Studies Of Inulinase Production And Characterization By Renicillium Sp.B01 Isolated From Soil

Inulin is a widespread polyfructan in plants, consisting of linear ofβ-(2,1)-linked fructose residues attached to a terminal glucose molecule. This storage polymer is accumulated in underground organs of several plants of Asteraceae and Compositae families such as Helianthus tuberosus L. 70%-80% of the dry material of the tube of jerusalem artichoke is inlulin. Inulin has received a great interest as it represents a relatively inexpensive and abundant substrate for production of high fructose syrup. It is a possible material for ethanol production. Microbial inulineses(2,1-β-D fructan fructano hydrolase, EC 3.2.1.7) are usually inducible and exo-acting enzymes, which catalyze the hydrolysis of inulins by splitting off terminal fructosyl units(D-fructose). Microbial inulineses have a very important function on commerce exploitation of inulin hydrolyzation.Different soil samples were collected from Jerusalem artichoke rhizosphere soil in the salt in the salt-affected lands of Laizhou, Shangdong province. More than 40 strains include bacterial, streptomyces, fungal were isolated. Through further screening, a fungal strain have a higher ability of producing inulinase. This strain was identified to be Penicillium sp.B01 primarily.After optimizing its fermentation condition, the optimum condition for higher inulinase production of Penicillium sp.B01 was determined as shown below(g·L-1): extract of Jerusalem artichoke 120, yeast extract 25, NHaH2PO4 2.5, NaCl 5.0, FeSO₄·7H₂O 0.1, ZnSO₄·7H₂O 0.1, pH 7, inoculated with 3.75% of spore suspensions(2.5×10⁶ spores). Maximal inulinase activity of Penicillium sp.B01 was 25.78 U·mL-1 and obtained after 72 h of growth in shake cultures (40 mL of medium in 250 mL flasks).We studied the fermentative dynamics of inulinase by flask shaking under the optimal condition mentioned above. The result demonstrates that growth of Penicillium sp.B01 entered into stationary phase after 48 h and entered into contabescence phase after 144 h. the highest yield of inulinase is obtained at 120 h. It declined when fermentation continues.The effect of pH, temperature, concentration of substrate, metal iron and reaction time on activity of inulinase was studied. From 3.6 to 5.4, the enzyme was stable and maintained high enzyme activity. The optimal pH is 4.6. The reaction rate varied under different temperature. The optimal temperature was 55℃, Inulinase could be stable at or below 60℃. Ca²⁺ induced the inulinase activity.The EJA were totally hydrolyzed on the conditions of concentration of substrate 5%, enzyme 8 U/g Jerusalem artichoke juice, temperature 55℃, pH4.6 and reaction time 6 h. When inulinase was used to produce high fructose syrups, the optimal conditions were: temperature 55℃, substrate concentration 10%-20%, enzyme 6 U/g Jerusalem artichoke juice and reaction time 8-10 hours.The products of the enzyme reaction were analysed by TLC. The result showed inulooligofructoses decreased in time course and the major resultants from enzymatic hydrolysis were fructose. The I/S ratio of inulinase was 1.21. the resultant analysis showed that the enzyme are exoinlinase.Native-PAGE was performed to separate crude enzyme. Protein was stained with Coomassie Brilliant Blue R-250. Eight protein bands were gained. Activity stained was carried out by TTC and three active piece were visualized. The protein bands were cut into 5mm for further utilization of the enzymes. The products of the enzyme digestion were tested by TLC and HPLC.There is pure exoinulinase activity from 3th band and 4th band because only monosaccharides are released from inulin by TLC.HPLC of the inulin hydrolysate showed that the inulin hydrolysate from bands 1,2,3,4 had an obvious effect on fructose increase. Bands 5,6 had no noticeable effect on inulin. It is therefore suggected that bands 1,2,3,4 were exoinulinases.Test of activity stained by TTC, TLC and HPLC all identified the 3th band showed high inulinase activity. It was demonstrated that the 3th band was exoinulinases.