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. 2021 Nov 15;11(12):496. doi: 10.1007/s13205-021-03053-7

Characteration of a novel arylesterase from probiotics Lacticaseibacillus rhamnosus GG with the preference for medium- and long-chain p-Nitrophenyl esters

Bin-Chun Li 1,✉, Tong-Tong Guo 1, Guo-Bin Ding 1
PMCID: PMC8593145  PMID: 34881159

Abstract

We prospected a novel arylesterase LggEst from the probiotics Lacticaseibacillus rhamnosus GG by genome mining strategy, and characterized the enzymatic properties in detail. Biochemical characterization revealed that arylesterase LggEst presented high activity at a wide range of temperatures from 25 to 65 °C with maximum activity at 50 °C. LggEst maintained high activity in the pH range from 5.5 to 7.5 with optimum pH of 6.5. LggEst might efficiently hydrolyze a series of aryl substrates p-nitrophenyl esters with different acyl chain lengths. LggEst displayed the Vmax from 2.8 to 77.3 μmol min−1 mg−1 protein and the kcat from 1.8 to 48.8 s−1 with the highest catalytic activity on pNPC6. The KM of LggEst on different substrates varied significantly from 4.9 μM to 5.6 mM with the highest affinity on pNPC10. LggEst exhibited the preference for medium- and long-chain p-nitrophenyl esters. LggEst showed remarkable thermostability at 45 °C. LggEst could be tolerant of several organic solvents at the concentration of 10% and DMSO and methanol at the concentration of 20%. Catalytic activity of LggEst was improved by 12% in the presence of 20% ethylene glycol. LggEst was resistant to high concentrations of sodium citrate and sodium chloride. Notably, enzymatic activity of LggEst was significantly enhanced in the presence of 0.1% sodium deoxycholate at high temperatures.

Supplementary Information

The online version contains supplementary material available at 10.1007/s13205-021-03053-7.

Keywords: Arylesterase, Lacticaseibacillus rhamnosus GG, Enzymatic properties, Sodium deoxycholate

Introduction

Esterases (EC 3.1.1.1 and EC 3.1.1.2), acting on the ester bonds, could catalyze the hydrolysis, alcoholysis, and esterification reactions (Bornscheuer 2002). Microbial esterases had become an important group of biotechnologically valuable enzymes for biocatalytic applications in pharmaceutical industry as resolution of racemates, environmental protection for recycling of waste oils and biodegradation of plastics and pesticides, and food field aimed for formation of flavor, nutrition improvements, and food fermentation. Microbial esterases were widely diversified in enzymatic properties and substrate specificity, which were determined by their origins. Owing to the functional versatility and potential applications in food industry, the interest in the salt-tolerant esterases was increasing.

Lactic acid bacteria (LAB) had been a potential source of the esterases used in food industry for cheese flavor (Holland et al. 2005) and vinification (Matthews et al. 2004, 2007; Pérez-Martín et al. 2013). Most recombinant esterases from LAB originated from the genus Lacticaseibacillus. To date, 25 esterases from Lacticaseibacillus species had been cloned and characterized (Table 1). The esterases from Lacticaseibacillus species mainly were arylesterases, and cinnamoyl and feruloyl esterases. Among them, the esterases Lp_0796, Lp_2631, and Lp_3562 from L. plantarum WCFS1 were suitable for food fermentations (Inmaculada et al. 2013; Esteban-Torres et al. 2014a, b). Esterase Lp_3505 from L. plantarum WCFS1, esterases EstB and EstC from L. casei LILA, and esterase EstA from L. helveticus CNRZ32 had potential application during cheese ripening (Esteban-Torres et al. 2014d; Fenster et al. 2013b; Fenster et al. 2013a; Fenster et al. 2000), and tributyrin esterase Lp_1760 from L. plantarum WCFS1 was suitable for cheese lipolysis (Esteban-Torres et al. 2014c). Arylesterase Lp_1002 from L. plantarum WCFS1 exhibited hydrolytic activity on wine esters at winemaking conditions (Esteban-Torres et al. 2014a), and EstC34 from L. hilgardii was stable and retained activity under wine conditions (Sumby et al. 2013). The cinnamoyl and feruloyl esterases from Lacticaseibacillus species might be used for fermentation of plant-derived food products. Nevertheless, most esterases from Lacticaseibacillus species displayed the highest activity on short-chain aryl esters p-nitrophenyl acetate and p-nitrophenyl butyrate (Table 1). However, the esterases from L. rhamnosus were rarely reported.

Table 1.

Enzymatic properties of recombinant esterases from the genus Lacticaseibacillus

Enzymes Organism GenBank accession Number of residues pHopt a Topt b (oC) pNPCopt c References
CE L. acidophilus DSM 20,079 YP_194675 247 7.5 20 pNPC2 Fritsch et al. (2017)
CE L. acidophilus F46 AIC34710 247 7.5 50 NA Kim and Baik (2015)
FAELac L. acidophilus KX545371 244 7.0 25 NA Liu et al. (2021)
EstI L. casei CL96 AAP02960 599 7.0 37 pNPC8 Choi et al. (2004)
EstB L. casei LILA AAP80677 318 7.0 50 pNPC6 Fenster et al. (2003b)
EstC L. casei LILA AAM90952 259 5.5 30 pNPC3 Fenster et al. (2003a)
FaeLcr L. crispatus S524 QBS13631 251 7.0 65 NA Xu et al. (2019)
CE L. fermentum WVS48/B4 YP_001844134 249 7.0 NA pNPC2 Fritsch et al. (2017)
CE L. gasseri DSM 20,243 YP_815563 260 7.5 30 pNPC2 Fritsch et al. (2017)
EstA L. helveticus CNRZ32 AAF64390 186 8.0 35 pNPC4 Fenster et al. (2000)
CE L. helveticus DSM 20,075 YP_001578032 251 8.5 NA pNPC2 Fritsch et al. (2017)
EstC34 L. hilgardii AFX62596 259 5.0 50 pNPC2 Sumby et al. (2012)
Lj0536 L. johnsonii N6.2 ADD11991 249 7.8 20 pNPC4 Lai et al. (2009)
Lj1228 L. johnsonii N6.2 AAS09049 248 6.7 30 pNPC4 Lai et al. (2009)
lp_0796 L. plantarum WCFS1 CCC78257 249 8.0 35 pNPC4

Inmaculada et al. (2013);

Esteban-Torres et al. (2013)
lp_0973 L. plantarum WCFS1 CCC78406 338 6.0 40 pNPC2 Brod et al. (2010)
lp_1002 L. plantarum WCFS1 CCC78426 264 7.0 40 pNPC2 Esteban-Torres et al. (2014a)
lp_1760 L. plantarum WCFS1 CCC79051 250 7.0 45 pNPC4 Esteban-Torres et al. (2014c)
lp_2631 L. plantarum WCFS1 CCC79769 261 6.5 20 pNPC2 Esteban-Torres et al. (2014b)
lp_3323 L. plantarum WCFS1 CCC80320 213 8.0 40 pNPC2 Kim et al. (2017)
lp_3505 L. plantarum WCFS1 CCC80473 263 6.0 5 pNPC2 Esteban-Torres et al. (2014d)
lp_3561 L. plantarum WCFS1 CCC80524 278 6.5 40 pNPC2 Esteban-Torres et al. (2016)
lp_3562 L. plantarum WCFS1 CCC80525 278 7.0 40 pNPC2 Esteban-Torres et al. (2015b)
CE L. plantarum WCSF1 YP_004890534 249 8.0 35 pNPC2 Fritsch et al. (2017)
Est_1092 L. plantarum JDM1 ACT61979 295 5.0 30 pNPC4 Esteban-Torres et al. (2015a)
CE L. reuteri JCM 1112 YP_001842568 249 8.0 20 pNPC2 Fritsch et al. (2017)
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apHopt indicates the optimum pH

bTopt indicates the optimum temperature

cpNPCopt indicates the optimal substrate for p-nitrophenyl esters. CE cinnamoyl esterase, NA not available

L. rhamnosus, a facultatively heterofermentative LAB, played a significant role during cheese ripening, leading to the formation of flavor. L. rhamnosus GG (LGG), as the L. rhamnosus strain, was isolated in 1983 by Barry R. Goldin and Sherwood L. Gorbach. LGG had become the most widely used probiotics for human health due to the ability to survive and to proliferate at gastric acid pH and in medium containing bile, and to adhere to enterocytes (Capurso 2019). LGG had been used in a variety of commercially probiotic products and various food products, usually dairy products such as yogurt, fermented milk, pasteurized milk, and semi-hard cheese. The complete genome of LGG had been determined and annotated in 2009 (Kankainen et al. 2009).

Herein, we focused on the putative esterase LGG_02908 from the probiotics LGG. The crystal structure of two homologues esterase B (AA7, PDB id 4PO3 and 4OUK) from L. rhamnosus HN001 and esterase B (PDB id 4N5I and 4N5H) from L. rhamnosus Lc705 had been determined and deposited into the Protein Data Bank (Berman et al. 2000), and the esterase AA7 had been biochemically characterized to a limited extent (Delabre et al. 2006; Holland et al. 2005). However, the enzymatic properties and hydrolytic activity against aryl esters had not yet been investigated. In this study, the arylesterase LGG_02908, designated as LggEst, was prospected by genome mining, heterologously over-expressed in E. coli BL21(DE3), and characterized in detail.

Materials and methods

Bacterial strains, enzymes and reagents

Lacticaseibacillus rhamnosus GG was obtained from China General Microbiological Culture Collection Center (No. 1.5132). E. coli DH5α was used for gene cloning, and E. coli BL21(DE3) was used for protein expression. TransStart® FastPfu Fly DNA polymerase and PCR related reagents were purchased from TransGen Biotech (Beijing, China). Oligonucleotide primers were synthesized by Sangon Biotech (Shanghai, China). T4 DNA ligase and restriction enzymes QuickCut Nde I and Xho I were obtained from TaKaRa (Otsu, Japan). Isopropyl-β-D-thiogalactopyranoside (IPTG), imidazole, and kanamycin were purchased from Sangon Biotech (Shanghai, China). The substrates p-nitrophenyl acetate (pNPC2), p-nitrophenyl butyrate (pNPC4), p-nitrophenyl caprylate (pNPC8), p-nitrophenyl caprate (pNPC10), and p-nitrophenyl laurate (pNPC12) used for arylesterase activity assays and kinetics were purchased from Sigma-Aldrich (St. Louis, USA) expect that the p-nitrophenyl caproate (pNPC6) was provided by Tokyo Chemical Industry (Shanghai, China). Other reagents were of analytical grade or higher.

Cloning, expression, and purification of arylesterase LggEst

The arylesterase gene LggEst (LGG_02908, GenBank accession: CAR88803) was amplified from genomic DNA of L. rhamnosus GG using the primers LggEst-Nde I-F and LggEst-Xho I-R (Table S1). PCR product of LggEst gene was digested with QuickCut Nde I and Xho I, and ligated into pET-28a digested by same restriction enzymes. The ligation product was transformed into E. coli DH5α. The sequence of LggEst gene was confirmed by DNA sequencing (Sangon Biotech, Shanghai, China).

The BL21(DE3) cells carrying the recombinant plasmid pET28a-LggEst were grown in 1 L 2 × YT medium containing 50 μg mL−1 kanamycin at 37 °C. Protein expression was induced for overnight at 16 °C by addition of 0.5 mM IPTG until OD600 reached 2.0. Then the cells were harvested and resuspended in 50 mM NaH2PO4-Na2HPO4 buffer (pH 8.0) containing 12 mM imidazole, and then disrupted by sonication for 50 min by working for 2 s with an interval of 3 s. After sonication, the cell lysate was cleared by centrifugation at 9000 × g for 30 min at 4 °C. The recombinant LggEst was purified by Ni–NTA affinity chromatography (Qiagen, Hilden, Germany). The purified LggEst was further dialyzed twice in 50 mM NaH2PO4-Na2HPO4 buffer (pH 8.0). Denaturing and nondenaturing gels were stained by coomassie brilliant blue. Activity staining was perfomred as described in that study (Zhou et al. 2012). Protein concentration of the purified LggEst was measured at 280 nm by NanoDrop 2000 UV–vis spectrophotometer (Thermo Fisher Scientific, Waltham, USA).

Enzyme activity assays

As p-nitrophenol showed the maximum absorption at the wavelength of 405 nm in alkaline condition, and catalytic activities of the esterases could be measured by instantaneously monitoring the release of p-nitrophenol at 405 nm in alkaline condition using an UV-5800PC spectrophotometer (METASH, Shanghai, China) equipped with a thermal controller. Therefore, the following standard assay was adopted in this study except for determining the effect of pH on enzyme activity. The reaction mixture of 1 mL consisted of 940 μL of 50 mM NaH2PO4-Na2HPO4 buffer (pH 7.5), 40 μL the substrate pNPC8 (predissolved in acetonitrile, initial 2.5 mM), and 20 μL enzyme solution (initial, 0.2 mg mL−1), and the reaction buffer containing the substrate was pre-incubated for 30 s before adding the enzyme. The released p-nitrophenol was determined at 405 nm for 30 s at 50 °C so that the relation of absorption value with reaction time was linear. One unit of enzyme activity (U) was defined as the amount of enzyme releasing 1 μmol p-nitrophenol from the substrate per min at 50 °C in pH 7.5. All assays were performed in triplicate.

Effect of pH and temperature on arylesterase activity

The effects of pH on enzymatic activities of arylesterase LggEst were determined at 45 °C under pH ranging from 4.0 to 11.0 using pNPC8 as the substrate. The reaction solution of 500 μL contained 470 µL 30 mM wide range buffer, 20 μL the substrate pNPC8 (predissolved in acetonitrile, initial, 10 mM), and 10 μL enzyme solution (initial, 0.2 mg mL−1). After the incubation for 30 s at 45 °C, the reaction was stopped by adding 0.5 M trichloroacetic acid of 500 μL. After adjusting reaction mixture to alkaline by adding 0.5 M Na2CO3 of 500 µL, the absorption value was determined at 405 nm. The wide range buffer contained 30 mM each of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 3-[[1,3-dihydroxy-2-(hydroxymethyl)propan-2-yl]amino]propa-ne-1-sulfonic acid (TAPS), 3-(cyclohexylamino)-1-propanesulfonicacid (CAPS), 2-(N-morpholino)ethanesulfonic acid (MES), and acetic acid, and was adjusted to the appropriate pH with 1 M NaOH at 45 °C.

The effects of temperatures on enzymatic activity of LggEst were determined by measuring catalytic activities at the temperature range from 20 °C to 65 °C in the standard assay described above.

Enzyme steady-state kinetics

Enzyme kinetics of LggEst for p-nitrophenyl esters pNPC2, pNPC4, pNPC6, pNPC8, pNPC10, and pNPC12 were determined in the standard assay described above for 5 s. For the p-nitrophenyl esters with different acyl chain lengths, appropriate substrates concentrations ranges and appropriate enzyme concentrations were used for measuring the initial rate of hydrolysis reaction. Kinetic parameters Vmax and KM were acquired by fitting catalytic activities as a function of substrate concentrations to the Michaelis–Menten equation using non-linear regression of the software GraphPad Prism 6. The parameter kcat was obtained by using the equation kcat = Vmax/[E], where [E] was molar concentration of the enzyme.

Effect of temperatures on arylesterase stability

Thermal inactivation experiments of arylesterase LggEst were performed by pre-incubating the enzyme (1.0 mg mL−1) for specified time intervals at 45 °C, 50 °C, or 55 °C. The enzyme samples were then cooled on ice and residual activities were measured in the standard assay described above.

Effect of organic solvents on arylesterase activity

The effects of organic solvents on enzymatic activity of arylesterase LggEst were determined by measuring catalytic activities with 10% and 20% (v/v) of various organic solvents in the standard assay described above.

Effect of salts on arylesterase activity

The effects of salts on enzymatic activity of arylesterase LggEst were determined by measuring catalytic activities in the standard assay described above with 0.25 M, 0.5 M, and 0.8 M sodium citrate or 0.5 M, 1.0 M, and 1.5 M NaCl.

Effect of detergents on arylesterase activity

The effects of ionic detergent sodium deoxycholate (DOC) on enzymatic activity of arylesterase LggEst were determined by measuring catalytic activities in the standard assay described above with 0.1% (w/v) DOC at different temperatures from 20 °C to 65 °C.

The effects of non-ionic detergents on enzymatic activity of arylesterase LggEst were determined by measuring catalytic activities in the standard assay described above with 0.1% and 0.5% (v/v) of Tween 20, Tween 80, or Triton X-100.

Results and discussion

Genome mining and sequence analysis

Lacticaseibacillus rhamnosus strains exhibited esterase activity. L. rhamnosus strains ETC14, ETC17, and ETC18 showed the activity on α-napthyl esters (Oliszewski et al. 2007). L. rhamnosus strains E/N, OXY, and PEN performed the limited ability to produce feruloyl esterase on cinnamic acids methyl esters (Szwajgier and Jakubczyk 2011). The complete genome of LGG had been sequenced and annotated (Kankainen et al. 2009). The genome of LGG with a single circular chromosome 3.01 Mbp in size contained 2944 proteins. Genome mining revealed that 6 genes were annotated into the esterases consisting of 213 to 336 amino acid residues (Table S2). Sequence analysis indicated that the putative esterase LGG_02908 shared the sequence identity of 100% with both esterase B (AA7) from L. rhamnosus HN001 and esterase B from L. rhamnosus Lc705. The crystal structures of two homologues had been resolved. Moreover, the esterase AA7 catalyzed both hydrolytic release of free fatty acids and alcoholytic synthesis of ethyl esters. AA7 was active on short-chain acyl esters and showed a preference for monoacylglycerols. AA7 was used in cheese manufacture and catalyzed an accumulation of butyric acid and ethyl esters in cheese (Delabre et al. 2006). However, the enzymatic properties and catalytic performance on aryl esters remained unclear. The putative esterase LGG_02908, designated as LggEst, was composed of 317 amino acid residues, and was similar in size with the arylesterase EstB from L. casei LILA containing 318 residues. Nevertheless, most esterases from Lacticaseibacillus species were close in the number of amino acid residues ranging from 244 to 278 (Table 1).

Production and purification of arylesterase LggEst

The recombinant LggEst was heterologously over-expressed in E. coli BL21 (DE3) as soluble form with high proportion in the supernatant (Fig. S2A). As the fusion protein with an N-terminal (His)6 tag, arylesterase LggEst had been purified with Ni–NTA affinity chromatography analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (Fig. S2A, B). Apparent molecular mass of about 37.9 kDa was consistent with the theoretical value. However, native-PAGE exhibited that this arylesterase appeared to be a tetramer (151.6 kDa) in solution as shown in Fig. S2C.

Optimum pH and temperature of arylesterase LggEst

LggEst exhibited the highest activity in pH 6.5 with a decline in enzymatic activity observed in acidic (< 5.0) and alkaline pH (> 8.0) (Fig. 1A). This was consistent with most esterases from Lacticaseibacillus species displaying optimal activity under pH 5.0–8.0 (Table 1). Moreover, LggEst showed high activity (> 60%) only in pH 5.5–7.5 (Fig. 1A). Many esterases from Lacticaseibacillus species exhibited high activity in a limited pH range, such as Lp_3505 from L. plantarum WCFS1 in 6.0–6.5 (Esteban-Torres et al. 2014d), Lp_3562 from L. plantarum WCFS1 in 6.5–7.0 (Esteban-Torres et al. 2015b), Lp_2631 from L. plantarum WCFS1 in 6–7 (Esteban-Torres et al. 2014b), FAELac from L. acidophilus in 6.5–7.5 (Liu et al. 2021), Lp_1760 from L. plantarum WCFS1 in 6–7.5 (Esteban-Torres et al. 2014c), Lp_1002 and Lp_0973 from L. plantarum WCFS1 in 5–7 (Esteban-Torres et al. 2014a; Brod et al. 2010), Lp_0796 from L. plantarum WCFS1 in 6–8 (Inmaculada et al. 2013), and FaeLcr from L. crispatus in 7–9 (Xu et al. 2019).

Fig. 1.

Fig. 1

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Effects of pH and temperature on enzymatic activities of arylesterase LggEst. A Effects of pH on enzymatic activities of LggEst at 45 °C under 4–11. Enzymatic activities are normalized as percentages of maximum activity in pH 6.5. B Effects of temperature on enzymatic activities of LggEst in 50 mM NaH2PO4–Na2HPO4 buffer (pH 7.5) at 20–65 °C. Enzymatic activities are normalized as percentages of maximum activity at 50 °C

The optimum temperature of LggEst was 50 °C, and more than 50% of enzymatic activity was maintained in the broad temperatures range from 25 oC to 65 °C (Fig. 1B). Most recombinant esterases from Lacticaseibacillus species displayed the maximal activity at the temperatures of 20–50 °C, except from FaeLcr from L. crispatus S524 (Table 1).

Acyl chain lengths specificity of arylesterase LggEst

Enzyme kinetic parameters were determined on the hydrolysis of p-nitrophenyl esters at 50 °C in pH 7.5 (Table 2, Fig. S3). Enzyme kinetics indicated that arylesterase LggEst presented high activity for all the tested p-nitrophenyl esters with different acyl chain lengths (Table 2). LggEst displayed the maximum reaction velocity Vmax from 2.8 to 77.3 μmol min−1 mg−1 protein and the corresponding turnover number kcat from 1.8 to 48.8 s−1, and the highest catalytic activity on pNPC6 (Table 2). The Michaelis constant KM of LggEst on different substrates varied significantly from 4.9 μM to 5.6 mM, and LggEst performed the highest affinity with pNPC10 (Table 2). The catalytic efficiency (kcat/KM) revealed that LggEst exhibited the preference for the medium- and long-chain p-nitrophenyl esters, and the most selective substrate was pNPC10 (Table 2). Nevertheless, most esterases from Lacticaseibacillus species presented the highest activity on short-chain aryl esters pNPC2 and pNPC4, apart from the esterases EstI from L. casei CL96 on pNPC8 (Choi et al. 2004) and EstB from L. casei LILA on pNPC6 (Fenster et al. 2003a, b) (Table 1).

Table 2.

Michaelis–Menten kinetic parameters of arylesterase LggEst on the hydrolysis of the substrates p-nitrophenyl esters

Substrate Vmax (µmol min−1 mg−1) kcat (s−1) KM (µM) kcat/KM (M−1 s−1)
pNPC2 58.7 ± 3.8 37.1 ± 2.4 5.6 × 103 ± 0.9 × 103 6.6 × 103
pNPC4 51.1 ± 2.9 32.3 ± 1.8 1.2 × 103 ± 0.1 × 103 2.7 × 104
pNPC6 77.3 ± 7.2 48.8 ± 4.5 260.8 ± 38.4 1.9 × 105
pNPC8 69.4 ± 4.3 43.8 ± 2.7 24.2 ± 3.6 1.8 × 106
pNPC10 23.1 ± 1.1 14.6 ± 0.7 4.9 ± 1.0 3.0 × 106
pNPC12 2.8 ± 0.1 1.8 ± 0.1 5.9 ± 0.4 3.1 × 105
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All reactions were performed in 50 mM NaH2PO4-Na2HPO4 buffer (pH 7.5) at 50 °C for 5 s

Thermal stability of arylesterase LggEst

Thermal inactivation curves for arylesterase LggEst at different temperatures (Fig. 2) showed that the arylesterase exhibited considerable thermostability at 45 °C. LggEst still kept 47% of initial activity even after the incubation of 6.0 h at 45 °C. The half-life of LggEst was approximately 60 min at 50 °C, and LggEst maintained 44% of initial activity after the incubation of 80 min at 50 °C. Enzymatic activity of LggEst was rapidly lost at 55 °C, and only 12% of initial activity was residual after the treatment for 0.5 h at 55 °C (Fig. 2). Most esterase from Lacticaseibacillus species were thermally unstable, and the esterases Lp_1002, Lp_1760, and Lp_3562 from L. plantarum WCFS1 were thermal stable after incubation for 5.0 h at 45 °C (Esteban-Torres et al. 2014a, c, 2015b).

Fig. 2.

Fig. 2

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Thermal inactivation profiles of arylesterase LggEst. The data for residual activities after pre-incubattion at 45 °C, 50 °C, and 55 °C are shown in square, circle, and triangle, respectively. Enzymatic activity of LggEst without incubation is taken as the 100%. All reactions were performed in triplicate, and error bars represent the standard deviations of mean

Effect of organic solvents on arylesterase LggEst activity

The effects of organic solvents on catalytic activity of arylesterase LggEst were measured. LggEst exhibited a good tolerance on a variety of organic solvents except for acetonitrile and maintained 61–105% of enzymatic activity at the concentration of 10% (v/v) (Fig. 3). LggEst displayed the tolerance on DMSO and methanol, and retained 64.5% and 49.3% of enzymatic activity at the concentration of 20% (v/v), respectively (Fig. 3). It was noteworthy that ethylene glycol improved catalytic activity of LggEst, and catalytic activity increased by 12% when the concentration reached 20% (v/v). In addition, this enzyme was dramatically inhibited by acetonitrile, and was almost inactive at the concentration of 20% (v/v) (Fig. 3).

Fig. 3.

Fig. 3

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Effects of organic solvents on enzymatic activity of arylesterase LggEst. Enzymatic activity by adding the corresponding volume of 50 mM NaH2PO4–Na2HPO4 buffer (pH 7.5) was taken as the control. All reactions were performed in triplicate, and error bars represent the standard deviations of mean

Effect of salts on arylesterase LggEst activity

The salts sodium citrate and NaCl were used as food additives. The effects of sodium citrate and NaCl on enzymatic activity of arylesterase LggEst had been shown in Fig. 4. It had been suggested that arylesterase LggEst was halo-tolerant to a certain extent. LggEst maintained 89% and 70% of catalytic activity at sodium citrate concentrations of 0.25 M and 0.5 M, respectively, and kept 97% and 83% of hydrolytic activity at NaCl concentrations of 0.5 M and 1.0 M, respectively (Fig. 4). Nevertheless, at higher salt concentration, this enzyme exhibited only weak activity. Many esterases from Lacticaseibacillus species was salt-tolerant and even salt-activated. NaCl concentrations of 2.5–15% increased activity of esterase Lp_3505 from L. plantarum WCFS1 (Esteban-Torres et al. 2014d). Arylesterases EstB from L. casei LILA (Fenster et al. 2003b) and EstA from L. helveticus CNRZ32 (Fenster et al. 2000) presented maximum activity at NaCl concentration of 15% and 3.5%, respectively, and maintained enzymatic activity of 85% and 64% at NaCl concentration of 20%, respectively. NaCl concentrations of 2.5–20% improved activity of esterase Lp_3562 from L. plantarum WCFS1 (Esteban-Torres et al. 2015b). NaCl concentrations of 2.5–5% increased activity of esterase Lp_1760 from L. plantarum WCFS1 and retained enzymatic activity of 90% at NaCl concentration of 10% (Esteban-Torres et al. 2014c).

Fig. 4.

Fig. 4

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Effects of sodium citrate and NaCl on enzyme activity of arylesterase LggEst. Enzymatic activity by adding the corresponding volume of 50 mM NaH2PO4–Na2HPO4 buffer (pH 7.5) was taken as the control. All reactions were performed in triplicate, and error bars represent the standard deviations of mean

Effect of detergents on arylesterase LggEst activity

Ionic detergent sodium deoxycholate (DOC) might increase the dissolution of lipid and promoted its absorption in the intestine. The effects of 0.1% (w/v) DOC on enzymatic activity of arylesterase LggEst at different temperatures were investigated. The results as shown in Fig. 5 indicated that the effects of 0.1% (w/v) DOC on esterase activity was temperature-dependent. 0.1% (w/v) DOC dropped catalytic activity of LggEst at low temperatures 20 °C and 25 °C. However, 0.1% (w/v) DOC improved enzymatic activity of LggEst at high temperatures ranging from 30 °C to 65 °C (Fig. 5). As the temperatures increased, the ratios of activity increasing also enhanced, and catalytic activity considerably increased by 2.4-fold at 65 °C. In addition, 0.1% (w/v) DOC raised the optimum temperature of LggEst from 50 °C to 55 °C, and the specific activity reached 52.6 μmol min−1 mg−1 at 55 °C (Fig. 5). The reason might be that DOC might inhibit protein aggregation and thermal inactivation of this enzyme at high temperatures by destroying the interactions between proteins. It had been indicated that DOC might have improved the catalytic activity of the lipases (Cai et al. 2011; Sekhon et al. 2005).

Fig. 5.

Fig. 5

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Effects of 0.1% (w/v) DOC on enzyme activity of arylesterase LggEst at different temperatures. Enzymatic activity by adding the corresponding volume of 50 mM NaH2PO4–Na2HPO4 buffer (pH 7.5) was taken as the control. All reactions were performed in triplicate, and error bars represent the standard deviations of mean

The effects of three non-ionic detergents on enzymatic activity of arylesterase LggEst were observed. It was suggested that that hydrolytic activity of arylesterase LggEst decreased to 64.8%, 49.1% and 40.5% in 0.1% Tween 20, Tween 80, and Triton X-100, respectively (Fig. 6).

Fig. 6.

Fig. 6

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Effects of non-ionic detergents on enzyme activity of arylesterase LggEst. Enzymatic activity by adding the corresponding volume of 50 mM NaH2PO4-Na2HPO4 buffer (pH 7.5) was taken as the control. All reactions were performed in triplicate, and error bars represent the standard deviations of mean

Conclusions

In this study, we characterized a novel arylesterase from the probiotics Lacticaseibacillus rhamnosus GG in detail. Arylesterase LggEst might efficiently hydrolyze a broad range of aryl substrates p-nitrophenyl esters and exhibited a preference for medium- and long-chain p-nitrophenyl esters. LggEst presented significant advantages, such as high activity at high temperatures, acceptable thermostability, salts tolerance, and ethylene glycol and DMSO tolerance. Furthermore, 0.1% (w/v) DOC markedly improved catalytic activity of LggEst at high temperatures.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 907 KB) (906.9KB, docx)

Acknowledgements

This work was supported by the Natural Science Foundation of Shanxi for Applied and Basic Research Program (No. 201901D111013).

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

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Supplementary Materials

Supplementary file1 (DOCX 907 KB) (906.9KB, docx)

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