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

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 55C), 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 45C, 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 65C.


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

Agar Plates preparation
The following amounts of chemical composition were used for the preparation of agar plates as by. [2]  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.

. 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]

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.

Preparation of Inoculum
For yeast medium the inocula was prepared according to the following composition (w/v): [3] Journal

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 o C 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. 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 (30 o C to 55 o C ) in fermentation process in order to investigate the optimized temperature for enzyme production. At 45 o C temperature the maximum production of extracellular enzyme was obtained. The extracellular enzyme production was (890 µmoles/min/g).   0  35  120  60  40  90  140  45  60  150  50  25  140  55  5  130  In table 5, the various temperatures (30 o C to 55 o C ) was applied from (30 o C to 55 o C ) in fermentation process in order to determine optimized temperature for production of enzyme. The best temperature was 45 o C for production of intercellular enzyme. The intercellular enzyme production was (150 µmoles/min/g).

CONCLUSION
It is concluded that the indigenous strain Kluyveromyces marxianus can work at high temperature up to 65 0 C 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.

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.