Recombinant Expression And Application Of Enzymes In The Resource Utilization Of Shells Waste

With the consumption of shrimp, crab and other aquatic products, the generation of shrimp and crab shell watste keeps booming. At president, resource utilization of shrimp and crab shells still stays on the preliminary processing. While shrimp and crab shell contains chitin, it can be trabsformed into highly valuable derivatives through deep processing. Undoubtedly the latter process represents the most effective ways in shell wastes treatment. At present, the deep processing of chitin relies on chemical methods, which will consume large amounts of acid and alkali, causing secondary environmental contamination. Besides the quality of the product varies. Therefore, eco-friendly enzymatic methods have aroused more and more attention, because of their lower pollution and mild reaction conditions.Three types of enzymeswas developed for enzymatic production of chitin derivatives as well as chitin oligosaccharides. The three enzymes were chitin deacetylase, chitinase and chitosanase. The recombinant strains were constructed, then the enzymes expression and enzyme activities were identified. The enzymatic properties were characterized, through which the optimum reaction temperature and pH were determined. For chitinase, the optimum reaction pH and temperature are 5.0 and 55 ℃ while they are 5.0 and 45 ℃ for chitosanase. For chitin deacetylase, the optimum reaction temperature and pH are 40 ℃ and 7.0. Enzymatic reaction kinetics studies show that Km and vmax of chitinase are 12.26 g/L and 5.33 μmol/(L?h). For chitosanase, they are 6.38 g/L and 7.53 μmol/(L?h) while they are 32.71 g/L and 15.55 μmol/(L?h)for chitin deacetylase. Chitosanase shows higher substrate affinity than the other two enzymes.A pretreatment route including “Demineralization-Deproteinization--Decolorization-Carboxymenthylation” has been proposed. On purpose of chitosan oligosaccharides(GlcN)n production, combination application of chitinase and chitin deacetylase is the optimal preparation method, using the carboxymethyl chitin as substrate. An oligosaccharides yield of 8.82% was realized under optimized conditons; On purpose of chitin oligosaccharides( GlcNAc)n, chitinase is utilized using carboxymethyl chitin as substrate, which resulted an oligosaccharides yield of 8.82%. The analysis of different product using TLC shows that oligosaccharides hydrolysis products were mainly disaccharide, trisaccharide, tetrasccharide, pentasaccharides and hexasaccharide. There were less hexasaccharide and more disaccharide, trisaccharide in the enzymatic hydrolysate.The expression of chitinase in shake flask culture was optimized. The optimal concentration of the inducer IPTG concentration is 0.3 mmol/L, the optimal timing of induction is the late logarithmic growth period(OD600=4.0 or so), the best inducing temperature and duration are 25 ℃ with 6 hours. Under fermenter culturing conditions, the highest enzyme activity of 90.22 U/L was achieved. The preservation of enzyme liquid was studied. Glycerol and PEG400 was suitbale for chitin enzyme langterm preservation. During chitinase transportation and storage, 5-10% PEG400 could be supplied in enzyme liquid and saved under different temperature to maintain the stability of the enzyme.