(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) > cellular organisms: NE > Bacteria: NE > Terrabacteria group: NE > Firmicutes: NE > Bacilli: NE > Bacillales: NE > Bacillaceae: NE > Anoxybacillus: NE > Anoxybacillus geothermalis: NE
LegendThis sequence has been compared to family alignement (MSA) red => minority aminoacid blue => majority aminoacid color intensity => conservation rate title => sequence position(MSA position)aminoacid rate Catalytic site Catalytic site in the MSA MKIVSPKPFTFEAGERAVLLLHGFTGNSSDVRMLGRFLESKGYTCHAPIY KGHGVPPEELVHTGPDDWWQDVMNAYEFLREKGYQKIAVVGLSLGGVFSL KLGYTVPVVGIVPMCAPMYIKSEETMYQGVLDYAREYKKREGKAPEQIEK EMEEFRKTPMKTLKALQALIAEVRNHIDLIYAPTFVVQARHDDMINTDSA NIIYNGVESPIKQIKWYEESGHVITLDKEKEQLHEDIYAFLESLDW
References
Title: Immobilization of Hyperthermostable Carboxylesterase EstD9 from Anoxybacillus geothermalis D9 onto Polymer Material and Its Physicochemical Properties Johan UUM, Rahman R, Kamarudin NHA, Latip W, Ali MSM Ref: Polymers (Basel), 15:, 2023 : PubMed
Carboxylesterase has much to offer in the context of environmentally friendly and sustainable alternatives. However, due to the unstable properties of the enzyme in its free state, its application is severely limited. The present study aimed to immobilize hyperthermostable carboxylesterase from Anoxybacillus geothermalis D9 with improved stability and reusability. In this study, Seplite LX120 was chosen as the matrix for immobilizing EstD9 by adsorption. Fourier-transform infrared (FT-IR) spectroscopy verified the binding of EstD9 to the support. According to SEM imaging, the support surface was densely covered with the enzyme, indicating successful enzyme immobilization. BET analysis of the adsorption isotherm revealed reduction of the total surface area and pore volume of the Seplite LX120 after immobilization. The immobilized EstD9 showed broad thermal stability (10-100 degreesC) and pH tolerance (pH 6-9), with optimal temperature and pH of 80 degreesC and pH 7, respectively. Additionally, the immobilized EstD9 demonstrated improved stability towards a variety of 25% (v/v) organic solvents, with acetonitrile exhibiting the highest relative activity (281.04%). The bound enzyme exhibited better storage stability than the free enzyme, with more than 70% of residual activity being maintained over 11 weeks. Through immobilization, EstD9 can be reused for up to seven cycles. This study demonstrates the improvement of the operational stability and properties of the immobilized enzyme for better practical applications.
Carboxylesterases are attractive biocatalysts for various industrial applications, especially hyperthermophilic carboxylesterases, due to their high tolerance towards extreme environments. Such ability confers many advantages, including cost-effectiveness and an increased manufacturing rate. In the current work, we first described the characterization of EstD9, a new carboxylesterase from thermophilic Anoxybacillus geothermalis D9, with a hyper-thermostability characteristic. In this study, the enzyme was expressed, purified and characterised. Additionally, the three-dimensional structure was predicted using Robetta software. Sequence analysis of EstD9 revealed a significant identity (80 %) with thermophilic carboxylesterase Est30 and a typical catalytic triad of carboxylesterase, composed of Ser93-His22-Asp193. As the protein sequence contained a highly conserved pentapeptide (GLSLG), carboxylesterase of A. geothermalis D9 could be proposed as a new member of family XIII. The putative carboxylesterase was recombinantly expressed in E. coli BL21 (DE3) with a molecular mass of 28 kDa and successfully purified via Ni(2+)-Sepharose affinity chromatography with recovery of 88.36 %. Using p-nitrophenyl butyrate (C4), EstD9 presented excellent stability at high temperature range (70 degreesC-100 degreesC) for 5 days of incubation and has broad pH tolerance (pH 6-9), with maximal activity at 80 degreesC and pH 7. Notably, EstD9 activity was stimulated in the presence of 1-propanol and DMSO with 107.8 % and 108.9 % relative activities, respectively. The purified EstD9 maintained 60 % residual activity after 30 min exposure to various surfactants and metal ions. Additionally, the inhibition studies demonstrated that EstD9 was strongly deactivated by phenylmethylsulfonyl fluoride, dithiothreitol, and beta-mercaptoethanol. Circular dichroism (CD) spectra analysis predicted Tm value at 72.12 degreesC. Unlike other carboxylesterases, in silico 3D model of EstD9 disclosed a topological alpha/beta hydrolase fold with a small alpha-helix cap below catalytic region. The enzymatic properties exhibited by EstD9 suggested this enzyme to be a highly suitable catalyst for industrial bioprocesses under harsh conditions.
