Purification and characterization of a chitosanase from Serratia marcescens TKU011

A chitosanase was purified from the culture supernatant of Serratia marcescens TKU011 with shrimp shell wastes as the sole carbon/nitrogen source. Zymogram analysis revealed the presence of chitosanolytic activity corresponding to one protein, which was purified by a combination of ion-exchange and gel-filtration chromatography. The molecular weight of the chitosanase was 21 kDa and 18 kDa estimated by SDS–PAGE and gel-filtration, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of the chitosanase were 5, 50 °C, pH 4–8, and <50 °C, respectively. The chitosanase was inhibited completely by EDTA, Mn²⁺, and Fe²⁺. The results of peptide mass mapping showed that three tryptic peptides of the chitosanase were identical to a chitin-binding protein Cbp21 from S. marcescens (GenBank accession number gi58177632) with 63% sequence coverage.     

球孢白僵菌高壳聚酶突变株的筛选

以球孢白僵菌（Beauveria bassiana)1316为出发菌株,通过紫外线和高能电子束分别诱变分生孢子,采用平板透明圆法初筛和摇瓶培养复筛的方法,经3轮诱变,筛选到5株高产突变株。其中一株最高产的定名为Beauveria bassiana 1316-V1,其壳聚糖酶活力是原始菌株的16倍;而且经传代培养,其高产特性能够稳定遗传。     

Purification of a constitutive chitosanase produced by Bacillus sp. MET 1299 with cloning and expression of the gene

A chitosanase produced constitutively by Bacillus sp. MET 1299 was purified by SP-Sephadex column chromatography. The molecular weight was estimated to be 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Optimal enzyme activity was observed at a pH of 5.5 and temperature of 60 degrees C. The purified chitosanase showed high activity on 90% deacetylated colloidal chitosan and beta-glucan, but not on hydrolyzed colloidal chitin, CMC, or their derivatives. The N-terminal amino acid sequence of the enzyme was determined. The cloned full length gene, 1362 bp in size, encoded a single peptide of 453 amino acids and had a conserved amino acid sequence of glycosyl hydrolase family 8. A search of the cDNA sequence with NCBI BLAST showed homology with chitosanase of Bacillus sp. KTCC 0377BP and Bacillus sp. No. 7-M. The recombinant protein was expressed in Escherichia coli, purified using affinity chromatography and characterized.     

Efficient digestion of chitosan using chitosanase immobilized on silica-gel for the production of multisize chitooligosaccharides

Chitosanase-coated silica-gels were prepared via cross-linking of the chitosanase onto silica-gels for the efficient production of multisize chitooligosaccharides (MCOs) in a continuous process. The kinetic aspects of immobilized chitosanase (IMMCTase) were investigated based on the reaction time, production of MCOs, and MALDI-TOF mass analyses to achieve maximum bioconversion of high molecular weight chitosan (HMWC) to MCOs. IMMCTase revealed a negligible loss of chitosanase activity after multi uses in continuous digestion of HMWC. The optimal temperature of IMMCTase was 37 °C, and kinetic parameters toward HMWC were determined to be K_m = 1.45 mM and V_max = 360 μmole/μg/min, respectively. Under optimal conditions, the recovery of enzyme activity of IMMCTase was determined to be 82.3%, thus indicating that it can still be reused few more times. In conclusion, use of IMMCTase resulted in rapid and efficient digestions of HMWC with consistent results to produce MCOs.     

Scale-up of PUF-immobilized fungal chitosanase–lipase preparation production

Mucor circinelloides IBT-83 mycelium that exhibits both lipolytic (A_L) and chitosanolytic (A_CH) activities was immobilized into polyurethane foam in a 30?L laboratory fermenter. The process of immobilization was investigated in terms of the carrier porosity, its type, amount, and shape, location inside the fermenter, mixing, and aeration parameters during the culture, as well as downstream processing operations. The selected conditions allowed for immobilization of approximately 7?g of defatted and dried mycelium in 1?g of carrier, i.e., seven times more than achievable in 1?L shake-flasks. Enzymatic preparation obtained by this method exhibited both the chitosanolytic (A_CH 432.5?±?6.8?unit/g) and lipolytic (A_L 150.0?±?9.3?U/g) activities. The immobilized preparation was successfully used in chitosan hydrolysis to produce chitooligosaccharides and low molecular weight chitosan, as well as in waste fats degradation and in esters synthesis in nonaqueous media. It was found that the half-life of immobilized preparations stored at room temperature is on average of 200 days.     

The bifunctional enzyme chitosanase-cellulase produced by the gram-negative microorganism Myxobacter sp. AL-1 is highly similar to Bacillus subtilis endoglucanases

The gram-negative bacterium Myxobacter sp. AL-1 produces chitosanase-cellulase activity that is maximally excreted during the stationary phase of growth. Carboxymethylcellulase zymogram analysis revealed that the enzymatic activity was correlated with two bands of 32 and 35 kDa. Ion-exchange-chromatography-enriched preparations of the 32-kDa enzyme were capable of degrading the cellulose fluorescent derivatives 4-methylumbelliferyl-β-d-cellobioside and 4-methylumbelliferyl-β-d-cellotrioside. These enzymatic preparations also showed a greater capacity at 70° C than at 42° C to degrade chitosan oligomers of a minimum size of six units. Conversely, the β-1,4 glucanolytic activity was more efficient at attacking carboxymethylcellulose and methylumbelliferyl-cellotrioside at 42° C than at 70° C. The 32-kDa enzyme was purified more than 800-fold to apparent homogeneity by a combination of ion-exchange and molecular-exclusion chromatography. Amino-terminal sequencing indicated that mature chitosanase-cellulase shares more than 70% identity with endocellulases produced by strains DLG, PAP115, and 168 of the gram-positive microorganism Bacillus subtilis. Received: 6 January 1997 / Accepted: 29 May 1997