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Production of Naringinase by Submerged Fermentation with Penicillium sp.1523 and Immobilization of the Enzyme

Author: CuiPeiWu
Tutor: LiJiLie
School: Central South University of Forestry Science and Technology
Course: Fermentation Engineering
Keywords: Naringinase Response Surface Optimization Fermentation kinetics Purification Immobilized
CLC: TQ925
Type: Master's thesis
Year: 2009
Downloads: 53
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Abstract

The the Naringinase (Naringinase) enzyme complex is composed of Al-rhamnosidase enzyme (aL-rhamnosidase, EC 3.2.1.40) and β-D-glucosidase (flavonoid-beta-D-glucosidase, EC 3.2.1.21) body citrus juice bitter substances naringin two steps the rhamnose hydrolysis into naringenin (Naringenin) and glucose so as to achieve the purpose of debittering; compared with the other debittering the the law debittering has The relatively mild reaction conditions, and can better maintain the quality of citrus juice, simple process, environmental pollution and other advantages thus ideal debittering method; enzymatic modification of natural plant flavonoids sugar can also be used in addition Naringinase chain to improve the biological activity of natural plant flavonoids to enhance the field of natural plant estrogen activity and wine flavoring. Lower production levels Naringinase, almost no reports of industrial production, which limits its application in industrial production, and therefore to carry out related research to a positive practical significance to improve Naringinase production levels. In this article the Penicillium sp.1523 laboratory breeding as the original strain optimize its fermentation condition of batch fermentation kinetics and the separation Naringinase purification and immobilization system, the main results are as follows: Single factor test Plackett-Burman design and response surface analysis methods, shake flask of Penicilliumsp.1523 production capacity Naringinase in fermentation conditions were optimized. The single factor test results show that the optimal carbon source in the fermentation medium corn flour optimal nitrogen source for soybean meal, seed age and inoculum size of 36 h and 5%, respectively. Steepest ascent was used to approach the maximum response region, based on the use of corn flour, cake flour, and naringin screened by Plackett-Burman design impact the production of Naringinase three important factors, and then using the Box-Behnken experimental design and The response surface analysis regression analysis to obtain the optimum medium: corn flour 31.14g / L, bean powder 31.53 g / L naringin 1.65 g / L of K2HPO4 on 1.00 g / L of ZnSO4 0.10 g / L, MgSO4 · 7H2O 0.06 g / L CaCl2 0.10 g / L. Fermentation the solution Naringinase enzyme activity under optimized fermentation conditions 891.79 ± 6.33 U / mL, with the predicted value is very close, and provided the basis for the subsequent fermentation to enlarge. 5L fermentor fermentation process of of Penicillium sp.1523 production of Naringinase, amplification, under optimized conditions the fermentation liquid Naringinase enzymatic activity can be reached 1229.40 U / mL, 41.0% higher than the shake flask fermentation; while the batch fermentation strains 5L fermenter is based on data, combined with the mathematical derivation of the establishment of its batch fermentation the production Naringinase of cell growth the synthesis of Naringinase and the substrate consumption Dynamics model, respectively, as follows: (1) cell growth kinetics model (2) the Kinetics Model Naringinase (3) substrate consumption dynamics model built model can basically to describe the Penicillium sp.1523 batch fermentation Naringinase dynamic process, for further amplification test to provide a reference. Separation and Purification Naringinase, followed by ultrafiltration, ammonium sulfate precipitation and SephadexG-200 gel filtration chromatography operation Naringinase the gradual purification of each step of the purification by SDS-PAGE electrophoresis The results were analyzed identification. The results showed that naringin purified enzyme specific activity reached 4478.85 U / mmg purification factor of 11.99 times, the activity recovery of 38.10%. Display two clear bands the purified Naringinase by SDS-PAGE gel electrophoresis, indicates that the enzyme is composed of two subunits, the molecular weight of 89 kDa and 72 kDa, respectively. The domestic TJS resin immobilized Naringinase. Single factor test and the Box-Behnken experimental design combine to optimize the impact the TJS resin immobilized Naringinase main factors. The results show that the best immobilization conditions: enzyme solution pH 6.5, fixed temperature of 26.6 ℃, the carrier put the amount to 0.79 g, immobilization time of 24 hours, this time the immobilized Naringinase enzyme activity reached 3949.32 U / g The activity yield increased to 44.79%, the protein fixed rate of 98.43%, will TJS resin used to the immobilized Naringinase is practical. Industrial production and the practical application of the above studies as Naringinase laid the foundation.

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