Screening And Optimization Of Fermentation Condition Of Bacterial Strain Producing Extracellular Elastase

In this study, elastase-producing strain was screened and breed. And then the cultivation process and technology of this elastase-producing strain were fully discussed. The batch process and dynamics model were determined. Meanwhile the immobilization of crude enzyme was studied.19 strains bacteria secreting extracellular elastase were isolated from soil and polluted water samples, 3 of them pocceessed considerably high elastiolytic activity of more than 15u/ml. The bacterial strain was preliminarily identified belong to Pseudomonas. The optimum elastase-producing conditions were studied by flask-shaking fermentation. The optimal combinations were as follow: casein 2%; glucose 0.5%; yeast extract 0.4%; K₂HPO₄ 0.2%; MgSO₄.7H₂O 0.01%; initial pH value was 7.0; fermentation temperature was 30℃, 25/250(ml) was the optimum amount in flask. The extracellular elastase activity reached 18 U/ml after 28 hours incubation.Elastase productivity was improved 65% after mutated by 30s exposure of mutation dose. And then it was treated with diethyl sulfate(DES) which is mutagenesis dose, Elastase productivity was improved 120%. Eventually, combined two means together, and after the strain was treated with UV-ray radiation in 30s, then treated with diethyl sulfate(DES) in 20min again, now the productivity will be improved 1.44 times,to 30u/ml. Results of futher study to the fermentation condition indicated that the strain need a high dissolved oxygen, Addition of oxygen-vector tween-80 to fermentation medium was recognized as a method of enhancing oxygen transfer and prorooting enzyme activity.Immobilization onto crude elastase were researched by means of embedding and covalent coupling,using PVA as the carrier and glutaradehyde as the crosslinking agent. Results showed that the activity of immobilized elastase reached to 21.6u/g by means of covalent coupling, the avtivity recovery was 48%, and the pH stability was increased also. After 6 brach of hydrolization to elastin, the elastolytic activity remained 25%, which showed that the immobilized enzyme possessed some working stability.To analyzing the kinetic property in fermentation reactor and researching the possibility of industrialized production, we did the experiment from flask-shaking fermentation to a 14L fermentation reactor.Based on the experimental data of batch fermentation, the kinetic model of cell growth, enzyme production and substrate consumption were proposed. The Logistic model for growth, the Luedeking-Piretequation equation for enzyme production and a simple equation for glucose consumption were constructed. Results showed that the production of elastase was growth-associated.