A novel recombinant chlorophyllase from cyanobacterium Cyanothece sp. ATCC 51142 for the production of bacteriochlorophyllide a.

Bacteriopheophorbide a (BPheid a) is used as a precursor for bacteriochlorin a (BCA), which can be used for photodynamic therapy in both in vitro and in vivo biochemical applications. This study successfully isolated and expressed a photosynthetic bacterium (Cyanothece sp. ATCC 51142) chlorophyllase called CyanoCLH, which can be used as a biocatalyst for the production of a BCA precursor by degrading bacteriochlorophyll a (BChl a). Substrate specificity and enzyme kinetic analyses were performed and the results verified that the recombinant CyanoCLH preferred hydrolyzing BChl a to produce bacteriochlorophyllide a (BChlide a), which can be converted to BPheid a by removing magnesium ion. The recombinant CyanoCLH was cloned and expressed in Escherichia coli BL-21 (DE3), and its molecular weight was 54.7 kDa. The deduced amino acid sequence of the recombinant CyanoCLH comprised a unique lipase-motif GHSLG, which differs from the GHSRG sequence of other plants and lacks a histidine of the typical and conserved catalytic triad Ser-Asp-His. The recombinant CyanoCLH was subjected to biochemical analyses, and the results indicated that its optimal pH and temperature were 7.0 and 60 °C, respectively.

Freeze-dried microalgae of Nannochloropsis oculata improve soybean oil's oxidative stability.

Marine microalga Nannochloropsis oculata possesses nutrients valuable for human health. In this study, we added freeze-dried N. oculata powder to soybean oil and observed a remarkable inhibition in oil oxidation. The amount of microalgae powder added was positively correlated to the increase in oil stability. The addition of 5.0 % (w/w) microalgae powder increased the oil stability index (OSI) values of soybean oil more than twofold at the tested temperatures 120 and 130 °C. N. oculata contains high levels of both phenolic compounds and α-tocopherols that could be the contributors to such an increase of the OSI. Two methods were conducted to assay the active ingredients released from microalgae: one employed three solvent systems to extract the microalgae and the other was the soybean oil added with microalgae. Analyses of free radical scavenging and reducing power suggested that the phenolic compounds dominated the antioxidation activities in soybean oil when it was infused with the microalgae powder. Our results suggest that N. oculata could potentially be used as an additive in cooking oil to increase the shelf life and nutritional value of the oil and to reduce the production of free radicals from lipid oxidation when the oil is used at high-temperature cooking processes.

Functional role of a non-active site residue Trp(23) on the enzyme activity of Escherichia coli thioesterase I/protease I/lysophospholipase L(1).

Escherichia coli possesses a versatile protein with the enzyme activities of thioesterase I, protease I, and lysophospholipase L(1). The protein is dubbed as TAP according to the chronological order of gene discovery (TesA/ApeA/PldC). Our previous studies showed that TAP comprises the catalytic triad Ser(10), Asp(154), and His(157) as a charge relay system, as well as Gly(44) and Asn(73) residues devoted to oxyanion hole stabilization. Geometrically, about 10 A away from the enzyme catalytic cleft, Trp(23) showed a stronger resonance shift than the backbone amide resonance observed in the nuclear magnetic resonance (NMR) analyses. In the present work, we conducted site-directed mutagenesis to change Trp into alanine (Ala), phenylalanine (Phe), or tyrosine (Tyr) to unveil the role of the Trp(23) indole ring. Biochemical analyses of the mutant enzymes in combination with TAP's three-dimensional structures suggest that by interlinking the residues participating in this catalytic machinery, Trp(23) could effectively influence substrate binding and the following turnover number. Moreover, it may serve as a contributor to both H-bond and aromatic-aromatic interaction in maintaining the cross-link within the interweaving framework of protein.

Enhanced preference for pi-bond containing substrates is correlated to Pro110 in the substrate-binding tunnel of Escherichia coli thioesterase I/protease I/lysophospholipase L(1).

Escherichia coli thioesterase I/protease I/lysophospholipase L(1) (TAP) possesses multifunctional enzyme with thioesterase, esterase, arylesterase, protease, and lysophospholipase activities. Leu109, located at the substrate-binding tunnel, when substituted with proline (Pro) in TAP, shifted the substrate-preference from medium-to-long acyl chains to shorter acyl chains of triglyceride and p-nitrophenyl ester, and increased the preference for aromatic-amino acid-derived esters. In the three-dimensional TAP structures, the only noticeable alteration of backbone and side chain conformation was located at the downstream Pro110-Ala123 region rather than at Pro109 itself. The residue Pro110, adjacent to Leu109 or Pro109, was found to contribute to the substrate preference of TAP enzymes for esters containing acyl groups with pi bond(s) or aromatic group(s). Some of the interactions between the enzyme protein and the substrate may be contributed by an attractive force between the Pro110 C-H donor and the substrate pi-acceptor.

Functional role of catalytic triad and oxyanion hole-forming residues on enzyme activity of Escherichia coli thioesterase I/protease I/phospholipase L1.

Escherichia coli TAP (thioesterase I, EC 3.1.2.2) is a multifunctional enzyme with thioesterase, esterase, arylesterase, protease and lysophospholipase activities. Previous crystal structural analyses identified its essential amino acid residues as those that form a catalytic triad (Ser10-Asp154-His157) and those involved in forming an oxyanion hole (Ser10-Gly44-Asn73). To gain an insight into the biochemical roles of each residue, site-directed mutagenesis was employed to mutate these residues to alanine, and enzyme kinetic studies were conducted using esterase, thioesterase and amino-acid-derived substrates. Of the residues, His157 is the most important, as it plays a vital role in the catalytic triad, and may also play a role in stabilizing oxyanion conformation. Ser10 also plays a very important role, although the small residual activity of the S10A variant suggests that a water molecule may act as a poor substitute. The water molecule could possibly be endowed with the nucleophilic-attacking character by His157 hydrogen-bonding. Asp154 is not as essential compared with the other two residues in the triad. It is close to the entrance of the substrate tunnel, therefore it predominantly affects substrate accessibility. Gly44 plays a role in stabilizing the oxyanion intermediate and additionally in acyl-enzyme-intermediate transformation. N73A had the highest residual enzyme activity among all the mutants, which indicates that Asn73 is not as essential as the other mutated residues. The role of Asn73 is proposed to be involved in a loop75-80 switch-move motion, which is essential for the accommodation of substrates with longer acyl-chain lengths.