Kgama Mathiba

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.

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.

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.

Hydrolysis of nitriles by soil bacteria: Variation with soil origin

The aim of this study was to explore bacterial soil diversity for nitrile biocatalysts, in particular, those for hydrolysis of β‐substituted nitriles, to the corresponding carboxamides and acids that may be incorporated into peptidomimetics. To achieve this, we needed to compare the efficiency of isolation methods and determine the influence of land use and geographical origin of the soil sample.

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.

Biocatalytic conversion of aloeresin A to aloesin.

Leaf exudates from Aloe species, such as the Southern African Aloe ferox, are used in traditional medicines for both humans and livestock. This includes aloesin, a skin bleaching product that inhibits the synthesis of melanin. Aloesin, (a C-glycoside-5-methylchromone) can be released from aloeresin A, an ester of aloesin, through hydrolysis. The objective of the current study was to identify an enzymatic hydrolysis method for converting aloeresin A to aloesin, resulting in increased concentrations of aloesin in the aloe bitters extract. More than 70 commercially available hydrolytic enzymes were screened for the conversion of aloeresin A. An esterase (ESL001-02) from Diversa, a lipase (Novozym 388) and a protease (Aspergillus oryzae) preparation were identified during screening as being capable of providing conversion of pure aloeresin A, with the protease giving the best conversion (~100%). It was found that a contaminating enzyme in Novo 388 was responsible for the conversion of aloeresin A to aloesin. This contaminating enzyme, possibly a protease, was able to give almost complete conversion using crude aloe bitters extract, doubling the concentration of aloesin in aloe bitters extract via the hydrolysis of aloeresin A.