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Recently, haloacid dehalogenases have gained a lot of interest because of their potential applications in bioremediation and synthesis of chemical products. The haloacid dehalogenase gene from Bacillus cereus IndB1 ( bcfd1 ) has been isolated, expressed, and Bcfd1 enzyme activity towards monochloroacetic acid has been successfully studied. However, the structure, enantioselectivity, substrate range, and essential residues of Bcfd1 have not been elucidated. This research performed computational studies to predict the Bcfd1 protein structure and analyse the interaction of Bcfd1 towards several haloacid substrates to comprehend their enantioselectivity and substrates' range. Structure prediction revealed that Bcfd1 protein consist of two domains. The main domain consists of seven β -sheets connected by six α -helices and four 3 10 -helices forming a Rossmannoid fold. On the other hand, the cap domain consists of five β -sheets connected by five α -helices. The docking simulation showed that 2-chloroalkanoic acids bind to the active site of Bcfd1 with docking energy decreases as the length of their alkyl chain increases. The docking simulation also indicated that the docking energy differences of two enantiomers of 2-chloroalkanoic acids substrates were not significant. Further analysis revealed the role of Met1, Asp2, Cys33, and Lys204 residues in orienting the carboxylic group of 2-chloroalkanoic acids in the active site of this enzyme through hydrogen bonds. This research proved that computational studies could be used to figure out the effect of substrates enantiomer and length of carbon skeleton to Bcfd1 affinity toward 2-chloroalkanoic acids., Competing Interests: The authors declare that there are no conflicts of interest regarding the publication of this paper., (Copyright © 2022 Enny Ratnaningsih and Saepulloh Saepulloh.)

In this paper, highly regioselective enzymatic acylations of 1-beta-D-arabinofuranosylcytosine (ara-C) with vinyl stearate (VS) in binary organic solvents were explored for the preparation of 5'-O-stearate of ara-C with potential antitumor activity. Twelve kinds of hydrolases were tested for the regioselective acylation reaction and the immobilized Candida antarctica lipase B (Novozym 435) showed the highest regioselectivity (>99.9%) towards the 5'-OH of ara-C. A comparative study showed that the lipase had much higher catalytic activity in the binary mixture of hexane and pyridine than in other tested co-solvent systems. To better understand lipase-mediated acylation conducted in the best binary organic solvent system, the effects of hydrophobic solvent content, molar ratio of VS to ara-C, initial water activity, and reaction temperature on the acylation reaction were studied. It was found that the most suitable hexane content, VS-ara-C molar ratio, initial water activity, and reaction temperature were shown to be 25% (v/v), 20:1, 0.07, and 50 degrees C, respectively. Under these reaction conditions, the initial reaction rate, the maximum substrate conversion, and regioselectivity were as high as 86.0 mmol.L(-1)h(-1), 96.6%, and >99.9%, respectively. The product of Novozym 435-catalyzed acylation was characterized by Carbon-13((13)C) NMR and confirmed to be 5'-O-stearate of ara-C.

Two fungal-secreted α-fucosidases and their genes were characterized. FoFCO1 was purified from culture filtrates of Fusarium oxysporum strain 0685 grown on l-fucose and its encoding gene identified in the sequenced genome of strain 4287. FoFCO1 was active on p-nitrophenyl-α-fucoside (pNP-Fuc), but did not defucosylate a nonasaccharide (XXFG) fragment of pea xyloglucan. A putative α-fucosidase gene ( FgFCO1) from Fusarium graminearum was expressed in Pichia pastoris. FgFCO1 was ∼1,800 times less active on pNP-Fuc than FoFCO1, but was able to defucosylate the XXFG nonasaccharide. Although FgFCO1 and FoFCO1 both belong to Glycosyl Hydrolase family 29, they share <25 % overall amino acid identity. Alignment of all available fungal orthologs of FoFCO1 and FgFCO1 indicated that these two proteins belong to two subfamilies of fungal GH29 α-fucosidases. Fungal orthologs of subfamily 1 (to which FoFCO1 belongs) are taxonomically more widely distributed than subfamily 2 (FgFCO1), but neither was universally present in the sequenced fungal genomes. Trichoderma reesei and most species of Aspergillus lack genes for either GH29 subfamily. [ABSTRACT FROM AUTHOR]