Strains, vectors, and chemical reagents
The strains used in this study were Escherichia coli (E. coli) DH5α and BL21 (DE3), and the vectors used were pUC118 (TaKaRa, Dalian, China) and pET32a (Novagen, Madison, WI, USA). The restriction endonuclease and ligase used in vector construction were purchased from the TaKaRa company, and DNA extraction and purification kits were purchased from Qiagen (Hilden, Germany). Other chemical reagents used were all analytically pure and were obtained from Sigma-Aldrich (St. Louis, MO, USA) or Sangon Biotech (Shanghai, China).
Metagenomic library construction and lipolytic enzyme screening
The sediment samples from papermaking wastewater used in this study were collected from a paper mill (34°02'15'' N, 113°51'28'' E) in Xuchang City, Henan Province, China. The genomic DNA of the samples was extracted using a QIAamp DNA Stool Mini Kit (Qiagen) according to the manufacturer’s instructions. The extracted DNA was partially digested with EcoRI, and 2–8 kb DNA fragments were recovered with a gel extraction kit (Qiagen). After ligating the recovered DNA fragment with the library construction vector pUC118, E. coli DH5α was transformed via electroporation. The transformed cells were incubated on LB selection medium containing 1.0 mM isopropyl-β-D-1-thiogalactopyranoside (IPTG), 1% tributyrin, and 100 µg/mL ampicillin to screen for lipolytic enzyme-positive clones. Clones with a transparent circle around the colony were considered lipolytic enzyme-positive clones. All positive clones were sent to Sangon Biotech for sequencing.
DNA sequence analysis of positive clones and lipase phylogenetic tree construction
An insertion fragment sequenced from a positive clone was used to search for possible open reading frames (ORFs) of lipolytic enzymes with ORF Finder software of the NCBI (http://www.ncbi.nlm.nih.gov/). Protein sequence similarity was searched using the NCBI BLAST program. The members of several lipolytic enzyme families that have certain homology with the identified lipase were first analyzed for sequence similarity using Clustal W software (http://www.ebilac.uk/clustalw/), and then the results were used to construct a phylogenetic tree according to the neighbor-joinning method with MEGA 7.0 software to obtain the species information of the identified lipase and its genetic relationships (Kumar et al. 2016).
Bioinformatics analysis of Lip486
The online analysis software ExPASy ProtParam tool (http://web.expasy.org/protparam/) was used to analyze the amino acid isoelectric point, molecular weight, half-life, instability and hydropathicity of Lip486. TMHMM 2.0 (Käll et al. 2004) and SignalP 4.0 servers (Petersen et al. 2011) were used to analyze the transmembrane region and signal peptide sequence of Lip486.
Heterologous expression of lip486 and purification of the recombinant enzyme
To study the enzymatic properties of Lip486, PCR technology was used to amplify lip486. The two primers used were lip486-forward (5'- CCG GAATTC Atg agc cgg atc gta gcc ctg ccg ggc gat g-3') and lip486-reverse (5'- CCC AAGCTT Acg gct gcc gac ggc cgc cgg gtc gag agc ttC-3'). The underlined parts in the two primers represent two restriction enzyme cleavage sites. After double digestion, the amplified lip486 was ligated with the same digested expression vector pET32a for 12 h, and then the ligation product was transformed into E. coli BL21(DE3) by electroporation. Next, the transformed cells were spread onto LB screening plates containing tributyrin, and cultured at 37 °C for 36 h to observe whether a transparent circle formed around the colony. To produce and purify the recombinant enzyme, the identified transformant was first inoculated into LB medium and cultured at 37°C until the OD600 reached 0.8. Then, the culture was induced with 0.28 mM IPTG at 30 °C and 180 rpm for 16 h. Cells were collected by centrifugation, washed twice with pure water and resuspended in PBS buffer. Cells were then broken by ultrasonic wave. Cellular lysates were centrifuged to obtain the crude enzyme solution, which was further purified by a Ni-NTA Sefinose Column (Sangon Biotech) according to the manufacturer’s manual. The resulting pure enzyme was stored at 4 °C for further study.
SDS-PAGE and western blot analysis of Lip486
The molecular weight of the recombinant Lip486 was analyzed by SDS-PAGE, and the molecular weight standard was obtained from Tiangen Biotech (Beijing, China). The western blot analysis is briefly described as follows: First, recombinant Lip486 was analyzed by SDS-PAGE, and then the protein bands from the gel were transferred to a PVDF membrane using a semidry tansfer system (Trans-Blot SD type, Bio-Rad, Hercules, California, USA). The recombinant Lip486 was then incubated at room temperature for 2 h with a 1,000-fold dilution of antibody I (mouse anti-histidine antibody), and after washing, the membrane was incubated with antibody II (IgG goat anti-mouse antibody, conjugated with HRP) diluted 5,000 times at room temperature for 2 h. Finally, NBT (nitro-blue-tetrazolium)-BCIP (5-bromo-4-chloro-3-indolyl-phosphate) reagent (Sangon Biotech) was added to develop color in the dark, and the band of the target protein was observed.
Lip486 enzyme activity analysis
First, 10 µL of appropriately diluted enzyme solution was added to a reaction system consisting of 390 µL of the following ingredients: 10 µL of 1 mM p-nitrophenol ester and 380 µL of 40 mM Britton-Robinson buffer (pH 8.0), which were reacted at 53 °C for 20 min, and 95% ethanol was added to stop the reaction. The absorbance at 405 nm was measured by a microplate reader. One unit of lipase activity is defined as the amount of enzyme required to hydrolyze the substrate to produce 1 µmol of p-nitrophenol per minute. Enzyme activity was measured three times, and the average value was taken.
Characterization of the recombinant Lip486
To detect the specificity of the recombinant Lip486 to substrates with different carbon chain lengths, different substrates with a final concentration of 1 mM were added into the reaction system at 53 °C and pH 8.0, including p-nitrophenyl butyrate (C4), p-nitrophenyl caproate (C6), p-nitrophenyl octanoatee (C8), p-nitrophenyl caprate (C10), p-nitrophenyl laurate (C12), p-nitrophenyl myristate (C14), and p-nitrophenyl palmitate (C16); 95% ethanol was added to terminate the reaction. The absorbance value of each reaction was detected at a wavelength of 405 nm, and the maximum value of enzyme activity was defined as 100%. The optimal substrate of Lip486 was determined after plotting the relative enzyme activity and then used to study the enzymatic properties of Lip486. The optimum pH was measured at a temperature of 53 °C, and the pH range was 5.0–12.0. pH stability was measured by incubating Lip486 at pH 8.0, 9.0 10.0, 11.0 and 12.0 for 1 h, 2 h, 3 h and 4 h, at 4 °C, and then the residual activity of the enzyme was measured. The initial enzyme activity was defined as 100%, and the relative enzyme activity after incubation was plotted against pH. The optimum temperature was investigated at pH 8.0, and the temperature range was 4 °C–70 °C. Temperature stability was investigated by incubating the enzyme at 4 °C, 30 °C, 40 °C, 50 °C and 60 °C for 1 h, 2 h, 3 h, and 4h at pH 8.0 and then measuring the residual enzyme activity. The initial enzyme activity was defined as 100%, and the relative enzyme activity after incubation was plotted against temperature.
Kinetic parameter determination
The optimal substrate was selected to measure the kinetic parameters of Lip486. Substrates with final concentrations of 0 mM, 0.1 mM, 0.25 mM, 0.5 mM, 0.75 mM, 1.0 mM, 1.25 mM, 1.5 mM and 1.75 mM were added and reacted with 20 µL pure enzyme in 0.1 M phosphate buffer (pH 8.0) for 20 min in the buffer solution with the optimal pH at the optimal temperature, and then 100 µL 20% SDS was added to terminate the reaction. The absorbance value of the reaction solution was determined at 405 nm. According to the enzyme activity, a Lineweaver-Burk double reciprocal diagram was constructed, and the kinetic parameters Km and Kcat of Lac486 were obtained. Then, the catalytic efficiency (Kcat/Km) of the enzyme for the optimal substrate was calculated.
Effects of metal ions, organic solvents, and other chemical reagents on Lip486
The effects of three concentrations (1 mM, 5 mM, and 10 mM) of metal ions (Zn2+, Mg2+, Ca2+, Mn2+, Cu2+, Ni2+, Fe2+, and Na+), three concentrations (1%, 15%, and 30%, v/v ) of organic solvents (acetonitrile, methanol, ethanol, and isopropanol), and different concentrations of chemical reagents, including EDTA (10 mM, 25 mM, and 50 mM), SDS (1 mM, 5 mM, and 10 mM), Tween-20 (0.1%, 1%, and 10%, w/v), Tween-80 (0.1%, 1%, and 10%, w/v), and Triton X-100 (0.1%, 1%, and 10%, w/v), were tested after the addition of each target reagent to enzyme solutions for 1 h at 53°C and pH 8.0. All tests were repeated three times, and enzyme activity without the target reagent was defined as 100%.
Compatibility test of the recombinant Lip486 with several commercial detergents
We selected the four most common, large-scale wash products in the Chinese market, including Tide detergent, Blue Moon detergent, Dettol hand sanitizer and Liby detergent for the test. According to the production instructions, the final concentration of Tide detergent was 7 mg/mL, and the final concentrations of Blue Moon detergent, Dettol hand sanitizer and Liby detergent were 1% (v/v). Then, the same volume of a lipase solution (10 U/mL) as the detergent was added to each solution to be tested. The pH values of the mixed solution were 9.50, 7.90, 8.06 and 7.84. After 1 h, 2 h, 3 h, 4 h, 6 h, and 24 h of incubation at room temperature, the residual enzyme activity was measured under standard conditions. The test was repeated three times, and the enzyme activity without detergent was defined as 100%.
Immersion test of the recombinant Lip486 in several commercial detergents
According to conventional practices, 10 g of detergent should be added to each kg of clothing in 5 L water for soaking. At this dose, 10 g of various detergents was added to 5 L of water, and then 10 mL of lipase solution (10 U/mL) was added for 10 min, 20 min and 30 min. Then, the residual enzyme activity of Lip486 was detected. The soaking test was repeated three times, and the enzymatic activity without detergent treatment was defined as 100%.
Determination of the ability of the recombinant Lip486 to remove oil stains from cotton cloth
Four pieces of white cotton cloth with a same size (4 cm ⋅ 4 cm) were cut, soaked in cooking oil for 24 h, removed and air-dried. The four pieces of cloth were divided into A, B, C, and D, and then the following different treatments were performed: A: oil-stained cloth + tap water; B: oil-stained cloth + tap water + thermally deactivated Blue Moon detergent (0.2%); C: oil-stained cloth + tap water + Blue Moon detergent (0.2%); and D: oil-stained cloth + tap water + thermally deactivated Blue Moon detergent (0.2%) + Lip486 solution (0.5 mL, 10 U). The four groups of white cloth were washed (150 rpm) at room temperature for 30 min, rinsed with tap water 3 times and air-dried. Photos were taken, and the percentage of oil stain removal from the four pieces of white cloth was recorded.
The accession number of lip486
The accession number of lip486 (GenBank database) is MZ396074.