Culture Conditions for the Production of Thermostable Lipase By

ARTICLE IN PRESS

beni-suef university journal of basic and applied sciences ■■ (2016) ■■– ■■

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1bs_bs_query Short Communication

2bs_bs_query

3bs_bs_query Culture conditions for the production of

4bs_bs_query

5bs_bs_query thermostable lipase by Thermomyces lanuginosus

6bs_bs_query a a,b, a a 7bs_bs_query Q1 B. Sreelatha , V. Koteswara Rao *, R. Ranjith Kumar , S. Girisham , a 8bs_bs_query S.M. Reddy

a 9bs_bs_query Department of Microbiology, Kakatiya University, Warangal, Telangana, India b 10bs_bs_query CSIR-Biochemical Sciences Division, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, India

11 bs_bs_query

12bs_bs_query

13bs_bs_query ARTICLE INFO ABSTRACT

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15bs_bs_query Article history: In the present investigation lipase production by three strains of thermophilic Thermomyces

16bs_bs_query Received 21 August 2016 lanuginosus (GSLMBKU-10, GSLMBKU-13 and GSLMBKU-14) was carried out in submerged fer-

17bs_bs_query Received in revised form 22 mentation process. Olive oil and triacetin (0.1%) were added to the basal medium, which Q5 18bs_bs_query November 2016 stimulated the lipase production. The maximum lipase was produced by GSLMBKU-10 and

19bs_bs_query Accepted 22 November 2016 GSLMBKU-13 in yeast extract starch medium supplemented with triacetin (0.1%). The optimum

20bs_bs_query Available online pH was recorded at 6.0, 6.5 and 7.0 by GSLMBKU-10, GSLMBKU-13 and GSLMBKU-14 respec- 21bs_bs_query

22bs_bs_query tively. T. lanuginosus GSLMBKU-10 strains failed to produce lipase at pH 8.0. The optimum

23bs_bs_query temperature for lipase production was observed at 45 °C by GSLMBKU-14 and GSLMBKU- 24bs_bs_query Keywords:

25bs_bs_query 10, while that for GSLMBKU-13 was at 50 °C. The marginal temperature ranged from 45 °C 26bs_bs_query Thermomyces lanuginosus

27bs_bs_query to 50 °C for both lipase production and vegetative growth by the three strains of T. lanuginosus 28bs_bs_query Oils

29bs_bs_query under study. In conclusion, the GSLMBKU-13 strain was comparatively superior in the pro- 30bs_bs_query Culture medium

31bs_bs_query duction of lipase than the other two strains under investigation. 32bs_bs_query pH

35bs_bs_query an open access article under the CC BY-NC-ND license (http://creativecommons.org/ 36bs_bs_query Lipase production

37bs_bs_query licenses/by-nc-nd/4.0/).

38bs_bs_query

39bs_bs_query acidolysis and aminolysis in non-aqueous media (Charles and 56bs_bs_query

40bs_bs_query 1. Introduction James, 2011). Lipases are used extensively in food and dairy 57bs_bs_query

41bs_bs_query industry for the hydrolysis of milk fat, cheese ripening, ﬂavour 58bs_bs_query

42bs_bs_query Lipases (triacylglycerol acylhydrolase, EC 3.1.1.3) are a enhancement and lipolysis of butterfat and cream (Ray, 2012). 59bs_bs_query

43bs_bs_query biotechnologically important group of enzymes which act on These are also used as additives in washing powder, for removal 60bs_bs_query Q6 44bs_bs_query the carboxyl ester bonds present in triacylglycerols and liber- of oil/fat stains, and to increase fabric absorbency in the textile 61bs_bs_query

45bs_bs_query ate fatty acids and glycerol (Abrunhosa et al., 2013). They are industry (Shivika and Shamsher, 2014). In addition, these 62bs_bs_query

46bs_bs_query also involved in a wide range of conversion reactions such as enzymes are used as a catalyst for the production of differ- 63bs_bs_query

47bs_bs_query esterification, interesterification, transesterification, alcoholysis, ent products used in the cosmetic industry, such as pulp and 64bs_bs_query

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49bs_bs_query Q2 50bs_bs_query * Corresponding author. V. Koteswara Rao, CSIR-Biochemical Sciences Division, National Chemical Laboratory, Dr. Homi Bhabha Road,

51bs_bs_query Pune 411008, India.

52bs_bs_query E-mail address: [email protected] (V. Koteswara Rao).

53bs_bs_query http://dx.doi.org/10.1016/j.bjbas.2016.11.010

55bs_bs_query CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: B. Sreelatha, V. Koteswara Rao, R. Ranjith Kumar, S. Girisham, S.M. Reddy, Culture conditions for the production of thermostable lipase by Thermomyces lanuginosus, Beni-Suef University Journal of Basic and Applied Sciences (2016), doi: 10.1016/j.bjbas.2016.11.010 ARTICLE IN PRESS 2 beni-suef university journal of basic and applied sciences ■■ (2016) ■■– ■■

125bs_bs_query 65bs_bs_query paper, synthesis of biodiesel, degreasing of leather and reso-

2. Materials and methods 126bs_bs_query 66bs_bs_query lution of racemic mixtures in the pharmaceutical industry

127bs_bs_query 67bs_bs_query (Ferreira-Dias et al., 2013). Lipases are also employed in waste-

2.1. Reagents and standards 128bs_bs_query 68bs_bs_query water treatment for degreasing of lipid clogged drains and in

129bs_bs_query 69bs_bs_query the medical industry as a diagnostic tool in blood triglyceride

Whatman ﬁlter paper No. 42, sodium chloride, citrate buffer 130bs_bs_query 70bs_bs_query assay (Verma and Prakash, 2014).

(pH 8.0), triacetin, toluene, absolute alcohol, olive oil, rhoda- 131bs_bs_query 71bs_bs_query Lipases are reported to be produced by many species of

mine B, phenolphthalein, yeast extract, starch, MgSO4·7H2O, 132bs_bs_query 72bs_bs_query animals, plants, bacteria, yeasts and fungi (Sztajer et al., 1998).

KH2PO4·7H2O, agar and all the chemicals employed were from 133bs_bs_query 73bs_bs_query Among these, microbial lipases have gained wide industrial ap-

Merck Chemicals (Mumbai), India. 134bs_bs_query 74bs_bs_query plications in view of high yields, rapid growth of microorganisms

135bs_bs_query 75bs_bs_query and ease of genetic manipulation (Andualema and Gessesse,

2.2. Basal medium 136bs_bs_query 76bs_bs_query 2012). Extracellular lipase production has been observed in

137bs_bs_query

77bs_bs_query several species of Mucor, Rhizopus, Geotrichum, Pencillium,

The yeast starch medium (basal medium) used in the present 138bs_bs_query

78bs_bs_query Acremonium, Candida, Humicola, Cunninghamella and Aspergil- study consists of yeast extract 5.0 g, starch 15.0 g, MgSO4·7H2O 139bs_bs_query

79bs_bs_query lus (Abrunhosa et al., 2013; Cihangir and Sarikaya, 2004; 0.5 g, KH2PO4·7H2O 1.0 g, pH 6.5 and Milli-Q ultrapure water 140bs_bs_query

80bs_bs_query Gopinath et al., 2000, 2002, 2003; Prabhakar et al., 2012). With

(Millipore). 141bs_bs_query

81bs_bs_query the advancement of molecular biology, fungal classiﬁcation and

142bs_bs_query

82bs_bs_query phylogenetic studies have shifted to DNA sequence based 2.3. Sampling 143bs_bs_query

83bs_bs_query methods (Shenoy et al., 2007). These methods play an impor- 144bs_bs_query

84bs_bs_query tant role in fungal identiﬁcation and in understanding of genetic Thermophilic fungi were isolated from dung of different animals 145bs_bs_query

85bs_bs_query diversity of fungi (Rajesh et al., 2013). The internal tran- (cow, sheep, turkey, pigeon, poultry, bear, duck, rabbit, monkey), 146bs_bs_query

86bs_bs_query scribed spacer (ITS) regions are more variable and used to zoo waste, banana peel and bird nest material collected from 147bs_bs_query

87bs_bs_query analyse interspecies and sometimes intra-species relations Zoo Park, Warangal, Telangana, India. 148bs_bs_query

88bs_bs_query (Divakara et al., 2015). The 5.8 S r-DNA and the ﬂanking ITS 149bs_bs_query

89bs_bs_query regions are conserved regions frequently used in phyloge- 2.4. Cultures used in the present study 150bs_bs_query

90bs_bs_query netic studies. A homology search by BLAST has shown that the 151bs_bs_query

91bs_bs_query ITS region and 5.8 S rDNA sequences were highly conserved Q9 Aspergillus fumigatus, A. ﬂavus, Chaetomium thermophilum, 152bs_bs_query

92bs_bs_query in thermophilic fungi, which suggests a possible recent taxo- Chrysosporium species, Humicola grisea, H. insolens, H. stellata, 153bs_bs_query

93bs_bs_query nomic divergence in the fungal community. Malbranchea pulchella, Mucor miehei, M. pusillus, Myriococcum 154bs_bs_query

94bs_bs_query Thermophilic fungi are widespread and its great impor- albomyces, Penicillium duponti, Rhizopus arrhizus, Torula thermophila 155bs_bs_query

95bs_bs_query tance in many natural processes has been reported (Ranjith and T. lanuginosus (formerly known as H. lanuginosus)wereused 156bs_bs_query

96bs_bs_query Kumar et al., 2010). Thermomyces lanuginosus is a thermo- Q10 in the present study. 157bs_bs_query

97bs_bs_query philic fungus formerly known as Humicola lanuginosa, frequently 158bs_bs_query

98bs_bs_query isolated from self-heating organic debris and a variety of de- 2.5. Morphological identiﬁcation 159bs_bs_query

99bs_bs_query caying plant materials (Anand et al., 1990; Gomes et al., 1993; 160bs_bs_query

100bs_bs_query Haarhoff et al., 1999; Hoq et al., 1994; Ranjith Kumar et al., 2010; The collected samples were analysed for the mycological ex- 161bs_bs_query

101bs_bs_query Sreelatha et al., 2013). Most of the mesophilic fungi grow at amination of the fungi using dilution plate technique (Apinis, 162bs_bs_query

102bs_bs_query temperatures between 10 °C and 40 °C and have an optimum 1963a) and paired petri plate technique (Waksman et al., 1939). 163bs_bs_query

103bs_bs_query growth at around 25–30 °C, while thermophilic fungi grow op- T. lanuginosus isolates were morphologically identiﬁed by stan- 164bs_bs_query

104bs_bs_query timally at 40 °C–50 °C and sometimes exhibit growth even at dard manuals and protocols (Apinis, 1963b; Barnett and Hunter, 165bs_bs_query

105bs_bs_query Q7 an elevated temperature of 60 °C, but fail to grow below 20 °C 1972; Cooney and Emerson, 1964). 166bs_bs_query

106bs_bs_query (Cooney and Emerson, 1964). A further characteristic feature 167bs_bs_query

107bs_bs_query of thermophiles is that their enzymes are more heat stable than 2.6. DNA extraction and PCR assay 168bs_bs_query

bs_bs_query 108 those of mesophiles when extracted and tested in cell-free 169bs_bs_query Q8 bs_bs_query 109 systems. In view of the potential biotechnological applica- Isolates of different strains of T. lanuginosus were cultured in 170bs_bs_query

bs_bs_query 110 tions of lipases in the oleochemical industry lead to the 100 mL yeast-extract broth in 250 mL Erlenmeyer ﬂasks 2–4d 171bs_bs_query

bs_bs_query 111 manufacturing of useful products such as cocoa butter sub- at 120 rpm on a rotary shaker. Mycelium was harvested over 172bs_bs_query

bs_bs_query 112 stitute. However, several comprehensive reviews and a Büchner funnel and freeze dried overnight, and total genomic 173bs_bs_query

bs_bs_query 113 information are available on lipases produced by mesophilic DNA was extracted from approximately 50–100 mg pulver- 174bs_bs_query

bs_bs_query 114 fungi (Almeida et al., 2013; de Almeida et al., 2016; Edwinoliver ized mycelium with a cetyl trimethyl-ammonium bromide 175bs_bs_query

bs_bs_query 115 et al., 2010; Fernanda et al., 2015; Gutarra et al., 2009; (CTAB) method as suggested (Ramana et al., 2012). Thus, ob- 176bs_bs_query

bs_bs_query μ Q11 116 Hosseinpour et al., 2012; Maldonado et al., 2016; Mukherjeea tained genomic DNA was dissolved in 50 Lof2mmol/L Tris– 177bs_bs_query

bs_bs_query 117 and Gupta, 2016; Mukhtar et al., 2016; Nwuche et al., 2013; EDTA and the concentration was estimated using a NanoDrop 178bs_bs_query

bs_bs_query 118 Punitha et al., 2016; Rajan and Nair, 2011; Ramos-Sánchez et al., 2000c spectrophotometer (Thermo Scientiﬁc, Waltham, MA, 179bs_bs_query

bs_bs_query − 119 2015; Santhosh Kumar and Ray, 2014; Sarkar and Laha, 2013; USA). The DNA samples and stored at 20 °C for further analysis. 180bs_bs_query

bs_bs_query 120 Yogesh and Prabhune, 2011). To date only limited informa- 181bs_bs_query

bs_bs_query 121 tion is available on lipases produced by T. lanuginosus 2.7. Molecular identiﬁcation 182bs_bs_query

122bs_bs_query (Ávila-Cisneros et al., 2014). Therefore, in the present investi- 183bs_bs_query

123bs_bs_query gation, production of thermostable lipase by three strains of The morphological identiﬁcation of T. lanuginosus strains was 184bs_bs_query

124bs_bs_query T. lanuginosus was studied. further conﬁrmed by molecular methods by polymerase chain 185bs_bs_query

Please cite this article in press as: B. Sreelatha, V. Koteswara Rao, R. Ranjith Kumar, S. Girisham, S.M. Reddy, Culture conditions for the production of thermostable lipase by Thermomyces lanuginosus, Beni-Suef University Journal of Basic and Applied Sciences (2016), doi: 10.1016/j.bjbas.2016.11.010 ARTICLE IN PRESS beni-suef university journal of basic and applied sciences ■■ (2016) ■■– ■■ 3

186bs_bs_query reaction (PCR) by 28S rRNA gene using universal primers LROR activity and biomass attained by the fungus were assessed. The 246bs_bs_query ′ ′ 187bs_bs_query as forward 5 -ACCCCGCTGAACTTAAGC-3 and LR3R as reverse best medium was selected for further studies for the maximum 247bs_bs_query ′ ′ 188bs_bs_query Q12 primer 5 -GGTCCGTGTTTCAAG ACGG-3 (http://sites.biology enzyme production. 248bs_bs_query

189bs_bs_query .duke.edu/fungi/mycolab/primers.htm). The reaction was carried 249bs_bs_query

190bs_bs_query out in a Master cycler thermal cycler (Eppendorf) under con- 2.12. Effect of pH on lipase production 250bs_bs_query

191bs_bs_query ditions of 30 cycles of denaturation at 94 °C for 1 min, primer 251bs_bs_query

192bs_bs_query annealing at 58 °C for 1 min and extension at 72 °C for 1.3 min. The inﬂuence of pH on lipase production by the three strains 252bs_bs_query

193bs_bs_query The DNA was initially denatured for 4 min and ﬁnally ex- of T. lanuginosus was studied by adjusting the pH of the medium 253bs_bs_query

194bs_bs_query tended at 72 °C for 8 min. The ampliﬁed DNA product was ranging from 5.0 to 8.0 (5.0, 5.5, 6.0, 6.5, 7.0, 7.5 and 8.0) and 254bs_bs_query Q17 195bs_bs_query resolved by 10g/kg agarose gel electrophoresis visualized under grown for 9 days. The pH which supported maximum lipase 255bs_bs_query

196bs_bs_query a UV transilluminator. production was selected for further investigations. 256bs_bs_query

197bs_bs_query 257bs_bs_query

198bs_bs_query 2.8. Nucleotide sequence submission and 2.13. Effect of temperature on lipase production 258bs_bs_query

bs_bs_query 199bs_bs_query phylogenetic analysis 259

bs_bs_query 200bs_bs_query To ﬁnd the suitable temperature for lipase production, T. 260

201bs_bs_query The contig obtained was deposited in the NCBI GenBank da- lanuginosus GSLMBKU-10, T. lanuginosus GSLMBKU-13 and T. 261bs_bs_query

202bs_bs_query tabase available under T. lanuginosus strains GSLMBKU-10, 13, lanuginosus GSLMBKU-14 were inoculated and incubated at dif- 262bs_bs_query Q18 203bs_bs_query and 14 with accession nos. HE984155, HF545596 and HF570957 ferent temperatures (35, 40, 45, 50 and 55°C) for 9 days. The 263bs_bs_query

204bs_bs_query respectively. Sequences with high query coverage and homol- incubation temperature which was most suitable for lipase pro- 264bs_bs_query

205bs_bs_query ogy were selected for phylogenetic analysis using the Neighbour- duction was employed in further investigations. 265bs_bs_query Q13 206bs_bs_query Joining method (Saitou and Nei, 1987). Multiple sequence 266bs_bs_query

207bs_bs_query alignment of the obtained nucleotide sequence was done using 2.14. Assay of lipase activity 267bs_bs_query Q14 208bs_bs_query ClustalX analysis (Larkin et al., 2007), the aligned sequences 268bs_bs_query

209bs_bs_query were trimmed using DAMBE and the phylogenetic tree was con- Lipase activity was quantitatively assayed by the method sug- 269bs_bs_query Q19 Q15 bs_bs_query 210bs_bs_query structed using MEGA version 5 (Tamura et al., 2011). gested by Urs et al. (1962) (32) with some modiﬁcations. To 270

bs_bs_query 211bs_bs_query 2.0 mL of triacetin substratum, 5 mL of citrate buffer (pH 8.0), 271

bs_bs_query 212bs_bs_query 2.9. Screening of thermophilic fungi on lipase production 2 mL of enzyme and 0.5 mL of toluene were added. The reac- 272 ± bs_bs_query 213bs_bs_query tion mixture was incubated at 45 2 °C for 3 hrs. The reaction 273

214bs_bs_query The thermophilic strains were primarily screened for lipase pro- was terminated by adding 10 mL of absolute alcohol. The 274bs_bs_query

215bs_bs_query duction on lipase screening medium containing rhodamine B amount of acid produced was titrated with 0.05 N NaOH to a 275bs_bs_query

216bs_bs_query agar medium at pH 7.0 (Bornscheuer et al., 2002). The agar perceptible pink colour using 1.0 mL (1.0%) of phenolphtha- 276bs_bs_query

217bs_bs_query medium plates thus prepared were inoculated with 2 d old lein solution as an indicator. Simultaneously the blank was run 277bs_bs_query

± bs_bs_query 218bs_bs_query fungal disc and incubated at 45 2 °C for 48 h. Lipase produc- with 2.0 mL of distilled water in place of enzyme extract. The 278

219bs_bs_query tion was detected by irradiating the plates with UV light activity was calculated from the difference between the control 279bs_bs_query

220bs_bs_query (350 nm). Fungal colonies showing orange ﬂuorescent halo were and experimental titre value and expressed as lipase units/ 280bs_bs_query

221bs_bs_query considered positive for lipase production. mL of culture broth. (0.1 mL of 0.05 N NaOH required for 281bs_bs_query

bs_bs_query 222bs_bs_query neutralizing acid produced by quantity was taken as 1.0 unit). 282

bs_bs_query 223bs_bs_query 2.10. Effect of incubation period on lipase production 283

224bs_bs_query 2.15. Estimation of biomass 284bs_bs_query

225bs_bs_query The effect of incubation period on lipase production was studied 285bs_bs_query

bs_bs_query 226bs_bs_query in 250 mL of sterile yeast extract starch medium in a 500 mL At the end of incubation very period, cultures were har- 286

bs_bs_query 227bs_bs_query Erlenmeyer flask. These flasks were inoculating with 1.0 mL vested on pre-weighed Whatman filter paper No. 42. The fungal 287 −5 × bs_bs_query 228bs_bs_query (1 10 )ofT. lanuginosus GSLMBKU-10, T. lanuginosus GSLMBKU- mycelium was squeezed in the folds of Whatman filter paper 288

bs_bs_query 229bs_bs_query 13 and T. lanuginosus GSLMBKU-14 strains and incubated at No. 42 and dried in an oven at 60–75 °C for 48 hrs and weighed 289 Q20 ± bs_bs_query 230bs_bs_query 45 2 °C for 12 days. At the end of 3, 6, 9 and 12 days of incu- to a constant weight after cooling to room temperature in a 290

bs_bs_query 231bs_bs_query bation period, cultures were harvested on pre-weighed dessicator. 291

232bs_bs_query Whatman ﬁlter paper No. 42. From each incubation period, 292bs_bs_query

233bs_bs_query 30 mL cell-free culture ﬁltrate was taken under sterile condi- 2.16. Statistical analysis 293bs_bs_query

bs_bs_query 234bs_bs_query tion and centrifuged and used for lipase assay. Out of different 294

bs_bs_query 235bs_bs_query incubation periods, the optimum incubation period for The results were statistically analysed to mean and standard 295

bs_bs_query 236bs_bs_query maximum enzyme production was ﬁxed for further studies. deviation by using GraphPad Prism 5.0 version software 296

bs_bs_query 237bs_bs_query (GraphPad Software Inc., USA). 297

bs_bs_query 238bs_bs_query 2.11. Effect of synthetic media on lipase production 298

bs_bs_query 239bs_bs_query 299

bs_bs_query 240 To ﬁnd a suitable substratum for lipase production, the three 3. Results 300bs_bs_query

bs_bs_query 241 strains of T. lanuginosus were grown in yeast extract starch 301bs_bs_query

bs_bs_query 242 medium and yeast extract glucose medium and pH was ad- 3.1. Mycological examination of thermophilic fungi 302bs_bs_query

243bs_bs_query justed at 6.5, and the media were supplemented with (0.1%) 303bs_bs_query ± 244bs_bs_query triacetin oil and olive oil and incubated at 45 2°Cfor9days A total of ten genera representing sixteen species listed above 304bs_bs_query Q16 245bs_bs_query for lipase production. At the end of incubation period, lipase were isolated from dung samples of different animals viz., cow, 305bs_bs_query

Please cite this article in press as: B. Sreelatha, V. Koteswara Rao, R. Ranjith Kumar, S. Girisham, S.M. Reddy, Culture conditions for the production of thermostable lipase by Thermomyces lanuginosus, Beni-Suef University Journal of Basic and Applied Sciences (2016), doi: 10.1016/j.bjbas.2016.11.010 ARTICLE IN PRESS 4 beni-suef university journal of basic and applied sciences ■■ (2016) ■■– ■■

306bs_bs_query Fig. 1 – Genetic diversity of T. lanuginosus used in the present study.

307bs_bs_query

308bs_bs_query sheep, turkey, pigeon, duck, poultry, bear, rabbit, monkey, bird using a BLAST algorithm and deposited in NCBI based on evo- 359bs_bs_query

309bs_bs_query nest material, banana peel and zoo dump collected from various lutionary analysis. Fig. 1 shows that the isolated T. lanuginosus 360bs_bs_query

310bs_bs_query places in Warangal, Telangana, India (Sreelatha et al., 2013). strains were genetically different from each other due to the 361bs_bs_query

bs_bs_query 311bs_bs_query isolation source. These strains were deposited in GenBank with 362

Accession No. GSLMBKU-10 (HE984155), GSLMBKU-13 (HF545596) 363bs_bs_query 312bs_bs_query 3.2. Morphological identiﬁcation T. lanuginosus

364bs_bs_query 313bs_bs_query and GSLMBKU-14 (HF570957) and were isolated from banana Q22 365bs_bs_query 314bs_bs_query The isolated different strains of T. lanuginosus appear white at peel, bird nest material and zoo waste respectively.

bs_bs_query 315bs_bs_query ﬁrst, but soon turn gray at the centre of the colony and gradu- 366

316bs_bs_query ally turn purple brown on YES medium at 45 °C. The agar 3.4. Screening of thermophilic fungi on lipase production 367bs_bs_query

317bs_bs_query medium stains deep-pink or wine colour, due to the secre- 368bs_bs_query

318bs_bs_query tion of diffusible pigments. Mature colonies appear dull–dark- Of the different species, A. fumigatus, A. ﬂavus, H. grisea, H. 369bs_bs_query μ 319bs_bs_query brown to black. Hyphae were colourless, septate, and 1.5–4.0 m stellata, Rhizomucor miehei, Rhizomucor pusillus, R. arrhizus, M. 370bs_bs_query

320bs_bs_query in diameter. Conidiogenous cell arises at a right angle to the pulchella, T. thermophila and Torula lanuginosus were positive for 371bs_bs_query μ 321bs_bs_query hyphae, 10–15 m long, generally unbranched or rarely branched lipase production. The three strains of T. lanuginosus (GSLMBKU- 372bs_bs_query

322bs_bs_query once or twice near the base forming clusters, often septate, 10, GSLMBKU-13 and GSLMBKU-14) produced the more lipase 373bs_bs_query

323bs_bs_query conidia single on each conidiogenous cell, colourless, spheri- activity as evidenced by maximum orange ﬂuorescent halo. 374bs_bs_query

324bs_bs_query cal, smooth walled when young, turn dark-brown and These selected three strains were transferred into yeast extract 375bs_bs_query μ 325bs_bs_query Q21 sculptured at maturity, and 6–10 m in diameter. Separating starch medium for quantitative estimation of lipase activity 376bs_bs_query

326bs_bs_query easily from the conidiogenous cell and commonly retains a for maximum production suggesting their potential to secrete 377bs_bs_query

327bs_bs_query short attachment piece. Mature spores are spherical, irregu- lipase of lipase enzyme (Table 1). 378bs_bs_query μ bs_bs_query 328 larly sculptured, and range from 6 to 10 m in diameter. 379bs_bs_query

bs_bs_query 329 3.5. Effect of incubation period on lipase production 380bs_bs_query

330bs_bs_query 3.3. Molecular identiﬁcation 381bs_bs_query

331bs_bs_query The results of the present study showed that GSLMBKU-10 and 382bs_bs_query

332bs_bs_query The morphologically identiﬁed T. lanuginosus strains were further GSLMBKU-14 produced maximum lipase production on the 9th 383bs_bs_query

333bs_bs_query conﬁrmed with molecular identiﬁcation by polymerase chain day of incubation, whereas GSLMBKU-13 secreted maximum 384bs_bs_query

334bs_bs_query reaction. The obtained sequences were analysed at the Na- lipase production on the 12th day of incubation. The longer an 385bs_bs_query

335bs_bs_query tional Centre for Biotechnology Information (Bethesda, MD) enzyme is incubated with its substrate, the greater the amount 386bs_bs_query

336bs_bs_query (http://www.nbi.nlm.nih.gov/BLAST) for closed homology to of product that will be formed. Thus, the rate of formation of 387bs_bs_query

337bs_bs_query

338bs_bs_query

339bs_bs_query Table 1 – Lipase producing ability of different species of thermophilic fungi isolated from different substrates.

340bs_bs_query S. No. Lab. deposition strain no. Name of the fungus Source of isolation Lipase activity + 341bs_bs_query 1 GSLMBKU-1 Aspergillus fumigatus Cow dung + 342bs_bs_query 2 GSLMBKU-2 A. ﬂavus Turkey dung − 343bs_bs_query 3 GSLMBKU-3 Chaetomium thermophilum Sheep dung − 344bs_bs_query 4 GSLMBKU-4 Chrysosporium species Pigeon dung + 345bs_bs_query 5 GSLMBKU-5 Humicola grisea Monkey dung − 346bs_bs_query 6 GSLMBKU-6 H. insolens Sheep dung + 347bs_bs_query 7 GSLMBKU-7 H. stellata Pigeon dung + 348bs_bs_query 8 GSLMBKU-8 Malbranchea pulchella Rabbit dung + 349bs_bs_query 9 GSLMBKU-9 Rhizomucor miehei Duck dung +++ 350bs_bs_query 10 GSLMBKU-10 Thermomyces lanuginosus Banana peel + 351bs_bs_query 11 GSLMBKU-11 R. pusillus Zoo dump − 352bs_bs_query 12 GSLMBKU-12 Myriococcum albomyces Cow dung +++ 353bs_bs_query 13 GSLMBKU-13 T. lanuginosus Bird nest materials +++ 354bs_bs_query 14 GSLMBKU-14 T. lanuginosus Zoo waste − 355bs_bs_query 15 GSLMBKU-15 Penicillium duponti Zoo waste + 356bs_bs_query 16 GSLMBKU-16 Rhizopus arrhizus Zoo waste + 357bs_bs_query 17 GSLMBKU-17 Torula thermophila Sheep dung + 358bs_bs_query 18 GSLMBKU-18 T. lanuginosus Zoo waste

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388bs_bs_query Fig. 2 – Effect of incubation period on lipase production of different isolated strains of T. lanuginosus.

389bs_bs_query

390bs_bs_query product slows down as the incubation proceeds, and if the in- supplemented with triacetin by GSLMBKU-10 and GSLMBKU- 432bs_bs_query

391bs_bs_query cubation time is too long, then the measured activity of the 13, while GSLMBKU-14 preferred YES containing olive oil. The 433bs_bs_query

392bs_bs_query enzyme is falsely low (Fig. 2). least amount of growth of the fungi under study was re- 434bs_bs_query

bs_bs_query 393bs_bs_query corded in YEG medium. The rest of the media were intermediate 435

bs_bs_query 394bs_bs_query 3.6. Effect of synthetic media on lipase production in the induction of vegetative growth. Positive correlation was 436

bs_bs_query 395bs_bs_query observed between growth and lipase secretion only in the 437

bs_bs_query 396bs_bs_query In the present study, the three strains of T. lanuginosus pro- GSLMBKU-10 and GSLMBKU-13 strains. 438

397bs_bs_query Q23 duced signiﬁcantly varied amounts of lipase. However, the 439bs_bs_query

398bs_bs_query degree of production varied both with the medium and the

399bs_bs_query fungal strain. The maximum lipase was produced by GSLMBKU- 3.7. Effect of pH on lipase production 440bs_bs_query

bs_bs_query 400bs_bs_query 10 and GSLMBKU-13 in yeast extract starch medium with 441

bs_bs_query 401bs_bs_query triacetin (0.1%) as shown in Table 2. Yeast extract glucose The results of present investigation showed that the pH of the 442

bs_bs_query 402bs_bs_query medium was the next preferred substrate for lipase activity. medium signiﬁcantly inﬂuenced the lipase activity and veg- 443

bs_bs_query 403bs_bs_query Among the two oils supplemented to the two-different media, etative growth of the three strains of T. lanuginosus (Table 3). 444

bs_bs_query 404bs_bs_query yeast extract sucrose medium plus triacetin and olive oil stimu- Of the different pH values studied, the stimulated lipase pro- 445

bs_bs_query 405bs_bs_query lated and produced the highest lipase production by the duction by GSLMBKU-10, GSLMBKU-13 and GSLMBKU-14 446

bs_bs_query 406bs_bs_query GSLMBKU-10 strain. The yeast extract glucose medium with respectively was recorded at 6.0, 6.5 and 7.0. However, GSLMBKU- 447

bs_bs_query 407bs_bs_query triacetin also increased the lipase production by the three 13 strain was superior in lipase production among the three 448

bs_bs_query 408bs_bs_query strains of the fungi under investigation. However, olive oil was strains studied and its degree of production signiﬁcantly varied 449

bs_bs_query 409bs_bs_query responsible for the production of lipase by the GSLMBKU-10 with the pH of the medium. Lipase production decreased with 450

bs_bs_query 410bs_bs_query and GSLMBKU-13 strains. The T. lanuginosus GSLMBKU13 strain the increase of acidity or alkalinity. Lipase production by the 451

bs_bs_query 411bs_bs_query was comparatively superior in production of lipase than the GSLMBKU-10 strain was not observed at pH 8.0. However, other 452

bs_bs_query 412bs_bs_query other two strains under investigation. Addition of lipid triac- strains secreted decreased lipase production. The highest veg- 453

bs_bs_query 413bs_bs_query etin to the medium did not make much difference in the etative growth of all strains under study was recorded at pH 454

bs_bs_query 414bs_bs_query production of lipase by all the three strains, suggesting their 6.0. On the other hand, the biomass of the tested strain de- 455

bs_bs_query 415bs_bs_query constitutive nature as compared to olive oil. The highest veg- creased with increased acidity or alkalinity. No growth was 456

bs_bs_query 416bs_bs_query etative growth of the fungi was recorded with YES medium observed at pH 8.0 by the GSLMBKU-10 strain. 457

417bs_bs_query

418bs_bs_query

419bs_bs_query Table 2 – Effect of culture medium on vegetative growth and lipase production by different isolated strains of a 420bs_bs_query T. lanuginosus.

421bs_bs_query Medium GSLMBKU-10 GSLMBKU-13 GSLMBKU-14 μ μ μ 422bs_bs_query Growth ( g/mL) Lipase (IU/mL) Growth ( g/mL) Lipase (IU/mL) Growth ( g/mL) Lipase (IU/mL) ± ± ± ± ± ± 423bs_bs_query YES 183.30 1.18 15.23 1.07 187.67 0.70 31.70 0.82 155.50 2.18 16.70 0.75 + ± ± ± ± ± ± 424bs_bs_query YES triacetin 205.80 1.13 29.33 0.85 225.30 1.18 38.73 0.75 165.23 1.07 14.63 0.93 ± ± ± ± ± ± 425bs_bs_query YEG 140.20 1.15 8.77 0.80 147.20 1.31 16.70 0.75 132.97 1.19 9.61 0.61 + ± ± ± ± ± ± 426bs_bs_query YEG triacetin 141.87 0.61 12.90 0.78 164.20 1.15 18.77 0.86 151.73 0.87 12.57 0.67 + ± ± ± ± ± ± 427bs_bs_query YES olive oil 175.87 0.76 20.67 0.83 196.70 0.82 28.70 0.82 167.33 0.76 16.67 0.31 + ± ± ± ± ± ± 428bs_bs_query YEG olive oil 171.30 1.28 11.73 0.81 188.67 0.70 23.90 0.82 162.30 1.18 8.40 0.36

429bs_bs_query YES, yeast extract sucrose; YEG, yeast extract glucose. Q30 a 430bs_bs_query Enzyme activity is expressed in units (0.1 mL, 0.05 N NaOH required quantity was taken as 1 unit of enzyme activity).

431bs_bs_query

Please cite this article in press as: B. Sreelatha, V. Koteswara Rao, R. Ranjith Kumar, S. Girisham, S.M. Reddy, Culture conditions for the production of thermostable lipase by Thermomyces lanuginosus, Beni-Suef University Journal of Basic and Applied Sciences (2016), doi: 10.1016/j.bjbas.2016.11.010 ARTICLE IN PRESS 6 beni-suef university journal of basic and applied sciences ■■ (2016) ■■– ■■

a 458bs_bs_query Table 3 – Effect of pH on vegetative growth and lipase production by different isolated strains of T. lanuginosus.

459bs_bs_query pH GSLMBKU-10 GSLMBKU-13 GSLMBKU-14 μ μ μ 460bs_bs_query Growth ( g/mL) Lipase (IU/mL) Growth ( g/mL) Lipase (IU/mL) Growth ( g/mL) Lipase (IU/mL) ± ± ± ± ± ± 461bs_bs_query 5.0 165.19 1.11 4.27 0.31 128.67 0.58 6.33 0.31 145.37 0.75 6.27 0.23 ± ± ± ± ± ± 462bs_bs_query 5.5 194.27 1.30 9.37 0.32 157.60 1.25 8.97 0.35 168.60 0.60 8.71 0.28 ± ± ± ± ± ± 463bs_bs_query 6.0 225.50 0.78 20.23 1.25 189.83 0.72 15.60 0.87 219.90 0.78 13.82 0.71 ± ± ± ± ± ± 464bs_bs_query 6.5 197.23 1.10 15.70 0.82 180.17 1.11 25.17 1.16 208.37 1.10 16.99 0.47 ± ± ± ± ± ± 465bs_bs_query 7.0 156.73 0.38 10.50 0.70 161.20 0.17 18.37 0.35 145.90 0.56 23.43 0.63 ± ± ± ± ± ± 466bs_bs_query 7.5 124.43 1.36 7.03 0.45 135.00 1.00 14.60 0.87 128.73 0.87 12.73 0.78 ± ± ± ± ± ± 467bs_bs_query 8.0 00.00 0.00 0.00 0.00 121.67 1.15 13.00 1.00 109.63 0.65 3.56 0.56

a 468bs_bs_query Enzyme activity is expressed in units (0.1 mL, 0.05 N NaOH required quantity was taken as 1 unit of enzyme activity).

469bs_bs_query

470bs_bs_query

488bs_bs_query

471bs_bs_query 3.8. Effect of temperature on lipase production

4. Discussion 489bs_bs_query 472bs_bs_query

490bs_bs_query 473bs_bs_query Result of the temporal studies on lipase production reveals that

474bs_bs_query the temperature range of 45–50 °C was optimum for both veg- Lipases are ubiquitous in nature and are produced by various 491bs_bs_query

475bs_bs_query etative growth and lipase production by the three strains of plants, animals, and microorganisms. However, to produce in- 492bs_bs_query

476bs_bs_query T. lanuginosus under study. The lipase production was de- dustrial lipases, microorganisms are the most preferred source 493bs_bs_query

477bs_bs_query creased with increase or decrease of incubation temperature (Shivika and Shamsher, 2014). Microbial lipases are currently 494bs_bs_query

478bs_bs_query (Fig. 3). Temperature of 45 °C was optimum for secretion of lipase receiving much attention with the rapid development of enzyme 495bs_bs_query

479bs_bs_query by GSLMBKU-14 and GSLMBKU-10, while that for GSLMBKU- technology and due to their ability to perform catalysis at ex- 496bs_bs_query

480bs_bs_query Q24 13 was at 50 °C. The rates of both the vegetative growth of fungi tremes of temperature, pH, and organic solvents. Also, lipase 497bs_bs_query

481bs_bs_query and lipase production decreased signiﬁcantly at an incuba- production by microorganisms varies according to the strains, 498bs_bs_query

482bs_bs_query tion temperature of 55 °C (Fig. 4). However, the maximum dry the composition of the growth medium, cultivation condi- 499bs_bs_query

483bs_bs_query weight of the three strains was recorded at 45 °C. tions, pH, temperature, and nutritional factors such as carbon 500bs_bs_query

484bs_bs_query

485bs_bs_query Fig. 3 – Effect of temperature on lipase production by different isolated strains of T. lanuginosus.

486bs_bs_query

487bs_bs_query Fig. 4 – Effect of temperature on vegetative growth of different isolated strains of T. lanuginosus.

Please cite this article in press as: B. Sreelatha, V. Koteswara Rao, R. Ranjith Kumar, S. Girisham, S.M. Reddy, Culture conditions for the production of thermostable lipase by Thermomyces lanuginosus, Beni-Suef University Journal of Basic and Applied Sciences (2016), doi: 10.1016/j.bjbas.2016.11.010 ARTICLE IN PRESS beni-suef university journal of basic and applied sciences ■■ (2016) ■■– ■■ 7

501bs_bs_query and nitrogen sources (Treichel et al., 2010). Fungi can produce no lipase production at pH 8.0 by GSLMBKU-10. Temperature 561bs_bs_query

502bs_bs_query several enzymes for their survival within a wide range of sub- is a variable that affects enzyme production, and in this case 562bs_bs_query Q27 503bs_bs_query strates. Among those enzymes, lipases are predominantly used an increase in temperature within the range. Results of the tem- 563bs_bs_query

504bs_bs_query in several applications (Abhijit, 2012). These fat-splitting perature study showed that 45 °C was optimum for secretion 564bs_bs_query

505bs_bs_query enzymes are attractive because of their applications in ﬁelds of lipase activity and showed an increasing trend of lipase pro- 565bs_bs_query

506bs_bs_query relevant to medicine and dairy industries (Kumar et al., 2016). duction up to 50 °C, and the enzyme activity decreased 566bs_bs_query

507bs_bs_query In the present study, screening of different thermophilic fungi signiﬁcantly at 55 °C (Falony et al., 2006). Thermophilic fungi 567bs_bs_query

508bs_bs_query for production of lipase enzyme revealed their potential to are a small assemblage in mycobiota, which have a minimum 568bs_bs_query

509bs_bs_query produce the enzyme. Further, the present study on the three temperature of growth at or above 20 °C and a maximum growth 569bs_bs_query

510bs_bs_query strains of T. lanuginosus also proved that they are an excellent at moderately high temperature yet limit their growth beyond 570bs_bs_query

511bs_bs_query source of thermostable lipase enzyme. Production of this 60–62 °C (Maheshwari et al., 2000). The optimum tempera- 571bs_bs_query

512bs_bs_query enzyme proved to be varying with the substratum and differ- ture for lipase production by Aspergillus terreus NCFT 4269.10 572bs_bs_query

513bs_bs_query ent environmental factors. Results of the investigation of the strain at 60 °C was reported (Sethi et al., 2016). The proper- 573bs_bs_query

514bs_bs_query lipolytic potential of the three selected strains showed that they ties of their enzymes show differences not only among species 574bs_bs_query

515bs_bs_query Q25 were also able to secrete lipase by the rhodamine B method but also among strains of the same species. Their extracellu- 575bs_bs_query

516bs_bs_query (Abrunhosa et al., 2013; Savitha et al., 2007). This method is lar lipases play a signiﬁcant effect on temperature optima for 576bs_bs_query

517bs_bs_query useful for the preliminary screening of lipase producing species. activity that are close to or above the optimum temperature 577bs_bs_query

518bs_bs_query The present fungal strains were stimulated to produce lipase for the growth of T. lanuginosus and, in general, are more heat 578bs_bs_query

519bs_bs_query in the presence of either triacetin or olive oil suggesting the stable than those of the mesophilic fungi. 579bs_bs_query

bs_bs_query 520bs_bs_query adaptive nature of their lipases. The adaptive nature of the po- 580

581bs_bs_query 521bs_bs_query tential of producing lipase by thermophilic fungi may probably

522bs_bs_query contribute to the deterioration of oil seeds during storage. Gen- 5. Conclusion 582bs_bs_query

bs_bs_query 523 erally, fungi can hydrolyse different lipid substrates (Charles 583bs_bs_query

524bs_bs_query and James, 2011), but a few fungi are incapable of degrading Data obtained from the present investigation showed that the 584bs_bs_query

525bs_bs_query lipids with the help of lipase. To achieve improved lipase pro- Q28 three strains isolated from diversiﬁed habitatshavewide genetic 585bs_bs_query

526bs_bs_query ductivity, variation in cultural and environmental parameters diversity. Also, efﬁcacy of culture medium, pH and tempera- 586bs_bs_query

527bs_bs_query has been investigated (Treichel et al., 2010; Veronica et al., 2010). ture stimulated lipase production by T. lanuginosus. These 587bs_bs_query

528bs_bs_query Fungal lipases which degrade lipids from palm oil have been isolated strains are capable of secreting novel thermostable 588bs_bs_query

529bs_bs_query reported (Wongwatanapaiboon et al., 2016). In general, it can lipase enzymes at high temperature. These lipases are candi- 589bs_bs_query

530bs_bs_query be stated that lipase production by fungi requires a some- dates for industrial applications for separation of racemic 590bs_bs_query

531bs_bs_query what higher concentration of nitrogen source than that of other mixtures, hydrolysis of toxigenic compounds and several trans- 591bs_bs_query

532bs_bs_query microbial nutrients. M. albomyces produced a substantial amount formation reactions. Further studies are also need to enhance 592bs_bs_query

533bs_bs_query of lipase and protease under submerged conditions at 45 °C lipase production by T. lanuginosus GSLMBKU13 strain in order 593bs_bs_query

534bs_bs_query in a medium containing soya meal, cornmeal and olive oil. The to determine the real level of lipase production. 594bs_bs_query

535bs_bs_query fungal species that were also isolated from oil-contaminated 595bs_bs_query

536bs_bs_query soil, compost heaps, coal tips, and industrial wastes were the 596bs_bs_query Q26 537bs_bs_query higher lipase producers (Cihangir and Sarikaya, 2004; D’Annibale Ethical statement 597bs_bs_query

538bs_bs_query et al., 2006; Gunasekaran and Das, 2005; Prabhakar et al., 2012). 598bs_bs_query

539bs_bs_query Trichoderma sp. produced the higher lipase out of twelve fungal

This article does not contain any studies with human or animal 599bs_bs_query

540bs_bs_query species isolated from oil-mill efﬂuent composts (Nwuche and

subjects performed by any of the authors. 600bs_bs_query

541bs_bs_query Ogbonna, 2011). Amin and Bhatti (2014) reported that canola 601bs_bs_query

542bs_bs_query oilseed used as substrates induced good lipase activity for the 602bs_bs_query

543bs_bs_query lipase production by Penicillium fellutanum. Acknowledgement 603bs_bs_query

544bs_bs_query The present investigations aimed at optimizing physical

604bs_bs_query 545bs_bs_query factors such as culture medium, pH and temperature for lipase

The authors are thankful to the Head, Department of Micro- 605bs_bs_query 546bs_bs_query production by three strains of T. lanuginosus revealed a signiﬁ-

biology, Kakatiya University, for providing the necessary 606bs_bs_query 547bs_bs_query cant role (Mukherjeea and Gupta, 2016). In the present

facilities. 607bs_bs_query 548bs_bs_query investigation, pH signiﬁcantly inﬂuenced both the biomass and

608bs_bs_query 549bs_bs_query lipase activity. The optimum pH for highest lipase produc-

609bs_bs_query

550bs_bs_query tion by T. lanuginosus was recorded at 6.5. However, its degree Q31 Uncited references 610bs_bs_query 551bs_bs_query of production varied with the incubation of initial pH. The mar-

611bs_bs_query 552bs_bs_query ginal pH for the highest lipase production by the three

553bs_bs_query thermophilic strains assayed was recorded at 6.0–7.0. The Singh et al, 2000 612bs_bs_query

bs_bs_query 554 present investigation was positively correlated with the reports 613bs_bs_query

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