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

Contents lists available at ScienceDirect

Journal of Molecular Catalysis B: Enzymatic

j ourna l ho me pa g e: www.elsev ier .com/ locate /molcatb

ovel lipase from basidiomycetes Schizophyllum commune ISTL04,roduced by solid state fermentation of Leucaena leucocephala seeds

anoj Kumar Singh, Jyoti Singh, Madan Kumar, Indu Shekhar Thakur ∗

chool of Environmental Sciences, Jawaharlal Nehru University, New Delhi, 110067, India

r t i c l e i n f o

rticle history:eceived 27 July 2014eceived in revised form8 September 2014ccepted 18 October 2014vailable online 24 October 2014

a b s t r a c t

The present investigation reports a novel thermoalkalotolerant lipase producing Basidiomycetes, Schizo-phyllum commune ISTL04 for the first time. Abundantly available Leucaena leucocephala seeds wereexploited as a cheap fermentable source for the feasible production of extracellular lipase. The isolateISTL04 grew vigorously on Leucaena seeds in comparison to Soybean meal and Wheat bran exhibiting alipase activity of 146.5 U/g. The S. commune ISTL04 (SCI) lipase was purified to 35.76 folds with a specificactivity of 238.13 U/mg and an estimated mass of 60 kDa. The temperature and pH optima for lipolytic

◦

eywords:ipasechizophyllum communeasidiomyceteseucaena leucocephala

activity were 60 C and 11, respectively. The enzyme exhibited remarkable stability in the presence ofnon-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%) andTween 80 (240%). The distinctive and broader operational properties of the SCI lipase make it a promisingbiocatalyst for various industrial processes.

SF

. Introduction

Lipases (triacylglycerol hydrolases E.C.3.1.1.3) are among theajor industrial enzymes that find their huge application in food,

airy, detergent, leather, biofuel and pharmaceutical industrieshich in turn have surged the production of lipases [1–3]. Though

ipases are ubiquitous in nature and are found in all forms of life,ungi and bacteria are mainly exploited as sources of commercialipases [4]. Fungal lipases are usually produced extracellularly and

idely used in industrial applications, especially in the food indus-ry. Major commercially important lipase producing fungi are:hizopus arrhizus, Rhizopus japonicus, Rhizopus niveus, Mucor miehei,andida rugosa, Aspergillus niger and Aspergillus terrues [5]. Basid-

omycetes fungi have been well documented for several enzymesnd proteins that are involved in the degradation of lignocellulosiciomass and other aromatic compounds responsible for environ-ental problems, however this group of edible mushrooms have

ot a little scientific attention till date for the possibility of find-ng potential lipolytic enzymes, except a few edible basidiomycetes

ncluding Agaricus bisporus [6] and Lentinus edodes [7] and Antrodiainnamomea [8].

∗ Corresponding author. Tel.: +91 11 26704321 10/26191370 (R);ax: +011 26717586.

E-mail addresses: isthakur@hotmail.com, isthakur@mail.jnu.ac.in (I.S. Thakur).

ttp://dx.doi.org/10.1016/j.molcatb.2014.10.010381-1177/© 2014 Elsevier B.V. All rights reserved.

© 2014 Elsevier B.V. All rights reserved.

Schizophyllum commune is one of the most widespreadmushroom-forming fungi, occurring on fallen branches and tim-ber of deciduous trees. In Asia and Africa, the mushrooms of S.commune are consumed as a food source [9]. S. commune is wellillustrated for the production of lignocellulose degrading enzymes,whereas the knowledge about the lipolytic enzymes from thismushroom is abridged and, thus, their biotechnological potentialunrealized [10,11]. Recently, sequence of a putative uncharacter-ized protein from S. commune strain H4-8/FGSC 9210 has beensubmitted to protein database as a hydrolase having triacyl-glyceride lipase activity (http://www.uniprot.org/uniprot/D8PRE5;http://evexdb.org/gene-family/ensembl/1636/). Regardless of thegreat interests in the application of lipases in various industries, theuse of lipases as biocatalysts is often restricted by multifaceted con-cerns over the economic viability of lipase production systems. Notonly that, properties like high thermal stability, wide pH tolerance,solvent tolerance, metal tolerance are still among the many bot-tlenecks for using lipases in industries [12]. Reducing productioncosts at large scale industrial level by employing different microor-ganisms, new supplements, substrates, culture conditions, in thebest possible permutations and combinations, discovering newstrains for the production of novel versatile enzymes with indus-trially useful properties has altogether emerged as a new interest

area of lipase research. One of the ways to substantially reducethe production cost is to use wastes/residues from agro-industryas culture media and optimization of production conditions [13].In this regard, Solid state fermentation (SSF) is gaining colossal

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M.K. Singh et al. / Journal of Molecula

ttention for enzyme production as enzyme titers are higher than inubmerged fermentation (SmF), when comparing the same strainnd fermentation broth, owing to; higher production of biomass,atabolite repression resistance and decreased enzyme breakdowny proteases [14].

In the search for a more practical, appropriate and economicaledium for the biotechnological production of lipase, we inves-

igated the possibility of utilizing Leucaena leucocephala seeds asn appropriate medium. Native to Central America and Mexico, L.eucocephala is a multi-utility legume, which is now found growingaturally in most tropical areas of the world. This legume tree has

ound a prime role in agro-forestry system owing to its ability to fixitrogen thus facilitating the growth of other plants. Not only that,

t helps in preventing soil erosion and can easily grow on marginalands with high biomass production [15]. L. leucocephala is has beensed widely as firewood, timber, fodder, green manure, bioethanolroduction, paper production etc. [16]. Nonetheless, L. leucocephalaas numerous precocious desirable growth traits which prospect

ts bright role as feedstock or substrate for enzyme production. Itowers and fruits all round a year, produces seed in galore, is self-

ertile, has hard coated seeds, re-germinates after fire or cutting,rought tolerant and is able to flourish on highly alkaline soils. Itas been reported that L. leucocephala can attain a seed produc-ivity of about 3–5 tonnes seeds ha−1 year−1 [17]. L. leucocephalaeeds are rich in lipids, crude proteins, carbohydrates along withmportant mineral nutrients including N, P, K, Ca, Mg, Mn, Fe, Cund Zn [18]. Use of L. leucocephala seeds as substrate for SSF for thistudy thus appeared logical and worth exploring. Although therere numerous reports on various agro-wastes/food residues beingsed as suitable substrates for SSF (Table 1), there is no report onSF of L. leucocephala seeds so far to the best of the knowledge ofhe authors.

In the present study, a screening procedure was followed thatdentified S. commune, an edible fungus for the first time as a potentipase producer grown on cheaply available seeds of L. leucocephalas solid substrate, under solid state fermentation conditions. Theartially purified lipase was thermoalkalotolerant and exhibitedemarkable stability in the presence of non-polar, polar organicolvents, metal ions and detergents. The distinctive and broaderperational properties of the SCI lipase make it a promising biocat-lyst for various industrial processes.

. Methods

.1. Materials

Tributyrin oil and bovine serum albumin (protein standard)ere purchased from HiMedia, India. para-Nitrophenyl Palmi-

ate (pNPP) was purchased from Sigma–Aldrich (St. Louis, MO,SA; isopropanol Merck (Darmstadt, Germany). Protein molecu-

ar weight marker (14.4–116.0 kDa) was procured Fermentas Lifecience (India). All other solvents and chemicals used during thexperiment were of analytical grade. Mature L. leucocephala podsontaining seeds were collected in February 2013 from forestrea of Jawaharlal Nehru University (New Delhi, India) locatedt latitude: 28◦32′50.496′′N; longitude: 71◦14′1.68′′E; altitude:23 m.

.2. Fungal sampling and isolation

Mushroom forming fungal species found fruiting on dead woodogs were collected from forest areas of Jawaharlal Nehru Universityampus (New Delhi, India) during monsoon period. Samples wereultured and purified as described by Hernández-Luna, 2008 [19].

lysis B: Enzymatic 110 (2014) 92–99 93

2.3. Screening of lipase producing fungi

Six fungal strains (F1, F2, F3, F4, F5 and F6) were isolatedand screened for lipase production by preparing phenol red chro-mogenic substrate plates [20]. The agar plates were inoculatedwith 0.7 cm diameter agar plugs colonised by the fungal isolatesand the plates were incubated at 30 ◦C for 3–5 days. Productionof extracellular lipase and lipase activity was further confirmedby p-NPP spectrophotometric method [20]. Fungal isolates werecultured in submerged fermentation conditions using Vogel Min-imum Salts Medium (VMSM) [21] and tributyrin (1%, v/v) as aninducer at pH 5.8–6.0 in 150 ml Erlenmeyer flasks. Each flask wasinoculated with four mycelium agar plugs and left for fermenta-tion for 5 days in orbital shaker (180 rpm) at 30 ◦C. Samples werecollected after 5 days and centrifuged at 10,000 rpm for 10 min.The filtrate was used as crude enzymatic extract. The isolate whichexhibited largest clear yellow zone and maximum lipolytic activitywas further identified using 18S rDNA sequencing.

2.4. Molecular identification of the fungal isolate

Molecular characterization was done via 18S rDNAsequencing. PCR amplification of the ITS1-5.8 S-ITS2 rDNAregion of the fungus was carried out using primer setpITS1 (5′-TCCGTAGGTGAACCTGCCG-3′) and pITS4 (5′-TCCTCCGCTTATTGATATGC-3′). Sequencing was carried out bySCIGENOM (INDIA). 18S rDNA sequences of the isolate were sub-jected to similarity search analysis via GenBanK BLAST function atNCBI electronic site (http://www.ncbi.nlm.nih.gov/). Phylogenetictree was constructed using software MEGA4.

2.5. Inoculums preparation for solid state fermentation

Kirk’s basal nutrient medium (100 ml) was used for the prepa-ration of inoculums [22]. The initial pH of inoculation media wasadjusted at pH 4.5 with 1 M NaOH or 1 M HCL. The inoculums flaskwas sterilized and supplemented with syringe filtered (0.22 �m)glucose (1%). Spores of S. commune ISTL04 were transferred asep-tically from the PDA slant under sterile conditions and the flaskwas incubated (120 rpm) at 30 ◦C for 5-days to get homogenousinoculums (1 × 106–108 spores/ml).

2.6. Substrate preparation and determination of nutritivecomposition

L. leucocephala, seeds were removed from the mature pods,damaged seeds were discarded, and the remaining seeds wereoven-dried at 60 ◦C for 24 h. The dried seeds were crushed in agrinder. 10 g of well milled L. leucocephala seeds was taken intotwo sets of 250 ml Erlenmeyer flasks, one set moistened with 40 mlof distilled water (1:4, w/v) and other with VMSM (1:4, w/v) andtributyrin (1%, w/v). The contents of the flask were mixed and auto-claved. More commonly studied, solid substrates Soybean mealand Wheat bran were also taken for comparative study. The lipid,carbohydrate, protein, fibre and ash content of the seeds weredetermined by the methods described by Ref. [18].

2.7. Lipase production through solid-state fermentation

Substrate prepared as mentioned above was allowed to coolat room temperature and inoculated with 3 ml of the homoge-nous inoculums of S. commune ISTL04. The inoculated flasks were

incubated at 30 ◦C for 5 days in a humidity (65%) controlled BODincubator. For extraction of lipase the solid fermented medium wasmixed with sodium phosphate buffer (50 mM, pH 8.0) in a ratio of1:10 (w/v). The mixture was shaken at 180 rpm, for 1 h at 30 ◦C.

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

Table 1Lipase 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 Glucose (4.8%), yeast extract(4%), and NaH2PO4 (4%)

[56]

630 U/g Olive oil [1]Gingelly oil cake 363.6 U/g No added supplement [32]

Penicillium simplicissimum Castor bean waste 44.8 U/g Sugar cane molasses (6.25%,w/w)

[12]

P. brevicompactum Caster meal 87.7 U/g Soybean oil and sugarcanemolasses

[12]

P. brevicompactum Babassu cake 48.6 U/g Soybean oil and sugarcanemolasses

[12]

30.3 U/g 2% olive oil [12]

Penicillium sp. Soybean meal 200 U/g 3 % of urea [57]Rhizopus rhizopodiformis Olive cake and sugarcane

bagasse79.6 U/g No added supplement [58]

R. chinensis wheat flour with wheat bran(3/2, w/w)

24.44 U/g Peptone (2%, w/w), additionalnitrogen source and olive oil(2%, v/w)

[58]

R. homothallicus IRD-13a Sugarcane bagasse 826 U/g Urea, olive oil andoligo-elements

[30]

R. oligosporous GCBR-3 Almond meal 48.0 U/g No added supplement [59]

Yarrowia lipolytica Niger seed oil cake 26.42 U/g Urea and glucose [12]Mustard seed cake 57.89 U/g Urea 1.5% w/w, glucose 7% w/w [60]

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Rhizomucor pusillus Olive cake and sugarcanebagasse

Schizophyllum commune ISTL04 L. leucocephala seeds

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

.8. Partial purification of lipases produced in SSF

Firstly, 1 mM of Phenylmethylsulfonyl fluoride (PMSF) wasdded to the crude enzyme extract in order to prevent proteoly-ic degradation taking place through the purification procedure.mmonium sulphate was added slowly, with continuous stirring,

o the supernatant to a final concentration of 70% (w/v) saturationt 4 ◦C. The precipitates were then harvested by centrifugation at◦C and 12,000 × g for 30 min. The precipitate therefore obtainedas dissolved in sodium phosphate buffer (pH 8) containing 1 mM

MSF and dialysed for 24 h with three changes against the sameuffer. The concentrated lipase preparation was then subjected toel permeation column chromatography by loading it onto to auperdexTM 200 gel filtration column (GE Healthcare Life Sciences)2.5 cm × 10 cm) pre-equilibrated with 50 mM sodium phosphateuffer containing 0.15 M NaCl (pH 8). The proteins were elutedith the same at a flow rate of 0.5 ml/min. Fractions of 0.5 ml were

ollected and assayed for both lipase activity and protein contentA280). All purification steps were carried out at 4 ◦C. Fractions con-aining active lipase were pooled and stored at −20 ◦C.

.9. Protein determination and gel electrophoresis

Protein concentration was determined by Bradford methodsing bovine serum albumin (BSA) as standard [23]. Sodiumodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)sing the method of Laemmli [24] on a 6% polyacrylamide stackingel and a 12% polyacrylamide-resolving gel. Protein bands were

isualized by staining with Coomassie brilliant blue R250. Activ-ty staining (Zymogram analysis) of the unstained gel was doneo determine the band corresponding to lipase, by the methodescribed by Ref. [25].

24 U/g No added supplement [30]

.67 U/g No added supplement This study

2.10. Characterization

2.10.1. Effects of temperature and pH on lipase activity andstability

The optimum temperature for partially purified lipase activitywas determined spectrophotometrically using p-NPP as substrate[26], over a temperature range of 4–70 ◦C with an incubation periodof 1 h at pH 8 using 50 mM Sodium Phosphate buffer. To exam-ine the thermo-stability, the enzyme was incubated at differenttemperatures (30–70 ◦C) for a period of 5 h and the activity wasdetermined at regular time intervals. The effect of pH on lipaseactivity and stability was determined spectrophotometrically usingthe following buffers: Na2HPO4–citric acid for pH 3.0–8.0, Tris–HClfor pH 8.0–10.0, and glycine–NaOH for pH 10.0–12.0 with an incu-bation period of 1 h and 24 h, respectively, at 37 ◦C.

2.10.2. Effects of metal ions, detergents on lipase activityEffect of different metal ions on the lipase activity was deter-

mined by incubating the enzyme with metal ions (Ca2+, Mg2+, Mn2+,Co2+, Hg2+, Fe2+, Zn2+) to a final concentration of 10 mM in SodiumPhosphate buffer (50 mM, pH 8.0) at 60 ◦C for 1 h. Similarly, theeffects of different surfactants, Triton X-100, Tween 20, Tween 80and SDS were determined by incubating the buffered enzyme withthe surfactant (1% v/v or w/v) for 1 h at 60 ◦C. After incubation,the residual activity of the enzyme was measured using spectro-photometric p-NPP enzyme assay.

2.10.3. Effect of organic solvents on the lipase activity andstability

The effect of various polar and non polar organic solvents on theenzyme activity and stability was determined by incubating thepartially purified enzyme in organic solvents (50%, v/v) at 37 ◦C for24 h with shaking at 200 rpm. Samples were withdrawn from aque-

ous phase and used for the determination of residual lipase activityby using spectrophotometric p-NPP enzyme assay. The organic sol-vents used were methanol, dimethyl sulphoxide (DMSO), acetone,toluene, chloroform, heptanes, hexane and acetonitrile.

r Catalysis B: Enzymatic 110 (2014) 92–99 95

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

Component Content (%)

Crude protein content (%) 9.28 ± 0.11Carbohydrate content (%) 42.56 ± 0.85Lipid content (%) 15.5 ± 1.25

TC

M.K. Singh et al. / Journal of Molecula

. Results and discussion

.1. Isolation and identification of fungus for producing lipase

Six mushroom forming fungal strains were isolated andcreened for the production of lipolytic enzyme by Phenol red platehromogenic assay with tributyrin as lipidic substrate. Out of theix isolates, F1, F2, F3, F4 (ISTL04), F5, F6, four isolates showedlearance zone formation (S1). Though four isolates showed clearydrolysis zone, isolate ISTL04 (F4) formed the largest yellowydrolysis zone. The results were further confirmed by spec-rophotometric method using p-NPP as substrate. Isolate ISTL04xhibited the highest lipase activity. Following screening, isolateSTL04 was identified as the potential lipase producer, was fur-her identified by 18S rDNA sequencing. Based on 18S rDNA data,solated strain was identified as S. commune ISTL04 and may beonsidered novel. BLAST results showed maximum similarity (99%)o S. commune strains (Fig. 1). The 18S rDNA sequence of ISTL04as been submitted to GenBank and has been assigned with theccession no. KF601697. S. commune is one of the most commonly

ound mushrooms which usually completes its life cycle in 10 days,nd is amenable to genetic manipulations. It is a model mushroomhose whole genome has been recently sequenced for gainingew insights of the otherwise underrated producers of valuablenzymes and pharmaceutical proteins [9]. Under the same genomeroject, a putative uncharacterized protein has been submittedaving lipolytic activity. The present study identified an indigenous. commune strain ISTL04 capable of appreciable lipolytic activity,hich could further add to the current knowledge of this modelushroom which has not got any attention till date for industrially

seful enzymes other than lingo-cellulolytic enzymes.

.2. Lipase production through solid-state fermentation ofeucaena leucocephala seeds

Extracellular lipase activities obtained in all the SSF mediare given in Table 2. Highest lipase activities (148.57 U/g) werebtained when isolate ISTL04 was grown on crushed L. leucocephalaeeds supplemented with VMSM (1:4, w/v) and 1% tributyrin asnducer for lipolytic activity as the solid substrate, as comparedo Soybean meal (95.19 U/g) and Wheat bran (70.23 U/g), supple-

ented with VMSM (1:4, w/v) and 1% tributyrin as inducer foripolytic activity. When grown on substrates only provided withistilled water, the extracellular lipase activity declined remarkably

n the case of Soybean meal (52.49 U/g) and Wheat bran (25.91 U/g)hereas, there was no significant effect on lipase production in L.

eucocephala seeds medium. S. commune ISTL04 produced substan-ial quantities of extracellular lipase (146.5 U/g) with L. leucocephalaeeds in presence of distilled water only in order to provideoisture. Physicochemical parameters such as pH, temperature,

gitation and nutritional factors (carbon and nitrogen sources) arehe significant factors influencing the lipase production by fungi.ipases, primarily being inducible enzymes are expressed largelyepending on the carbon source. It has been reported that lipases

able 2omparison of lipase production by S. commune ISTL04 in SSF, using different substrates.

Vogel’s Minimal salts medium (VMSM) (1:4, w/v) Inducer for lipase, tribut

Presence Presence

None* None

* Replaced by distilled water.

Crude fibre content 4.53 ± 0.16Ash content mg/100 g 19.58 ± 2.30

production is induced by the presence of some lipidic source suchas tributyrin, oils, etc. In this study, it was found that L. leuco-cephala seeds, in presence of distilled water only were able tosupport vigorous growth and induce appreciable lipase produc-tion (146.5 U/g) by the isolate even in the absence of any externalinducer supplement. As determined by nutritive value analysis, theL. leucocephala seeds contained 15.5% lipids, 42.56% carbohydrates,9.28% proteins, 4.53% crude fibre (Table 3). Whereas, Soybean mealis reported to contain, on an average 44% crude protein, 3% crudefibre and only 0.5% fat, and 12% moisture, [27] and Wheat branon an average contains 45–70% carbohydrates, 15–18% proteins,4–5% fats [28]. The higher extracellular lipase production in caseof L. leucocephala seeds could be attributed to the fact that L. leu-cocephala seeds contain high amount of lipids (15.5%) along withcarbohydrates, proteins and other important nutrients includingminerals N, P, K, Ca, Mg, Mn, Fe, Cu and Zn, which could whollysupport the growth of the microorganism, without the additionof extra nutrients other than a little moisture [18]. Various rawmaterials such as Olive oil cake, Babassu oil cake, Egg yolk, Almondmeal, Sunflower oil, Soybean bran, Olive oil, Wheat bran and Soycake have been exploited as fermentable sources for extracellu-lar lipase production from fungi [29] (Table 1). Though, all suchfermentable substrates are generally supplemented with nutri-ent medium or an inducer to induce lipase production, there arefew reports on extracellular lipase production by fungus throughsolid state fermentation of wastes/agro-industrial residues with-out any supplementation. Rodriguez et al., 2006 [30] reportedlipase production (20.24 U/g) from Rhizomucor pusillus and Cor-dova et al., 1998 [31] reported lipase production (79.6 U/g) fromRhizopus rhizopodiformis using Olive cake and sugarcane bagassewithout any extra nutrient supplementation, Kamini et al., 2000[32] reported lipase production (363.6 U/g) from Aspergillus nigerusing Gingelly oil cake without extra nutrient supplementation.In this study, it was found that L. leucocephala seeds alone, evenin the absence of any external inducer supplement, were ableto support vigorous growth and induce appreciable lipase pro-duction (146.5 U/g) by the isolate which falls within the range ofmost reported lipase activities (20–363 U/g), produced by solidstate fermentation of agro-industrial wastes without supplemen-tation (Table 1). Nonetheless, the year round abundant availability(3–5 tonnes seeds ha−1 year−1) of this cheap, nutrient rich sub-

strate makes L. leucocephala seeds a very promising candidate to beexplored as fermentable nutrient source for the growth of microor-ganisms for the production of enzymes and other bio-products.

Experiments were carried out in triplicate (mean ± SD).

yrin (1%, v/w) Solid substrate (10 g) Lipase activity (U/g)

L. leucocephala seeds 148.57 ± 2.5Wheat bran 70.23 ± 1.71Soybean meal 95.19 ± 5.53

L. leucocephala seeds 146.5 ± 6.32Wheat bran 25.91 ± 3.51Soybean meal 52.49 ± 4.21

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

Schizophyllum commune ISTL04 Schizophyll um comm une strain P3- B

Schizophyllum commune strain 1-19 Auricularia polytricha voucher Dai1045 1

Basidiomycete sp. RM4ac Schizoph yllum comm une strain MJ03

Agaricaceae sp. 72 Agaricace ae sp. 71 0

Schizop hyllum commun e isolate BDG2-1- 1 Agaricaceae sp. 64 7

Schizophyllum commune isolate HN21 Pochonia suchlaspo ria strain NS-1 7

Schizop hyllum commun e strain P8-B Schizophyllum commune isolate BCC22128 Schizophyllum commun e isolate BCC264 07 Schizophyll um comm une strain NF58

Schizophyll um comm une strain Sc1 Schizoph yllum comm une strain MX4

Trametes ro biniophil a strain CFCC 68 39 Schizophyllum commun e st rain D

Bas idiomycete sp. LC2 Schizophyllum commun e st rain DMRF-7

Rhizopus or yzae strain xsd08 049 Schizoph yllum comm une strain xsd0803 6 Schizophyllum commun e IFM 4609 7

Schizophyllum commun e isolate HLJ 20 Schizophyllum commune isolate HE2740

Schizop hyllum commune isolate BCC26 414 Asc hersonia sp. DY115-21 -2-M5

Aschersonia aley rodis st rain GZZ KB Schizoph yllum sp. P DD 10338 0 Schizoph yllum comm une strain FBst04

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Schizophyll um comm une ISTL04 (NCBI Access ion no. KF601697 )

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ig. 1. Phylogenetic tree showing the relationships among 18S rDNA sequences of isootstrap consensus tree (1000 replicates) was drawn by multiple sequence alignm

ore culture optimization experiments with different nutrientupplements will definitely lead to realizing the full potential ofhe isolate ISTL04 and the substrate, however, the primary resultsre more than promising.

.3. Partial purification of SCI lipase produced by SSF of L.eucocephala seeds

The supernatant containing the extracellular lipase was sub-ected to enzyme purification to exclude other undesirablenzymes/proteins which may hamper/hinder the properties ofipase [33]. The supernatant was subjected to ammonium sulfaterecipitation (70%), the precipitates were then concentrated byialysis against sucrose. The concentrated sample was then loadednto a SuperdexTM 200 column (Table 4) followed by SDS-PAGE.

35.76 fold partial purification of SCI lipase was achieved afterel permeation chromatography with a specific enzyme activityf 238.13 U/mg of protein. The partially purified lipase fractionppeared as a single major protein band of 60 kDa after SuperdexTM

00 gel permeation chromatography on SDS-PAGE Gel (Fig. 2a).he activity staining analysis also revealed the lipolytic activityo be associated with the 60 kDa band in both ammonium sul-ate precipitation proteins and the active fraction concentrate after

uperdexTM 200 gel permeation chromatography (Fig. 2b). Till dateany lipases from different microorganisms have been reported

alling under a wide range of molecular mass from 29 to 92 kDancluding Pseudomonas sp lipases 29–92 kDa [34–38], Burkholderia

STL04 and the most similar sequences retrieved from the NCBI nucleotide database.ith neighbour joining method using software MEGA 4.

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 lipase60 kDa [42]. The molecular mass of the SCI lipase was found to be60 kDa, which falls within magnitude range of previously reportedbacterial and fungal lipases.

3.4. Determination of optimum reaction conditions

3.4.1. Effect of temperature on partially purified lipase activityand stability

The purified lipase was active over a temperature range of10–70 ◦C, retaining more than 50% of its relative activity with amaximal activity at 60 ◦C (Fig. 3a). However, the activity showed asharp 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) wasdetermined at regular time intervals after incubating the enzymefor up to 5 h (Fig. 3b). SCI lipase was stable after pre-incubation at50 ◦C and 60 ◦C for 5 h, retaining more than 90% of its original activ-ity. Although, the lipase was fairly stable at 70 ◦C, retaining morethan 60% residual activity after preincubation for 1 h, the activ-ity declined to 30% after a period of 5 h. The results imply that

the SCI lipase is remarkably thermostable. Although, moderatelythermostable lipases (stable at 40–50 ◦C) have been reported fromRhizopus homothallicus, Aspergillus niger, Mucor sp., Geotrichum sp.,Mucor pusillus, Aspergillus terreus, Rhizomucor sp., Aspergillus sp.,

M.K. Singh et al. / Journal of Molecular Catalysis B: Enzymatic 110 (2014) 92–99 97

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 pre-incubation 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) usingpNP-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 withvarying 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 differentbuffers 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 ofany metal ions was considered as control (100%). Experiments were carried out in triplicate (mean ± SD).

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

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

Purification steps Volume (ml) Totalunits

Totalproteins (mg)

Lipase activity(U/ml)

Protein content(mg/ml)

Specific activity(U/mg)

Fold Recovery (%)

Hh[ehihTi

3

ooo8fruSlppiitrmrpfdljaptb

3

ptao(oamnamrpcbo

could be attributed to a state of open conformation of the enzymemaintained through some changes in enzyme conformation, whichopens up the lid covering the active site of the enzyme [54].

Table 5After incubating ISTL04 Lipase for 1 h and 24 h in different organic solvents, theresidual activity was determined in 50 mM Sodium Phosphate buffer (pH 8.0) at60 ◦C using pNP-palmitate as the substrate. The residual activity is defined as theactivity remaining relative to the non-solvent-containing control. Experiments werecarried out in triplicate (mean ± SD).

Organic solvents (50%, v/v) Log Pow value Residual activity (%)

1 h 24 h

DMSO −1.3 99.45 ± 1.4 92.53 ± 3.6Methanol −0.50 45.04 ± 2.3 40.41 ± 4.25Acetone −0.23 61.06 ± 1.8 62.56 ± 1.95Acetonitrile −0.15 63.97 ± 4.7 55.28 ± 2.8Chloroform 2.2 92.87 ± 7.6 92.04 ± 7.5

Supernatant 100 1465 220

Ammonium sulfate precipitation (0–70%) 4.5 1030 12.32

Gel permeation chromatography 2 535.79 2.25

umicola sp., Candida sp., and Penicillium sp., reports on lipases withigh stability at high temperatures and pH from fungi are scarce29,43,44]. Thermostable biocatalysts are desirable as they allow anlevated operation temperature, hence is advantageous providingigher reaction rates, higher stability, higher yields, lower viscos-

ty and lesser contamination problems. Thermostable lipases finduge application potential in detergent and leather industries [2].hus, remarkable thermostability exhibited by the SCI lipase makest a very promising candidate to be exploited commercially.

.4.2. Effect of pH on partially purified lipase activity and stabilityThe S. commune ISTL04 lipase exhibited optimal lipolytic activity

ver a broad alkaline pH range (pH 7.0–12.0), attaining a maximalbserved activity at pH 11.0 (Fig. 3c). The lipase was found activever a broad alkaline pH range (pH 7.0–11.0), retaining more than0% of its maximum activity at pH 10.0 and enzyme activity wasound to be highest at pH 11 (Fig. 3c). The SCI lipase retained 43%elative activity at pH 8 and 57% at pH 12. However, at low pH val-es (pH < 5.0), the SCI lipase was found to be inactive. Though theCI lipase was active at pH 5.0–6.0, the relative activity was veryow as compared to that with higher pH values. After incubation atH values ranging from 3.0 to 12.0 for 24 h at 37 ◦C, the partiallyurified SCI lipase retained more than 70% of the control activity

n the alkaline pH range 8.0–12.0 (Fig. 3c). The remarkable activ-ty and stability of the S. commune lipase at high pH values implieshat it is a potential alkaline lipase. Dandavante et al., 2009 [25]eported lipase from Burkholderia multivorans V2 showing maxi-um activity at pH 8. Likewise, Pseudomonas sp. lipases have been

eported to be fairly stable upto 14 h at 37 ◦C over a wide range ofH from 5.6 to 9.0 and upto 3 h at 30 ◦C over a pH range of 5.5–10.5or P. aeruginosa san-ai [34,36]. Similarly lipase from Rhizopus sp.isplayed maximum lipolytic activity at pH 7.5 [39]. Alkalotolerant

ipases have also been reported in Humicola lanuginosa, Rhizopusaponicus, Aspergillus terreus and Mucor sp. [29]. The high activitynd stability of S. commune lipase at alkaline conditions make itromising for the use in processes which require alkaline condi-ions such as synthesis of biopolymers, cosmetics, pharmaceuticals,iodiesel, detergents and leather [45].

.4.3. Effect of metal ionsThe effect of different metal ions on the enzyme activity of

artially purified of SCI lipase was estimated (Fig. 3d). The lipoly-ic activity increased considerably in the presence of Ca2+, Mg2+

nd Mn2+ as compared to control activity (without any metal),f which Mg2+ showed the maximal increased relative activity154%), followed by and Ca2+ (109.7%) and Mn2+ (106%). On thether hand, metal ions Co2+, Fe2+, Zn2+ inhibited the SCI lipasectivity. Some metal ions are well reported to play a key role inaintaining the active conformation of an enzyme. There are a

umber of reports confirming the activity enhancing effect of Ca2+

nd Mg2+ ions [38,46]. The side chains of the catalytic triads ofost of the lipases have negatively charged aspartyl or glutamyl

esidues at their carboxylate ends, which come together by the

olypeptide chain folding. Ca2+/Mg2+ are known to bridge andross-link such polypeptide chains, thus providing rigidity and sta-ility of the enzyme [47]. For industrial applications, the knowledgef metal ion activation and inhibition is important in order to get

14.65 2.20 6.66 1 100228.89 2.74 83.60 12.55 70.31

47.58 1.13 238.13 35.76 36.57

maximum catalysis efficiencies [48]. Heavy metals such as Co2+,Fe2+, and Zn2+ inhibited the lipase activity. The inhibitory effectcould be accounted to the interaction of ions with charged sidechain groups of surface amino acids that eventually affect the ter-tiary structure and stability of the enzyme [49]. Similar results werereported 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 lipaseactivity and stability

One of the crucial factors, influencing the applicability of lipaseenzyme in various industrial processes is the stability and activityof the enzyme in organic solvents. The effect of organic solventson partially purified SCI lipase was tested with a range of polarand 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 parti-tion coefficient, P, of the solvent between n-octanol and water,serves as the best measure of the solvent polarity [52]. Interest-ingly, SCI lipase showed remarkable activity in all the tested organicsolvents (Table 5). SCI lipase retained 40–92% and 88–127% of itsoriginal activity after incubation in 50% (v/v) organic solvents withlow polarities (log P < 0) and organic solvents with high polarities(log P > 2) respectively, at 37 ◦C for 24 h (Table 4). In general, SCIshowed more stability in hydrophobic organic solvents than inhydrophilic organic solvents, which is a common phenomenon formost lipases [53]. Hydrophobic solvents (high log Pow) are less ableto strip the necessary tightly bound water molecules, off enzymethan hydrophilic solvents (low log Pow) and thus are more prefer-able under anhydrous conditions [4]. Very few bacterial and fungallipases have been reported having good stability in hydrophilicorganic solvents [53]. In this study the enzyme retained its activityby more than 45% in the presence of methanol (50%, v/v), morethan 60% in acetone and acetonitrile, and 99% in DMSO, whichindicates SCI lipase to be quite promising especially for biodieselproduction. Retained activity of SCI lipase in hydrophilic solvents

Toluene 2.5 93.31 ± 5.2 88.47 ± 8.2Hexane 3.5 131.02 ± 3.4 127.1 ± 1.67Heptane 4.0 107.45 ± 8.1 104.22 ± 4.35None 100 100

M.K. Singh et al. / Journal of Molecular Cata

Table 6Effect of surfactants on ISTL04 lipase activity. Experiments were carried out in trip-licate (mean ± SD).

Surfactants (1%, v/v) Residual activity (%)

Tween 20 83.59 ± 5.21Tween 80 240.38 ± 7.6

3

TabthtesglPPwilp

4

7iScepiTliuam

A

oaG

A

i2

R

[

[

[

[

[

[[

[[[

[

[[[[[

[

[[[[

[

[[

[[[

[[

[

[[

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[

[[

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[[[[[

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Triton X 100 77.44 ± 3.4SDS 23.28 ± 2.5None 100

.4.5. Effect of detergents on lipase activityThe SCI lipase exhibited fair activity with non-ionic surfactants

riton X 100, Tween 80 and Tween 20 (Table 6). Tween 80 hadn exceptional stimulatory effect on lipase activity which maye due to change in the enzyme conformation thus facilitatinghe substrate interaction [4]. Whereas SDS, an ionic surfactantad inhibitory effect on lipase activity, which may be accountedo disrupting electrostatic interactions of the surfactant with thenzyme conformation [55]. Most of the reports on microbial lipaseshow similar pattern of enzyme activity in presence of these deter-ents. Similar pattern of relative activities have been reported foripases from thermotolerant fungus Rhizopus homothallicus [39],seudomonas gessardi [38], P. aeruginosa AAU2 [4], P. aeruginosaseA [34]. In most of the studies, Tween 80 had a stimulatory effecthile SDS showed an inhibitory effect. The stability in surfactants

s a desirable property for lipase application in detergent formu-ations [4]. The intrinsic ability of SCI lipase to remain active inresence in detergents makes it suitable for such applications.

. Conclusion

A thermoalkalotolerant lipase, retaining remarkable activity at0 ◦C temperature and pH 12, from Basidiomycetes, S. commune

s reported for the first time. The extracellular lipase produced bySF of L. leucocephala seeds, was purified 35.76 fold showing spe-ific activity 238.13 U/mg, with an estimated mass of 60 kDa. Thenzyme exhibited significant stability in a range of polar and non-olar organic solvents including methanol. Ca2+, Mg2+and Mn2+

ons and surfactant Tween 80 showed stimulated lipase activity.he study demonstrates the enormous untapped potential of L.

eucocephala seeds to be used as fermentable growth source mak-ng the process feasible. Most importantly, the study highlights thentapped potential of this group of fungi for lipolytic enzymes sos to provide a new dimension to the research dedicated to com-ercial lipases.

cknowledgements

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

ppendix A. Supplementary data

Supplementary data associated with this article can be found,n the online version, at http://dx.doi.org/10.1016/j.molcatb.014.10.010.

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