Utilizing chitooligosaccharides from shrimp waste biodegradation via recombinant chitinase A: a promising approach for emulsifying hydrocarbon and bioremediation.

BACKGROUND: Hydrocarbon pollution stemming from petrochemical activities is a significant global environmental concern. Bioremediation, employing microbial chitinase-based bioproducts to detoxify or remove contaminants, presents an intriguing solution for addressing hydrocarbon pollution. Chitooligosaccharides, a product of chitin degradation by chitinase enzymes, emerge as key components in this process. Utilizing chitinaceous wastes as a cost-effective substrate, microbial chitinase can be harnessed to produce Chitooligosaccharides. This investigation explores two strategies to enhance chitinase productivity, firstly, statistical optimization by the Plackett Burman design approach to  evaluating the influence of individual physical and chemical parameters on chitinase production, Followed by  response surface methodology (RSM) which delvs  into the interactions among these factors to optimize chitinase production. Second, to further boost chitinase production, we employed heterologous expression of the chitinase-encoding gene in E. coli BL21(DE3) using a suitable vector. Enhancing chitinase activity not only boosts productivity but also augments the production of Chitooligosaccharides, which are found to be used as emulsifiers. RESULTS: In this study, we focused on optimizing the production of chitinase A from S. marcescens using the Plackett Burman design and response surface methods. This approach led to achieving a maximum activity of 78.65 U/mL. Subsequently, we cloned and expressed the gene responsible for chitinase A in E. coli BL21(DE3). The gene sequence, named SmChiA, spans 1692 base pairs, encoding 563 amino acids with a molecular weight of approximately 58 kDa. This sequence has been deposited in the NCBI GenBank under the accession number "OR643436". The purified recombinant chitinase exhibited a remarkable activity of 228.085 U/mL, with optimal conditions at a pH of 5.5 and a temperature of 65 °C. This activity was 2.9 times higher than that of the optimized enzyme. We then employed the recombinant chitinase A to effectively hydrolyze shrimp waste, yielding chitooligosaccharides (COS) at a rate of 33% of the substrate. The structure of the COS was confirmed through NMR and mass spectrometry analyses. Moreover, the COS demonstrated its utility by forming stable emulsions with various hydrocarbons. Its emulsification index remained stable across a wide range of salinity, pH, and temperature conditions. We further observed that the COS facilitated the recovery of motor oil, burned motor oil, and aniline from polluted sand. Gravimetric assessment of residual hydrocarbons showed a correlation with FTIR analyses, indicating the efficacy of COS in remediation efforts. CONCLUSIONS: The recombinant chitinase holds significant promise for the biological conversion of chitinaceous wastes into chitooligosaccharides (COS), which proved its potential in bioremediation efforts targeting hydrocarbon-contaminated sand.

Electroactive Brevundimonas diminuta consortium mediated selenite bioreduction, biogenesis of selenium nanoparticles and bio-electricity generation.

In this study, highly selenite-resistant strains belonging to Brevundimonas diminuta (OK287021, OK287022) genus were isolated from previously operated single chamber microbial fuel cell (SCMFC). The central composite design showed that the B. diminuta consortium could reduce selenite. Under optimum conditions, 15.38 Log CFU mL-1 microbial growth, 99.08% Se(IV) reduction, and 89.94% chemical oxygen demand (COD) removal were observed. Moreover, the UV-visible spectroscopy (UV) and Fourier transform infrared spectroscopy (FTIR) analyses confirmed the synthesis of elemental selenium nanoparticles (SeNPs). In addition, transmission electron microscopy (TEM) and scanning electron microscope (SEM) revealed the formation of SeNPs nano-spheres. Besides, the bioelectrochemical performance of B. diminuta in the SCMFC illustrated that the maximum power density was higher in the case of selenite SCMFCs than those of the sterile control SCMFCs. Additionally, the bioelectrochemical impedance spectroscopy and cyclic voltammetry characterization illustrated the production of definite extracellular redox mediators that might be involved in the electron transfer progression during the reduction of selenite. In conclusion, B. diminuta whose electrochemical activity has never previously been reported could be a suitable and robust biocatalyst for selenite bioreduction along with wastewater treatment, bioelectricity generation, and economical synthesis of SeNPs in MFCs.