase-catalyzed transesterification and interesterification for the synthesis of flavor esters, sugar esters, thiol esters, and fatty amides. Moreover, Lip4346 retained more than 60% residual activity in methanol, ethanol, trichloromethane, petroleum ether, and hexane at a concentration of 50% (v/v) (Fig.7).

Fig.7 Effects of organic solvents on the lipase activity of Lip4346.

Compared with plant and animal lipases, bacterial and fungal lipases show higher thermal and organic solvent tolerance. Many solvent-stable lipases have been reported.For example, thePseudomonas aeruginosaAAU2 lipase is stable in organic solvents and retains more than 70% of its activity even after 24 h of incubation (Bose and Keharia, 2013), and a lipase fromStreptomycessp. CS133 retains high stability in 25% (v/v) n-hexane and octane after 48 h of incubation (Manderet al., 2012). Lipases that have high tolerance to organic solvents can be widely applied in the food, pharmaceutical, and green manufacturing industries.

3.6 Substrate Specificity of Lip4346

The recombinant Lip4346 showed different lipolytic activities toward a wide range of substrates, includingpnitrophenyl esters with different chain lengths (C2, C4,C6, C8, C12, C14, C16, and C18) (Table 2). Lip4346 was highly active onp-nitrophenyl esters with medium-chain lengths (C6–C14);p-nitrophenyl caproate (C6) was the most suitable substrate among thep-nitrophenyl esters tested, and lipase activity was low (0–14.77%) with the short-chain (C2) and long-chain (C16, C18) substrates.With the triglyceride substrates, Lip4346 exhibited the highest activity toward tributyrin (C4), followed by tricaproin (C6) and tricaprin (C10), and the lowest lipase activity (0%) was achieved when the chain lengths of triacylglycerols are C14, C16, and C18 (Table 2).

Table 2 Substrate specificity of the lipase Lip4346

The recombinant Lip4346 efficiently hydrolyzedp-nitrophenyl esters with medium-chain lengths (C6–C14) and triglycerides with short-chain lengths (C4) but exhibited very low activity towards all the long-chain substrates (C16,C18). These results show that Lip4346 shows optimum activity with short- and medium-chain substrates. Similarly,Bacilluslipases show high specificity toward fatty acid substrates with short- and medium-chain lengths, and a recombinant lipase fromBacillus megaterium370 preferentially hydrolyzes substrates with short- and medium-chain lengths (C4–C8) rather than a C16 substrate (Ruizet al.,2002).

3.7 Data Availability

The nucleotide sequence oflip4346obtained in this study has been deposited in the GenBank database under accession number MK990595.

4 Conclusions

This study characterized a novel lipase that belongs to lipase family IV detected by the genome fine mapping ofMicrobulbifersp. YNDZ01, a bacterial strain on the surface of macroalgae collected from Indonesia. The recombinant lipase Lip4363 features tolerance to high alkaline and high temperature, rendering it suitable for different biotechnological applications, including the manufacture of drugs, detergents, and cosmetics.

Acknowledgements

This study was financially supported by the China Ocean Mineral Resources R & D Association project (No. DY 135-B2-11), and the China-ASEAN Maritime Cooperation Fund.