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  • Original article
  • Open Access

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

AMB Express20188:95

https://doi.org/10.1186/s13568-018-0618-z

  • Received: 23 January 2018
  • Accepted: 23 May 2018
  • Published:

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 activities 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 comparison 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.
Graphical Abstract image

Keywords

  • Trichoderma lentiforme
  • Alkaline lipase
  • Heterologous expression
  • Detergent

Introduction

Lipase (EC 3.1.1.3) is regarded as one of the most important commercial enzymes, and has been attracting enormous attention in the rapidly growing biotechnological area. It catalyzes the hydrolysis of triacylglycerols to release diacylglyceride, monoacylglycerol, long-chain fatty acids (> 8 carbons) and glycerol at the interface of oil and water (Brockerhoff 1974). According to the protein structure similarity, lipase belongs to the family of α/β hydrolases, in which a catalytic triad (usually serine, histidine, and aspartic or glutamic acid) and an oxyanion hole (just like a catalytic pocket) are crucial for catalysis (Gupta et al. 2015), and a lid structure involves in the substrate accessibility and binding in the active site (Woolley and Petersen 1996). During hydrolysis, the hydroxy group of the catalytic serine attacks the carbonyl carbon of the ester bond of the substrate, while the catalytic histidine acts as a general-base catalyst and abstracts a proton from the catalytic serine. The alcohol group of the substrate is released and an acyl-enzyme intermediate is formed, which is stabilized in the oxyanion hole by hydrogen bonds. The acyl-enzyme intermediate can be attacked by a water or alcohol molecule, leading to the formation of acid or new ester, respectively (Beer et al. 1996).

Lipases are widespread in nature and have been reported in microbes, plants, and animals. Nevertheless, bacterial and fungal lipases are of special interest as they are easily produced and favorable for industrial purposes due to the high yields and great versatility and stability under harsh conditions (Schmid 2016). Microbial lipases vary in structures and enzymatic properties. For example, the lipases from Candida rugosa with different hydrophobic zones at the central channel showed different activities (Mancheño et al. 2003; Domínguez et al. 2006). At the entrance of the channel in close proximity to the catalytic site and the substrate binding site, there is a phenylalanine-rich region associated with substrate binding. The phenylalanine content is negatively correlated with the catalytic activity towards cholesterol ester. In addition, the lipase activity is also related to the size and orientation of the channel. For example, the lipase from Aspergillus niger having a narrow and curved channel shows relatively low activity, while those from Ophiostoma piceae, Nectria haematococca and Trichoderma reesei have straightforward and wider channels and much higher activities (Barriuso et al. 2016).

Microbial lipases are widely used in various industries, especially in the detergent (Saxena et al. 2004). Since the 1960s, enzyme-based detergent has been introduced into the market, and lipase that efficiently removes acylglycerols has been one of the major additives in cleaning agent (Abo 1990). The industrial and environmental significances of lipase include but are not limited to: 1 lipase provides an increasable detergency (comparing with the detergent alone), especially at low temperatures and neutral to alkaline pH; 2 lipase with low substrate specificity is highly effective to remove stubborn stains, such as blood and fat; and 3 lipase not only has high biodegradation ability but also brings harmless effect on aquatic ecosystems (Jurado et al. 2007). However, several bottlenecks have limited lipase application in detergent, such as their broad substrate specificity, stringent washing conditions (low temperature and alkaline conditions), and sensitivity to chemicals in detergents (Sharma et al. 2001, 2002). For example, a large number of microbial lipases produced by bacteria and yeast show the maximum activities at high temperatures, such as the lipases from Pseudomonas aeruginosa, thermophilic Bacillus sp. and yeast Kurtzmanomyces sp. that have temperature optima of 60–75 °C. Although some bacterial lipases are neutral to alkaline, but they lose most of the activities at pHs higher than 9.0 (Karadzic et al. 2006; Nawani et al. 2007). Moreover, the sensitivity to surfactant deters numerous lipases from their application in laundry. Therefore, it’s of great value to obtain an alkaline mesophilic lipase with a high tolerance to surfactants in the washing industry (Gutarra et al. 2009).

Trichoderma is a common filamentous fungus in soil and root ecosystems, and sometimes in air, water, sand, etc. It shows antagonistic, symbiotic and parasitic capabilities to interact with other microbes and is used more extensively than any other single microbe in agriculture (Benítez et al. 2005). Moreover, Trichoderma has significant lignocellulose-degrading capability because it can produce a variety of hydrolytic, lytic and auxiliary enzymes including cellulase, xylanase, chitinase, laccase, lipase, etc. (Zhang and Xia 2017). Besides, Trichoderma represents one of the most important expression systems that is widely used to produce industrial enzymes on large scale (Singh et al. 2015). Up to now, the genome sequences of 13 Trichoderma strains have been completed (Halliwell and Griffin 1973; Martinez et al. 2008; Kubicek et al. 2011; Studholme et al. 2013; Xie et al. 2014; Baroncelli et al. 2015, 2016; Shikunne et al. 2015; Yang et al. 2015; Lee et al. 2017), and the number keeps increasing. Sequence analysis of the genomes of T. viride, T. reesei, T. harzianum and T. gamsii indicated that Trichoderma harbors a great variety of lipase genes. In our preliminary studies, four Trichoderma strains demonstrated lipase-producing capabilities. One of them, T. lentiforme ACCC30425, showed the highest lipase-producing capability under alkaline conditions and thus was selected for draft genome sequencing. In this study, we reported on the gene cloning, heterologous expression, and biochemical characterization of an alkaline mesophilic lipase from T. lentiforme ACCC30425. Its application potential as an additive in detergent was assessed as well.

Materials and methods

Strains

Trichoderma lentiforme ACCC30425 was supplied by the Agricultural Culture Collection of China. Escherichia coli Trans1-T1 was purchased from TransGen (China). The heterologous expression system containing the vector pPIC9 and Pichia pastoris GS115 competent cells were obtained from the Invitrogen.

Induction and detection of the lipase production by T. lentiforme ACCC30425

Trichoderma lentiforme ACCC30425 was grown in the lipase-inducing medium (5 g/L glucose, 5 g/L NaNO3, 5 g/L K2HPO4, 0.3 g/L MgSO4, 0.01 g/L FeSO4, and 4 g/L olive oil as the sole carbon source) with the agitation rate of 180 rpm at 28 °C for 8 days. The culture supernatants were collected every day and subject to lipase activity assay (spectrophotometric method as described below).

Cloning of the gene TllipA

Seven-day-old mycelia of T. lentiforme ACCC30425 were collected, flash-frozen in liquid nitrogen, and ground into a fine powder. Total RNA was extracted using the Trizol method (Chomczynski and Sacchi 1987), and cDNAs were synthesized by reverse transcription. According to the whole genome sequence of T. lentiforme ACCC30425 (accomplished by the Majorbio, China), a lipase-encoding gene, TllipA, was identified. The nucleotide and amino acid sequences of TllipA were analyzed by using the BLASTx and BLASTp programs (https://blast.ncbi.nlm.nih.gov/Blast.cgi), respectively. The signal peptide was predicted using the SignalP 4.0 (http://www.cbs.dtu.dk/services/SignalP/). The prediction of molecular mass and isoelectric point (pI) value was performed using the Vector NTI Advance 10.0 software (Invitrogen). Multiple sequence alignment of TlLipA and other lipase representatives was conducted by using the ClustalX 1.81 and presented by ESPript 3.0 (http://espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi). The putative three-dimensional structure was built by SWISS-MODEL (https://www.swissmodel.expasy.org/) with the lipase from Ophiostoma piceae (PDB: 4BE4) as the template.

PCR was then conducted to obtain the DNA fragment coding for mature TlLipA with an expression primer set (TllipA-expF: 5′-GGGGAATTCGCTCAAGGCCAAGTCAACGTTACCATTCCC-3′ and TllipA-expR: 5′-GGGGCGGCCGCCTAGAAGATCAGTGAATCGATATGCTCCTTGATAAAG-3′, the EcoRI/NotI sites underlined). The amplification was performed at 94 °C for 5 min followed by 35 cycles of denaturation (1 min at 94 °C), annealing (1 min at 62 °C) and extension (1.5 min at 72 °C), and a final extension of 72 °C for 10 min. The PCR products of the appropriate size were sequenced by Biomed (China).

Expression of the recombinant TlLipA in P. pastoris

The correct PCR products were digested with EcoRI and NotI and ligated into the EcoRI/NotI-digested pPIC9 vector to construct the recombinant plasmid pPIC9-TllipA. Colony PCR was conducted using the AOX primers to screen positive clones, which were further verified by DNA sequencing. The correct recombinant plasmid was then linearized with BglII and transformed into P. pastoris GS115 competent cells by the electroporation method with the Gene Pulser Xcell Electroporation apparatus (Bio-Rad), following the instructions of Invitrogen’s protocol (2000 V, 200 Ω, 25 μF, and 5 ms). The transformants were grown on minimal dextrose (MD) agar plates at 32°C for 48 h. Ninety-six colonies were randomly selected to grow in 2-mL buffered glycerol complex medium (BMGY) at 30°C for 48 h. The cells were collected by centrifugation (12,000g) and resuspended in 2-mL buffered methanol complex medium (BMMY). After 48-h induction with the 0.5% (v/v) methanol at 30°C, the culture supernatants were collected by centrifugation for the lipase activity assay. The positive transformant showing the highest lipase activity was grown in 1-L Erlenmeyer flasks containing 200 mL medium for large-scale fermentation.

Purification of recombinant TllipA

The culture supernatants were collected by centrifugation at 12,000g, 4°C for 10 min and concentrated through Vivaflow 200 membrane of 5-kDa molecular weight cutoff (Vivascience, Germany). The crude enzyme was loaded onto the HiPrepTm 26/10 Desalting column and HiTrapQHP column (GE Healthcare). Fractions containing lipase activity were pooled and concentrated by ultrafiltration (5-kDa molecular weight cutoff) for further characterization. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out with the 5% stacking gel and 12% separation gel. Protein concentration was determined using the Bradford method with bovine serum albumin as the standard.

Lipase activity assays

The lipase activity was determined by the alkali titration method, using olive oil as the substrate. Olive oil was emulsified in 4% (w/v) polyvinyl alcohol solution at the ratio of 1:3. The reaction mixture contained 2.5 mL of 20 mM citric acid-Na2HPO4 (pH 7.5), 2.0 mL of emulsified olive oil, and 0.5 mL of properly diluted enzyme solution. After incubation at 40 °C for 15 min in a shaking water bath, 7.5 mL of 95% ethanol was added to terminate the reaction. The amount of liberated fatty acids was measured by titration with 50 mM NaOH, using phenolphthalein as an indicator. One unit (U) of lipase activity was defined as the amount of lipase to liberate 1 μmol of fatty acids per min from the olive oil. All determinations were performed in triplicate.

The spectrophotometric method was also used to determine the lipase activity of purified recombinant TlLipA. The reaction system consisted of 0.1 mL of appropriately diluted enzyme and 2.4 mL of substrate solution containing 0.8 mM p-nitrophenyl octanoate (p-NPO, C8; dissolved in isopropanol at first) in 20 mM Tris–HCl (specific pH). After incubation at 37 °C (or the optimum temperature of 50 °C) for 15 min, the reactions were terminated by addition of 2.0 mL of 95% ethanol. After centrifugation at 5000g for 10 min, 200 μL of each reaction supernatant was transferred to 96-well microplate for absorbance measurement at OD410. One unit of lipase activity was defined as the amount of enzyme that produced 1 μmol of p-nitrophenol (pNP) per min under standard reaction conditions. All determinations were performed in triplicate.

Effects of pH and temperature on the TlLipA activity

The optimal pH of the recombinant TlLipA was determined at 37 °C for 15 min in the following buffers: 25 mM citric acid-Na2HPO4 for pH 5.0–7.0 and 20 mM Tris–HCl for pH 7.0–10.0. The optimal temperature was determined over the temperature range of 20–60 °C in 20 mM Tris–HCl (pH 9.5) for 15 min.

Effects of pH and temperature on the TlLipA stability

The pH stability of TlLipA was determined by measuring the residual lipase activity under the optimal conditions (pH 9.5, 50 °C and 15 min) after pre-incubation of the enzyme at 37 °C for 1 h in the same buffers (pH 5.0–10.0) mentioned above. Thermal stability of TlLipA was determined by measuring the residual lipase activity under the optimal condition after incubation of the enzyme at 40 and 50 °C, respectively, for various periods (0–120 min). The TlLipA activities under optimal conditions (pH 9.5, 50 °C and 15 min) were defined as 100% relative activity.

Effects of metal ions and chemical reagents on the TlLipA activity

To find out the effects of different metal ions and chemical reagents on TlLipA activity, 5 mM of Na+, K+, Ca2+, Ag+, Mg2+, Mn2+, Zn2+, Ni2+, EDTA or β-mercaptoethanol was added into the reaction system, respectively. TlLipA activity was determined at pH 9.5 and 50 °C for 15 min with p-NPO as the substrate. The TlLipA activities without any chemical addition were defined as 100%.

Kinetic parameters

p-Nitrophenyl esters with different acyl chains (C4–C16) including p-nitrophenol butyrate (pNPB, C4), pNPO (C8), p-nitrophenol decanoic acid (pNPD, C10), p-nitrophenol dodecanoate (pNPDD, C12), p-nitrophenol myristate (pNPM, C14) and p-nitrophenol palmitate (pNPP, C16) at the concentrations of 0.2–1.6 mM were used as the substrates. The Michaelis–Menten kinetic parameters K m and V max of TlLipA were determined at pH 9.5 and 50 °C for 10 min (shorter reaction time for pseudo-first order kinetic analysis). The experiments were repeated for three times, and each experiment included triplicate. GraphPad Prism 5 (GraphPad Software) was used to calculate the K m (substrate affinity) and V max (maximum velocity) values. The k cat (the turnover rate per second) and k cat /K m (catalytic efficiency) values were then calculated to measure the efficiency of an enzyme that converts substrate to product at sub-saturating substrate concentration and catalytic efficiency (Chinaglia 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 TlLipA 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 containing pNPO as the substrate, and the relative activities were tested under optimum reaction conditions (pH 9.5 and 50 °C for 15 min). The reaction systems without any surfactant were treated as controls. To assess the application potential of TlLipA in detergent industry, four commercial lipases (HA, HB, HD and HE) from Xinhuayang Co. (China) were selected as references, and their enzymatic properties were determined as described above.

To assess the stability in the presence of different surfactants, TlLipA was incubated in 20 mM Tris–HCl (pH 9.5) at 37 °C containing 50, 20, 10 or 1% (v/v) of Triton 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 determined under the optimum reaction conditions of each enzyme.

Results

Olive oil-degrading capability of T. lentiforme ACCC30425

By using olive oil as the sole carbon source, T. lentiforme ACCC30425 showed detectable lipase activity at day 4 and afterwards (Fig. 1). Using p-NPO as the substrate, the lipase activity in the culture supernatants reached maximum 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 has the capability of producing lipases to degrade olive oil in the medium.
Fig. 1
Fig. 1

Lipase activities of the culture supernatants of T. lentiforme ACCC30425 growing in the inducing medium with olive oil as the sole carbon source. The lipase activities were assayed using spectrophotometric method with pNPP as the substrate

Sequence analysis of the TllipA

Genome sequence analysis indicated that the lipase-encoding gene, TllipA (GenBank accession number: MF460438), contains 1707 bp. Deduced TlLipA consists of a putative signal peptide of 20 residues and a mature protein of 548 residues. The molecular mass and pI of mature TlLipA were estimated to be 60.0 kDa and 4.56, respectively. Multiple sequence alignment and homology modeling analysis indicated that deduced TlLipA contains the typical α/β-hydrolase fold structure with an N-terminal 3-stranded β-sheet, a major 12-stranded β-sheet, and 19 helices (Fig. 2, 3). The putative catalytic triad consists of S215, E346 and H464. These catalytic residues are responsible for the nucleophilic attack on the carbonyl carbon atom of the ester bond. The putative lid consists of one α-helix (residues 76–81) and two 310-helics (residues 83–87 and 89–91) flanked by two loops that end in a disulfide hinge (residues C62 and C101).
Fig. 2
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

Fig. 3
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

Production and purification of the recombinant TlLipA

The DNA fragment coding for the mature TlLipA was obtained with primers TllipA-expF and TllipA-expR, and transformed into E. coli Trans1-T1 for sequencing. The correct PCR product was digested with EcoRI and NotI and then cloned into the pPIC9 vector in-frame fusion of the α-factor signal peptide to construct the recombinant plasmid pPIC9-TllipA. The recombinant plasmid was linearized by BglII, and transformed into the P. pastoris GS115 competent cells by electroporation. TlLipA was successfully produced according to the Pichia expression kit and secreted into the culture. After centrifugation, concentration and exchange chromatography, the crude enzyme was purified to electrophoretic homogeneity, showing a single band of approximately 60 kDa in SDS-PAGE (Fig. 4). This molecular mass was similar to the theoretical value (60.0 kDa), indicating that the band was purified recombinant TlLipA indeed. The lipase activity of purified recombinant TlLipA was determined to be 10.4 ± 0.5 U/mL by using the alkali titration method.
Fig. 4
Fig. 4

SDS-PAGE analysis of purified recombinant TlLipA. Lane M, the molecular weight standard markers; lane 1, the purified recombinant TlLipA at 0.3 mg/mL; and lane 2, the purified recombinant TlLipA at 3.0 mg/mL

Effect of pH and temperature on TlLipA activity

pNPO was used as the substrate for biochemical characterization of purified recombinant TlLipA. Over the range of pH 5.0–10.0, the TlLipA had poor activity under acidic conditions and showed maximum activity at pH 9.5 (Fig. 5a). Under the optimum pH (9.5), the temperature-activity profile of TlLipA was determined over the temperature range from 20 to 60 °C. The enzyme had a temperature optimum at 50 °C and remained 20–40% of the maximum activity at 20–40 °C (Fig. 5b). These results indicated that the purified recombinant TlLipA is a mesophilic alkaline lipase with great adaptation to low moderate temperature.
Fig. 5
Fig. 5

Biochemical characterization of TlLipA. a The pH-activity profiles determined in citric acid-Na2HPO4 (black square) and Tris–HCl (black 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

Thermal and pH stability of TlLipA

After incubation at 40 and 50 °C respectively for various periods, aliquots of TlLipA were withdrawn for residual activity assay. As shown in Fig. 5c, TlLipA was relatively stable at 40 °C, retaining more than 60% of the initial activity after 60-min incubation; when extended to 120 min, more than 50% activity was still retained. In contrast, it lost stability at 50 °C, losing more than 50% activity within 5-min incubation and almost all activity within 120-min. The pH stability of TlLipA was also assessed. The enzyme was stable at pH 6.0–9.0, retaining more than 80% of the initial activity after 60-min pre-incubation at 37 °C (Fig. 5d). These results indicated that TlLipA was stable over the cold (≤ 40 °C) and neutral to alkaline conditions.

Kinetic parameters

The kinetic parameters K m , V max , k cat and k cat /k m of TlLipA were determined using the six p-nitrophenyl esters of various acyl chain lengths as substrates. As shown in Table 1, TlLipA showed higher affinities (decreased K m values) and catalytic efficiencies (increased k cat /k m values) towards short-chain substrates with (C4 to C10). pNPO (C8) as the preferred substrate was catalyzed with the highest efficiency of 41.0/s mM.
Table 1

The kinetic values of purified recombinant TlLipA towards esters of different lengths

Substrate

V max (μmol/min mg)

k cat (/s)

K m (mM)

k cat /K m (/s mM)

pNPO (C8)

27.3 ± 4.4

27.4 ± 4.4

0.67 ± 0.23

41.0

pNPD (C10)

12.5 ± 0.7

12.5 ± 0.7

0.35 ± 0.07

35.8

pNPB (C4)

12.2 ± 0.8

12.2 ± 0.8

0.40 ± 0.07

30.7

pNPDD (C12)

16.8 ± 2.4

16.8 ± 2.4

1.59 ± 0.42

10.6

pNPM (C14)

4.3 ± 1.0

4.3 ± 1.0

3.29 ± 1.21

1.32

pNPP (C16)

4.8 ± 1.0

4.8 ± 1.0

4.22 ± 1.28

1.14

Effect of metal ions and chemical reagents on TlLipA activity

Of the ten chemicals tested in this study (Table 2), Ni2+, Zn2+, Mn2+ and EDTA strongly inhibited the TlLipA activity, leading to the activity loss of more than 50%, while other chemicals had no or little effects (0–32%). None of the chemical addition resulted in an improvement of lipase activity. The results indicated that TlLipA was tolerant to most tested metal ions and chemical reagents.
Table 2

Effects of metal ions and chemical reagents on the TlLipA activity

Chemicals

Relative activity (%)a

Chemical

Relative activity (%)

CK

100.0 ± 2.4

Mn2+

13.9 ± 0.8

Mg2+

97.6 ± 2.5

Zn2+

11.9 ± 0.9

Ca2+

92.4 ± 4.0

Ni2+

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

  

aValues are given as the mean ± standard deviations (n = 3)

TlLipA tolerance to surfactants

To find out the application potentials of TlLipA in detergent industry, we selected four commercial lipases as references and compared their activities with TlLipA in the presence of 0.05–0.50% of Tween 20, Tween 80, Triton X100 or SDS. As shown in Fig. 6, Tween 20, SDS and Triton X100 at higher concentrations (0.10–0.50%) significantly enhanced the TlLipA activity up to 2.24-fold, while 0.05–0.50% of Tween 80 and 0.05% of Tween 20, SDS and Triton X100 inhibited the lipase activity of TlLipA by 20‒60%. It indicated that high concentrations of Tween 20, Triton X100 and SDS may emulsify the substrate to enlarge the interface area between TlLipA and substrate, consequently improving the lipase activities. On the contrary, the activities of the four commercial lipases were mostly inhibited by the surfactants under each optimum 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 (approximately 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.
Fig. 6
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

TlLipA stability to surfactants

Considering the long shelf life of laundry detergent, lipase stability in the presence of surfactants is a key factor 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 and 1‒50% SDS (Fig. 7a). In contrast, both HA and HB lost stability in the presence of 1‒50% of Triton X100 or SDS (Fig. 7b, c), retaining less than 30% activity after incubation at 3 h.
Fig. 7
Fig. 7

Stability of TlLipA (a) and commercial lipases HA (b) and HB (c) after incubation with SDS or Triton X100 for various durations

Discussion

The genus Trichoderma contains a very large group of important microorganisms. It is not only a genetic resource of various functional proteins (Freitas et al. 2014) but also a key workhorse for enzyme production on commercial scale (i.e. T. reesei) (Jørgensen et al. 2014). In the present study, we reported an alkaline, mesophilic lipase-producing strain (ACCC30425) of T. lentiforme. Along with the rapid progress of genome sequencing (Yang et al. 2015), to obtain objective genes with special characters is very simple and efficient. Based on the sequence analysis and annotation of the genome of T. lentiforme ACCC30425, the full-length TllipA was identified and its structure and functions were predicted. Although TllipA shows high sequence identity (100%) to the hypotheoretical lipase from T. guizhouense, its identities to lipases with function verified or structure resolved are much lower (< 50%). Thus it is of importance and novelty to clone the gene and produce the gene product for potential applications in various industries.

Most fungal lipases act over a broad pH range, with the pH optima of 4.0–8.0 (Sharma et al. 2011; Singh and Mukhopadhyay 2012), and are mesophilic with thermolability at > 40 °C (Gutarra et al. 2009). The pH optimum of TlLipA was 9.5, which is higher than most fungal lipases characterized so far. Moreover, it showed great adaptability and stability under neutral to alkaline conditions (pH 7.0–10.0). On the other hand, TlLipA showed maximum activity at 50 °C and thermolability at > 40 °C. These enzymatic properties make TlLipA potential for application in the alkaline and low to moderate temperature fields, especially the washing industry (Jurado et al. 2007; Grbavčić et al. 2011). Some lipases are resistant to heavy metals, such as Ca2+, Fe2+, and Mg2+ (Jurado et al. 2007; Gaur et al. 2008; Rao et al. 2009; Sethi et al. 2016). TlLipA showed similar tolerance to Ca2+ and Mg2+, but showed sensitivity to heavy metals Ni2+, Mn2+ and Zn2+ and chemical reagents EDTA and β-mercaptoethanol. Thus the effects of metal ions and chemical reagents on TlLipA should be considered for future application.

Lipase is usually considered as an enzyme that hydrolyzes the cleavage of long-chain acylglycerols; however, most known lipases are also active on shorter acyl chain esters (Li and Zong 2010). TlLipA having lipase activity as determined by the alkali titration method is a true lipase, but it prefers medium-chain fatty acid esters. The catalytic efficiencies (k cat /K m ) of TlLipA towards pNPO (C8) and pNPD (C10) were higher than that towards other esters of different lengths. Similar results had been reported on the two highly thermophilic alkaline lipases from Thermosyntropha lipolytica (Moh’d and Wiegel 2007).

The resistance to surfactant is a big challenge for lipase’s application in washing industry. In general, the surfactant has negative effects on enzymatic hydrolysis and represents a competitive inhibiter in the reaction system (Tatara et al. 1985). TlLipA remained highly active in the presence of both anionic and non-ionic surfactants, including SDS, Triton X100, Tween 20 and Tween 80. Moreover, the resistance of TlLipA to surfactants showed improvement along with increased concentration. For 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 tested in this study and the previous study that the lipase activity would drop down along with the increased concentration of SDS (Rathi et al. 2001). Moreover, incubation with the increased concentrations of Triton X100 simulated the TlLipA activities but strongly inhibited the enzymatic activities of commercial lipases HA and HB. In comparison to the stabilities of other lipases with surfactants (1-h incubation) (Rathi et al. 2001; Bora and Kalita 2010), TlLipA retained similar or higher activity in the presence of 10% Triton X100 even over 24 h-incubation (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.

Abbreviations

BMGY: 

buffered glycerol-complex medium

BMMY: 

buffered methanol-complex medium

MD: 

minimal dextrose

PAGE: 

polyacrylamide gel electrophoresis

SDS: 

sodium dodecyl sulfonate

pNP: 

p-Nitrophenol

pNPB: 

p-Nitrophenol butyrate

pNPD: 

p-Nitrophenol decanoic acid

pNPDD: 

p-nitrophenol dodecanoate

pNPM: 

p-Nitrophenol myristate

pNPO: 

p-Nitrophenyl octanoate

pNPP: 

p-Nitrophenol palmitate

pI: 

isoelectric point

Declarations

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.

Competing interests

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.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Not applicable. This article does not contain any studies with human participants 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 Agricultural Sciences (IARRP-2015-24) and the Fundamental Research Funds for Central Non-profit Scientific Institution of China (1610132016050).

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Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/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.

Authors’ Affiliations

(1)
Key Laboratory of Microbial Resources of the Ministry of Agriculture, Institute of Agricultural Resources and Regional Planning, Chinese Academy of Agricultural Sciences, Beijing, 100081, People’s Republic of China
(2)
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing, 100081, People’s Republic of China

References

  1. Abo M (1990) Method of purifying dry-cleaning solvent by decomposing liquid contaminants with a lipase. World Organ Patent 9(007):606Google Scholar
  2. Baroncelli R, Piaggeschi G, Fiorini L, Bertolini E, Zapparata A, Pè ME, Sarrocco S, Vannacci G (2015) Draft whole-genome sequence of the biocontrol agent Trichoderma harzianum T6776. Genome Announc 3:e00647–e00715View ArticlePubMedPubMed CentralGoogle Scholar
  3. Baroncelli R, Zapparata A, Piaggeschi G, Sarrocco S, Vannacci G (2016) Draft whole-genome sequence of Trichoderma gamsii T6085, a promising biocontrol agent of Fusarium head blight on wheat. Genome Announc 4:e01747–e01815View ArticlePubMedPubMed CentralGoogle Scholar
  4. Barriuso J, Vaquero ME, Prieto A, Martínez MJ (2016) Structural traits and catalytic versatility of the lipases from the Candida rugosa-like family: a review. Biotechnol Adv 34:874–885View ArticlePubMedGoogle Scholar
  5. Beer HD, Wohlfahrt G, Mccarthy JEG, Schomburg D, Schmid RD (1996) Analysis of the catalytic mechanism of a fungal lipase using computer-aided design and structural mutants. Protein Eng Des Sel 9:507–517View ArticleGoogle Scholar
  6. Benítez T, Rincón AM, Limón MC, Codón AC (2005) Biocontrol mechanisms of Trichoderma strains. Int Microbiol 7:249–260Google Scholar
  7. Bora L, Kalita MC (2010) Production of thermostable alkaline lipase on vegetable oils from a thermophilic Bacillus sp. DH4, characterization and its potential applications as detergent additive. J Chem Technol Biotechnol 83:688–693View ArticleGoogle Scholar
  8. Brockerhoff H (1974) Model of interaction of polar lipids, cholesterol, and proteins in biological membranes. Lipids 9:645–650View ArticlePubMedGoogle Scholar
  9. Chinaglia S, Chiarelli LR, Maggi M, Rodolfi M, Valentini G, Picco AM (2014) Biochemistry of lipolytic enzymes secreted by Penicillium solitum and Cladosporium cladosporioides. Biosci Biotechnol Biochem 78:245–254View ArticlePubMedGoogle Scholar
  10. Chomczynski P, Sacchi N (1987) Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159View ArticlePubMedGoogle Scholar
  11. Domínguez MP, Sánchezmontero JM, Sinisterra JV, Alcántara AR (2006) Understanding Candida rugosa lipases: an overview. Biotechnol Adv 24:180–196View ArticleGoogle Scholar
  12. Freitas RS, Steindorff AS, Ramada MHS, de Siqueira SJL, Noronha EF, Ulhoa CJ (2014) Cloning and characterization of a protein elicitor Sm1 gene from Trichoderma harzianum. Biotechnol Lett 36:783–788View ArticlePubMedGoogle Scholar
  13. Gaur R, Gupta A, Khare S (2008) Purification and characterization of lipase from solvent tolerant Pseudomonas aeruginosa PseA. Process Biochem 43:1040–1046View ArticleGoogle Scholar
  14. Grbavčić S, Bezbradica D, Izrael-Živković L, Avramović N, Milosavić N, Karadžić I, Knežević-Jugović Z (2011) Production of lipase and protease from an indigenous Pseudomonas aeruginosa strain and their evaluation as detergent additives: compatibility study with detergent ingredients and washing performance. Bioresour Technol 102:11226–11233View ArticlePubMedGoogle Scholar
  15. Gupta R, Kumari A, Syal P, Singh Y (2015) Molecular and functional diversity of yeast and fungal lipases: their role in biotechnology and cellular physiology. Prog Lipid Res 57:40–54View ArticlePubMedGoogle Scholar
  16. Gutarra ML, Godoy MG, Maugeri F, Rodrigues MI, Freire DM, Castilho LR (2009) Production of an acidic and thermostable lipase of the mesophilic fungus Penicillium simplicissimum by solid-state fermentation. Bioresour Technol 100:5249–5254View ArticlePubMedGoogle Scholar
  17. Halliwell G, Griffin M (1973) The nature and mode of action of the cellulolytic component C1 of Trichoderma koningii on native cellulose. Biochem J 135:587–594View ArticlePubMedPubMed CentralGoogle Scholar
  18. Jørgensen MS, Skovlund DA, Johannesen PF, Mortensen UH (2014) A novel platform for heterologous gene expression in Trichoderma reesei (Teleomorph Hypocrea jecorina). Microb Cell Fact 13:33View ArticlePubMedPubMed CentralGoogle Scholar
  19. Jurado E, Bravo V, Luzon G, Fernández-Serrano M, García-Román M, Altmajer-Vaz D, Vicaria JM (2007) Hard-surface cleaning using lipases: enzyme-surfactant interactions and washing tests. J Surfactants Deterg 10:61–70View ArticleGoogle Scholar
  20. Karadzic I, Masui A, Zivkovic LI, Fujiwara N (2006) Purification and characterization of an alkaline lipase from Pseudomonas aeruginosa isolated from putrid mineral cutting oil as component of metalworking fluid. J Biosci Bioeng 102:82–89View ArticlePubMedGoogle Scholar
  21. Kubicek CP, Herrera-Estrella A, Seidl-Seiboth V, Martinez DA, Druzhinina IS, Thon M, Zeilinger S, Casas-Flores S, Horwitz BA, Mukherjee PK (2011) Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma. Genome Biol 12:R40View ArticlePubMedPubMed CentralGoogle Scholar
  22. Lee SH, Yun SH, Chun J, Kim DH (2017) Characterization of a novel dsRNA mycovirus of Trichoderma atroviride NFCF028. Arch Virol 162:1073–1077View ArticlePubMedGoogle Scholar
  23. Li N, Zong MH (2010) Lipases from the genus Penicillium: production, purification, characterization and applications. J Mol Catal B Enzym 66:43–54View ArticleGoogle Scholar
  24. Mancheño JM, Pernas MA, Martínez MJ, Ochoa B, Rúa ML, Hermoso JA (2003) Structural insights into the lipase/esterase behavior in the Candida rugosa lipases family: crystal structure of the lipase 2 isoenzyme at 1.97A resolution. J Mol Biol 332:1059–1069View ArticlePubMedGoogle Scholar
  25. Martinez D, Berka RM, Henrissat B, Saloheimo M, Arvas M, Baker SE, Chapman J, Chertkov O, Coutinho PM, Cullen D (2008) Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nat Biotechnol 26:553View ArticlePubMedGoogle Scholar
  26. Moh’d AS, Wiegel J (2007) Purification and characterization of two highly thermophilic alkaline lipases from Thermosyntropha lipolytica. Appl Environ Microbiol 73:7725–7731View ArticleGoogle Scholar
  27. Nawani N, Singh R, Kaur J (2007) Immobilization and stability studies of a lipase from thermophilic Bacillus sp: the effect of process parameters on immobilization of enzyme. Electron J Biotechnol 9:559–565Google Scholar
  28. Rao CS, Sathish T, Ravichandra P, Prakasham RS (2009) Characterization of thermo-and detergent stable serine protease from isolated Bacillus circulans and evaluation of eco-friendly applications. Process Biochem 44:262–268View ArticleGoogle Scholar
  29. Rathi P, Saxena R, Gupta R (2001) A novel alkaline lipase from Burkholderia cepacia for detergent formulation. Process Biochem 37:187–192View ArticleGoogle Scholar
  30. Saxena RK, Davidson WS, Sheoran A, Giri B (2004) Purification and characterization of an alkaline thermostable lipase from Aspergillus carneus. Process Biochem 39:239–247View ArticleGoogle Scholar
  31. Schmid KM (2016) Lipid metabolism in plants. In: Ridgway N, McLeod R (eds) Biochemistry of lipids, lipoproteins and membranes, 6th edn. Elsevier B.V, Amsterdam, pp 113–147View ArticleGoogle Scholar
  32. Sethi BK, Nanda PK, Sahoo S (2016) Characterization of biotechnologically relevant extracellular lipase produced by Aspergillus terreus NCFT 4269.10. Braz J Microbiol 47:143–149View ArticlePubMedPubMed CentralGoogle Scholar
  33. Sharma R, Chisti Y, Banerjee UC (2001) Production, purification, characterization, and applications of lipases. Biotechnol Adv 19:627–662View ArticlePubMedGoogle Scholar
  34. Sharma R, Soni SK, Vohra RM, Gupta LK, Gupta JK (2002) Purification and characterisation of a thermostable alkaline lipase from a new thermophilic Bacillus sp. rsj-1. Process Biochem 37:1075–1084View ArticleGoogle Scholar
  35. Sharma D, Sharma B, Shukla AK (2011) Biotechnological approach of microbial lipase: a review. Biotechnology 10:23–40View ArticleGoogle Scholar
  36. Shikunne X, Seidl MF, Faino L, Thomma BP (2015) Draft genome sequence of a strain of cosmopolitan fungus Trichoderma atroviride. Genome Announc 3:e00287–e00315Google Scholar
  37. Singh AK, Mukhopadhyay M (2012) Overview of fungal lipase: a review. Appl Biochem Biotechnol 166:486–520View ArticlePubMedGoogle Scholar
  38. Singh A, Vander Wall TA, Linger J, Himmel ME, Podkaminer K, Adney WS (2015) Heterologous protein expression in Hypocrea jecorina: a historical perspective and new developments. Biotechnol Adv 33:142–154View ArticlePubMedGoogle Scholar
  39. Studholme DJ, Harris B, Cocq KL, Winsbury R, Perera V, Ryder L (2013) Investigating the beneficial traits of Trichoderma hamatum gd12 for sustainable agriculture-insights from genomics. Front Plant Sci 4:258View ArticlePubMedPubMed CentralGoogle Scholar
  40. Tatara T, Fujii T, Kawase T, Minagawa M (1985) Studies on applications of lypolytic enzymes in detergency ii. Evaluation of adaptability of various kinds of lipases in practical laundry conditions. J Am Oil Chem Soc 62:1053–1058View ArticleGoogle Scholar
  41. Woolley P, Petersen SB (1996) Lipases: their structure, biochemistry and application. In: Woolley P, Petersen SB (eds) Biochem cell bio, vol 28. Elsevier Scicence Ltd., Cambridge, pp 831–835Google Scholar
  42. Xie BB, Qin QL, Shi M, Chen LL, Shu YL, Luo Y, Wang XW, Rong JC, Gong ZT, Li D (2014) Comparative genomics provide insights into evolution of Trichoderma nutrition style. Genome Biol Evol 6:379–390View ArticlePubMedPubMed CentralGoogle Scholar
  43. Yang D, Pomraning K, Kopchinskiy A, Aghcheh RK, Atanasova L, Chenthamara K, Baker SE, Zhang R, Shen Q, Freitag M (2015) Genome sequence and annotation of Trichoderma parareesei, the ancestor of the cellulase producer Trichoderma reesei. Genome Announc 3:e00885–e00915PubMedPubMed CentralGoogle Scholar
  44. Zhang X, Xia L (2017) Expression of Talaromyces thermophilus lipase gene in Trichoderma reesei by homologous recombination at the cbh1 locus. J Ind Microbiol Biotechnol 44:1–9View ArticleGoogle Scholar

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© The Author(s) 2018

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