Enzymatic Synthesis And Characterization Of Chitosan-Gelatin Copolymer By MTGase

Chitosan is an important natural raw material with many biologicao functions, which has a wide application prosperity in the field of biomedicine and medicin. Chitosan-gelatin copolymer is a new reactive polymers. Theoretically, it has not only properties like film forming, gas permeation, antibiosis of chitosan, but also skin affinity, flexibility, nourishment of gelatin. So it's a degradable biomaterial with huge applied potential. This paper studied the process conditions of AOT reverse micelle extraction MTGase and main influencing factors using raw MTGase, and the optimum condition of catalytic synthesis chitosan-gelatin copolymer using purified MTGase and its characterization. This study possesses certain academic significance and applications.Main factors affecting specific activity in MTGase purification were discussed by AOT-isooctane reversed micellar extraction from raw MTGase. The experimental results showed that the optimum operating conditions are phase ratio 1:1, pH 4.8～5.2, 0.12mol/L NaCl, raw MTGase concentration 20.0 mg/mL, 20min, and the re-extraction conditions are 2.0mol/L KCl, pH11.00 and reacting 20min by 400r/min magnetic stirring. By isoelectric precipitation (pH4.28), ultrafiltration(molecular cut off 10 000) and AOT reverse micelle extraction MTGase, MTGase specific activity enhanced to 14.2 U/g from 0.16U/g, increasing by a factor of 88.75. The yield of activity is 100%.MTGase ultraviolet absorption band appear blue shift in AOT reverse micelle due to MTGase's Phe, Tyr and Trp residues conjugative effect is attenuated. And the emerging hydrogen bond between -OH, -NH2 of MTGase and oxygen carbonyl of AOT induce electric dipole moment augmention so infrared absorption shifte to low wave number,i.e. 1136 cm-1 to 1127 cm-1 , 1113 cm-1 to 1105 cm-1.Chitosan-gelatin copolymer was synthesized by primary purified MTGase using elatin and chitosan. The graft rate achieve 65.51% when gelatin/chitosan (m/m) ratio is 1.3:1, reaction temperature is 50℃, pH is 6.00,reaction time is 45 min. The yield of chitosan-gelatin copolymer is 21.74% by TCA multi-stage precipitation and lyophilization. Compared with the mixture of chitosan and gelatin, chitosan-gelatin copolymer's infrare absorption peak displace to high wave number in two places, i.e. 1627 cm-1 of–C=O in amide shifte to 1654 cm-1, 1502 cm-1 of primary amine shifte to 1546 cm-1, on account of inductive effect by lead-in electro-attracting group. The reducing sugar content reache to 0.75mmol/L in chitosan-gelatin copolymer by lysozyme...