ARTICLE IN PRESS beni-suef university journal of basic and applied sciences ■■ (2016) ■■– ■■ HOSTED BY Available online at www.sciencedirect.com ScienceDirect journal homepage: www.elsevier.com/locate/bjbas 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 14bs_bs_query 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 33bs_bs_query © 2016 Production and hosting by Elsevier B.V. on behalf of Beni-Suef University. This is 34bs_bs_query Temperature 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 48bs_bs_query 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 54bs_bs_query 2314-8535/© 2016 Production and hosting by Elsevier B.V. on behalf of Beni-Suef University. This is an open access article under the 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