Immobilization of Chitosanase on Magnetic Nanoparticles: Preparation, Characterization and Properties

Chitosanase could cleaveβ-1,4-linkage of chitosan to produce chitooligosaccharides(COS)with diverse biological activities.However,there are many limitations on the use of free chitosanase in industrial production.Enzyme immobilization is generally considered a valuable strategy in industrial-scale applications.In this study,the chitosanase Csn-BAC from Bacillus sp.MD-5 was immobilized on Fe_(3)O_(4)-SiO_(2) magnetic nanoparticles(MNPs)to enhance its properties,which could be recovered easily from reaction media using magnetic separation.The activities of Csn-BAC immobilized with MNPs(MNPs@Csn-BAC)were de-termined with temperature and pH,and the thermal-and pH-stabilities,respectively.The reusability of the MNPs@Csn-BAC was determined in repeated reaction cycles.Immobilization enhanced the thermal and pH stability of Csn-BAC compared with the free enzyme.After eight reaction cycles using MNPs@Csn-BAC,the residual enzyme activity was 72.15%.Finally,the amount of COS released by MNPs@Csn-BAC was 1.86 times higher than that of the free Csn-BAC in the catalytic performance experiment.The immobilized Csn-BAC exhibits broad application prospects in the production of COS.

A Study of the Structural Properties and Thermal Stability of Chitosanases EAG1 by Molecular Dynamics Simulations

Chitosanases EAG1, a classical glycoside hydrolase from Bacillus ehimensis, is relatively unstable with higher temperature. This shortcoming seriously restricts its industrial application. Therefore, it is crucial to clarify the theoretical basis of thermo stability and to produce enzymes with high activity and stability. Using the structural modeling and molecular dynamical simulation, residues Leu84, Gly113, Asp116, Ala207 and Leu286 were believed to be the key residues for structural stability. Then the predicted residue Leu84 was mutated to ALA. It was shown that the L84A mutation can improve the thermal stability of chitosanases EAG1. Together with previous studies, mutations of G113C, D116C, A207C and L286C forms two sulfur bonds can change the thermal stability of EAG1. The results suggest that the thermal stability of EAG1 could be engineered by site-directed mutagenesis on the conserved residues. This protocol could be employed for improving thermal stability of other chitosanases EAG1.

Enhancement of Gongronella sp.JG Chitosanase Production by Ion Beam Implantation

Gongronella sp. JG was a fungal strain which expressed extracellular chitosanase of about 800 U/L during its growth in production medium. To improve its enzyme production, low energy N＋ implantation was employed to mutate spores of JG. The implantation condition was optimized and the parameters of 15 keV and 60 × breeding experiments. A mutant designated as SG 2.6 × 10^13 ions/cm^2 were selected for further was obtained. It showed increased chitosanase production （1800 U/L） and shortened cultivation period （from 72 h to 60 h）. Five-generation cultivation of SG indicated that its chitosanase production was stable at about 1800 U/L.

Solid-State Fermentation Production of Chitosanase by <i>Streptomyces</i>with Waste Mycelia of <i>Aspergillus niger</i>

Solid-state fermentation was carried out using mycelium powder of Aspergillus niger as substrate for the production of chitosanase of Streptomyces. Results of the experiments indicated that the optimal medium consisted of wheat bran and mycelium powder of Aspergillus niger with initial moisture content of 60% - 70%. The enzyme activity reached 41.33 U per gram dry medium after cultured for 5 days at 28°C - 30°C and an initial pH 6.5. Chitosanase was detected on the second day of incubation and had maximal activity at 5 days and decreased gradually within a 1 month period. Solid-state fermentation is maybe an economic alternative in the production.

Virtual Screening of Inhibitors for Chitosanases EAG1

Chitosanases EAG1 is a classical glycoside hydrolase from Bacillus ehimensis. The previous researches showed that this Chitosanases can not only hydrolyze the b1,4-glycosidic bonds of chitosan to COS in different sizes but also keep a high catalytic activity in organic, which was useful for producing chitooligosaccharides and GlcN for use in the food and pharmacological industries. While it is instable in the liquid state. This shortcoming seriously restricts its industrial application. Here we used the modeled structure of EAG1 and the molecular modeling software package to screen the free chemical database ZINC. Moreover, the strategies including “initial filter” and consensus scoring were applied to accelerate the process and improve the success rate of virtual screening. Finally, five compounds were screened and they were purchased or synthetized to test their binding affinity against EAG1. The test results showed that one of them could inhibit the enzyme with an apparent Ki of 1.5 μM. The result may take the foundation for further inhibitor screening and design against EAG1 and the screened compound may also help to improve the liquid stability of EAG1 and expand its industrial application.

固定化技术在壳聚糖酶生产中的应用

以产酶活性与产酶稳定性为指标评价3种固定化细胞产壳聚糖酶(Chitosanase,EC3.2.1.99)方法,以酶比活力、酶结合效率、酶活力回收率及稳定性评价4种固定化壳聚糖酶方法,以筛选最优固定化方案应用于壳聚糖酶的生产。结果表明,海藻酸钠壳聚糖包埋法固定化细胞的产壳聚糖酶活性较高且稳定性好;海藻酸钠壳聚糖吸附包埋固定化壳聚糖酶的酶比活力、酶结合效率与酶活力回收率均为最高,且稳定性强。