An alkaline and surfactant-tolerant lipase from Trichoderma lentiforme ACCC30425 with high application potential in the detergent industry

An alkaline and surfactant-tolerant lipase from Trichoderma lentiforme ACCC30425 with high... Alkaline lipases with adaptability to low temperatures and strong surfactant tolerance are favorable for application in the detergent industry. In the present study, a lipase-encoding gene, TllipA, was cloned from Trichoderma lentiforme ACCC30425 and expressed in Pichia pastoris GS115. The purified recombinant TlLipA was found to have optimal activi- ties at 50 °C and pH 9.5 and retain stable over the pH range of 6.0–10.0 and 40 °C and below. When using esters of different lengths as substrates, TlLipA showed preference for the medium length p-nitrophenyl octanoate. In com- parison to commercial lipases, TlLipA demonstrated higher tolerance to various surfactants (SDS, Tween 20, and Triton X100) and retained more activities after incubation with Triton X100 for up to 24 h. These favorable characteristics make TlLipA prospective as an additive in the detergent industry. Keywords: Trichoderma lentiforme, Alkaline lipase, Heterologous expression, Detergent Introduction site (Woolley and Petersen 1996). During hydrolysis, the Lipase (EC 3.1.1.3) is regarded as one of the most impor- hydroxy group of the catalytic serine attacks the carbonyl tant commercial enzymes, and has been attracting carbon of the ester bond of the substrate, while the cata- enormous attention in the rapidly growing biotechno- lytic histidine acts as a general-base catalyst and abstracts logical area. It catalyzes the hydrolysis of triacylglycerols a proton from the catalytic serine. The alcohol group of to release diacylglyceride, monoacylglycerol, long-chain the substrate is released and an acyl-enzyme intermedi- fatty acids (> 8 carbons) and glycerol at the interface of ate is formed, which is stabilized in the oxyanion hole by oil and water (Brockerhoff 1974). According to the pro- hydrogen bonds. The acyl-enzyme intermediate can be tein structure similarity, lipase belongs to the family of attacked by a water or alcohol molecule, leading to the α/β hydrolases, in which a catalytic triad (usually ser- formation of acid or new ester, respectively (Beer et  al. ine, histidine, and aspartic or glutamic acid) and an oxy- 1996). anion hole (just like a catalytic pocket) are crucial for Lipases are widespread in nature and have been catalysis (Gupta et  al. 2015), and a lid structure involves reported in microbes, plants, and animals. Neverthe- in the substrate accessibility and binding in the active less, bacterial and fungal lipases are of special interest as they are easily produced and favorable for industrial pur- poses due to the high yields and great versatility and sta- *Correspondence: baiyingguo@caas.cn; gujingang@caas.cn bility under harsh conditions (Schmid 2016). Microbial Key Laboratory of Microbial Resources of the Ministry of Agriculture, lipases vary in structures and enzymatic properties. For Institute of Agricultural Resources and Regional Planning, Chinese example, the lipases from Candida rugosa with different Academy of Agricultural Sciences, Beijing 100081, People’s Republic of China hydrophobic zones at the central channel showed differ - Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, ent activities (Mancheño et  al. 2003; Domínguez et  al. Feed Research Institute, Chinese Academy of Agricultural Sciences, 2006). At the entrance of the channel in close proximity Beijing 100081, People’s Republic of China © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Wang et al. AMB Expr (2018) 8:95 Page 2 of 11 to the catalytic site and the substrate binding site, there that is widely used to produce industrial enzymes on is a phenylalanine-rich region associated with substrate large scale (Singh et  al. 2015). Up to now, the genome binding. The phenylalanine content is negatively corre - sequences of 13 Trichoderma strains have been com- lated with the catalytic activity towards cholesterol ester. pleted (Halliwell and Griffin 1973; Martinez et  al. 2008; In addition, the lipase activity is also related to the size Kubicek et  al. 2011; Studholme et  al. 2013; Xie et  al. and orientation of the channel. For example, the lipase 2014; Baroncelli et  al. 2015, 2016; Shikunne et  al. 2015; from Aspergillus niger having a narrow and curved chan- Yang et  al. 2015; Lee et  al. 2017), and the number keeps nel shows relatively low activity, while those from Ophi- increasing. Sequence analysis of the genomes of T. vir- ostoma piceae, Nectria haematococca and Trichoderma ide, T. reesei, T. harzianum and T. gamsii indicated that reesei have straightforward and wider channels and much Trichoderma harbors a great variety of lipase genes. In higher activities (Barriuso et al. 2016). our preliminary studies, four Trichoderma strains dem- Microbial lipases are widely used in various industries, onstrated lipase-producing capabilities. One of them, T. especially in the detergent (Saxena et al. 2004). Since the lentiforme ACCC30425, showed the highest lipase-pro- 1960s, enzyme-based detergent has been introduced into ducing capability under alkaline conditions and thus was the market, and lipase that efficiently removes acylg - selected for draft genome sequencing. In this study, we lycerols has been one of the major additives in cleaning reported on the gene cloning, heterologous expression, agent (Abo 1990). The industrial and environmental sig - and biochemical characterization of an alkaline meso- nificances of lipase include but are not limited to: 1 lipase philic lipase from T. lentiforme ACCC30425. Its applica- provides an increasable detergency (comparing with the tion potential as an additive in detergent was assessed as detergent alone), especially at low temperatures and neu- well. tral to alkaline pH; 2 lipase with low substrate specific - ity is highly effective to remove stubborn stains, such as Materials and methods blood and fat; and 3 lipase not only has high biodegrada- Strains tion ability but also brings harmless effect on aquatic eco - Trichoderma lentiforme ACCC30425 was supplied by the systems (Jurado et al. 2007). However, several bottlenecks Agricultural Culture Collection of China. Escherichia coli have limited lipase application in detergent, such as their Trans1-T1 was purchased from TransGen (China). The broad substrate specificity, stringent washing conditions heterologous expression system containing the vector (low temperature and alkaline conditions), and sensitivity pPIC9 and Pichia pastoris GS115 competent cells were to chemicals in detergents (Sharma et al. 2001, 2002). For obtained from the Invitrogen. example, a large number of microbial lipases produced by bacteria and yeast show the maximum activities at high Induction and detection of the lipase production by T. temperatures, such as the lipases from Pseudomonas aer- lentiforme ACCC30425 uginosa, thermophilic Bacillus sp. and yeast Kurtzmano- Trichoderma lentiforme ACCC30425 was grown in the myces sp. that have temperature optima of 60–75  °C. lipase-inducing medium (5  g/L glucose, 5  g/L NaNO , Although some bacterial lipases are neutral to alkaline, 5 g/L K HPO , 0.3 g/L M gSO , 0.01 g/L F eSO , and 4 g/L 2 4 4 4 but they lose most of the activities at pHs higher than olive oil as the sole carbon source) with the agitation rate 9.0 (Karadzic et  al. 2006; Nawani et  al. 2007). Moreover, of 180 rpm at 28 °C for 8 days. The culture supernatants the sensitivity to surfactant deters numerous lipases from were collected every day and subject to lipase activity their application in laundry. Therefore, it’s of great value assay (spectrophotometric method as described below). to obtain an alkaline mesophilic lipase with a high toler- ance to surfactants in the washing industry (Gutarra et al. Cloning of the gene TllipA 2009). Seven-day-old mycelia of T. lentiforme ACCC30425 were Trichoderma is a common filamentous fungus in soil collected, flash-frozen in liquid nitrogen, and ground into and root ecosystems, and sometimes in air, water, sand, a fine powder. Total RNA was extracted using the Trizol etc. It shows antagonistic, symbiotic and parasitic capa- method (Chomczynski and Sacchi 1987), and cDNAs bilities to interact with other microbes and is used more were synthesized by reverse transcription. According to extensively than any other single microbe in agriculture the whole genome sequence of T. lentiforme ACCC30425 (Benítez et  al. 2005). Moreover, Trichoderma has sig- (accomplished by the Majorbio, China), a lipase-encod- nificant lignocellulose-degrading capability because it ing gene, TllipA, was identified. The nucleotide and can produce a variety of hydrolytic, lytic and auxiliary amino acid sequences of TllipA were analyzed by using enzymes including cellulase, xylanase, chitinase, laccase, the BLASTx and BLASTp programs (https ://blast .ncbi. lipase, etc. (Zhang and Xia 2017). Besides, Trichoderma nlm.nih.gov/Blast .cgi), respectively. The signal peptide represents one of the most important expression systems was predicted using the SignalP 4.0 (http://www.cbs.dtu. Wang et al. AMB Expr (2018) 8:95 Page 3 of 11 dk/servi ces/Signa lP/). The prediction of molecular mass loaded onto the HiPrepTm 26/10 Desalting column and and isoelectric point (pI) value was performed using the HiTrapQHP column (GE Healthcare). Fractions contain- Vector NTI Advance 10.0 software (Invitrogen). Multi- ing lipase activity were pooled and concentrated by ultra- ple sequence alignment of TlLipA and other lipase rep- filtration (5-kDa molecular weight cutoff) for further resentatives was conducted by using the ClustalX 1.81 characterization. Sodium dodecyl sulfate-polyacrylamide and presented by ESPript 3.0 (http://espri pt.ibcp.fr/ESPri gel electrophoresis (SDS-PAGE) was carried out with the pt/cgi-bin/ESPri pt.cgi). The putative three-dimensional 5% stacking gel and 12% separation gel. Protein concen- structure was built by SWISS-MODEL (https ://www. tration was determined using the Bradford method with swiss model .expas y.org/) with the lipase from Ophios- bovine serum albumin as the standard. toma piceae (PDB: 4BE4) as the template. PCR was then conducted to obtain the DNA fragment Lipase activity assays coding for mature TlLipA with an expression primer The lipase activity was determined by the alkali titration set (TllipA-expF: 5′-GGG GAA TTC GCT CAA GGC method, using olive oil as the substrate. Olive oil was CAA GTC AAC GTT ACC ATT CCC -3′ and TllipA-expR: emulsified in 4% (w/v) polyvinyl alcohol solution at the 5′ -GGG GC G GC C GC C TA GA A GAT C A G T GA AT C ratio of 1:3. The reaction mixture contained 2.5  mL of GAT ATG CTC CTT GAT AAA G-3′, the EcoRI/NotI sites 20 mM citric acid-Na HPO (pH 7.5), 2.0 mL of emulsi- 2 4 underlined). The amplification was performed at 94  °C fied olive oil, and 0.5 mL of properly diluted enzyme solu - for 5 min followed by 35 cycles of denaturation (1 min at tion. After incubation at 40  °C for 15  min in a shaking 94 °C), annealing (1 min at 62 °C) and extension (1.5 min water bath, 7.5  mL of 95% ethanol was added to termi- at 72  °C), and a final extension of 72  °C for 10  min. The nate the reaction. The amount of liberated fatty acids was PCR products of the appropriate size were sequenced by measured by titration with 50 mM NaOH, using phenol- Biomed (China). phthalein as an indicator. One unit (U) of lipase activity was defined as the amount of lipase to liberate 1 μmol of Expression of the recombinant TlLipA in P. pastoris fatty acids per min from the olive oil. All determinations The correct PCR products were digested with EcoRI and were performed in triplicate. NotI and ligated into the EcoRI/NotI-digested pPIC9 vec- The spectrophotometric method was also used to tor to construct the recombinant plasmid pPIC9-TllipA. determine the lipase activity of purified recombinant Colony PCR was conducted using the AOX primers to TlLipA. The reaction system consisted of 0.1  mL of screen positive clones, which were further verified by appropriately diluted enzyme and 2.4  mL of substrate DNA sequencing. The correct recombinant plasmid solution containing 0.8  mM p-nitrophenyl octanoate was then linearized with BglII and transformed into (p-NPO, C8; dissolved in isopropanol at first) in 20  mM P. pastoris GS115 competent cells by the electropora- Tris–HCl (specific pH). After incubation at 37 °C (or the tion method with the Gene Pulser Xcell Electroporation optimum temperature of 50 °C) for 15 min, the reactions apparatus (Bio-Rad), following the instructions of Invit- were terminated by addition of 2.0  mL of 95% ethanol. rogen’s protocol (2000  V, 200 Ω, 25 μF, and 5  ms). The After centrifugation at 5000g for 10 min, 200 μL of each transformants were grown on minimal dextrose (MD) reaction supernatant was transferred to 96-well micro- agar plates at 32°C for 48 h. Ninety-six colonies were ran- plate for absorbance measurement at OD . One unit of domly selected to grow in 2-mL buffered glycerol com - lipase activity was defined as the amount of enzyme that plex medium (BMGY) at 30°C for 48  h. The cells were produced 1 μmol of p-nitrophenol (pNP) per min under collected by centrifugation (12,000g) and resuspended standard reaction conditions. All determinations were in 2-mL buffered methanol complex medium (BMMY). performed in triplicate. After 48-h induction with the 0.5% (v/v) methanol at 30°C, the culture supernatants were collected by cen- Eec ff ts of pH and temperature on the TlLipA activity trifugation for the lipase activity assay. The positive trans - The optimal pH of the recombinant Tl LipA was deter- formant showing the highest lipase activity was grown mined at 37  °C for 15  min in the following buffers: in 1-L Erlenmeyer flasks containing 200 mL medium for 25  mM citric acid-Na HPO for pH 5.0–7.0 and 20  mM 2 4 large-scale fermentation. Tris–HCl for pH 7.0–10.0. The optimal temperature was determined over the temperature range of 20–60  °C in Purification of recombinant TllipA 20 mM Tris–HCl (pH 9.5) for 15 min. The culture supernatants were collected by centrifuga - tion at 12,000g, 4°C for 10 min and concentrated through Eec ff ts of pH and temperature on the TlLipA stability Vivaflow 200 membrane of 5-kDa molecular weight The pH stability of Tl LipA was determined by measuring cutoff (Vivascience, Germany). The crude enzyme was the residual lipase activity under the optimal conditions Wang et al. AMB Expr (2018) 8:95 Page 4 of 11 (pH 9.5, 50  °C and 15  min) after pre-incubation of the commercial lipases (HA, HB, HD and HE) from Xin- enzyme at 37 °C for 1 h in the same buffers (pH 5.0–10.0) huayang Co. (China) were selected as references, and mentioned above. Thermal stability of Tl LipA was deter- their enzymatic properties were determined as described mined by measuring the residual lipase activity under the above. optimal condition after incubation of the enzyme at 40 To assess the stability in the presence of different sur - and 50 °C, respectively, for various periods (0–120 min). factants, TlLipA was incubated in 20 mM Tris–HCl (pH The TlLipA activities under optimal conditions (pH 9.5, 9.5) at 37  °C containing 50, 20, 10 or 1% (v/v) of Triton 50 °C and 15 min) were defined as 100% relative activity. X100, or 20, 10 or 1% (w/v) of SDS for various periods (0, 3, 6, 12, and 24 h). Mesophilic alkaline lipases HA and HB were used as references. The residual activities were Eec ff ts of metal ions and chemical reagents on the TlLipA determined under the optimum reaction conditions of activity each enzyme. To find out the effects of different metal ions and chemical reagents on TlLipA activity, 5  mM of N a , Results + 2+ + 2+ 2+ 2+ 2+ K, Ca, Ag, Mg, Mn, Zn, Ni , EDTA or Olive oil‑degrading capability of T. lentiforme ACCC30425 β-mercaptoethanol was added into the reaction system, By using olive oil as the sole carbon source, T. lentiforme respectively. TlLipA activity was determined at pH 9.5 ACCC30425 showed detectable lipase activity at day 4 and 50  °C for 15  min with p-NPO as the substrate. The and afterwards (Fig. 1). Using p-NPO as the substrate, the TlLipA activities without any chemical addition were lipase activity in the culture supernatants reached maxi- defined as 100%. mum at day 7, which was up to 1.7 U/mL (pH 8.0, 37 °C and 15  min). It indicated that T. lentiforme ACCC30425 Kinetic parameters has the capability of producing lipases to degrade olive oil p-Nitrophenyl esters with different acyl chains (C4– in the medium. C16) including p-nitrophenol butyrate (pNPB, C4), pNPO (C8), p-nitrophenol decanoic acid (pNPD, C10), Sequence analysis of the TllipA p-nitrophenol dodecanoate (pNPDD, C12), p-nitrophe- Genome sequence analysis indicated that the lipase- nol myristate (pNPM, C14) and p-nitrophenol palmitate encoding gene, TllipA (GenBank accession number: (pNPP, C16) at the concentrations of 0.2–1.6  mM were MF460438), contains 1707  bp. Deduced TlLipA consists used as the substrates. The Michaelis–Menten kinetic of a putative signal peptide of 20 residues and a mature parameters K and V of TlLipA were determined at protein of 548 residues. The molecular mass and pI of m max pH 9.5 and 50  °C for 10  min (shorter reaction time for mature TlLipA were estimated to be 60.0  kDa and 4.56, pseudo-first order kinetic analysis). The experiments respectively. Multiple sequence alignment and homol- were repeated for three times, and each experiment ogy modeling analysis indicated that deduced TlLipA included triplicate. GraphPad Prism 5 (GraphPad Soft- contains the typical α/β-hydrolase fold structure with ware) was used to calculate the K (substrate affinity) and an N-terminal 3-stranded β-sheet, a major 12-stranded V (maximum velocity) values. The k (the turnover max cat rate per second) and k /K (catalytic efficiency) val - cat m ues were then calculated to measure the efficiency of an enzyme that converts substrate to product at sub-saturat- ing substrate concentration and catalytic efficiency (Chi - naglia et al. 2014). TlLipA tolerance and stability to various surfactants Surfactant is a common and indispensable ingredient in detergents. To find out the effect of surfactant on Tl LipA activity, four concentrations (0.50, 0.20, 0.10 and 0.05%) of Tween 20, Tween 80, Triton X100 or 50 μM SDS (v/v) were individually added into the reaction system con- taining pNPO as the substrate, and the relative activities were tested under optimum reaction conditions (pH 9.5 Fig. 1 Lipase activities of the culture supernatants of T. lentiforme ACCC30425 growing in the inducing medium with olive oil as and 50 °C for 15 min). The reaction systems without any the sole carbon source. The lipase activities were assayed using surfactant were treated as controls. To assess the appli- spectrophotometric method with pNPP as the substrate cation potential of TlLipA in detergent industry, four Wang et al. AMB Expr (2018) 8:95 Page 5 of 11 (See figure on next page.) Fig. 2 Multiple sequence alignment of deduced TlLipA with structure-resolved lipases 4BE4 from Ophiostoma piceae and 1LLF from Candida cylindracea as well as biochemically characterized lipases TbLipA from Trichophyton benhamiae and DrLipA from Diutina rugosa. The secondary structural elements are indicated β-sheet, and 19 helices (Fig.  2, 3). The putative catalytic residual activity assay. As shown in Fig.  5c, TlLipA was triad consists of S215, E346 and H464. These catalytic relatively stable at 40 °C, retaining more than 60% of the residues are responsible for the nucleophilic attack on initial activity after 60-min incubation; when extended the carbonyl carbon atom of the ester bond. The puta - to 120 min, more than 50% activity was still retained. In tive lid consists of one α-helix (residues 76–81) and two contrast, it lost stability at 50  °C, losing more than 50% 3 -helics (residues 83–87 and 89–91) flanked by two activity within 5-min incubation and almost all activ- loops that end in a disulfide hinge (residues C62 and ity within 120-min. The pH stability of Tl LipA was also C101). assessed. The enzyme was stable at pH 6.0–9.0, retaining more than 80% of the initial activity after 60-min pre- Production and purification of the recombinant TlLipA incubation at 37 °C (Fig. 5d). These results indicated that The DNA fragment coding for the mature Tl LipA was TlLipA was stable over the cold (≤ 40 °C) and neutral to obtained with primers TllipA-expF and TllipA-expR, and alkaline conditions. transformed into E. coli Trans1-T1 for sequencing. The correct PCR product was digested with EcoRI and NotI Kinetic parameters and then cloned into the pPIC9 vector in-frame fusion of The kinetic parameters K , V , k and k /k of m max cat cat m the α-factor signal peptide to construct the recombinant TlLipA were determined using the six p-nitrophe- plasmid pPIC9-TllipA. The recombinant plasmid was nyl esters of various acyl chain lengths as substrates. linearized by BglII, and transformed into the P. pastoris As shown in Table  1, TlLipA showed higher affinities GS115 competent cells by electroporation. TlLipA was (decreased K values) and catalytic efficiencies (increased successfully produced according to the Pichia expression k /k values) towards short-chain substrates with (C4 cat m kit and secreted into the culture. After centrifugation, to C10). pNPO (C8) as the preferred substrate was cata- concentration and exchange chromatography, the crude lyzed with the highest efficiency of 41.0/s mM. enzyme was purified to electrophoretic homogeneity, showing a single band of approximately 60  kDa in SDS- Eec ff t of metal ions and chemical reagents on TlLipA PAGE (Fig.  4). This molecular mass was similar to the activity 2+ theoretical value (60.0 kDa), indicating that the band was Of the ten chemicals tested in this study (Table  2), Ni , 2+ 2+ purified recombinant Tl LipA indeed. The lipase activity Zn, Mn and EDTA strongly inhibited the TlLipA of purified recombinant TlLipA was determined to be activity, leading to the activity loss of more than 50%, 10.4 ± 0.5 U/mL by using the alkali titration method. while other chemicals had no or little effects (0–32%). None of the chemical addition resulted in an improve- Eec ff t of pH and temperature on TlLipA activity ment of lipase activity. The results indicated that Tl LipA pNPO was used as the substrate for biochemical char- was tolerant to most tested metal ions and chemical acterization of purified recombinant Tl LipA. Over the reagents. range of pH 5.0–10.0, the TlLipA had poor activity under acidic conditions and showed maximum activity at pH TlLipA tolerance to surfactants 9.5 (Fig.  5a). Under the optimum pH (9.5), the tempera- To find out the application potentials of Tl LipA in deter- ture-activity profile of Tl LipA was determined over the gent industry, we selected four commercial lipases as ref- temperature range from 20 to 60  °C. The enzyme had a erences and compared their activities with TlLipA in the temperature optimum at 50 °C and remained 20–40% of presence of 0.05–0.50% of Tween 20, Tween 80, Triton the maximum activity at 20–40 °C (Fig. 5b). These results X100 or SDS. As shown in Fig. 6, Tween 20, SDS and Tri- indicated that the purified recombinant Tl LipA is a mes- ton X100 at higher concentrations (0.10–0.50%) signifi - ophilic alkaline lipase with great adaptation to low mod- cantly enhanced the TlLipA activity up to 2.24-fold, while erate temperature. 0.05–0.50% of Tween 80 and 0.05% of Tween 20, SDS and Triton X100 inhibited the lipase activity of TlLipA by Thermal and pH stability of TlLipA 20‒60%. It indicated that high concentrations of Tween After incubation at 40 and 50  °C respectively for vari- 20, Triton X100 and SDS may emulsify the substrate to ous periods, aliquots of TlLipA were withdrawn for enlarge the interface area between TlLipA and substrate, Wang et al. AMB Expr (2018) 8:95 Page 6 of 11 Wang et al. AMB Expr (2018) 8:95 Page 7 of 11 Fig. 3 Multiple sequence alignment of deduced TlLipA with structure-resolved lipases 4BE4 from Ophiostoma piceae and 1LLF from Candida cylindracea as well as biochemically characterized lipases TbLipA from Trichophyton benhamiae and DrLipA from Diutina rugosa. The secondary structural elements are indicated condition, respectively (pH 9.0 and 50 °C for HA and HB, pH 8.0 and 50 °C for HD, and pH 8.0 and 40 °C for HE), with the only exception of increased HB activity (approx- imately 13.4%) by 0.05% SDS. The results indicated that TlLipA was highly tolerant to all tested surfactants and retained most or even enhanced activities. TlLipA stability to surfactants Considering the long shelf life of laundry detergent, lipase stability in the presence of surfactants is a key fac- tor of commercialization. Therefore we also determined the TlLipA stability after pre-incubation with 1–20% SDS or 1–50% Triton X100 for various periods. In comparison to the surfactant-untreated controls, TlLipA incubated with 1‒20% Triton X100 for 0–24 h showed significantly enhanced activities of 1.3‒1.7-fold, but lost more than 50% activity when incubated with 50% of Triton X100 Fig. 4 SDS-PAGE analysis of purified recombinant Tl LipA. Lane M, the and 1‒50% SDS (Fig.  7a). In contrast, both HA and HB molecular weight standard markers; lane 1, the purified recombinant lost stability in the presence of 1‒50% of Triton X100 TlLipA at 0.3 mg/mL; and lane 2, the purified recombinant Tl LipA at or SDS (Fig.  7b, c), retaining less than 30% activity after 3.0 mg/mL incubation at 3 h. Discussion consequently improving the lipase activities. On the con- The genus Trichoderma contains a very large group trary, the activities of the four commercial lipases were of important microorganisms. It is not only a genetic mostly inhibited by the surfactants under each optimum resource of various functional proteins (Freitas et  al. Wang et al. AMB Expr (2018) 8:95 Page 8 of 11 Fig. 5 Biochemical characterization of TlLipA. a The pH-activity profiles determined in citric acid-Na HPO (black square) and Tris–HCl (black 2 4 circle) buffers. b The temperature-activity profile determined in Tris–HCl buffer at pH 9.5. c The thermal stability at 50 °C (black circle) and 40 °C (blackup-pointing triangle). d The pH stability after 1-h incubation at 37 °C and pH 5.0–10.0 lentiforme ACCC30425, the full-length TllipA was iden- Table 1 The kinetic values of  purified recombinant TlLipA towards esters of different lengths tified and its structure and functions were predicted. Although TllipA shows high sequence identity (100%) to Substrate V (μmol/ k (/s) K (mM) k /K (/s mM) max cat m cat m the hypotheoretical lipase from T. guizhouense, its identi- min mg) ties to lipases with function verified or structure resolved pNPO (C8) 27.3 ± 4.4 27.4 ± 4.4 0.67 ± 0.23 41.0 are much lower (< 50%). Thus it is of importance and pNPD (C10) 12.5 ± 0.7 12.5 ± 0.7 0.35 ± 0.07 35.8 novelty to clone the gene and produce the gene product pNPB (C4) 12.2 ± 0.8 12.2 ± 0.8 0.40 ± 0.07 30.7 for potential applications in various industries. pNPDD (C12) 16.8 ± 2.4 16.8 ± 2.4 1.59 ± 0.42 10.6 Most fungal lipases act over a broad pH range, with pNPM (C14) 4.3 ± 1.0 4.3 ± 1.0 3.29 ± 1.21 1.32 the pH optima of 4.0–8.0 (Sharma et al. 2011; Singh and pNPP (C16) 4.8 ± 1.0 4.8 ± 1.0 4.22 ± 1.28 1.14 Mukhopadhyay 2012), and are mesophilic with thermola- bility at > 40 °C (Gutarra et al. 2009). The pH optimum of TlLipA was 9.5, which is higher than most fungal lipases 2014) but also a key workhorse for enzyme production characterized so far. Moreover, it showed great adapt- on commercial scale (i.e. T. reesei) (Jørgensen et al. 2014). ability and stability under neutral to alkaline conditions In the present study, we reported an alkaline, mesophilic (pH 7.0–10.0). On the other hand, TlLipA showed maxi- lipase-producing strain (ACCC30425) of T. lentiforme. mum activity at 50 °C and thermolability at > 40 °C. These Along with the rapid progress of genome sequencing enzymatic properties make TlLipA potential for appli- (Yang et  al. 2015), to obtain objective genes with spe- cation in the alkaline and low to moderate temperature cial characters is very simple and efficient. Based on the fields, especially the washing industry (Jurado et al. 2007; sequence analysis and annotation of the genome of T. Grbavčić et al. 2011). Some lipases are resistant to heavy Wang et al. AMB Expr (2018) 8:95 Page 9 of 11 Table 2 Eec ff ts of metal ions and chemical reagents on the TlLipA activity Chemicals Relative activity (%) Chemical Relative activity (%) 2+ CK 100.0 ± 2.4 Mn 13.9 ± 0.8 2+ 2+ Mg 97.6 ± 2.5 Zn 11.9 ± 0.9 2+ 2+ Ca 92.4 ± 4.0 Ni 7.6 ± 0.8 K 91.1 ± 3.0 β-Mercaptoethanol 60.5 ± 0.9 Na 85.7 ± 6.8 EDTA 33.8 ± 2.0 Ag 68.6 ± 3.8 Values are given as the mean ± standard deviations (n = 3) ab 0.05% 0.10% 0.20% 0.50% 0.05%0.10% 0.20%0.50% HA HB HD HE TlLipA HA HB HD HE TlLipA cd 0.05% 0.10% 0.20% 0.50% 0.05%0.10% 0.20%0.50% 0 0 HA HB HD HE TlLipA HA HB HD HE TlLipA Fig. 6 Tolerance of TlLipA and four commercial lipases HA, HB, HD and HE to Tween 20 (a), Tween 80 (b), Triton X100 (c) and SDS (d) at different concentrations, respectively 2+ 2+ 2+ (C8) and pNPD (C10) were higher than that towards metals, such as Ca, Fe , and Mg (Jurado et al. 2007; other esters of different lengths. Similar results had been Gaur et al. 2008; Rao et al. 2009; Sethi et al. 2016). TlLipA 2+ 2+ reported on the two highly thermophilic alkaline lipases showed similar tolerance to C a and M g , but showed 2+ 2+ 2+ from Thermosyntropha lipolytica (Moh’d and Wiegel sensitivity to heavy metals Ni, Mn and Zn and 2007). chemical reagents EDTA and β-mercaptoethanol. u Th s The resistance to surfactant is a big challenge for the effects of metal ions and chemical reagents on Tl LipA lipase’s application in washing industry. In general, the should be considered for future application. surfactant has negative effects on enzymatic hydrolysis Lipase is usually considered as an enzyme that hydro- and represents a competitive inhibiter in the reaction sys- lyzes the cleavage of long-chain acylglycerols; however, tem (Tatara et al. 1985). TlLipA remained highly active in most known lipases are also active on shorter acyl chain the presence of both anionic and non-ionic surfactants, esters (Li and Zong 2010). TlLipA having lipase activ- including SDS, Triton X100, Tween 20 and Tween 80. ity as determined by the alkali titration method is a true Moreover, the resistance of TlLipA to surfactants showed lipase, but it prefers medium-chain fatty acid esters. The improvement along with increased concentration. For catalytic efficiencies (k /K ) of TlLipA towards pNPO cat m Wang et al. AMB Expr (2018) 8:95 Page 10 of 11 the enzymatic activities of commercial lipases HA and SDS 1% SDS 10% SDS 20% HB. In comparison to the stabilities of other lipases with TX100 10% TX100 20% TX100 50% surfactants (1-h incubation) (Rathi et  al. 2001; Bora and control TX100 1% Kalita 2010), TlLipA retained similar or higher activity in the presence of 10% Triton X100 even over 24 h-incu- bation (150% vs. 93‒164%). Thus the alkaline mesophilic TlLipA with adaptability and stability to broad pH and temperature ranges and high tolerance to surfactants is favorable for potential application in the washing industry. 036 12 24 Incubaon me (hr) Abbreviations BMGY: buffered glycerol-complex medium; BMMY: buffered methanol-com- plex medium; MD: minimal dextrose; PAGE: polyacrylamide gel electrophore- sis; SDS: sodium dodecyl sulfonate; pNP: p-Nitrophenol; pNPB: p-Nitrophenol SDS 1% SDS 10% SDS 20% butyrate; pNPD: p-Nitrophenol decanoic acid; pNPDD: p-nitrophenol dode- TX100 10% TX100 20% TX100 50% canoate; pNPM: p-Nitrophenol myristate; pNPO: p-Nitrophenyl octanoate; TX100 1% control pNPP: p-Nitrophenol palmitate; pI: isoelectric point. Authors’ contributions YW, YB and JG designed the research; YW, RM and SL performed the research; YW, MG, BY and JG analyzed the data and wrote the paper. All authors read and approved the final manuscript. Acknowledgements We are grateful to the Agricultural Culture Collection of China (ACCC) for providing the Trichoderma strain ACCC30425. 03 61224 Competing interests Incubaon me (hr) The authors declare that they have no competing interests. Availability of data and materials Data can be shared. Please send email to gujingang@caas.cn. SDS 1% SDS 10% SDS 20% TX100 10% TX100 20% TX100 50% Consent for publication TX100 1% control Not applicable. Ethics approval and consent to participate 100 Not applicable. This article does not contain any studies with human partici- pants or animals performed by any of the authors. Funding This work was supported by the National High Technology Research and Development Program of China (863 Program, 2013AA102805-05), the National Non-profit Institute Research Grant of Chinese Academy of Agri- 0 cultural Sciences (IARRP-2015-24) and the Fundamental Research Funds for 03 61224 Central Non-profit Scientific Institution of China (1610132016050). Incubaon me (hr) Fig. 7 Stability of TlLipA (a) and commercial lipases HA (b) and HB (c) Publisher’s Note after incubation with SDS or Triton X100 for various durations Springer Nature remains neutral with regard to jurisdictional claims in pub- lished maps and institutional affiliations. Received: 23 January 2018 Accepted: 23 May 2018 example, when increased the concentration of SDS from 0.05 to 0.50%, the relative activity was enhanced from 60 to 224%. This result is contrary to the commercial lipases References tested in this study and the previous study that the lipase Abo M (1990) Method of purifying dry-cleaning solvent by decomposing activity would drop down along with the increased con- liquid contaminants with a lipase. World Organ Patent 9(007):606 Baroncelli R, Piaggeschi G, Fiorini L, Bertolini E, Zapparata A, Pè ME, Sar- centration of SDS (Rathi et  al. 2001). 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An alkaline and surfactant-tolerant lipase from Trichoderma lentiforme ACCC30425 with high application potential in the detergent industry

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Abstract

Alkaline lipases with adaptability to low temperatures and strong surfactant tolerance are favorable for application in the detergent industry. In the present study, a lipase-encoding gene, TllipA, was cloned from Trichoderma lentiforme ACCC30425 and expressed in Pichia pastoris GS115. The purified recombinant TlLipA was found to have optimal activi- ties at 50 °C and pH 9.5 and retain stable over the pH range of 6.0–10.0 and 40 °C and below. When using esters of different lengths as substrates, TlLipA showed preference for the medium length p-nitrophenyl octanoate. In com- parison to commercial lipases, TlLipA demonstrated higher tolerance to various surfactants (SDS, Tween 20, and Triton X100) and retained more activities after incubation with Triton X100 for up to 24 h. These favorable characteristics make TlLipA prospective as an additive in the detergent industry. Keywords: Trichoderma lentiforme, Alkaline lipase, Heterologous expression, Detergent Introduction site (Woolley and Petersen 1996). During hydrolysis, the Lipase (EC 3.1.1.3) is regarded as one of the most impor- hydroxy group of the catalytic serine attacks the carbonyl tant commercial enzymes, and has been attracting carbon of the ester bond of the substrate, while the cata- enormous attention in the rapidly growing biotechno- lytic histidine acts as a general-base catalyst and abstracts logical area. It catalyzes the hydrolysis of triacylglycerols a proton from the catalytic serine. The alcohol group of to release diacylglyceride, monoacylglycerol, long-chain the substrate is released and an acyl-enzyme intermedi- fatty acids (> 8 carbons) and glycerol at the interface of ate is formed, which is stabilized in the oxyanion hole by oil and water (Brockerhoff 1974). According to the pro- hydrogen bonds. The acyl-enzyme intermediate can be tein structure similarity, lipase belongs to the family of attacked by a water or alcohol molecule, leading to the α/β hydrolases, in which a catalytic triad (usually ser- formation of acid or new ester, respectively (Beer et  al. ine, histidine, and aspartic or glutamic acid) and an oxy- 1996). anion hole (just like a catalytic pocket) are crucial for Lipases are widespread in nature and have been catalysis (Gupta et  al. 2015), and a lid structure involves reported in microbes, plants, and animals. Neverthe- in the substrate accessibility and binding in the active less, bacterial and fungal lipases are of special interest as they are easily produced and favorable for industrial pur- poses due to the high yields and great versatility and sta- *Correspondence: baiyingguo@caas.cn; gujingang@caas.cn bility under harsh conditions (Schmid 2016). Microbial Key Laboratory of Microbial Resources of the Ministry of Agriculture, lipases vary in structures and enzymatic properties. For Institute of Agricultural Resources and Regional Planning, Chinese example, the lipases from Candida rugosa with different Academy of Agricultural Sciences, Beijing 100081, People’s Republic of China hydrophobic zones at the central channel showed differ - Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, ent activities (Mancheño et  al. 2003; Domínguez et  al. Feed Research Institute, Chinese Academy of Agricultural Sciences, 2006). At the entrance of the channel in close proximity Beijing 100081, People’s Republic of China © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Wang et al. AMB Expr (2018) 8:95 Page 2 of 11 to the catalytic site and the substrate binding site, there that is widely used to produce industrial enzymes on is a phenylalanine-rich region associated with substrate large scale (Singh et  al. 2015). Up to now, the genome binding. The phenylalanine content is negatively corre - sequences of 13 Trichoderma strains have been com- lated with the catalytic activity towards cholesterol ester. pleted (Halliwell and Griffin 1973; Martinez et  al. 2008; In addition, the lipase activity is also related to the size Kubicek et  al. 2011; Studholme et  al. 2013; Xie et  al. and orientation of the channel. For example, the lipase 2014; Baroncelli et  al. 2015, 2016; Shikunne et  al. 2015; from Aspergillus niger having a narrow and curved chan- Yang et  al. 2015; Lee et  al. 2017), and the number keeps nel shows relatively low activity, while those from Ophi- increasing. Sequence analysis of the genomes of T. vir- ostoma piceae, Nectria haematococca and Trichoderma ide, T. reesei, T. harzianum and T. gamsii indicated that reesei have straightforward and wider channels and much Trichoderma harbors a great variety of lipase genes. In higher activities (Barriuso et al. 2016). our preliminary studies, four Trichoderma strains dem- Microbial lipases are widely used in various industries, onstrated lipase-producing capabilities. One of them, T. especially in the detergent (Saxena et al. 2004). Since the lentiforme ACCC30425, showed the highest lipase-pro- 1960s, enzyme-based detergent has been introduced into ducing capability under alkaline conditions and thus was the market, and lipase that efficiently removes acylg - selected for draft genome sequencing. In this study, we lycerols has been one of the major additives in cleaning reported on the gene cloning, heterologous expression, agent (Abo 1990). The industrial and environmental sig - and biochemical characterization of an alkaline meso- nificances of lipase include but are not limited to: 1 lipase philic lipase from T. lentiforme ACCC30425. Its applica- provides an increasable detergency (comparing with the tion potential as an additive in detergent was assessed as detergent alone), especially at low temperatures and neu- well. tral to alkaline pH; 2 lipase with low substrate specific - ity is highly effective to remove stubborn stains, such as Materials and methods blood and fat; and 3 lipase not only has high biodegrada- Strains tion ability but also brings harmless effect on aquatic eco - Trichoderma lentiforme ACCC30425 was supplied by the systems (Jurado et al. 2007). However, several bottlenecks Agricultural Culture Collection of China. Escherichia coli have limited lipase application in detergent, such as their Trans1-T1 was purchased from TransGen (China). The broad substrate specificity, stringent washing conditions heterologous expression system containing the vector (low temperature and alkaline conditions), and sensitivity pPIC9 and Pichia pastoris GS115 competent cells were to chemicals in detergents (Sharma et al. 2001, 2002). For obtained from the Invitrogen. example, a large number of microbial lipases produced by bacteria and yeast show the maximum activities at high Induction and detection of the lipase production by T. temperatures, such as the lipases from Pseudomonas aer- lentiforme ACCC30425 uginosa, thermophilic Bacillus sp. and yeast Kurtzmano- Trichoderma lentiforme ACCC30425 was grown in the myces sp. that have temperature optima of 60–75  °C. lipase-inducing medium (5  g/L glucose, 5  g/L NaNO , Although some bacterial lipases are neutral to alkaline, 5 g/L K HPO , 0.3 g/L M gSO , 0.01 g/L F eSO , and 4 g/L 2 4 4 4 but they lose most of the activities at pHs higher than olive oil as the sole carbon source) with the agitation rate 9.0 (Karadzic et  al. 2006; Nawani et  al. 2007). Moreover, of 180 rpm at 28 °C for 8 days. The culture supernatants the sensitivity to surfactant deters numerous lipases from were collected every day and subject to lipase activity their application in laundry. Therefore, it’s of great value assay (spectrophotometric method as described below). to obtain an alkaline mesophilic lipase with a high toler- ance to surfactants in the washing industry (Gutarra et al. Cloning of the gene TllipA 2009). Seven-day-old mycelia of T. lentiforme ACCC30425 were Trichoderma is a common filamentous fungus in soil collected, flash-frozen in liquid nitrogen, and ground into and root ecosystems, and sometimes in air, water, sand, a fine powder. Total RNA was extracted using the Trizol etc. It shows antagonistic, symbiotic and parasitic capa- method (Chomczynski and Sacchi 1987), and cDNAs bilities to interact with other microbes and is used more were synthesized by reverse transcription. According to extensively than any other single microbe in agriculture the whole genome sequence of T. lentiforme ACCC30425 (Benítez et  al. 2005). Moreover, Trichoderma has sig- (accomplished by the Majorbio, China), a lipase-encod- nificant lignocellulose-degrading capability because it ing gene, TllipA, was identified. The nucleotide and can produce a variety of hydrolytic, lytic and auxiliary amino acid sequences of TllipA were analyzed by using enzymes including cellulase, xylanase, chitinase, laccase, the BLASTx and BLASTp programs (https ://blast .ncbi. lipase, etc. (Zhang and Xia 2017). Besides, Trichoderma nlm.nih.gov/Blast .cgi), respectively. The signal peptide represents one of the most important expression systems was predicted using the SignalP 4.0 (http://www.cbs.dtu. Wang et al. AMB Expr (2018) 8:95 Page 3 of 11 dk/servi ces/Signa lP/). The prediction of molecular mass loaded onto the HiPrepTm 26/10 Desalting column and and isoelectric point (pI) value was performed using the HiTrapQHP column (GE Healthcare). Fractions contain- Vector NTI Advance 10.0 software (Invitrogen). Multi- ing lipase activity were pooled and concentrated by ultra- ple sequence alignment of TlLipA and other lipase rep- filtration (5-kDa molecular weight cutoff) for further resentatives was conducted by using the ClustalX 1.81 characterization. Sodium dodecyl sulfate-polyacrylamide and presented by ESPript 3.0 (http://espri pt.ibcp.fr/ESPri gel electrophoresis (SDS-PAGE) was carried out with the pt/cgi-bin/ESPri pt.cgi). The putative three-dimensional 5% stacking gel and 12% separation gel. Protein concen- structure was built by SWISS-MODEL (https ://www. tration was determined using the Bradford method with swiss model .expas y.org/) with the lipase from Ophios- bovine serum albumin as the standard. toma piceae (PDB: 4BE4) as the template. PCR was then conducted to obtain the DNA fragment Lipase activity assays coding for mature TlLipA with an expression primer The lipase activity was determined by the alkali titration set (TllipA-expF: 5′-GGG GAA TTC GCT CAA GGC method, using olive oil as the substrate. Olive oil was CAA GTC AAC GTT ACC ATT CCC -3′ and TllipA-expR: emulsified in 4% (w/v) polyvinyl alcohol solution at the 5′ -GGG GC G GC C GC C TA GA A GAT C A G T GA AT C ratio of 1:3. The reaction mixture contained 2.5  mL of GAT ATG CTC CTT GAT AAA G-3′, the EcoRI/NotI sites 20 mM citric acid-Na HPO (pH 7.5), 2.0 mL of emulsi- 2 4 underlined). The amplification was performed at 94  °C fied olive oil, and 0.5 mL of properly diluted enzyme solu - for 5 min followed by 35 cycles of denaturation (1 min at tion. After incubation at 40  °C for 15  min in a shaking 94 °C), annealing (1 min at 62 °C) and extension (1.5 min water bath, 7.5  mL of 95% ethanol was added to termi- at 72  °C), and a final extension of 72  °C for 10  min. The nate the reaction. The amount of liberated fatty acids was PCR products of the appropriate size were sequenced by measured by titration with 50 mM NaOH, using phenol- Biomed (China). phthalein as an indicator. One unit (U) of lipase activity was defined as the amount of lipase to liberate 1 μmol of Expression of the recombinant TlLipA in P. pastoris fatty acids per min from the olive oil. All determinations The correct PCR products were digested with EcoRI and were performed in triplicate. NotI and ligated into the EcoRI/NotI-digested pPIC9 vec- The spectrophotometric method was also used to tor to construct the recombinant plasmid pPIC9-TllipA. determine the lipase activity of purified recombinant Colony PCR was conducted using the AOX primers to TlLipA. The reaction system consisted of 0.1  mL of screen positive clones, which were further verified by appropriately diluted enzyme and 2.4  mL of substrate DNA sequencing. The correct recombinant plasmid solution containing 0.8  mM p-nitrophenyl octanoate was then linearized with BglII and transformed into (p-NPO, C8; dissolved in isopropanol at first) in 20  mM P. pastoris GS115 competent cells by the electropora- Tris–HCl (specific pH). After incubation at 37 °C (or the tion method with the Gene Pulser Xcell Electroporation optimum temperature of 50 °C) for 15 min, the reactions apparatus (Bio-Rad), following the instructions of Invit- were terminated by addition of 2.0  mL of 95% ethanol. rogen’s protocol (2000  V, 200 Ω, 25 μF, and 5  ms). The After centrifugation at 5000g for 10 min, 200 μL of each transformants were grown on minimal dextrose (MD) reaction supernatant was transferred to 96-well micro- agar plates at 32°C for 48 h. Ninety-six colonies were ran- plate for absorbance measurement at OD . One unit of domly selected to grow in 2-mL buffered glycerol com - lipase activity was defined as the amount of enzyme that plex medium (BMGY) at 30°C for 48  h. The cells were produced 1 μmol of p-nitrophenol (pNP) per min under collected by centrifugation (12,000g) and resuspended standard reaction conditions. All determinations were in 2-mL buffered methanol complex medium (BMMY). performed in triplicate. After 48-h induction with the 0.5% (v/v) methanol at 30°C, the culture supernatants were collected by cen- Eec ff ts of pH and temperature on the TlLipA activity trifugation for the lipase activity assay. The positive trans - The optimal pH of the recombinant Tl LipA was deter- formant showing the highest lipase activity was grown mined at 37  °C for 15  min in the following buffers: in 1-L Erlenmeyer flasks containing 200 mL medium for 25  mM citric acid-Na HPO for pH 5.0–7.0 and 20  mM 2 4 large-scale fermentation. Tris–HCl for pH 7.0–10.0. The optimal temperature was determined over the temperature range of 20–60  °C in Purification of recombinant TllipA 20 mM Tris–HCl (pH 9.5) for 15 min. The culture supernatants were collected by centrifuga - tion at 12,000g, 4°C for 10 min and concentrated through Eec ff ts of pH and temperature on the TlLipA stability Vivaflow 200 membrane of 5-kDa molecular weight The pH stability of Tl LipA was determined by measuring cutoff (Vivascience, Germany). The crude enzyme was the residual lipase activity under the optimal conditions Wang et al. AMB Expr (2018) 8:95 Page 4 of 11 (pH 9.5, 50  °C and 15  min) after pre-incubation of the commercial lipases (HA, HB, HD and HE) from Xin- enzyme at 37 °C for 1 h in the same buffers (pH 5.0–10.0) huayang Co. (China) were selected as references, and mentioned above. Thermal stability of Tl LipA was deter- their enzymatic properties were determined as described mined by measuring the residual lipase activity under the above. optimal condition after incubation of the enzyme at 40 To assess the stability in the presence of different sur - and 50 °C, respectively, for various periods (0–120 min). factants, TlLipA was incubated in 20 mM Tris–HCl (pH The TlLipA activities under optimal conditions (pH 9.5, 9.5) at 37  °C containing 50, 20, 10 or 1% (v/v) of Triton 50 °C and 15 min) were defined as 100% relative activity. X100, or 20, 10 or 1% (w/v) of SDS for various periods (0, 3, 6, 12, and 24 h). Mesophilic alkaline lipases HA and HB were used as references. The residual activities were Eec ff ts of metal ions and chemical reagents on the TlLipA determined under the optimum reaction conditions of activity each enzyme. To find out the effects of different metal ions and chemical reagents on TlLipA activity, 5  mM of N a , Results + 2+ + 2+ 2+ 2+ 2+ K, Ca, Ag, Mg, Mn, Zn, Ni , EDTA or Olive oil‑degrading capability of T. lentiforme ACCC30425 β-mercaptoethanol was added into the reaction system, By using olive oil as the sole carbon source, T. lentiforme respectively. TlLipA activity was determined at pH 9.5 ACCC30425 showed detectable lipase activity at day 4 and 50  °C for 15  min with p-NPO as the substrate. The and afterwards (Fig. 1). Using p-NPO as the substrate, the TlLipA activities without any chemical addition were lipase activity in the culture supernatants reached maxi- defined as 100%. mum at day 7, which was up to 1.7 U/mL (pH 8.0, 37 °C and 15  min). It indicated that T. lentiforme ACCC30425 Kinetic parameters has the capability of producing lipases to degrade olive oil p-Nitrophenyl esters with different acyl chains (C4– in the medium. C16) including p-nitrophenol butyrate (pNPB, C4), pNPO (C8), p-nitrophenol decanoic acid (pNPD, C10), Sequence analysis of the TllipA p-nitrophenol dodecanoate (pNPDD, C12), p-nitrophe- Genome sequence analysis indicated that the lipase- nol myristate (pNPM, C14) and p-nitrophenol palmitate encoding gene, TllipA (GenBank accession number: (pNPP, C16) at the concentrations of 0.2–1.6  mM were MF460438), contains 1707  bp. Deduced TlLipA consists used as the substrates. The Michaelis–Menten kinetic of a putative signal peptide of 20 residues and a mature parameters K and V of TlLipA were determined at protein of 548 residues. The molecular mass and pI of m max pH 9.5 and 50  °C for 10  min (shorter reaction time for mature TlLipA were estimated to be 60.0  kDa and 4.56, pseudo-first order kinetic analysis). The experiments respectively. Multiple sequence alignment and homol- were repeated for three times, and each experiment ogy modeling analysis indicated that deduced TlLipA included triplicate. GraphPad Prism 5 (GraphPad Soft- contains the typical α/β-hydrolase fold structure with ware) was used to calculate the K (substrate affinity) and an N-terminal 3-stranded β-sheet, a major 12-stranded V (maximum velocity) values. The k (the turnover max cat rate per second) and k /K (catalytic efficiency) val - cat m ues were then calculated to measure the efficiency of an enzyme that converts substrate to product at sub-saturat- ing substrate concentration and catalytic efficiency (Chi - naglia et al. 2014). TlLipA tolerance and stability to various surfactants Surfactant is a common and indispensable ingredient in detergents. To find out the effect of surfactant on Tl LipA activity, four concentrations (0.50, 0.20, 0.10 and 0.05%) of Tween 20, Tween 80, Triton X100 or 50 μM SDS (v/v) were individually added into the reaction system con- taining pNPO as the substrate, and the relative activities were tested under optimum reaction conditions (pH 9.5 Fig. 1 Lipase activities of the culture supernatants of T. lentiforme ACCC30425 growing in the inducing medium with olive oil as and 50 °C for 15 min). The reaction systems without any the sole carbon source. The lipase activities were assayed using surfactant were treated as controls. To assess the appli- spectrophotometric method with pNPP as the substrate cation potential of TlLipA in detergent industry, four Wang et al. AMB Expr (2018) 8:95 Page 5 of 11 (See figure on next page.) Fig. 2 Multiple sequence alignment of deduced TlLipA with structure-resolved lipases 4BE4 from Ophiostoma piceae and 1LLF from Candida cylindracea as well as biochemically characterized lipases TbLipA from Trichophyton benhamiae and DrLipA from Diutina rugosa. The secondary structural elements are indicated β-sheet, and 19 helices (Fig.  2, 3). The putative catalytic residual activity assay. As shown in Fig.  5c, TlLipA was triad consists of S215, E346 and H464. These catalytic relatively stable at 40 °C, retaining more than 60% of the residues are responsible for the nucleophilic attack on initial activity after 60-min incubation; when extended the carbonyl carbon atom of the ester bond. The puta - to 120 min, more than 50% activity was still retained. In tive lid consists of one α-helix (residues 76–81) and two contrast, it lost stability at 50  °C, losing more than 50% 3 -helics (residues 83–87 and 89–91) flanked by two activity within 5-min incubation and almost all activ- loops that end in a disulfide hinge (residues C62 and ity within 120-min. The pH stability of Tl LipA was also C101). assessed. The enzyme was stable at pH 6.0–9.0, retaining more than 80% of the initial activity after 60-min pre- Production and purification of the recombinant TlLipA incubation at 37 °C (Fig. 5d). These results indicated that The DNA fragment coding for the mature Tl LipA was TlLipA was stable over the cold (≤ 40 °C) and neutral to obtained with primers TllipA-expF and TllipA-expR, and alkaline conditions. transformed into E. coli Trans1-T1 for sequencing. The correct PCR product was digested with EcoRI and NotI Kinetic parameters and then cloned into the pPIC9 vector in-frame fusion of The kinetic parameters K , V , k and k /k of m max cat cat m the α-factor signal peptide to construct the recombinant TlLipA were determined using the six p-nitrophe- plasmid pPIC9-TllipA. The recombinant plasmid was nyl esters of various acyl chain lengths as substrates. linearized by BglII, and transformed into the P. pastoris As shown in Table  1, TlLipA showed higher affinities GS115 competent cells by electroporation. TlLipA was (decreased K values) and catalytic efficiencies (increased successfully produced according to the Pichia expression k /k values) towards short-chain substrates with (C4 cat m kit and secreted into the culture. After centrifugation, to C10). pNPO (C8) as the preferred substrate was cata- concentration and exchange chromatography, the crude lyzed with the highest efficiency of 41.0/s mM. enzyme was purified to electrophoretic homogeneity, showing a single band of approximately 60  kDa in SDS- Eec ff t of metal ions and chemical reagents on TlLipA PAGE (Fig.  4). This molecular mass was similar to the activity 2+ theoretical value (60.0 kDa), indicating that the band was Of the ten chemicals tested in this study (Table  2), Ni , 2+ 2+ purified recombinant Tl LipA indeed. The lipase activity Zn, Mn and EDTA strongly inhibited the TlLipA of purified recombinant TlLipA was determined to be activity, leading to the activity loss of more than 50%, 10.4 ± 0.5 U/mL by using the alkali titration method. while other chemicals had no or little effects (0–32%). None of the chemical addition resulted in an improve- Eec ff t of pH and temperature on TlLipA activity ment of lipase activity. The results indicated that Tl LipA pNPO was used as the substrate for biochemical char- was tolerant to most tested metal ions and chemical acterization of purified recombinant Tl LipA. Over the reagents. range of pH 5.0–10.0, the TlLipA had poor activity under acidic conditions and showed maximum activity at pH TlLipA tolerance to surfactants 9.5 (Fig.  5a). Under the optimum pH (9.5), the tempera- To find out the application potentials of Tl LipA in deter- ture-activity profile of Tl LipA was determined over the gent industry, we selected four commercial lipases as ref- temperature range from 20 to 60  °C. The enzyme had a erences and compared their activities with TlLipA in the temperature optimum at 50 °C and remained 20–40% of presence of 0.05–0.50% of Tween 20, Tween 80, Triton the maximum activity at 20–40 °C (Fig. 5b). These results X100 or SDS. As shown in Fig. 6, Tween 20, SDS and Tri- indicated that the purified recombinant Tl LipA is a mes- ton X100 at higher concentrations (0.10–0.50%) signifi - ophilic alkaline lipase with great adaptation to low mod- cantly enhanced the TlLipA activity up to 2.24-fold, while erate temperature. 0.05–0.50% of Tween 80 and 0.05% of Tween 20, SDS and Triton X100 inhibited the lipase activity of TlLipA by Thermal and pH stability of TlLipA 20‒60%. It indicated that high concentrations of Tween After incubation at 40 and 50  °C respectively for vari- 20, Triton X100 and SDS may emulsify the substrate to ous periods, aliquots of TlLipA were withdrawn for enlarge the interface area between TlLipA and substrate, Wang et al. AMB Expr (2018) 8:95 Page 6 of 11 Wang et al. AMB Expr (2018) 8:95 Page 7 of 11 Fig. 3 Multiple sequence alignment of deduced TlLipA with structure-resolved lipases 4BE4 from Ophiostoma piceae and 1LLF from Candida cylindracea as well as biochemically characterized lipases TbLipA from Trichophyton benhamiae and DrLipA from Diutina rugosa. The secondary structural elements are indicated condition, respectively (pH 9.0 and 50 °C for HA and HB, pH 8.0 and 50 °C for HD, and pH 8.0 and 40 °C for HE), with the only exception of increased HB activity (approx- imately 13.4%) by 0.05% SDS. The results indicated that TlLipA was highly tolerant to all tested surfactants and retained most or even enhanced activities. TlLipA stability to surfactants Considering the long shelf life of laundry detergent, lipase stability in the presence of surfactants is a key fac- tor of commercialization. Therefore we also determined the TlLipA stability after pre-incubation with 1–20% SDS or 1–50% Triton X100 for various periods. In comparison to the surfactant-untreated controls, TlLipA incubated with 1‒20% Triton X100 for 0–24 h showed significantly enhanced activities of 1.3‒1.7-fold, but lost more than 50% activity when incubated with 50% of Triton X100 Fig. 4 SDS-PAGE analysis of purified recombinant Tl LipA. Lane M, the and 1‒50% SDS (Fig.  7a). In contrast, both HA and HB molecular weight standard markers; lane 1, the purified recombinant lost stability in the presence of 1‒50% of Triton X100 TlLipA at 0.3 mg/mL; and lane 2, the purified recombinant Tl LipA at or SDS (Fig.  7b, c), retaining less than 30% activity after 3.0 mg/mL incubation at 3 h. Discussion consequently improving the lipase activities. On the con- The genus Trichoderma contains a very large group trary, the activities of the four commercial lipases were of important microorganisms. It is not only a genetic mostly inhibited by the surfactants under each optimum resource of various functional proteins (Freitas et  al. Wang et al. AMB Expr (2018) 8:95 Page 8 of 11 Fig. 5 Biochemical characterization of TlLipA. a The pH-activity profiles determined in citric acid-Na HPO (black square) and Tris–HCl (black 2 4 circle) buffers. b The temperature-activity profile determined in Tris–HCl buffer at pH 9.5. c The thermal stability at 50 °C (black circle) and 40 °C (blackup-pointing triangle). d The pH stability after 1-h incubation at 37 °C and pH 5.0–10.0 lentiforme ACCC30425, the full-length TllipA was iden- Table 1 The kinetic values of  purified recombinant TlLipA towards esters of different lengths tified and its structure and functions were predicted. Although TllipA shows high sequence identity (100%) to Substrate V (μmol/ k (/s) K (mM) k /K (/s mM) max cat m cat m the hypotheoretical lipase from T. guizhouense, its identi- min mg) ties to lipases with function verified or structure resolved pNPO (C8) 27.3 ± 4.4 27.4 ± 4.4 0.67 ± 0.23 41.0 are much lower (< 50%). Thus it is of importance and pNPD (C10) 12.5 ± 0.7 12.5 ± 0.7 0.35 ± 0.07 35.8 novelty to clone the gene and produce the gene product pNPB (C4) 12.2 ± 0.8 12.2 ± 0.8 0.40 ± 0.07 30.7 for potential applications in various industries. pNPDD (C12) 16.8 ± 2.4 16.8 ± 2.4 1.59 ± 0.42 10.6 Most fungal lipases act over a broad pH range, with pNPM (C14) 4.3 ± 1.0 4.3 ± 1.0 3.29 ± 1.21 1.32 the pH optima of 4.0–8.0 (Sharma et al. 2011; Singh and pNPP (C16) 4.8 ± 1.0 4.8 ± 1.0 4.22 ± 1.28 1.14 Mukhopadhyay 2012), and are mesophilic with thermola- bility at > 40 °C (Gutarra et al. 2009). The pH optimum of TlLipA was 9.5, which is higher than most fungal lipases 2014) but also a key workhorse for enzyme production characterized so far. Moreover, it showed great adapt- on commercial scale (i.e. T. reesei) (Jørgensen et al. 2014). ability and stability under neutral to alkaline conditions In the present study, we reported an alkaline, mesophilic (pH 7.0–10.0). On the other hand, TlLipA showed maxi- lipase-producing strain (ACCC30425) of T. lentiforme. mum activity at 50 °C and thermolability at > 40 °C. These Along with the rapid progress of genome sequencing enzymatic properties make TlLipA potential for appli- (Yang et  al. 2015), to obtain objective genes with spe- cation in the alkaline and low to moderate temperature cial characters is very simple and efficient. Based on the fields, especially the washing industry (Jurado et al. 2007; sequence analysis and annotation of the genome of T. Grbavčić et al. 2011). Some lipases are resistant to heavy Wang et al. AMB Expr (2018) 8:95 Page 9 of 11 Table 2 Eec ff ts of metal ions and chemical reagents on the TlLipA activity Chemicals Relative activity (%) Chemical Relative activity (%) 2+ CK 100.0 ± 2.4 Mn 13.9 ± 0.8 2+ 2+ Mg 97.6 ± 2.5 Zn 11.9 ± 0.9 2+ 2+ Ca 92.4 ± 4.0 Ni 7.6 ± 0.8 K 91.1 ± 3.0 β-Mercaptoethanol 60.5 ± 0.9 Na 85.7 ± 6.8 EDTA 33.8 ± 2.0 Ag 68.6 ± 3.8 Values are given as the mean ± standard deviations (n = 3) ab 0.05% 0.10% 0.20% 0.50% 0.05%0.10% 0.20%0.50% HA HB HD HE TlLipA HA HB HD HE TlLipA cd 0.05% 0.10% 0.20% 0.50% 0.05%0.10% 0.20%0.50% 0 0 HA HB HD HE TlLipA HA HB HD HE TlLipA Fig. 6 Tolerance of TlLipA and four commercial lipases HA, HB, HD and HE to Tween 20 (a), Tween 80 (b), Triton X100 (c) and SDS (d) at different concentrations, respectively 2+ 2+ 2+ (C8) and pNPD (C10) were higher than that towards metals, such as Ca, Fe , and Mg (Jurado et al. 2007; other esters of different lengths. Similar results had been Gaur et al. 2008; Rao et al. 2009; Sethi et al. 2016). TlLipA 2+ 2+ reported on the two highly thermophilic alkaline lipases showed similar tolerance to C a and M g , but showed 2+ 2+ 2+ from Thermosyntropha lipolytica (Moh’d and Wiegel sensitivity to heavy metals Ni, Mn and Zn and 2007). chemical reagents EDTA and β-mercaptoethanol. u Th s The resistance to surfactant is a big challenge for the effects of metal ions and chemical reagents on Tl LipA lipase’s application in washing industry. In general, the should be considered for future application. surfactant has negative effects on enzymatic hydrolysis Lipase is usually considered as an enzyme that hydro- and represents a competitive inhibiter in the reaction sys- lyzes the cleavage of long-chain acylglycerols; however, tem (Tatara et al. 1985). TlLipA remained highly active in most known lipases are also active on shorter acyl chain the presence of both anionic and non-ionic surfactants, esters (Li and Zong 2010). TlLipA having lipase activ- including SDS, Triton X100, Tween 20 and Tween 80. ity as determined by the alkali titration method is a true Moreover, the resistance of TlLipA to surfactants showed lipase, but it prefers medium-chain fatty acid esters. The improvement along with increased concentration. For catalytic efficiencies (k /K ) of TlLipA towards pNPO cat m Wang et al. AMB Expr (2018) 8:95 Page 10 of 11 the enzymatic activities of commercial lipases HA and SDS 1% SDS 10% SDS 20% HB. In comparison to the stabilities of other lipases with TX100 10% TX100 20% TX100 50% surfactants (1-h incubation) (Rathi et  al. 2001; Bora and control TX100 1% Kalita 2010), TlLipA retained similar or higher activity in the presence of 10% Triton X100 even over 24 h-incu- bation (150% vs. 93‒164%). Thus the alkaline mesophilic TlLipA with adaptability and stability to broad pH and temperature ranges and high tolerance to surfactants is favorable for potential application in the washing industry. 036 12 24 Incubaon me (hr) Abbreviations BMGY: buffered glycerol-complex medium; BMMY: buffered methanol-com- plex medium; MD: minimal dextrose; PAGE: polyacrylamide gel electrophore- sis; SDS: sodium dodecyl sulfonate; pNP: p-Nitrophenol; pNPB: p-Nitrophenol SDS 1% SDS 10% SDS 20% butyrate; pNPD: p-Nitrophenol decanoic acid; pNPDD: p-nitrophenol dode- TX100 10% TX100 20% TX100 50% canoate; pNPM: p-Nitrophenol myristate; pNPO: p-Nitrophenyl octanoate; TX100 1% control pNPP: p-Nitrophenol palmitate; pI: isoelectric point. Authors’ contributions YW, YB and JG designed the research; YW, RM and SL performed the research; YW, MG, BY and JG analyzed the data and wrote the paper. All authors read and approved the final manuscript. Acknowledgements We are grateful to the Agricultural Culture Collection of China (ACCC) for providing the Trichoderma strain ACCC30425. 03 61224 Competing interests Incubaon me (hr) The authors declare that they have no competing interests. Availability of data and materials Data can be shared. Please send email to gujingang@caas.cn. SDS 1% SDS 10% SDS 20% TX100 10% TX100 20% TX100 50% Consent for publication TX100 1% control Not applicable. Ethics approval and consent to participate 100 Not applicable. This article does not contain any studies with human partici- pants or animals performed by any of the authors. Funding This work was supported by the National High Technology Research and Development Program of China (863 Program, 2013AA102805-05), the National Non-profit Institute Research Grant of Chinese Academy of Agri- 0 cultural Sciences (IARRP-2015-24) and the Fundamental Research Funds for 03 61224 Central Non-profit Scientific Institution of China (1610132016050). 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AMB ExpressSpringer Journals

Published: Jun 5, 2018

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