Lipase production by botryosphaeria ribis EC-01 on soybean meal supplemented with amino acids, and some physicochemical properties of the enzyme.
Lipases (EC 22.214.171.124; triacylglycerol acylhydrolases) belong to a group of enzymes of great technological interest and are produced by plants, mammals, and microorganisms that include bacteria, actinobacteria, yeasts and fungi. Fungal lipases are versatile enzymes because of their properties stability, cofactors not required, and wide substrate specificity--that make them attractive from an industrial standpoint [1, 2].
Microbial lipases are produced mostly by submerged fermentation (SmF) in a complex nutrient medium influenced by the type and concentration of carbon (C) and nitrogen (N) sources, nutrients, pH and temperature [1, 3, 4]. Vegetable oils and animal fats appear to be the preferable C sources to induce lipase production [5-7], however, some reports have demonstrated good lipase production in the absence of lipids as inducers [8, 9]. Inorganic and organic sources ofN can also influence lipase production . Nitrate and ammonium salts, peptone, soybean meal, urea, hydrolyzed proteins and amino acids are the most commonly used N sources for lipase production, although amino acids and proteins can also act as C sources by certain fungi .
Lipases have a broad spectrum of applications  but their use to convert vegetable oils to methyl, or other short chain alcohol esters, in a single transesterification reaction has been one of the most active fields of recent research. The increasing interest in using lipases as biocatalysts for biofuels production is related to the easy separation of the product, fatty acyl alkyl esters that constitute biodiesel, their purification, minimal wastewater treatment, and facile recovery of glycerol in comparison to the chemical catalyzed processes of making biodiesel [15-17]. The cost of lipase still disfavors this application, but the enzyme production cost and its possible immobilization could be the key to an economical process for making biodiesel.
Botryosphaeria ribis EC-01, an ascomycete, was previously selected as a good producer of lipase when grown on different vegetable oils, including soybean oil . This fungal isolate produced high levels of lipase in submerged cultivation when grown only on soybean meal in the presence of distilled water, proving that agro-industrial waste residues can be used as a nutrient source for producing lipases and thereby reducing the cost of production. The addition of KH2PO4 enhanced lipase activity by this fungal isolate when cultivated on soybean meal in terms of specific activity .
In this work, the amino acids serine, histidine, glutamic and aspartic acid and glycine when added to soybean meal were evaluated in order to enhance lipase production by B. ribis EC-01 under SmF. These amino acids are not present in the composition of soybean meal, or when present, appear in small amounts. The influence of several metal salts, solvents and detergents on B. ribis EC-01 lipase activity, as well as the substrate specificities, together with the apparent Km for substrates of 2 to 18 carbon chain lengths, were also evaluated. Considering that the main objective is the future immobilization of lipase and its application to produce biodiesel, this study was performed using the crude enzyme extract which is the norm in commercial processes employing enzymes.
2. MATERIAL AND METHODS Materials
Soybean meal was kindly donated by Importacao, Exportacao e Industria de Oleos S.A. (Cambe-PR, Brasil). p-Nitrophenyl esters (acetate, butyrate, caproate, caprate, palmitate and stearate) were purchased from Sigma-Aldrich (USA). All the other reagents were of analytical grade.
Cultivation of Botryosphaeria ribis EC-01
Botryosphaeria ribis EC-01 (GenBank Accession Number DQ852308) was maintained on potato-dextrose agar (PDA) slants at 4.0 [+ or -] 2[degrees]C and sub-cultured at three-monthly intervals. B. ribis EC01 was transferred to agar plates containing glucose (10 g [L.sup.-1]), Vogel minimum salts medium (VMSM) , agar (20 g [L.sup.-1]), and left at 28 [+ or -] 2[degrees]C for 5 days. Following growth, four plugs of 0.7 [cm.sup.-1] diameter were taken from the mat of mycelial-colonized agar plates. Inoculum were prepared in 125 mL Erlenmeyer flasks, containing 1% (w/v) soybean meal in 25 mL distilled water, and incubated at 28[degrees]C (180 rpm) for 120 h. Five-day old cultures of B. ribis EC01 were harvested by centrifugation (1500 x g/15 min), and the supernatants recovered (CLE) were used as the source of crude lipase to determine some enzyme properties. All experiments were carried out in replicates of three, and results are expressed as the means [+ or -] SD.
Production of lipase by Botryosphaeria ribis EC01: effect of amino acids added to soybean meal
The effect of soybean meal supplemented separately with different amino acids (serine, glycine, histidine, glutamic acid, aspartic acid) were examined as substrates for lipase production by B. ribis EC-01 in SmF, using 1% (w/v) soybean meal and concentrations ranging from 0.01 to 0.5% (w/v) of these amino acids.
Proteins were determined according to Bradford , using bovine albumin as the standard.
Assay of lipase activity
All assays for lipase activity were determined using p-nitrophenyl palmitate (p-NPP) as the standard substrate. The reaction was carried out in 0.05 M sodium phosphate buffer (pH 8.0) at 55[degrees]C for 2 min. Absorbance was measured at 410 nm using the coefficient of molar extinction for p-nitrophenol (pNP) of 1.5 x [10.sup.4] [M.sup.-1] [cm.sup.-1] . One unit (1 U) of lipase activity is defined as the release of 1 [micro]mol of p-NP per min per mL of enzyme solution.
Lipase activity was tested against various synthetic substrates consisting of fatty-acyl esters of p-nitrophenyl such as acetate (C2), butyrate (C4), caproate (C6), caprate (C10), palmitate (C16) and stearate (C18). The reactions were performed at 55[degrees]C (pH 8.0) for 2 min.
Effect of pH and temperature on lipase activity
B. ribis EC-01 lipase was assayed using p-NPP as substrate by incubating the crude lipase extract (CLE) in 0.05 M of the different buffer systems at pH ranging from 1-10, and at temperatures ranging from 30[degrees] to 65[degrees]C (0.05 M sodium phosphate buffer, pH 8.0) for 2 min at 55[degrees]C. The different buffers used were citrate-phosphate (pH 3-7), phosphate (pH 6-8), Tris-HCl (pH 8-9), boric acid-borax (pH 8- 9)
and glycine-NaOH (pH 9-10).
Lipase temperature and pH stability
CLE was incubated at 30, 40, 45, 50, 55, 60, 70 and 80[degrees]C for times ranging from 5 min to 3 h. For temperatures between 30 and 55[degrees]C, the period of incubation was 96 h. At the pre-established time intervals, aliquots were removed and assayed for lipase activity under standard assay conditions. The influence of pH on enzyme stability was evaluated by incubating CLE (in the proportion of 1:1 in different buffer solutions with pH ranging from 3 to 10 as well as in distilled water) for 1 h at 30[degrees]C followed by assay of lipase activity under standard assay conditions.
Influence of metal salts, detergents and organic solvents on lipase activity
CLE was incubated with different metal salt solutions (1:1 ratio) at concentrations of 1, 5, 10, 25, 50 and 100 mM at 30[degrees]C in water for 1 h. Similarly, CLE was incubated at 30[degrees]C for 1 h in the presence of the surfactants: Tween 80, SDS (sodium dodecylsulfate) and Triton X-100, at a final concentration of 0.01% (w/v). Relative lipase activity was measured under standard assay conditions.
Stability of lipase in organic solvents was evaluated by the protocol described by Sztajer et al. , but in this case, the CLE was incubated at 30[degrees]C. Several organic solvents were tested. They included: glycerol (50%, v/v); ethanol and methanol (each at 1, 2, 5, 10, 25, 50 and 99.3%, v/v) as stability in these solvents is desirable for transesterification in biodiesel production from plant seed-oils. Residual lipase activity was assayed against p-NPP as described above.
The apparent Michaelis-Menten constant ([K.sub.m]) and maximum velocity ([V.sub.max]) of the crude enzyme were calculated by Michaelis-Menten method using GraphPad Prism v.5.03 for Windows, GraphPad Software, Inc. (www.graphpad.com). Enzyme assay was carried out at optimal conditions at concentrations of esters of p-nitrophenols that ranged from 0.1 to 12 mM and at constant enzyme concentration (100 [micro]L).
3. RESULTS AND DISCUSSION
Influence of amino acids on lipase production by Botryosphaeria ribis EC-01 in distilled water with soybean meal as substrate
Soybean meal is a rich source of proteins and amino acids, especially arginine, leucine and lysine , and few studies [10, 24, 25] have described the use of this agro-industrial waste residue as fermentative substrates for lipase production by SmF. Most studies have used rich nutrient media with addition of vegetable oils (soybean, olive), sources of sulfates and phosphates, peptone, glucose, yeast extract and corn steep liquor. Although soybean is rich in some amino acids, it does not have all those present that are found in the catalytic triad of lipases, particularly serine, histidine, glutamic or aspartic acid . Our aim was therefore to evaluate the addition of each of the above amino acids in the concentration range (0.01 to 0.5% w/v), using only distilled water and soybean meal (1%, w/v), in order to evaluate the effect of the respective amino acids on lipase production. Glycine was also included in this study considering that this amino acid is part of the lipase composition, although it does not belong to the catalytic triad. Lipase production was determined in U [mL.sup.-1], and U [g.sup.-1] of dry substrate (Figure 1a and 1b). The comparison of all amino acids evaluated at 0.01 % (w/v) concentration, revealed that glutamic acid stimulated lipase production by 60% (31 [+ or -] 3.8 U [mL.sup.-1]) in relation to the control (19 [+ or -] 2.0 U [mL.sup.-1]). Glycine (0.05%, w/v) increased enzyme production by 31% (25 [+ or -] 0.3 U [mL.sup.-1]). At both concentrations all the other amino acids showed no significant difference when compared to the control. Kathiravan et al.  evaluated the effect of various amino acids on lipase production by the bacterium, Pseudomonas aeruginosa, by SmF and observed that glutamic acid and glycine at 0.2% concentration in a minimal medium, without soybean meal, increased lipase production by 240 and 200%, respectively. Makhzoum et al.  tested amino acids at 5 mM as sole nitrogen source in minimal medium for P. fluorescens 2D, and glutamic acid and glycine were the amino acids that showed a moderate stimulatory effect on lipase production.
Glutamic acid at concentrations of 0.01 and 0.1% was the only amino acid that showed a significant increased difference (p <0.05) on lipase production by B. ribis EC-01 in U [g.sup.-1] of dry substrate (Figure 1b) in relation to the control (1,690 [+ or -] 181 U [g.sup.-1] of dry substrate). Glutamic acid at 0.01% concentration promoted a 60% increase in lipase activity (2,684 [+ or -] 336 U [g.sup.-1] of dry substrate), while at 0.1% the increase was 80% (3,039 [+ or -] 771 U [g.sup.-1] of dry substrate) compared to the control.
Physicochemical parameters of Botryosphaeria ribis EC-01 lipase
Botryosphaeria ribis EC-01 was grown on 1% (w/v) soybean cake with only distilled water, and the supernatant used as the source of lipases to determine the properties listed below.
Effects of pH and temperature
Lipases have promising commercial applications: in detergent formulations, production of oleochemicals, dairy and food products, pitch removal from paper, use in cosmetics and pharmaceuticals . Every application requires unique properties in specificity, stability, temperature and pH of the lipase preparation.
The B. ribis EC-01 lipase was shown to be active in the range of the pH evaluated (3-10), and highest activity was detected at pH 8 in phosphate buffer (data not shown), which is in accordance to other fungal lipases [28, 29] that have generally been described to vary the between 6 to 9 [30, 31].
Table 1 presents the results on lipase stability of B. ribis EC-01 in the range of pH (3-10) after 1 h incubation at 30[degrees]C. The enzyme was stable at all of the pH values evaluated, and retained from 61 to 93% ofits original enzyme activity.
Fungal lipases exhibit optimum temperature usually between 25-30[degrees]C , except for thermophilic fungi that exhibit higher optima; 45-70[degrees]C [32, 33]. B. ribis EC-01 lipase demonstrated optimum temperature at 55[degrees]C as has been observed for some bacterial [34, 35] and other fungal lipases . Thermal stability (at least 65%) was observed with B. ribis EC-01 lipase at temperatures ranging from 30 to 55[degrees]C after 96 h incubation. At 48 h, the activity remained 74-90% at these temperatures. At 60[degrees]C, B. ribis EC-01 lipase retained 65% of its initial activity after 30 min of incubation. During 10 min, this lipase was stable at 70[degrees]C (data not shown). Similar results were reported for the lipase of Aspergillus carneus, which was stable for 5 min at this temperature .
Effect of surfactants, metal cations and organic solvents
The surfactants tested did not exert a negative effect on B. ribis EC-01 lipase activity. Tween 80 and SDS increased lipase activity by 23% (34.7 [+ or -] 1.41; 34.9 [+ or -] 0.89 U [mL.sup.-1], respectively) compared to the control (28.3 [+ or -] 0.38 U [mL.sup.-1]). Triton X-100 did not affect lipase activity (28.9 [+ or -] 0.75 U [mL.sup.-1]). Similar results for Triton X-100 and Tween 80 were reported by Liu et al. . In contrast, Triton X-100, Tween 80 and SDS reduced Galactomyces geotrichum Y05 lipase activity by 33, 5 and 5%, respectively .
The effects of some metal salts on the activity of B. ribis EC-01 lipase are listed in Table 2. The cations [Mn.sup.2+], [Mg.sup.2+] and [Ba.sup.2+] were the most significant activators (p < 0.05), with a difference to the control of 14.10 to 33.54% increasing lipase activity in almost all of the concentrations evaluated. The lipase activity of Rhizopus homothallicus  and Penicillium sp. DS-39  also increased in the presence of [Mn.sup.2+], while lipase activity decreased for a lipase from Rhizopus delemar . [Mg.sup.2+] ion increased the activities of lipases for Aspergillus carneus  and Mucor hiemalis f. hiemalis . [Ba.sup.2+] had no significant effect on Galactomyces geotrichum Y05 lipase , while Fusarium solani N4-2 lipase was inhibited .
[Ca.sup.2+] did not affect B. ribis EC-01 lipase (1-50 mM), and at 100 mM lipase activity decreased by 16.88% (p <0.05). Dheeman et al.  found that [Ca.sup.2+] at 1 and 10 mM increased Penicillium sp. DS-39 lipase activity. [Na.sup.+] at 1 mM increased significantly the activity of B. ribis EC-01 lipase (13.88% (p <0.05) compared to the control, but showed no activity increases at the other concentrations examined. Na+ also was reported to have minimal effect on Fusarium solani N4-2 lipase , but enhanced Rhizopus delemar lipase activity . Other cations [K.sup.+], [Zn.sup.2+] and [Ag.sup.+] did not affect significantly (p < 0.05) B. ribis EC-01 lipase. K+ and Na+ had little influence on Galactomyces geotrichum Y05 lipase activity, but [Zn.sup.2+] partially inhibited and Ag+ totally inhibited at 1 mM . B. ribis EC-01 lipase was partially inhibited (p < 0.05) by [Co.sup.2+] only at concentrations over 50 mM (37.61 to 47.65% of significant difference to control). [Cu.sup.2+,] [Hg.sup.2+] and [Fe.sup.3+] inhibited at concentrations greater than 5 mM (13.68 to 66.45% inhibition). [Cu.sup.2+], [Hg.sup.2+] and [Fe.sup.3+] also inhibited the activity of Rhizopus oryzae lipase at 5 mM . [Hg.sup.2+] partially inhibited lipase activity from Penicillium sp. DS-39 , and Galactomyces geotrichum Y05 totally  at 1 mM concentration. Abbas et al.  found that [Co.sup.2+] at 1 mM enhanced the activity of Mucor sp. Lipase, and [Fe.sup.3+] totally inhibited at the same concentration.
The activity of B. ribis EC-01 lipase was evaluated in the presence of water-miscible solvents such as acetone, butanol, isopropanol, methanol and ethanol at concentration up to 99.3% (v/v), and was incubated for 1 h at 30[degrees]C (Table 3).
The relative residual activity of the lipase ranged from 18.7 to 68% (v/v), and the enzyme remained stable in the presence of 25% (v/v) methanol and 10% (v/v) ethanol, retaining more than 82% of its initial activity. The lipase from Rhizopus oryzae in presence of 30% (v/v) of methanol and ethanol exhibited, respectively, 2 and 33% of its initial activity (1 h at 25[degrees]C) . Saxena et al.  reported that Aspergillus carneus lipase retained of 59.16 and 55.27% of its original activity in 40% methanol and ethanol, respectively, after 30 min incubation. B. ribis EC-01 lipase similarly retained 61.5% and 47.6% activity in methanol and ethanol at 50% concentration, respectively, after 1 h incubation. This lipase was also stable in the presence of 50% (v/v) glycerol, retaining its total activity. These results demonstrate the applicability of B. ribis EC-01 lipase in biodiesel production. B. ribis EC-01 lipase exhibited low stability in 99.3% (v/v) immiscible solvents, while about 25.5% of its initial activity was retained in hexane and 1.1% in toluene.
Substrate specificity--apparent [K.sub.m] and [V.sub.max]
B. ribis EC-01 lipase hydrolyzed all tested substrates at different degrees (see Table 4). Lipase activity was lower for p-nitrophenyl acetate (0.08 [+ or -] 0.04 U [mL.sup.-1]) and increased as the fatty acid chain length increased, such as p-nitrophenyl palmitate (35.53 [+ or -] 0.57 U [mL.sup.-1]). The relative lipase activities (%) on the substrates C2, C4, C6, C10, C16 and C18 were 0.22 [+ or -] 0.10, 13.6 [+ or -] 0.99, 0.46 [+ or -] 0.09, 12.57 [+ or -] 1.31, 100.0 [+ or -] 1.59 and 68.29 [+ or -] 1.72, respectively. Similar results were observed with lipases produced by Streptomyces sp., CS326 , and Pseudomonas aeruginosa PseA , both of which preferred longer carbon chain fatty acid substrates, such as pnitrophenyl palmitate. Table 4 also presents the [K.sub.m] (apparent) and [V.sub.max] of crude B. ribis EC-01 lipase. Low values of Km represent a high affinity for a specific substrate. The [K.sub.m] values for enzymes vary widely, but for most industrial enzymes used, they range from [10.sup.-1] to [10.sup.-5] M . Considering that the lipase from B. ribis EC-01 was produced on a solid substrate by SmF, the future goal will be to immobilize this enzyme and apply the immobilized lipase in transesterification for biodiesel production.
The affinity of B. ribis EC-01 lipase was evaluated on p-nitrophenyl esters of varying chain length (Table 4), and showed that this lipase exhibited a high affinity for p-NPP with a [K.sub.m(app)] of 0.372 [+ or -] 0. 08 mM and a [V.sub.max(app)] of 107 [+ or -] 4.09 [micro]mol [min.sup.-1] [mg.sup.-1] 1, and is considered a true lipase . For the same substrate, Streptomyces sp. CS326  and Pseudomonas aeruginosa PseA  lipases exhibited [K.sub.m] and [V.sub.m], respectively, of 0.24 mM and 4.6 mM [min.sup.-1] m[g.sup.-1] and 71.4 mM and 2.24 [micro]mol [min.sup.-1] [mg.sup.-1]. A Fusarium solani lipase showed a [K.sub.m] and [V.sub.max] of 1.8 mM and 1.7 [micro]mol [min.sup.-1] [mg.sup.-1], respectively for p-NPP .
This work demonstrated that adding 0.01% (w/v) glutamic acid to distilled water containing soybean meal (1%, w/v) effectively increased lipase production by 60 and 80% in terms of U [mL.sup.-1] and U [g.sup.-1] of dry substrate, respectively. Glycine also stimulated lipase production on this simple nutrient medium increasing enzyme production by 30% in comparison to the control in U [mL.sup.-1]. The lipase produced showed good thermal stability, as well as stability in the presence of detergents, salts and solvents, and displayed high affinity for long-chain fatty acyl esters. These properties make B. ribis EC01 lipase attractive for use in biodiesel production.
The authors acknowledge Fundacao Araucaria do Parana for financial support. M. M. Andrade is grateful to CAPES-Brazil for the award of a scholarship.
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Milena M. Andrade (a), Aneli M. Barbosa (a), *, Robert F. H. Dekker (a), Maria I. Rezende (b), Josana M. Messias (b), # and Luiz H. Dall'antonia (a)
(a) Departamento de Quimica, CCE, Universidade Estadual de Londrina, CEP: 86051-990, Londrina--PR, Brazil
(b) Departamento de Bioquimica e Biotecnologia, CCE, Universidade Estadual de Londrina, CEP: 86051-990, Londrina--PR, Brazil
# Present address: Departamento de Bioquimica e Imunologia, FMRP--Universidade de Sao Paulo, CEP: 14049900, Ribeirao Preto--SP, Brazil
Article history: Received: 23 March 2014; revised: 13 July 2014; accepted: 11 August 2014. Available online: 01 September 2014.
* Corresponding author. E-mail: anelibarbosa@gmail. com
Table 1. Botryosphaeria ribis EC-01 lipase stability in different buffers. Buffers (0.05 M) pH Relative residual activity (%) (a) Control -- 100 [+ or -] 1.35 citrate-phosphate 3 72.4 [+ or -] 2.69 citrate-phosphate 4 66.7 [+ or -] 1.35 citrate-phosphate 5 75.2 [+ or -] 0.67 citrate-phosphate 6 63.2 [+ or -] 3.20 citrate-phosphate 7 61.4 [+ or -] 3.70 sodium phosphate 6 88.0 [+ or -] 0.84 sodium phosphate 7 65.7 [+ or -] 0.67 sodium phosphate 8 79.2 [+ or -] 2.19 boric acid-borax 8 62.6 [+ or -] 4.38 boric acid-borax 9 79.2 [+ or -] 1.85 Tris-HCl 8 93.0 [+ or -] 1.85 Tris-HCl 9 73.0 [+ or -] 2.53 glycine-NaOH 9 76.8 [+ or -] 3.20 glycine-NaOH 10 88.8 [+ or -] 5.39 (a) 1 h at 30[degrees]C. p-NPP was used as substrate for lipase activity determination Table 2. Influence of metal cations on lipase activity from Botryosphaeria ribis EC-01 (1). Metal cations Relative lipase activity (%) (2) 1mM 5mM Control 100 [+ or -] 3.0 (a) [Mn.sup.2+] 123 [+ or -] 3.0 (bA) 124 [+ or -] 0.6 (bA) (Mn[Cl.sub.2]) [Mg.sup.2+] 117 [+ or -] 3.4 (bA) 114 [+ or -] 0.9 (bA) (Mg[Cl.sub.2]) [Ca.sup.2+] 107 [+ or -] 3.3 (aA) 112 [+ or -] 4.2 (aA) (Ca[Cl.sub.2]) [K.sup.+] (KCl) 99.8 [+ or -] 1.5 (aAB) 106 [+ or -] 3.3 (aA) [Zn.sup.2+] 99.4 [+ or -] 0.9 (aA) 102 [+ or -] 3.3 (aA) (Zn[Cl.sub.2]) [Na.sup.+](NaCl) 114 [+ or -] 1.51 (A) 103 [+ or -] 4.5 (aAB) [Ba.sup.2+] 95.5 [+ or -] 4.5 (aB) 124 [+ or -] 2.1 (bA) (Ba[Cl.sub.2]) [Ag.sup.+](AgNO3) 97.6 [+ or -] 1.5 (aA) 97.4 [+ or -] 4.8 (aA) [Co.sup.2+] 98.3 [+ or -] 3.0 (aAC) 98.5 [+ or -] 3.3 (aAC) (Co[Cl.sub.2]) [Cu.sup.2+] 95.5 [+ or -] 2.7 (aA) 81.6 [+ or -] 3.6 (bB) (CuS[O,.sub.4]) [Hg.sup.2+] 93.2 [+ or -] 3.6 (aA) 86.3 [+ or -] 0.0 (bAB) (Hg[Cl.sub.2]) [Fe.sup.3+] 96.4 [+ or -] 3.3 (aA) 72.6 [+ or -] 3.6 (bB) Metal cations Relative lipase activity (%) (2) 10 mM 25 mM Control 100 [+ or -] 3.0 (a) [Mn.sup.2+] 120 [+ or -] 3.9 (bA) 117 [+ or -] 4.2 (bA) (Mn[Cl.sub.2]) [Mg.sup.2+] 111 [+ or -] 5.4 (aA) 116 [+ or -] 3.6 (bA) (Mg[Cl.sub.2]) [Ca.sup.2+] 101 [+ or -] 3.3 (aA) 103 [+ or -] 4.2 (aA) (Ca[Cl.sub.2]) [K.sup.+] (KCl) 107 [+ or -] 3.9 (aA) 105 [+ or -] 3.9 (aAB) [Zn.sup.2+] 98.3 [+ or -] 4.2 (aA) 93.6 [+ or -] 1.2 (aA) (Zn[Cl.sub.2]) [Na.sup.+](NaCl) 102 [+ or -] 3.6 (aAB) 104 [+ or -] 3.9 (aAB) [Ba.sup.2+] 134 [+ or -] 2.1 (bA) 114 [+ or -] 1.2 (bAC) (Ba[Cl.sub.2]) [Ag.sup.+](AgNO3) 96.6 [+ or -] 3.9 (aA) 101 [+ or -] 4.2 (aA) [Co.sup.2+] 105 [+ or -] 3.9 (aA) 91.5 [+ or -] 2.4 (aC) (Co[Cl.sub.2]) [Cu.sup.2+] 75.4 [+ or -] 0.9 (bB) 40.2 [+ or -]4.8 (bC) (CuS[O,.sub.4]) [Hg.sup.2+] 72.9 [+ or -] 4.5 (bBC) 69.4 [+ or -]3.9 (bC) (Hg[Cl.sub.2]) [Fe.sup.3+] 71.6 [+ or -] 5.1 (bB) 81.4 [+ or -] 0.3 (bAB) Metal cations Relative lipase activity (%) (2) 50 mM 100 mM Control 100 [+ or -] 3.0 (a) [Mn.sup.2+] 101 [+ or -] 1.8 (aB) 99.6 [+ or -] 0.6 (aB) (Mn[Cl.sub.2]) [Mg.sup.2+] 107 [+ or -] 0.9 (aA) 105 [+ or -] 1.8 (aA) (Mg[Cl.sub.2]) [Ca.sup.2+] 94.9 [+ or -] 0.6 (aAB) 83.1 [+ or -] 6.3 (bB) (Ca[Cl.sub.2]) [K.sup.+] (KCl) 106 [+ or -] 4.8 (aAB) 92.5 [+ or -] 0.9 (aB) [Zn.sup.2+] 96.4 [+ or -] 5.1 (aA) 92.5 [+ or -] 2.7 (Aa) (Zn[Cl.sub.2]) [Na.sup.+](NaCl) 98.1 [+ or -] 4.5 (aB) 98.1 [+ or -] 3.9 (aB) [Ba.sup.2+] 110 [+ or -] 4.8 (aC) 97.6 [+ or -] 0.3 (aB) (Ba[Cl.sub.2]) [Ag.sup.+](AgNO3) 95.3 [+ or -]3.0 (aA) 98.1 [+ or -] 0.9 (aA) [Co.sup.2+] 62.4 [+ or -] 0.6 (bB) 52.4 [+ or -] 2.7 (bB) (Co[Cl.sub.2]) [Cu.sup.2+] 36.8 [+ or -] 3.3 (bC) 33.5 [+ or -] 0.3 (bC) (CuS[O,.sub.4]) [Hg.sup.2+] 65.2 [+ or -] 3.9 (bC) 65.2 [+ or -] 5.7 (bC) (Hg[Cl.sub.2]) [Fe.sup.3+] 79.1 [+ or -]3.6 (bB) 57.7 [+ or -] 5.4 (bBC) (Fe[Cl.sub.3]) (1) Values with different lowercase letters in the column indicate significant difference (p < 0.05) compared with the control by the Tukey test. Values with different uppercase letters in the row indicate significant difference (p < 0.05) between cation concentrations by the Tukey test. (2) 1 h at 30[degrees]C. p-NPP was used to assay lipase activity Table 3. Influence of different solvents on the activity of the crude lipase preparation from Botryosphaeria ribis EC-01. Solvent (% v/v) Relative residual activity (%) (a) Control 100 [- or +] 5.26 Propanone (99.3) 31.1 [- or +] 1.23 n- Butanol (99.3) 45.9 [- or +] 0.35 Isopropyl alcohol (99.3) 68.0 [- or +] 7.02 Hexane (b) (99.3) 25.5 [- or +] 2.31 Toluene (c) (99.3) 1.1 [- or +] 0.00 Glycerol (50.0) 104 [- or +] 8.95 Methanol (1.00) 110 [- or +] 1.58 (2.00) 110 [- or +] 0.35 (5.00) 104 [- or +] 6.84 (10.0) 95.0 [- or +] 2.11 (25.0) 96.0 [- or +] 0.70 (50.0) 61.5 [- or +] 7.02 (99.3) 18.7 [- or +] 0.88 Ethanol (1.00) 107 [- or +] 1.05 (2.00) 102 [- or +] 0.00 (5.00) 86.7 [- or +] 5.44 (10.0) 82.3 [- or +] 4.04 (25.0) 32.4 [- or +] 0.18 (50.0) 47.6 [- or +] 2.81 (99.3) 37.2 [- or +] 2.46 (a) 1 h at 30[degrees]C. p-NPP was used to assay lipase activity. (b) Complete solvent evaporation at room temperature for 4.5 h. (c) Complete solvent evaporation at room temperature for 30 h. Table 4. Substrate specificity, [K.sub.m] (apparent) and [V.sub.max] of the crude lipase produced by Botryosphaeria ribis EC-01 grown on soybean meal. Substrates (a) Lipase activity (U [mL.sup.-1]) (b) p-nitrophenyl acetate (2:0) (d) 0.08 [+ or -] 0.04 p-nitrophenyl butyrate (4:0) 4.48 [+ or -] 0.35 p-nitrophenyl caproate (6:0) 0.18 [+ or -] 0.02 p-nitrophenyl caprate (10:0) 4.47 [+ or -] 0.47 p-nitrophenyl palmitate 16:0) 35.5 [+ or -] 0.57 p-nitrophenyl stearate (18:0) 24.3 [+ or -] 0.61 Substrates (a) [K.sub.m(app)] (mM) (c) p-nitrophenyl acetate (2:0) (d) 3.20 [+ or -] 0.78 p-nitrophenyl butyrate (4:0) 0.972 [+ or -] 0.11 p-nitrophenyl caproate (6:0) 0.943 [+ or -] 0.35 p-nitrophenyl caprate (10:0) 0.819 [+ or -] 0.22 p-nitrophenyl palmitate 16:0) 0.372 [+ or -] 0.08 p-nitrophenyl stearate (18:0) 0.512 [+ or -] 0.18 Substrates (a) [V.sub.max(app)] ([micro]mol [min.sup.-1] [mg.sup.-1] protein) p-nitrophenyl acetate (2:0) (d) 15.4 [+ or -] 1.34 p-nitrophenyl butyrate (4:0) 23.2 [+ or -] 0.59 p-nitrophenyl caproate (6:0) 0.640 [+ or -] 0.05 p-nitrophenyl caprate (10:0) 30.9 [+ or -] 1.90 p-nitrophenyl palmitate 16:0) 107 [+ or -] 4.09 p-nitrophenyl stearate (18:0) 97.9 [+ or -] 6.88 (a) p-nitrophenyl esters were used as substrates. (b) p-nitrophenyl esters used as substrates at 8 mM. (c) p-nitrophenyl esters varied from 0.1 to 12 mM. (d) Carbon chain length in parentheses.
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|Title Annotation:||Full Paper|
|Author:||Andrade, Milena M.; Barbosa, Aneli M.; Dekker, Robert F.H.; Rezende, Maria I.; Messias, Josana M.; D|
|Publication:||Orbital: The Electronic Journal of Chemistry|
|Date:||Jul 1, 2014|
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