This study is focused on enzymolgy regarding enzyme purification technique and application of thermotolerant specie Kluyveromyces marxianus yeast in bio reactions for the research and optimization of fermentation temperature for intracellular and extracellular enzyme production. Fermentation studies were carried out in shake flask level to optimize intracellular and extracellular enzyme production by changing  process conditions like temperature range from (30 to 550C), the pH (5.5), speed (350).  The substrate type sugar cane molasses 15% added as a carbon and ammonium sulphate (0.75%) as nitrogen source. The optimized fermentation temperature was found 45oC, at pH 5.5, and rpm was 350. The production of  intracellular invertase was (890µmoles/min/g) while the extracellular (120µmoles/min/g) Kluyveromyces marxianus was greater efficiency as compared to other specie because of its metabolic activity, which express more heat stability and Invertase activty upto 650C. Keywords: Yeast, Fermentation, K.marxianus DOI: 10.7176/JNSR/9-9-02 Publication date:May 31st 201

Laghari, A.Q

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Journal of Natural Sciences Research                                                                                                                                                www.iiste.org ISSN 2224-3186 (Paper)   ISSN 2225-0921 (Online) DOI: 10.7176/JNSR Vol.9, No.9, 2019  12 Effect of Temperature on Production Intracellular and Extracellular Invertase by Potential Indigenous Strain Kluyveromyces Marxianus Using Sugarcane Molasses  A.Q Laghari Chemical Engineering Department, Mehran University of Engineering & Technology, Jamshoro, Pakistan  S.Aziz Chemical Engineering Department, Mehran University of Engineering & Technology, Jamshoro, Pakistan  Arshad Iqbal Chemical Engineering Department, Mehran University of Engineering & Technology, Jamshoro, Pakistan  A.Sami Chemical Engineering Department, Quaid-e-Awam University of Engineering Science & Technology,Nawbshah, Pakistan  Farman Ali Shah Mehran University of Engineering & Technology, Jamshoro, Pakistan  Abstract This study is focused on enzymolgy regarding enzyme purification technique and application of thermotolerant specie Kluyveromyces marxianus yeast in bio reactions for the research and optimization of fermentation temperature for intracellular and extracellular enzyme production. Fermentation studies were carried out in shake flask level to optimize intracellular and extracellular enzyme production by changing  process conditions like temperature range from (30 to 550C), the pH (5.5), speed (350).  The substrate type sugar cane molasses 15% added as a carbon and ammonium sulphate (0.75%) as nitrogen source. The optimized fermentation temperature was found 45oC, at pH 5.5, and rpm was 350. The production of  intracellular invertase was (890µmoles/min/g) while the extracellular (120µmoles/min/g) Kluyveromyces marxianus was greater efficiency as compared to other specie because of its metabolic activity, which express more heat stability and Invertase activty upto 650C. Keywords: Yeast, Fermentation, K.marxianus DOI: 10.7176/JNSR/9-9-02 Publication date:May 31st 2019  1.Introduction  Nowadays, most of the people are suffer from daises due insufficient nutrient supply in the diets of human and feed of animals food[10]. Because of to this inconvenience, it is essential to propagate the manufacturing of protein with the help of all available technique. The situation of malnutrition in people living in developed and developing countries have become a growing concern as a result of deficiencies in food protein[11]. With the rapid growth of the world's population, high-pressure effects on the industry of food and the feed for producing sufficient human food and animal feed to meet demands of nutrition. The continual population resource of developing countries calls for increased and improved supply of human food and animal feed. The world's growing demand for protein rich foods has affected the formulation of alternative protein sources to be counterpart to conventional protein sources. This growing world demand for food and feed proteins has led to the search for unconventional protein sources for supplementing conventional protein source. The companies dealing with animals feed are also facing under priced and over affected by conventional components, [1]   2. MATERIALS AND METHODS 2.1  Purification of Strain Purification of current strain Kluyveromyces marxianus was done at biochemical engineering laboratory of Chemical engineering Department Mehran University of Engineering and Technology Jamshoro. [2]    Journal of Natural Sciences Research                                                                                                                                                www.iiste.org ISSN 2224-3186 (Paper)   ISSN 2225-0921 (Online) DOI: 10.7176/JNSR Vol.9, No.9, 2019  13 2.2  Maintenance of Culture  The practical work was carried out at biochemical laboratory of Department Chemical Engineering Mehran University of Engineering & Technology Jamshoro. All analytical greade chemicals, glassware were purchased, oxide, and Dae-Jung companies from AL-Bourne and Shabbir Scientific Store Hyderabad. The black strip liquid (molasses) for fermentation was purchased from Khairpur and Rani pur District hairpur Mir's. Kluyveromyces marxianus culture was maintained as per methood [5,6] on Saboraud’s Dextrose agar (SDA) slants and plates. Medium was prepared by the mixing of different analytical grade chemicals into distilled water one by one. The shaking volume was made up to 100 ml in an Erlenmeyer flask of 250 ml capacity. the pH of the medium was maintained up to 5.5 by using HCl and NaOH  2.3  Agar Plates preparation The following amounts of chemical composition were used for the preparation of agar plates as by. [2] TABLE I.  CHEMICALS OF NUTRIENT AGAR PLATE Chemicals % (w/v) Agar 3.0 Glucose 2.0 Peptone 0.5 Sodium chloride 0.5 Yeast extract 1.0 Above chemical composition were used for the preparation of nutrient  agar plates method mentioned by. [2,3]. These chemicals used as medium for the growth of microorganisms. about 50 ml of distilled water was poured in 250 ml Erlenmeyer flask followed their shaking. After that further water is added maintained up to 100 ml and HCL  and Sodium NaOH used for proper maintenance of pH upto5.5.  Fig. 1. Kluyveromyces marxians strain before purification   Fig. 2. Kluyveromyces marxians strain after purification Purity of culture was checked in compound microscope before preparing inoculum the Fig. 1 shows the growth of  Kluyveromyces marxians strain before purification when it was at raw state, after treatment the strain was purified shown in Fig. 2 for the proper application in the fermentation process for the production intracellular and extracellular enzyme production.[7,8,9]  2.4  Sterilization The media was sterilized, at 121°C, 15 psi pressure for 15 min. then purity of medium was confirmed after different time interval ,24,48,72 hours. (Madihah et al., 2008; Madigan and Martinko, 2005).  2.5  Preparation of Inoculum For  yeast medium the inocula was prepared according to the following composition (w/v):[3]   Journal of Natural Sciences Research                                                                                                                                                www.iiste.org ISSN 2224-3186 (Paper)   ISSN 2225-0921 (Online) DOI: 10.7176/JNSR Vol.9, No.9, 2019  14 TABLE II. CHEMICAL COMPOSITION OF INOCULUM COMPOUND Chemical  % (w/v) Yeast Extract 1.0  Sodium Chloride   0.5 Glucose 2.0 Peptone 0.5 PH 5.5  2.6  Preparation of Fermentation Medium Following composition of chemicals are use for the media preparation for fermentation.[2] TABLE III: COMPOSITION OF CHEMICAL FOR FERMENTATION MEDIA Chemical (%)age Carbon source (Sugar cane molasses)  15 Nitrogen source (Di ammonium sulphate) 0.75 PH 5.5 Inoculums 0.5  3.  RESULT AND DISCUSSION Fermantation   studies  were undertaken  to  optimize  the temperature  by  changing process temperature. fermentation for  growth of K. marxianus, in  the presence of various temperatures, (30°C to 65°t C) speed (350 rpm) pH (5.5) for the substrate consumption and invertase production. Molasses were employed to study  their  effect  on  growth  and  production. The intracellular and the extracellular enzyme production at 45 oC by indigenous strain K. marxianus with 15 %  sugar  concentration of substrate gave the maximum amount of intracellular (890 µmoles/min/g) and the extracellular (150 µmoles/min/g) enzyme. The optimum intracellular and the extracellular enzyme was  observed  after  48h of fermentation with media containing blackstrap molasses (15%  total reducing sugars), the optimized temperature was 45°C, pH 5.5 and speed 300 rpm. Table IV:  Effect of temperature on the extracellular enzyme production at pH 5.5, 350rpm and 48 hours. Temp= oC Sugar/Molasses g/l Extracellular Activity (µmoles/min/g) 30 150 0 35 120 450 40 90 700 45 60 890 50 25 850 55 5 800 The above mentioned table IV, the various temperatures was applied from (30oC  to 55oC ) in fermentation process in order to investigate the optimized temperature for enzyme production. At 45oC temperature the maximum production of extracellular enzyme was obtained. The  extracellular enzyme production was (890 µmoles/min/g).   Fig. 3. Temperature Effect On The Production Of Intracellular Activity at various temperatures.      Journal of Natural Sciences Research                                                                                                                                                www.iiste.org ISSN 2224-3186 (Paper)   ISSN 2225-0921 (Online) DOI: 10.7176/JNSR Vol.9, No.9, 2019  15 Table V.  Effect of temperature on the extracellular enzyme production at pH 5.5, 350rpm and 48 hours. Temp= oC Sugar/Molasses g/l Intracellular activity (µmoles/min/g) 30 150 0 35 120 60 40 90 140 45 60 150 50 25 140 55 5 130 In table 5, the various temperatures (30oC  to 55oC ) was applied from (30oC  to 55oC ) in fermentation process in order to determine optimized temperature for production of enzyme. The best temperature was 45oC for production of intercellular enzyme. The intercellular enzyme production was (150 µmoles/min/g).   Fig. 4. Temperature Effect On The Production Of Intracellular Activity at various temperatures.  4. CONCLUSION It is concluded that the indigenous strain Kluyveromyces marxianus can work at high temperature up to 65 0C and at optimum temperature 40°C, pH 5.5, and speed 300rpm it gives maximum intracellular and the extracellular enzyme production. It is economically feasible for large scale production because it reduces the cooling cost. In Pakistan it can be use to produce the enzyme which can be utilize as food supplement to overcome on the malnutrition problem.  5. ACKNOWLEDGEMENT My deepest feeling of thanks and appreciations are to my Department of Chemical Engineering, Mehran University of Engineering and Technology Jamshoro, Pakistan for providing me best research facilities and a great deal of knowledge that helped me to achieve my goals and Objective of my research. I feel a great pleasure and honor to express my heart full gratitude to my supervisor Prof. Dr. Shaheen Aziz, and Co-supervisor, Prof. Dr. Syed Farman Ali Shah, for their proper guide line, technical support and sympathetic attitude throughout my experimental work at MUET.  References [1] G. Suman, M. Nupur, S. Anuradha, B. Pradeep"International Journal of current micro biology and applied science" Single Cell Protein Production, Vol. 4, No. 9, pp. 251-262, 2015 [2] S. Aziz, H. Rehman Memon, F. Ali Shah,M.Ibrahim Rajoka, S. Ahmed Soomro” Production of Ethanol by Indigenous Wild and Mutant Strain of Thermotolerant Kluyveromyces Marxianus Under Optimized Fermentation Conditions” Pak. J. Anal. Environ. Chem. Vol. 10, No. 1 & 2 2533, 2009 [3] F. Ali Shah, S. Aziz, H. Rahman Memon, Z. M Ali, M.Ibrahim Rajoka ”Enhanced Production of Invertase from Thermoltolerant Yeast Through Black Strap Molasses a Waste Prodcut of Sugar Industry” Australian Journal of Basic and Applied Sciences, Vol.5 No.1, 48-54, 2011  [4] M. Shuler "Investigation of the Performance and Kinetics of Anaerobic Digestion at 45°C "Journal of Water Resource and Protection. Vol.7 No.14, 2015 [5] M. Ibrahim Rajoka, F. Latif, S. Khan and R. Shahid, "Kinetics of improved productivity of ß galactosidase by a cycloheximide resistant mutant of Kluyveromyces marxianus. Biotechnol" Lett. 26 (2004) 714. [6] M. Ibrahim Rajoka, S. Khan, R. Shahid" Regulation of Galactosidase Production from K. marxianus" Food Technol. Biotechnol. Vol.41, No.4, pp.315–320 2003 [7] I. Banat, R.Merchant, “Isolation of thermotolerant, fermentative yeasts growing at 52 oC and producing ethanol Journal of Natural Sciences Research                                                                                                                                                www.iiste.org ISSN 2224-3186 (Paper)   ISSN 2225-0921 (Online) DOI: 10.7176/JNSR Vol.9, No.9, 2019  16 at 45 o C and 50 o C”. World J. Microbial Biotech.V.8, pp. 259-263, 1992. [8] S. Limtong, W.Yongmanitchai, “Production of fuel ethanol at high temperature from sugar cane juice by a newly isolated Kluyveromyces marxianus,” Letters in App: Microbial. V. 98, pp.3367-3374. 2007 [9] Brady, D. Logan, S.R, McHale, “The effect of soluble alginate and calcium on b-galactosidase activity produced by the thermotolerant, ethanol-producing yeast strain Kluyveromyces marxianus” Bioproc Eng, V.18, pp. 101–104, 2007 [10] U. Bacha, M. Nasir, A. Khalique, A. A. Anjum, and M. A. Jabbar" comparative assessment of various agro-industrial wastes for saccharomyces cerevisiae biomass and its quality evaluation as single cell protein" The Journal of Animal & Plant Sciences, Vol. 21, No.4, pp. 844-849, 2011, Page:  [11] K. Hanim, A.Rahman, S. J Hanim, M. Yusof Z. Zakaria"Bioproteins Production from Palm Oil Agro-Industrial Waste"Pertanika J. Trop. Agric. Sci. Vol.39, No. 1, pp. 29 - 39, 2016 

Effect of Temperature on Production Intracellular and Extracellular Invertase by Potential Indigenous Strain Kluyveromyces Marxianus Using Sugarcane Molasses

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Effect of Temperature on Production Intracellular and Extracellular Invertase by Potential Indigenous Strain Kluyveromyces Marxianus Using Sugarcane Molasses

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