Hydrolytic enzymes consist of esterases and lipases together and are called lipolytic enzymes. These enzymes have been classified into eight families based on the conserved amino acid sequences and biochemical properties. Among them, lipolytic family IV is called microbial hormone-sensitive lipase because microbial proteins share amino acid sequence(s) with mammalian hormone-sensitive lipase. HGGG and GDSAG amino acid motifs are the hallmarks of microbial HSLs. Microbial HSLs have larger industrial applications in biosensors, pharmaceuticals, and cosmetic industries. Unfortunately, the applications of microbial HSLs have not been explored, hence there is a search and demand for the new microbial HSLs with new biotechnological applications. In this study, the industrial microbe Clostridium acetobutylicum (ATCC 824) was used to produce ABE by the fermentation process. Gene CA_C0816 codes for lipase/esterase protein that belongs to a member of the serine hydrolase family. Multiple amino acid sequence alignment showed that this protein belongs to a member of hormone-sensitive lipase of lipolytic family IV, still, it is a variant of microbial HSL due to the presence of catalytic serine residue around the GYSMG, a pentapeptide motif instead of GDSAG and GTSAG. Upstream to this pentapeptide motif HGSG instead of HGGG plays an important role in the hydrolysis of a tetrahedral intermediate during catalysis. The primary sequence of Clostridium acetobutylicum esterase (Ca-Est) is 243 amino acids in length. It exists as a homodimer with a predicted molecular mass of 55 kDa. The tertiary structure showed that it has a cap and catalytic domain. The cap domain was made of four helices that play a major role in substrate specificity, thermal stability, and regioselectivity. The catalytic domain had α/β hydrolase fold. It consists of 7 β sheets and 13 α helices, Ser 89, Asp 196, and His 224 are the catalytic amino acids conserved in loops. Phylogenetic analysis showed that it has a separate branch in the lipolytic family IV, this information was further supported by superposition analysis, as the Ca-Est (3E0X) lacks one β sheet.
This esterase gene of Clostridium acetobutylicum was isolated and then inserted into the pMCSG7 expression vector transformed into E. coli strain DH5α cells. This clone was purchased from the DNASU plasmid repository with clone ID caCD00398108. From this clone, plasmid DNA was isolated and overexpressed in E. coli strain BL21 and purified using Ni2+-NTA chromatography. The protein was purified to homogeneity with a molecular mass of 29 kDa by SDS PAGE. Size exclusion chromatography revealed that the protein is a dimer in solution. The enzyme activity was determined by measuring the release of p-nitrophenol continuously by UV spectrophotometer at isosbestic point 348 nm. Assay conditions were optimized with optimum pH and temperature for recombinant Clostridium acetobutylicum esterase (Ca-Est) were found to be 7.0 and 60 °C and 25 ng of purified protein respectively. The purified enzyme showed a high preference for carboxyl esters. This enzyme was able to hydrolyze less than 10 carbon water-soluble esters but exhibited a high preference for p-nitrophenyl butyrate. Docking analysis further showed that the substrate binding pocket was surrounded by bulky amino acids such as leucine and phenylalanine that make the funnel-shaped pocket that limits the substrate length. The KM and kcat/KM of the enzyme with p-nitrophenyl butyrate were 24.90 µM and 25.13 s-1µM-1 respectively. Among the tested solvents, the presence of methanol showed a marginal increase in enzyme activity. In the presence of metal ions like Cu2+ and Fe2+, there is a significant loss of enzyme activity. The thermal stability of the protein was studied at 30 ℃, 60 ℃, and 80 ℃. The secondary structure of the protein was stable at 25 ℃ and 60 ℃ confirming that the enzyme was stable at optimized assay conditions. The melting temperature of the protein was found to be 67.7 ℃ by using CD spectroscopy. Amino acid modifiers showed inhibitory loss of activity with PMSF, a serine modifier, and diethyl pyrocarbonate a histidine modifier. Site-directed mutagenesis confirmed that Ser 89 and His 224 play a major role in catalysis. This study reports that Ca-Est belongs to lipolytic family IV, but it is a variant of microbial HSL. It showed that low Km and high Vmax values, showing thermostability at 60 ℃ and an increase in the activity in the presence of methanol make a suitable catalyst for industrial applications.