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Studies on Screening and Identification of Chondroitinase Producing Strain and Medthods of the Enzyme Immobilization

Author: NiuHaiBin
Tutor: YanHaoLin
School: Shenyang Pharmaceutical University
Course: Microbial and Biochemical Pharmacy
Keywords: Chondroitin sulfate lyase Mild Aeromonas Immobilized Gelatin Sodium alginate Macroporous resin Magnetic PF microspheres
CLC: R91
Type: Master's thesis
Year: 2009
Downloads: 29
Quote: 0
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Abstract


Chondroitin sulfate lyase (chondroitinase or chondroitin sulfate lyase, ChSase) with a variety of medicinal value, especially in the preparation of low molecular weight chondroitin sulfate has a broad application prospects. This paper ChSase producing strains and strains and identification of enzyme production characteristics, enzymatic properties and enzyme immobilization methods do a systematic study. Quick screened a production ChSase higher activity strain N235, from carp fish sausage samples screened by morphological, physiological and biochemical tests, and this strain 16srRNA gene sequence analysis, the strains N235 attributed to the mild air Aeromonas; the expression of ChSase strains enzyme production characteristics, ChSase extracellular enzymes to strains N235 fermentation at 30 ℃ the 36h production ChSase activity to achieve the highest; using a crude enzyme solution is prepared by ammonium sulfate precipitation, and investigated enzymatic properties of ChSase. Using crude enzyme solution prepared as raw materials, gelatin and sodium alginate the macroporous resin magnetic PF microspheres as a carrier for ChSase immobilization method. The results show that: (1) gelatin and sodium alginate co-embedded the immobilized ChSase optimal conditions is: 10% of the gelatin concentration, concentration of 2% sodium alginate, calcium chloride concentration of 4%, plus the enzyme amount 1U/ml system, cross linking agent glutaraldehyde concentration of 0.05% -0.07% crosslinking time 10min; take advantage of this condition to obtain the enzymatic properties of the immobilized enzyme: the optimum temperature of 45 ° C, the optimum pH of 7.5, the apparent Michaelis constant of 7.06 × 10 -2g / L, with a certain degree of stability in operation. (2) macroporous resin adsorption the immobilized ChSase optimal conditions is: adsorption temperature 15 ° C, adsorption time 6 h, adsorption pH 7.0, plus the amount of enzyme 150U / g resin, glutaraldehyde concentration 0.01%, and cross-linking 3h, crosslinking temperature 4 ° C; take advantage of this condition to obtain the enzymatic properties of the immobilized enzyme: the optimum temperature of 40 ° C -50 ° C, the optimum pH 7.0, the apparent Michaelis constant of 1.46 × 10-1 g / L better operational stability. (3) Magnetic PF microspheres the immobilized ChSase optimization conditions: 8.2g hydroquinone, formaldehyde 15ml hydrochloric 2ml prepared the PF gel; Preparation of magnetic PF PF gel 1.5g the microsphere; fixed temperature 20 ℃, fixed time the 8h, plus the amount of enzyme 67.3U / g magnetic PF microsphere preparation immobilized enzyme; enzymatic properties of the immobilized enzyme prepared to take advantage of this condition: the optimum temperature of 50 ° C, the optimum pH 8.0, the apparent Michaelis constant 5.23 × 10-1g / L, has a good stability in operation.

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