Konanani Rashamuse

A novel recombinant ethyl ferulate esterase from Burkholderia multivorans

Aims: Isolation and identification of bacterial isolates with specific ferulic acid (FA) esterase activity and cloning of a gene encoding activity.

Functional characterisation of a metagenome derived family VIII esterase with a deacetylation activity on β-lactam antibiotics.

Family VIII esterases represent a poorly characterised esterase family, with high sequence identity to class C β-lactamases, peptidases and penicillin binding proteins. This study reports on the metagenomic library screening and biochemical characterisation of a novel esterase (Est22) derived from an acidic Leachate environment. The enzyme is 423 amino acids in length and contained 22 aa signal peptide. The Est22 primary structure revealed the presence of N-terminus S-x-x-K sequence, which is also highly conserved in class C β-lactamases, peptidases as well as carboxylesterases belonging to family VIII. Phylogenetic analysis using the representative sequences from class C β-lactamases and family VIII esterases indicated that Est22 is a member of family VIII esterases. Substrate specificity profiling using p-nitrophenyl esters (C2-C16) indicated that Est22 preferred shorter chain p-nitrophenyl esters (C2-C5), a characteristic that is typical for true carboxylesterases. In addition of hydrolysing Nitrocefin, Est22 also hydrolysed first generation cephalosporin based derivatives. Detailed selectivity study using different cephalosporin based substrates indicated that Est22 selectively hydrolyse the ester bond of a cephalosporin derivatives leaving the amide bond of the β-lactam ring intact. The selective nature of Est22 makes this enzyme a potential candidate for the use in the synthesis and modification cephalosporin based molecules.

Discovery of a novel carboxylesterase through functional screening of a pre-enriched environmental library

Aims:  The aim of this study was to demonstrate the application of environmental sample pre‐enrichment to access novel carboxylesterases from environmental genomes, along with subsequent heterologous expression and characterization of the discovered enzyme(s).

Molecular Characterization of a Novel Family VIII Esterase from Burkholderia multivorans UWC10

An esterase producing Burkholderia multivorans UWC10 strain was isolated by culture enrichment. A shotgun library of B. multivorans UWC10 genomic DNA was screened for esterase activity and a recombinant clone conferring an esterolytic phenotype was identified. Full-length sequencing of the DNA insert showed that it consisted of a single open reading frame (ORF1) encoding a predicted protein of 398 amino acids. ORF1 (termed EstBL) had a high protein sequence identity to family VIII esterases. The EstBL primary structure showed two putative serine motifs, G-V-S149-D-G and S74-V-T-K. The estBL gene was successfully over-expressed in E. coli and the encoded protein purified by a combination of ammonium sulphate fractionation, hydrophobic interaction, ion exchange and size exclusion chromatographies. Biochemical assays confirmed EstBL esterase activity and revealed a preference for short-chain p-nitrophenyl and β-naphthyl esters (C2-C4) with no activity against β-lactam substrates. Secondary structure predictions indicated that EstBL adopts the α/β fold, which is common to all esterases.

High-yielding cascade enzymatic synthesis of 5-methyluridine using a novel combination of nucleoside phosphorylases

Abstract A novel combination of Bacillus halodurans purine nucleoside phosphorylase (BhPNP1) and Escherichia coli uridine phosphorylase (EcUP) has been applied to a dual-enzyme, sequential, biocatalytic one-pot synthesis of 5-methyluridine from guanosine and thymine. A 5-methyluridine yield of >79% on guanosine was achieved in a reaction slurry at a 53 mM (1.5% w/w) guanosine concentration. 5-Methyluridine is an intermediate in synthetic routes to thymidine and the antiretroviral drugs zidovudine and stavudine.

Accessing carboxylesterase diversity from termite hindgut symbionts through metagenomics.

A shotgun metagenomic library was constructed from termite hindgut symbionts and subsequently screened for esterase activities. A total of 68 recombinant clones conferring esterolytic phenotypes were identified, of which the 14 most active were subcloned and sequenced. The nucleotide lengths of the esterase-encoding open reading frames (ORFs) ranged from 783 to 2,592 bp and encoded proteins with predicted molecular masses of between 28.8 and 97.5 kDa. The highest identity scores in the GenBank database, from a global amino acid alignment ranged from 39 to 83%. The identified ORFs revealed the presence of the G-X-S-X-D, G-D-S-X, and S-X-X-K sequence motifs that have been reported to harbour a catalytic serine residue in other previously reported esterase primary structures. Five of the ORFs (EstT5, EstT7, EstT9, EstT10, and EstT12) could not be classified into any of the original eight esterase families. One of the ORFs (EstT9) showed a unique primary structure consisting of an amidohydrolase-esterase fusion. Six of the 14 esterase-encoding genes were recombinantly expressed in Escherichia coli and the purified enzymes exhibited temperature optima of between 40–50°C. Substrate-profiling studies revealed that the characterised enzymes were ‘true’ carboxylesterases based on their preferences for short to medium chain length p-nitrophenyl ester substrates. This study has demonstrated a successful application of a metagenomic approach in accessing novel esterase-encoding genes from the gut of termites that could otherwise have been missed by classical culture enrichment approaches.

Metagenomic mining of glycoside hydrolases from the hindgut bacterial symbionts of a termite, Trinervitermise trinervoides and the characterisation of a multimodular β-1, 4-Xylanase (GH11).

In recent years, there have been particular emphases worldwide on the development and optimization of bioprocesses for the utilization of biomass. An essential component of the biomass processing conduit has been the need for robust biocatalysts as high‐performance tools for both the depolymerization of lignocellulosic biomass and synthesis of new high‐value bio‐based chemical entities. Through functional screening of the metagenome of the hindgut bacterial symbionts of a termite, Trinervitermes trinervoides, we discovered open reading frames for 25 cellulases and hemicellulases. These were classified into 14 different glycoside hydrolase (GH) families: eight GH family 5; four GH9, two GH13, and one each in GH2, GH10, GH11, GH26, GH29, GH43, GH44, GH45, GH67, and GH94 families. Of these, eight were overexpressed and partially characterized to be shown to be endocellulases (GH5C, GH5E, GH5F, and GH5G), an exocellulase (GH5D), endoxylanases (GH5H and GH11), and an α‐fucosidase (GH29). The GH11 (Xyl1) was of particular interest as it was discovered to be a multimodular β‐1,4‐xylanase, consisting of a catalytic domain and two carbohydrate‐binding modules (CBMs). The CBM functions to selectively bind insoluble xylan and increases the rate of hydrolysis. The primary structure of GH11 showed a classical catalytic dyad of glutamic acid residues that generally forms part of the active site in GH11 enzyme family. This endoxylanase was optimal at pH 6 and 50 °C, and generated xylobiose and xylotriose from various xylan sources, including beechwood, birchwood, and wheat arabinoxylan. The catalytic ability of GH11 against natural substrate (e.g., wheat arabinoxylan) renders GH11 as a potential useful biocatalyst in the effective dismantling of complex plant biomass architecture.

Application of termite hindgut metagenome derived carboxyl ester hydrolases in the modification of cephalosporin substrates

In the pharmaceutical industry, de-acetylated cephalosporins are highly valuable starting materials for producing semi-synthetic β-lactam antibiotics. In this study a fosmid metagenome library from termite hindgut symbionts was screened for carboxyl ester hydrolases capable of de-acetylating cephalosporins. Recombinant Escherichia coli clones with esterolytic phenotypes on tributyrin agar plates were selected and further tested for de-acetylating activity against Cephalothin and 7-aminocephalosporanic acid (7-ACA). Two clones displaying de-acetylating activity were sequenced and the corresponding two carboxyl ester hydrolase encoding genes (axeA and axeB) belonging to the carbohydrate esterase family 7 (CE7) were identified. The primary structure of both the axeA and axeB revealed the presence of G-X-S-X-G sequence motif and respective subunit molecular masses of 40 kDa. In addition to de-acetylating cephalosporin based molecules, the two enzymes were also shown to be true esterases based on their preferences for short chain length fatty acid esters.