Novel lipase from basidiomycetes Schizophyllum commune ISTL04, produced by solid state fermentation of Leucaena leucocephala seeds

Journal of Molecular Catalysis B: Enzymatic 110 (2014) 92–99

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Novel lipase from basidiomycetes Schizophyllum commune ISTL04, produced by solid state fermentation of Leucaena leucocephala seeds Manoj Kumar Singh, Jyoti Singh, Madan Kumar, Indu Shekhar Thakur ∗ School of Environmental Sciences, Jawaharlal Nehru University, New Delhi, 110067, India

a r t i c l e

i n f o

Article history: Received 27 July 2014 Received in revised form 18 September 2014 Accepted 18 October 2014 Available online 24 October 2014 Keywords: Lipase Schizophyllum commune Basidiomycetes Leucaena leucocephala SSF

a b s t r a c t The present investigation reports a novel thermoalkalotolerant lipase producing Basidiomycetes, Schizophyllum commune ISTL04 for the first time. Abundantly available Leucaena leucocephala seeds were exploited as a cheap fermentable source for the feasible production of extracellular lipase. The isolate ISTL04 grew vigorously on Leucaena seeds in comparison to Soybean meal and Wheat bran exhibiting a lipase activity of 146.5 U/g. The S. commune ISTL04 (SCI) lipase was purified to 35.76 folds with a specific activity of 238.13 U/mg and an estimated mass of 60 kDa. The temperature and pH optima for lipolytic activity were 60 ◦ C and 11, respectively. The enzyme exhibited remarkable stability in the presence of non-polar and polar organic solvents (50%, v/v), retaining more than 90% and 40% activity, respectively. The lipase activity increased significantly in the presence of Mg2+ (154%), Ca2+ (109.7%), Mn2+ (106%) and Tween 80 (240%). The distinctive and broader operational properties of the SCI lipase make it a promising biocatalyst for various industrial processes. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Lipases (triacylglycerol hydrolases E.C.3.1.1.3) are among the major industrial enzymes that find their huge application in food, dairy, detergent, leather, biofuel and pharmaceutical industries which in turn have surged the production of lipases [1–3]. Though lipases are ubiquitous in nature and are found in all forms of life, fungi and bacteria are mainly exploited as sources of commercial lipases [4]. Fungal lipases are usually produced extracellularly and widely used in industrial applications, especially in the food industry. Major commercially important lipase producing fungi are: Rhizopus arrhizus, Rhizopus japonicus, Rhizopus niveus, Mucor miehei, Candida rugosa, Aspergillus niger and Aspergillus terrues [5]. Basidiomycetes fungi have been well documented for several enzymes and proteins that are involved in the degradation of lignocellulosic biomass and other aromatic compounds responsible for environmental problems, however this group of edible mushrooms have got a little scientific attention till date for the possibility of finding potential lipolytic enzymes, except a few edible basidiomycetes including Agaricus bisporus [6] and Lentinus edodes [7] and Antrodia cinnamomea [8].

∗ Corresponding author. Tel.: +91 11 26704321 10/26191370 (R); fax: +011 26717586. E-mail addresses: [email protected], [email protected] (I.S. Thakur). http://dx.doi.org/10.1016/j.molcatb.2014.10.010 1381-1177/© 2014 Elsevier B.V. All rights reserved.

Schizophyllum commune is one of the most widespread mushroom-forming fungi, occurring on fallen branches and timber of deciduous trees. In Asia and Africa, the mushrooms of S. commune are consumed as a food source [9]. S. commune is well illustrated for the production of lignocellulose degrading enzymes, whereas the knowledge about the lipolytic enzymes from this mushroom is abridged and, thus, their biotechnological potential unrealized [10,11]. Recently, sequence of a putative uncharacterized protein from S. commune strain H4-8/FGSC 9210 has been submitted to protein database as a hydrolase having triacylglyceride lipase activity (http://www.uniprot.org/uniprot/D8PRE5; http://evexdb.org/gene-family/ensembl/1636/). Regardless of the great interests in the application of lipases in various industries, the use of lipases as biocatalysts is often restricted by multifaceted concerns over the economic viability of lipase production systems. Not only that, properties like high thermal stability, wide pH tolerance, solvent tolerance, metal tolerance are still among the many bottlenecks for using lipases in industries [12]. Reducing production costs at large scale industrial level by employing different microorganisms, new supplements, substrates, culture conditions, in the best possible permutations and combinations, discovering new strains for the production of novel versatile enzymes with industrially useful properties has altogether emerged as a new interest area of lipase research. One of the ways to substantially reduce the production cost is to use wastes/residues from agro-industry as culture media and optimization of production conditions [13]. In this regard, Solid state fermentation (SSF) is gaining colossal

M.K. Singh et al. / Journal of Molecular Catalysis B: Enzymatic 110 (2014) 92–99

attention for enzyme production as enzyme titers are higher than in submerged fermentation (SmF), when comparing the same strain and fermentation broth, owing to; higher production of biomass, catabolite repression resistance and decreased enzyme breakdown by proteases [14]. In the search for a more practical, appropriate and economical medium for the biotechnological production of lipase, we investigated the possibility of utilizing Leucaena leucocephala seeds as an appropriate medium. Native to Central America and Mexico, L. leucocephala is a multi-utility legume, which is now found growing naturally in most tropical areas of the world. This legume tree has found a prime role in agro-forestry system owing to its ability to fix nitrogen thus facilitating the growth of other plants. Not only that, it helps in preventing soil erosion and can easily grow on marginal lands with high biomass production [15]. L. leucocephala is has been used widely as firewood, timber, fodder, green manure, bioethanol production, paper production etc. [16]. Nonetheless, L. leucocephala has numerous precocious desirable growth traits which prospect its bright role as feedstock or substrate for enzyme production. It flowers and fruits all round a year, produces seed in galore, is selffertile, has hard coated seeds, re-germinates after fire or cutting, drought tolerant and is able to flourish on highly alkaline soils. It has been reported that L. leucocephala can attain a seed productivity of about 3–5 tonnes seeds ha−1 year−1 [17]. L. leucocephala seeds are rich in lipids, crude proteins, carbohydrates along with important mineral nutrients including N, P, K, Ca, Mg, Mn, Fe, Cu and Zn [18]. Use of L. leucocephala seeds as substrate for SSF for this study thus appeared logical and worth exploring. Although there are numerous reports on various agro-wastes/food residues being used as suitable substrates for SSF (Table 1), there is no report on SSF of L. leucocephala seeds so far to the best of the knowledge of the authors. In the present study, a screening procedure was followed that identified S. commune, an edible fungus for the first time as a potent lipase producer grown on cheaply available seeds of L. leucocephala as solid substrate, under solid state fermentation conditions. The partially purified lipase was thermoalkalotolerant and exhibited remarkable stability in the presence of non-polar, polar organic solvents, metal ions and detergents. The distinctive and broader operational properties of the SCI lipase make it a promising biocatalyst for various industrial processes.

2. Methods 2.1. Materials Tributyrin oil and bovine serum albumin (protein standard) were purchased from HiMedia, India. para-Nitrophenyl Palmitate (pNPP) was purchased from Sigma–Aldrich (St. Louis, MO, USA; isopropanol Merck (Darmstadt, Germany). Protein molecular weight marker (14.4–116.0 kDa) was procured Fermentas Life Science (India). All other solvents and chemicals used during the experiment were of analytical grade. Mature L. leucocephala pods containing seeds were collected in February 2013 from forest area of Jawaharlal Nehru University (New Delhi, India) located at latitude: 28◦ 32 50.496 N; longitude: 71◦ 14 1.68 E; altitude: 823 m.

2.2. Fungal sampling and isolation Mushroom forming fungal species found fruiting on dead wood logs were collected from forest areas of Jawaharlal Nehru University campus (New Delhi, India) during monsoon period. Samples were cultured and purified as described by Hernández-Luna, 2008 [19].

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2.3. Screening of lipase producing fungi Six fungal strains (F1, F2, F3, F4, F5 and F6) were isolated and screened for lipase production by preparing phenol red chromogenic substrate plates [20]. The agar plates were inoculated with 0.7 cm diameter agar plugs colonised by the fungal isolates and the plates were incubated at 30 ◦ C for 3–5 days. Production of extracellular lipase and lipase activity was further confirmed by p-NPP spectrophotometric method [20]. Fungal isolates were cultured in submerged fermentation conditions using Vogel Minimum Salts Medium (VMSM) [21] and tributyrin (1%, v/v) as an inducer at pH 5.8–6.0 in 150 ml Erlenmeyer flasks. Each flask was inoculated with four mycelium agar plugs and left for fermentation for 5 days in orbital shaker (180 rpm) at 30 ◦ C. Samples were collected after 5 days and centrifuged at 10,000 rpm for 10 min. The filtrate was used as crude enzymatic extract. The isolate which exhibited largest clear yellow zone and maximum lipolytic activity was further identified using 18S rDNA sequencing. 2.4. Molecular identification of the fungal isolate Molecular characterization was done via 18S rDNA sequencing. PCR amplification of the ITS1-5.8 S-ITS2 rDNA region of the fungus was carried out using primer set (5 -TCCGTAGGTGAACCTGCCG-3 ) and pITS4 (5 pITS1  TCCTCCGCTTATTGATATGC-3 ). Sequencing was carried out by SCIGENOM (INDIA). 18S rDNA sequences of the isolate were subjected to similarity search analysis via GenBanK BLAST function at NCBI electronic site (http://www.ncbi.nlm.nih.gov/). Phylogenetic tree was constructed using software MEGA4. 2.5. Inoculums preparation for solid state fermentation Kirk’s basal nutrient medium (100 ml) was used for the preparation of inoculums [22]. The initial pH of inoculation media was adjusted at pH 4.5 with 1 M NaOH or 1 M HCL. The inoculums flask was sterilized and supplemented with syringe filtered (0.22 ␮m) glucose (1%). Spores of S. commune ISTL04 were transferred aseptically from the PDA slant under sterile conditions and the flask was incubated (120 rpm) at 30 ◦ C for 5-days to get homogenous inoculums (1 × 106 –108 spores/ml). 2.6. Substrate preparation and determination of nutritive composition L. leucocephala, seeds were removed from the mature pods, damaged seeds were discarded, and the remaining seeds were oven-dried at 60 ◦ C for 24 h. The dried seeds were crushed in a grinder. 10 g of well milled L. leucocephala seeds was taken into two sets of 250 ml Erlenmeyer flasks, one set moistened with 40 ml of distilled water (1:4, w/v) and other with VMSM (1:4, w/v) and tributyrin (1%, w/v). The contents of the flask were mixed and autoclaved. More commonly studied, solid substrates Soybean meal and Wheat bran were also taken for comparative study. The lipid, carbohydrate, protein, fibre and ash content of the seeds were determined by the methods described by Ref. [18]. 2.7. Lipase production through solid-state fermentation Substrate prepared as mentioned above was allowed to cool at room temperature and inoculated with 3 ml of the homogenous inoculums of S. commune ISTL04. The inoculated flasks were incubated at 30 ◦ C for 5 days in a humidity (65%) controlled BOD incubator. For extraction of lipase the solid fermented medium was mixed with sodium phosphate buffer (50 mM, pH 8.0) in a ratio of 1:10 (w/v). The mixture was shaken at 180 rpm, for 1 h at 30 ◦ C.

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Table 1 Lipase production by various fungi in SSF of different agro-industrial wastes. Fungus

Solid substrate used

Lipase yield

Supplement

Reference

Aspergillus niger

Wheat bran

33.03 U/g

[56]

Gingelly oil cake

630 U/g 363.6 U/g

Glucose (4.8%), yeast extract (4%), and NaH2 PO4 (4%) Olive oil No added supplement

Penicillium simplicissimum

Castor bean waste

44.8 U/g

[12]

P. brevicompactum

Caster meal

87.7 U/g

Sugar cane molasses (6.25%, w/w) Soybean oil and sugarcane molasses

P. brevicompactum

Babassu cake

48.6 U/g

[12]

30.3 U/g

Soybean oil and sugarcane molasses 2% olive oil

Penicillium sp. Rhizopus rhizopodiformis

Soybean meal Olive cake and sugarcane bagasse wheat flour with wheat bran (3/2, w/w)

200 U/g 79.6 U/g

3 % of urea No added supplement

[57] [58]

24.44 U/g

[58]

R. homothallicus IRD-13a

Sugarcane bagasse

826 U/g

R. oligosporous GCBR-3

Almond meal

48.0 U/g

Peptone (2%, w/w), additional nitrogen source and olive oil (2%, v/w) Urea, olive oil and oligo-elements No added supplement

Yarrowia lipolytica

Niger seed oil cake Mustard seed cake

26.42 U/g 57.89 U/g

Urea and glucose Urea 1.5% w/w, glucose 7% w/w

[12] [60]

Rhizomucor pusillus

Olive cake and sugarcane bagasse L. leucocephala seeds

20.24 U/g

No added supplement

[30]

146.67 U/g

No added supplement

This study

R. chinensis

Schizophyllum commune ISTL04

The suspension was then centrifuged at 12,000 × g for 15 min at 4 ◦ C and the supernatant was used as crude lipase extract.

2.8. Partial purification of lipases produced in SSF Firstly, 1 mM of Phenylmethylsulfonyl fluoride (PMSF) was added to the crude enzyme extract in order to prevent proteolytic degradation taking place through the purification procedure. Ammonium sulphate was added slowly, with continuous stirring, to the supernatant to a final concentration of 70% (w/v) saturation at 4 ◦ C. The precipitates were then harvested by centrifugation at 4 ◦ C and 12,000 × g for 30 min. The precipitate therefore obtained was dissolved in sodium phosphate buffer (pH 8) containing 1 mM PMSF and dialysed for 24 h with three changes against the same buffer. The concentrated lipase preparation was then subjected to gel permeation column chromatography by loading it onto to a SuperdexTM 200 gel filtration column (GE Healthcare Life Sciences) (2.5 cm × 10 cm) pre-equilibrated with 50 mM sodium phosphate buffer containing 0.15 M NaCl (pH 8). The proteins were eluted with the same at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were collected and assayed for both lipase activity and protein content (A280 ). All purification steps were carried out at 4 ◦ C. Fractions containing active lipase were pooled and stored at −20 ◦ C.

[1] [32]

[12]

[12]

[30] [59]

2.10. Characterization 2.10.1. Effects of temperature and pH on lipase activity and stability The optimum temperature for partially purified lipase activity was determined spectrophotometrically using p-NPP as substrate [26], over a temperature range of 4–70 ◦ C with an incubation period of 1 h at pH 8 using 50 mM Sodium Phosphate buffer. To examine the thermo-stability, the enzyme was incubated at different temperatures (30–70 ◦ C) for a period of 5 h and the activity was determined at regular time intervals. The effect of pH on lipase activity and stability was determined spectrophotometrically using the following buffers: Na2 HPO4 –citric acid for pH 3.0–8.0, Tris–HCl for pH 8.0–10.0, and glycine–NaOH for pH 10.0–12.0 with an incubation period of 1 h and 24 h, respectively, at 37 ◦ C. 2.10.2. Effects of metal ions, detergents on lipase activity Effect of different metal ions on the lipase activity was determined by incubating the enzyme with metal ions (Ca2+ , Mg2+ , Mn2+ , Co2+ , Hg2+ , Fe2+ , Zn2+ ) to a final concentration of 10 mM in Sodium Phosphate buffer (50 mM, pH 8.0) at 60 ◦ C for 1 h. Similarly, the effects of different surfactants, Triton X-100, Tween 20, Tween 80 and SDS were determined by incubating the buffered enzyme with the surfactant (1% v/v or w/v) for 1 h at 60 ◦ C. After incubation, the residual activity of the enzyme was measured using spectrophotometric p-NPP enzyme assay.

2.9. Protein determination and gel electrophoresis Protein concentration was determined by Bradford method using bovine serum albumin (BSA) as standard [23]. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using the method of Laemmli [24] on a 6% polyacrylamide stacking gel and a 12% polyacrylamide-resolving gel. Protein bands were visualized by staining with Coomassie brilliant blue R250. Activity staining (Zymogram analysis) of the unstained gel was done to determine the band corresponding to lipase, by the method described by Ref. [25].

2.10.3. Effect of organic solvents on the lipase activity and stability The effect of various polar and non polar organic solvents on the enzyme activity and stability was determined by incubating the partially purified enzyme in organic solvents (50%, v/v) at 37 ◦ C for 24 h with shaking at 200 rpm. Samples were withdrawn from aqueous phase and used for the determination of residual lipase activity by using spectrophotometric p-NPP enzyme assay. The organic solvents used were methanol, dimethyl sulphoxide (DMSO), acetone, toluene, chloroform, heptanes, hexane and acetonitrile.

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3. Results and discussion 3.1. Isolation and identification of fungus for producing lipase Six mushroom forming fungal strains were isolated and screened for the production of lipolytic enzyme by Phenol red plate chromogenic assay with tributyrin as lipidic substrate. Out of the six isolates, F1, F2, F3, F4 (ISTL04), F5, F6, four isolates showed clearance zone formation (S1). Though four isolates showed clear hydrolysis zone, isolate ISTL04 (F4) formed the largest yellow hydrolysis zone. The results were further confirmed by spectrophotometric method using p-NPP as substrate. Isolate ISTL04 exhibited the highest lipase activity. Following screening, isolate ISTL04 was identified as the potential lipase producer, was further identified by 18S rDNA sequencing. Based on 18S rDNA data, isolated strain was identified as S. commune ISTL04 and may be considered novel. BLAST results showed maximum similarity (99%) to S. commune strains (Fig. 1). The 18S rDNA sequence of ISTL04 has been submitted to GenBank and has been assigned with the Accession no. KF601697. S. commune is one of the most commonly found mushrooms which usually completes its life cycle in 10 days, and is amenable to genetic manipulations. It is a model mushroom whose whole genome has been recently sequenced for gaining new insights of the otherwise underrated producers of valuable enzymes and pharmaceutical proteins [9]. Under the same genome project, a putative uncharacterized protein has been submitted having lipolytic activity. The present study identified an indigenous S. commune strain ISTL04 capable of appreciable lipolytic activity, which could further add to the current knowledge of this model mushroom which has not got any attention till date for industrially useful enzymes other than lingo-cellulolytic enzymes. 3.2. Lipase production through solid-state fermentation of Leucaena leucocephala seeds Extracellular lipase activities obtained in all the SSF media are given in Table 2. Highest lipase activities (148.57 U/g) were obtained when isolate ISTL04 was grown on crushed L. leucocephala seeds supplemented with VMSM (1:4, w/v) and 1% tributyrin as inducer for lipolytic activity as the solid substrate, as compared to Soybean meal (95.19 U/g) and Wheat bran (70.23 U/g), supplemented with VMSM (1:4, w/v) and 1% tributyrin as inducer for lipolytic activity. When grown on substrates only provided with distilled water, the extracellular lipase activity declined remarkably in the case of Soybean meal (52.49 U/g) and Wheat bran (25.91 U/g) whereas, there was no significant effect on lipase production in L. leucocephala seeds medium. S. commune ISTL04 produced substantial quantities of extracellular lipase (146.5 U/g) with L. leucocephala seeds in presence of distilled water only in order to provide moisture. Physicochemical parameters such as pH, temperature, agitation and nutritional factors (carbon and nitrogen sources) are the significant factors influencing the lipase production by fungi. Lipases, primarily being inducible enzymes are expressed largely depending on the carbon source. It has been reported that lipases

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Table 3 Nutritive values of L. Leucocephala seeds. Observations were taken in triplicate (mean ± SD). Component

Content (%)

Crude protein content (%) Carbohydrate content (%) Lipid content (%) Crude fibre content Ash content mg/100 g

9.28 ± 0.11 42.56 ± 0.85 15.5 ± 1.25 4.53 ± 0.16 19.58 ± 2.30

production is induced by the presence of some lipidic source such as tributyrin, oils, etc. In this study, it was found that L. leucocephala seeds, in presence of distilled water only were able to support vigorous growth and induce appreciable lipase production (146.5 U/g) by the isolate even in the absence of any external inducer supplement. As determined by nutritive value analysis, the L. leucocephala seeds contained 15.5% lipids, 42.56% carbohydrates, 9.28% proteins, 4.53% crude fibre (Table 3). Whereas, Soybean meal is reported to contain, on an average 44% crude protein, 3% crude fibre and only 0.5% fat, and 12% moisture, [27] and Wheat bran on an average contains 45–70% carbohydrates, 15–18% proteins, 4–5% fats [28]. The higher extracellular lipase production in case of L. leucocephala seeds could be attributed to the fact that L. leucocephala seeds contain high amount of lipids (15.5%) along with carbohydrates, proteins and other important nutrients including minerals N, P, K, Ca, Mg, Mn, Fe, Cu and Zn, which could wholly support the growth of the microorganism, without the addition of extra nutrients other than a little moisture [18]. Various raw materials such as Olive oil cake, Babassu oil cake, Egg yolk, Almond meal, Sunflower oil, Soybean bran, Olive oil, Wheat bran and Soy cake have been exploited as fermentable sources for extracellular lipase production from fungi [29] (Table 1). Though, all such fermentable substrates are generally supplemented with nutrient medium or an inducer to induce lipase production, there are few reports on extracellular lipase production by fungus through solid state fermentation of wastes/agro-industrial residues without any supplementation. Rodriguez et al., 2006 [30] reported lipase production (20.24 U/g) from Rhizomucor pusillus and Cordova et al., 1998 [31] reported lipase production (79.6 U/g) from Rhizopus rhizopodiformis using Olive cake and sugarcane bagasse without any extra nutrient supplementation, Kamini et al., 2000 [32] reported lipase production (363.6 U/g) from Aspergillus niger using Gingelly oil cake without extra nutrient supplementation. In this study, it was found that L. leucocephala seeds alone, even in the absence of any external inducer supplement, were able to support vigorous growth and induce appreciable lipase production (146.5 U/g) by the isolate which falls within the range of most reported lipase activities (20–363 U/g), produced by solid state fermentation of agro-industrial wastes without supplementation (Table 1). Nonetheless, the year round abundant availability (3–5 tonnes seeds ha−1 year−1 ) of this cheap, nutrient rich substrate makes L. leucocephala seeds a very promising candidate to be explored as fermentable nutrient source for the growth of microorganisms for the production of enzymes and other bio-products.

Table 2 Comparison of lipase production by S. commune ISTL04 in SSF, using different substrates. Experiments were carried out in triplicate (mean ± SD). Vogel’s Minimal salts medium (VMSM) (1:4, w/v)

Inducer for lipase, tributyrin (1%, v/w)

Solid substrate (10 g)

Lipase activity (U/g)

Presence

Presence

L. leucocephala seeds Wheat bran Soybean meal

148.57 ± 2.5 70.23 ± 1.71 95.19 ± 5.53

None*

None

L. leucocephala seeds Wheat bran Soybean meal

146.5 ± 6.32 25.91 ± 3.51 52.49 ± 4.21

*

Replaced by distilled water.

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Schizophyllum commune ISTL04 (NCBI Accession no. KF601697)

75

Schizophyllum commune ISTL04

13

5 0

Schizophyllum commune strain P3-B Schizophyllum commune strain 1-19 Auricularia polytricha voucher Dai10451 Basidiomycete sp. RM4ac Schizophyllum commune strain MJ03 Agaricaceae sp. 72 63 Agaricaceae sp. 710 Schizophyllum commune isolate BDG2-1-1 11

0

16

Agaricaceae sp. 647 Schizophyllum commune isolate HN21 Pochonia suchlasporia strain NS-17 Schizophyllum commune strain P8-B 5

15

2

11 88

0

Schizophyllum commune isolate BCC22128 Schizophyllum commune isolate BCC26407 Schizophyllum commune strain NF58

Schizophyllum commune strain Sc1 Schizophyllum commune strain MX4 Trametes robiniophila strain CFCC 6839 2 Schizophyllum commune strain D 0

Basidiomycete sp. LC2 Schizophyllum commune strain DMRF-7 Rhizopus oryzae strain xsd08049 Schizophyllum commune strain xsd08036 4

0 0

0

2

0

Schizophyllum commune IFM 46097 93 Schizophyllum commune isolate HLJ 20 14 Schizophyllum commune isolate HE2740 Schizophyllum commune isolate BCC26414 0 2

Aschersonia sp. DY115-21-2-M5 Aschersonia aleyrodis strain GZZKB Schizophyllum sp. PDD 103380 6 94 Schizophyllum commune strain FBst04

2

Fig. 1. Phylogenetic tree showing the relationships among 18S rDNA sequences of isolate ISTL04 and the most similar sequences retrieved from the NCBI nucleotide database. Bootstrap consensus tree (1000 replicates) was drawn by multiple sequence alignment with neighbour joining method using software MEGA 4.

More culture optimization experiments with different nutrient supplements will definitely lead to realizing the full potential of the isolate ISTL04 and the substrate, however, the primary results are more than promising. 3.3. Partial purification of SCI lipase produced by SSF of L. leucocephala seeds

multivorans V2 lipase, 44 kDa [25], Rhizopus homothallicus lipase, 29 kDa [39], Penicillium camembertii Thom PG-3 lipase, 28.18 kDa [40], Pichia burtonii lipase, 51 kDa [41], Ophiostoma piliferum lipase 60 kDa [42]. The molecular mass of the SCI lipase was found to be 60 kDa, which falls within magnitude range of previously reported bacterial and fungal lipases. 3.4. Determination of optimum reaction conditions

The supernatant containing the extracellular lipase was subjected to enzyme purification to exclude other undesirable enzymes/proteins which may hamper/hinder the properties of lipase [33]. The supernatant was subjected to ammonium sulfate precipitation (70%), the precipitates were then concentrated by dialysis against sucrose. The concentrated sample was then loaded onto a SuperdexTM 200 column (Table 4) followed by SDS-PAGE. A 35.76 fold partial purification of SCI lipase was achieved after gel permeation chromatography with a specific enzyme activity of 238.13 U/mg of protein. The partially purified lipase fraction appeared as a single major protein band of 60 kDa after SuperdexTM 200 gel permeation chromatography on SDS-PAGE Gel (Fig. 2a). The activity staining analysis also revealed the lipolytic activity to be associated with the 60 kDa band in both ammonium sulfate precipitation proteins and the active fraction concentrate after SuperdexTM 200 gel permeation chromatography (Fig. 2b). Till date many lipases from different microorganisms have been reported falling under a wide range of molecular mass from 29 to 92 kDa including Pseudomonas sp lipases 29–92 kDa [34–38], Burkholderia

3.4.1. Effect of temperature on partially purified lipase activity and stability The purified lipase was active over a temperature range of 10–70 ◦ C, retaining more than 50% of its relative activity with a maximal activity at 60 ◦ C (Fig. 3a). However, the activity showed a sharp decline with an increase in temperature from 60 ◦ C to 70 ◦ C. The SCI lipase was found to be inactive at lower temperatures (4 ◦ C). The stability of SCI lipase at different temperatures (30–70 ◦ C) was determined at regular time intervals after incubating the enzyme for up to 5 h (Fig. 3b). SCI lipase was stable after pre-incubation at 50 ◦ C and 60 ◦ C for 5 h, retaining more than 90% of its original activity. Although, the lipase was fairly stable at 70 ◦ C, retaining more than 60% residual activity after preincubation for 1 h, the activity declined to 30% after a period of 5 h. The results imply that the SCI lipase is remarkably thermostable. Although, moderately thermostable lipases (stable at 40–50 ◦ C) have been reported from Rhizopus homothallicus, Aspergillus niger, Mucor sp., Geotrichum sp., Mucor pusillus, Aspergillus terreus, Rhizomucor sp., Aspergillus sp.,

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Fig. 2. (a) SDS-PAGE of partially purified Lipase from Schizophyllum commune ISTL04. Lane 1, Cell free culture supernatant; Lane 2, Ammonium Sulfate precipitation sample; Lane 3, SuperdexTM 200 chromatography sample. (b) Zymogram analysis of partially purified SCI lipase.

Fig. 3. (a) The lipase activity was determined at different temperatures in 50 mM Sodium phosphate buffer (pH 8.0) using pNP-palmitate as the substrate. (b) After preincubation of the lipase at 30 ◦ C, 40 ◦ C, 50 ◦ C, 60 ◦ C, 70 ◦ C for up to 5 h, the remaining activity was determined at 37 ◦ C mM in Sodium phosphate buffer (pH 8.0) using pNP-palmitate as the substrate. The lipase activity is represented as a percentage of the maximum activity. (c) The lipase activity was determined in different buffers with varying pH values at 37 ◦ C using pNP-palmitate as the substrate. To examine the stability, the lipase activity was determined after pre-incubation of the lipase in different buffers with varying pH values for 24 h. (d) Effect of different metal ions at 10 mM concentration on Schizophyllum commune ISTL04 lipase. Enzyme activity in the absence of any metal ions was considered as control (100%). Experiments were carried out in triplicate (mean ± SD).

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Table 4 Partial purification of extracellular lipase produced by S. commune ISTL04. Purification steps

Volume (ml)

Total units

Total proteins (mg)

Lipase activity (U/ml)

Protein content (mg/ml)

Specific activity (U/mg)

Fold

Recovery (%)

Supernatant Ammonium sulfate precipitation (0–70%) Gel permeation chromatography

100 4.5 2

1465 1030 535.79

220 12.32 2.25

14.65 228.89 47.58

2.20 2.74 1.13

6.66 83.60 238.13

1 12.55 35.76

100 70.31 36.57

Humicola sp., Candida sp., and Penicillium sp., reports on lipases with high stability at high temperatures and pH from fungi are scarce [29,43,44]. Thermostable biocatalysts are desirable as they allow an elevated operation temperature, hence is advantageous providing higher reaction rates, higher stability, higher yields, lower viscosity and lesser contamination problems. Thermostable lipases find huge application potential in detergent and leather industries [2]. Thus, remarkable thermostability exhibited by the SCI lipase makes it a very promising candidate to be exploited commercially. 3.4.2. Effect of pH on partially purified lipase activity and stability The S. commune ISTL04 lipase exhibited optimal lipolytic activity over a broad alkaline pH range (pH 7.0–12.0), attaining a maximal observed activity at pH 11.0 (Fig. 3c). The lipase was found active over a broad alkaline pH range (pH 7.0–11.0), retaining more than 80% of its maximum activity at pH 10.0 and enzyme activity was found to be highest at pH 11 (Fig. 3c). The SCI lipase retained 43% relative activity at pH 8 and 57% at pH 12. However, at low pH values (pH < 5.0), the SCI lipase was found to be inactive. Though the SCI lipase was active at pH 5.0–6.0, the relative activity was very low as compared to that with higher pH values. After incubation at pH values ranging from 3.0 to 12.0 for 24 h at 37 ◦ C, the partially purified SCI lipase retained more than 70% of the control activity in the alkaline pH range 8.0–12.0 (Fig. 3c). The remarkable activity and stability of the S. commune lipase at high pH values implies that it is a potential alkaline lipase. Dandavante et al., 2009 [25] reported lipase from Burkholderia multivorans V2 showing maximum activity at pH 8. Likewise, Pseudomonas sp. lipases have been reported to be fairly stable upto 14 h at 37 ◦ C over a wide range of pH from 5.6 to 9.0 and upto 3 h at 30 ◦ C over a pH range of 5.5–10.5 for P. aeruginosa san-ai [34,36]. Similarly lipase from Rhizopus sp. displayed maximum lipolytic activity at pH 7.5 [39]. Alkalotolerant lipases have also been reported in Humicola lanuginosa, Rhizopus japonicus, Aspergillus terreus and Mucor sp. [29]. The high activity and stability of S. commune lipase at alkaline conditions make it promising for the use in processes which require alkaline conditions such as synthesis of biopolymers, cosmetics, pharmaceuticals, biodiesel, detergents and leather [45]. 3.4.3. Effect of metal ions The effect of different metal ions on the enzyme activity of partially purified of SCI lipase was estimated (Fig. 3d). The lipolytic activity increased considerably in the presence of Ca2+ , Mg2+ and Mn2+ as compared to control activity (without any metal), of which Mg2+ showed the maximal increased relative activity (154%), followed by and Ca2+ (109.7%) and Mn2+ (106%). On the other hand, metal ions Co2+ , Fe2+ , Zn2+ inhibited the SCI lipase activity. Some metal ions are well reported to play a key role in maintaining the active conformation of an enzyme. There are a number of reports confirming the activity enhancing effect of Ca2+ and Mg2+ ions [38,46]. The side chains of the catalytic triads of most of the lipases have negatively charged aspartyl or glutamyl residues at their carboxylate ends, which come together by the polypeptide chain folding. Ca2+ /Mg2+ are known to bridge and cross-link such polypeptide chains, thus providing rigidity and stability of the enzyme [47]. For industrial applications, the knowledge of metal ion activation and inhibition is important in order to get

maximum catalysis efficiencies [48]. Heavy metals such as Co2+ , Fe2+ , and Zn2+ inhibited the lipase activity. The inhibitory effect could be accounted to the interaction of ions with charged side chain groups of surface amino acids that eventually affect the tertiary structure and stability of the enzyme [49]. Similar results were reported for lipases from Bacillus sp. VITL8 [48], Bacillus subtilis Pa2 [50], Rhizopus homothallicus [39], Thermosyntropha lipolytica [51].

3.4.4. Effect of organic solvents on partially purified SCI lipase activity and stability One of the crucial factors, influencing the applicability of lipase enzyme in various industrial processes is the stability and activity of the enzyme in organic solvents. The effect of organic solvents on partially purified SCI lipase was tested with a range of polar and non-polar solvents (50%, v/v) having log Pow (polarity measure) values from −1.3 to 4.0. The log Pow , the logarithm of the partition coefficient, P, of the solvent between n-octanol and water, serves as the best measure of the solvent polarity [52]. Interestingly, SCI lipase showed remarkable activity in all the tested organic solvents (Table 5). SCI lipase retained 40–92% and 88–127% of its original activity after incubation in 50% (v/v) organic solvents with low polarities (log P < 0) and organic solvents with high polarities (log P > 2) respectively, at 37 ◦ C for 24 h (Table 4). In general, SCI showed more stability in hydrophobic organic solvents than in hydrophilic organic solvents, which is a common phenomenon for most lipases [53]. Hydrophobic solvents (high log Pow ) are less able to strip the necessary tightly bound water molecules, off enzyme than hydrophilic solvents (low log Pow ) and thus are more preferable under anhydrous conditions [4]. Very few bacterial and fungal lipases have been reported having good stability in hydrophilic organic solvents [53]. In this study the enzyme retained its activity by more than 45% in the presence of methanol (50%, v/v), more than 60% in acetone and acetonitrile, and 99% in DMSO, which indicates SCI lipase to be quite promising especially for biodiesel production. Retained activity of SCI lipase in hydrophilic solvents could be attributed to a state of open conformation of the enzyme maintained through some changes in enzyme conformation, which opens up the lid covering the active site of the enzyme [54].

Table 5 After incubating ISTL04 Lipase for 1 h and 24 h in different organic solvents, the residual activity was determined in 50 mM Sodium Phosphate buffer (pH 8.0) at 60 ◦ C using pNP-palmitate as the substrate. The residual activity is defined as the activity remaining relative to the non-solvent-containing control. Experiments were carried out in triplicate (mean ± SD). Organic solvents (50%, v/v)

DMSO Methanol Acetone Acetonitrile Chloroform Toluene Hexane Heptane None

Log Pow value

−1.3 −0.50 −0.23 −0.15 2.2 2.5 3.5 4.0

Residual activity (%) 1h

24 h

99.45 ± 1.4 45.04 ± 2.3 61.06 ± 1.8 63.97 ± 4.7 92.87 ± 7.6 93.31 ± 5.2 131.02 ± 3.4 107.45 ± 8.1 100

92.53 ± 3.6 40.41 ± 4.25 62.56 ± 1.95 55.28 ± 2.8 92.04 ± 7.5 88.47 ± 8.2 127.1 ± 1.67 104.22 ± 4.35 100

M.K. Singh et al. / Journal of Molecular Catalysis B: Enzymatic 110 (2014) 92–99 Table 6 Effect of surfactants on ISTL04 lipase activity. Experiments were carried out in triplicate (mean ± SD). Surfactants (1%, v/v)

Residual activity (%)

Tween 20 Tween 80 Triton X 100 SDS None

83.59 ± 5.21 240.38 ± 7.6 77.44 ± 3.4 23.28 ± 2.5 100

[4] [5] [6] [7] [8] [9] [10] [11] [12] [13]

3.4.5. Effect of detergents on lipase activity The SCI lipase exhibited fair activity with non-ionic surfactants Triton X 100, Tween 80 and Tween 20 (Table 6). Tween 80 had an exceptional stimulatory effect on lipase activity which may be due to change in the enzyme conformation thus facilitating the substrate interaction [4]. Whereas SDS, an ionic surfactant had inhibitory effect on lipase activity, which may be accounted to disrupting electrostatic interactions of the surfactant with the enzyme conformation [55]. Most of the reports on microbial lipases show similar pattern of enzyme activity in presence of these detergents. Similar pattern of relative activities have been reported for lipases from thermotolerant fungus Rhizopus homothallicus [39], Pseudomonas gessardi [38], P. aeruginosa AAU2 [4], P. aeruginosa PseA [34]. In most of the studies, Tween 80 had a stimulatory effect while SDS showed an inhibitory effect. The stability in surfactants is a desirable property for lipase application in detergent formulations [4]. The intrinsic ability of SCI lipase to remain active in presence in detergents makes it suitable for such applications. 4. Conclusion A thermoalkalotolerant lipase, retaining remarkable activity at temperature and pH 12, from Basidiomycetes, S. commune is reported for the first time. The extracellular lipase produced by SSF of L. leucocephala seeds, was purified 35.76 fold showing specific activity 238.13 U/mg, with an estimated mass of 60 kDa. The enzyme exhibited significant stability in a range of polar and nonpolar organic solvents including methanol. Ca2+, Mg2+ and Mn2+ ions and surfactant Tween 80 showed stimulated lipase activity. The study demonstrates the enormous untapped potential of L. leucocephala seeds to be used as fermentable growth source making the process feasible. Most importantly, the study highlights the untapped potential of this group of fungi for lipolytic enzymes so as to provide a new dimension to the research dedicated to commercial lipases. 70 ◦ C

[14] [15] [16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] [33] [34] [35] [36] [37] [38] [39] [40] [41] [42] [43] [44]

Acknowledgements

[45]

The authors thank Department of Biotechnology, Government of India, New Delhi, India, University Grants Commission (UGC)), and Council of Scientific and Industrial Research (CSIR), New Delhi, Government of India for providing Research Grants.

[46] [47]

Appendix A. Supplementary data

[50] [51] [52] [53] [54]

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.molcatb. 2014.10.010. References

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