| Chitin is the second natural resources in nature after cellulose,and widely distributed in the exoskeleton of shrimp crabs and insects,and in the cell wall of fungi and plants.Chitin is still an unwidely utilized organic substance,since its highly crystalline and bulk structure formed by numerous intra-and intermolecular hydrogen bonds,which limits its solubility in water.Ionic liquid have many advantages,especially their powerful ability to dissolve biopolymers such as cellulose and chitin.Therefore,we can pretreat shrimp crab shells with ionic liquid to weak intra-and intermolecular hydrogen bonds,and then hydrolyze the β-1,4glycosidic bond in chitin by chitinase to obtain chitooligosaccharide and N-acetylglucosamine.Compared to the preparation of chitooligosaccharide with chemical method,enzymatic method has the advantages of simple process,mild reaction condition,environmental friendliness,and thus shows good application prospect.In the study,the strain Paenibacillus pasadenensis CS0611 was cultured under optimal fermentation conditions for 48 h,and using crab shell powder as the sole carbon source.A novel extracellular chitinase produced by Paenibacillus pasadenensis CS0611 was purified by ammonium sulfate precipitation,HiTrap DEAE FF and Hi Load 26/600 Superdex 200 pg column chromatography.The purified chitinase achieved a 5.30-fold purification and 15.70%finally yield.The apparent molecular was 69.0 kDa determined by SDS-PAGE.By analyzing the MALDI-TOF-MS data,the Paenibacillus pasadenensis CS0611 chitinase showed higher homology with chitinase from other Paenibacillus pasadenensis(accession No: gi655151624),further confirming the purified protein is chitinase.Then,the enzymatic properties of the purified chitinase were studied.The result showed the optimum pH and optimum temperature of the chitinase were 5.0 and 50 °C respectively.The enzyme showed high stability under alkaline pH environment and temperature below 40 °C.Additionally,the tested metal ions had no obvious active affect on the catalytic activity of the enzyme,indicating that the chitinase was non-metal enzyme.Mn2+,Mg2+ and Co2+ inhibited the chitinase activity.Substrate spectrum analysis indicated that the chitinase reacted preferably with the glucosidic bond between GlcNAc-GlcNAc.The chitinase was active on colloidal chitin with an apparent Km value of 4.41 mg/mL and Vmaxvalue of 1.08 mg/min,the hydrolysis efficiency higher than chitin.The enzymatic hydrolysate,analyzed by HPLC and TLC,clearly exhibited that a subunit of(GlcNAc)2 was the main hydrolysis product.Among the imidazole-based ionic liquids,[BMIm]Ac was found to be effective for crab shells pretreatment,the optimal pretreatment temperature and time were 100 °C and 1 h,respectively.Chitinase could efficiently hydrolyze the regenerated crab shells under 35°C,7 h,and the hydrolysis product was up to 2.88 mg/mL,while the hydrolysate of colloidal chitin was only 0.86 mg/mL.The product was proved to be(GlcNAc)2 by mass spectrometry.The analysis from FTIR,XRD and SEM showed that,in contrast to the decrease of crystallinity,[BMIm]Ac,increased the deacetylation degree,destroyed the original tissue,increased the dispersibility in the water and facilitated the contact with enzyme,while the crystallinity didn’t change.This study not only enriches fundamental enzymology knowledge of chitinase,but also provides a novel and efficient route to obtain N,N’-diacetylchitobiose. |
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