Screening, Identification And Research On Enzyme Production Of The Chitosanase Produced From Marine Bacteria Renibacterium Sp. QD1

Chitosan, an important marine polysaccharide, is limited to be used due to its low solubility,high molecular weight, and absorption rate. Chitooligosaccharides can be prepared throughchemical method and physical method. However, the hydrolysis product is not homogenous andthe hydrolyzing reaction is uncontrollable. Those methods also causing serious environmentalpollution. Enzymatic degradation of chitosan should be a promising alternative to chemicalmethod and physical method with high specificity and under mild conditions. Especially for it’slow cost and no pollution, It is significante to do research of chitosanase. Currently, it was founda wide range of chitosanase. But because most of the chitosanase activity is low, and poordegradation efficiency, it is difficult to be applied to the industry for the preparation ofchitooligosaccharides. Therefore, finding and screening yield novel and high activity chitosanasestrains become a hot topic in the field of chitosan research..Renibacterium sp. QD1, a bacteria strain capable of hydrolyzing chitosan was isolated fromthe homogenate of small crustaceans in QingDao. Based on the16srDNA sequence, QD1wasassessed to be Renibacterium sp., and is thus named Renibacterium sp. QD1. Isolate QD1is agram-positive strain that formed a colorless colony on a screening plate. An extracellular chitosanase,Csn-A, was purified from the QD1fermentation broth by （NH₄）₂SO₄salting and Phenyl SepharoseHP. Csn-A is a monomeric enzyme and had a molecular weight of26.1kDa. The optimum pH andtemperature of Csn-A were5.3to6.5and55°C, respectively. The enzyme is stable under theconditions of <50℃and pH4-8. The enzyme was significantly activated by Mn²⁺but wasinhibited by Fe³⁺, Cu²⁺, Al³⁺, Zn²⁺, and SDS. The enzyme was purified7.89-fold with a yield of67%, and a specific activity of1574.5U/mg proteins by performing two consecutive procedures.The strain QD1which produced the largest clearing zone on the chitosan plate, show good signs ofchitosanase production. Csn-A hydrolyzed N-deacetylated polymeric glucosamine （degree of polymerization>3） into chito-biose and-triose. Endo-chitosanlytic activity was demonstratedthrough a time course analysis of the hydrolysis products. The higher specific activity and simplyisolation procedure presage the potential use of Csn-A in industrial application.We attempts to construct the genomic library of Renibacterium sp. QD1strains to get thegene of chitosanase. The functional screening tablet is LB solid medium containing chitosan.Results showed that chitosan can inhibite the growth of host strain DH5α seriously, the tablet cannot play the role of functional screening. Therefore this method is proved to be infeasible.In orderto clarify its gene sequence and the protein sequence, The protein N-terminal sequencingtechnique was used. The15amino acids of the protein N-terminal was got by sequencing of purechitosanase. Comparison and analysis of the amino acid sequence found that the enzyme havehigh similarity with Renibacterium salmoninarum and Renibacterium salmoninarum are morethan99%homology through16S sequence comparison, so we speculate that the chitosanase of thetwo strains is very similar. Therefore we designed gene primers according to the known sequencesof Renibacterium salmoninarum for PCR testing. The result show that it was the chitosanase geneindeed by analysising a period of944bases of sequence. The chitosanase gene of QD1have95%similarity with Renibacterium salmoninarum,67%similarity with the sequence of enzyme inStreptomyces N174, and the sequence belong to the glycoside hydrolase46family. It wasundoubtedly enriched chitosanase gene resources, and promote the study of the structure-activityrelationship of chitosanase by cloning and analysis the csn-A gene.