Sparse Epistatic Patterns in the Evolution of Terpene Synthases.

We explore sequence determinants of enzyme activity and specificity in a major enzyme family of terpene synthases. Most enzymes in this family catalyze reactions that produce cyclic terpenes-complex hydrocarbons widely used by plants and insects in diverse biological processes such as defense, communication, and symbiosis. To analyze the molecular mechanisms of emergence of terpene cyclization, we have carried out in-depth examination of mutational space around (E)-β-farnesene synthase, an Artemisia annua enzyme which catalyzes production of a linear hydrocarbon chain.

Discovery of germacrene A synthases in Barnadesia spinosa: The first committed step in sesquiterpene lactone biosynthesis in the basal member of the Asteraceae.

The Andes-endemic Barnadesioideae lineage is the oldest surviving and phylogenetically basal subfamily of the Asteraceae (Compositae), a prolific group of flowering plants with world-wide distribution (∼24,000 species) marked by a rich diversity of sesquiterpene lactones (STLs). Intriguingly, there is no evidence that members of the Barnadesioideae produce STLs, specialized metabolites thought to have contributed to the adaptive success of the Asteraceae family outside South America. The biosynthesis of STLs requires the intimate expression and functional integration of germacrene A synthase (GAS) and germacrene A oxidase (GAO) to sequentially cyclize and oxidize farnesyl diphosphate into the advanced intermediate germacrene A acid leading to diverse STLs.

Food Matrix Effects of Polyphenol Bioaccessibility from Almond Skin during Simulated Human Digestion.

The goal of the present study was to quantify the rate and extent of polyphenols released in the gastrointestinal tract (GIT) from natural (NS) and blanched (BS) almond skins. A dynamic gastric model of digestion which provides a realistic simulation of the human stomach was used. In order to establish the effect of a food matrix on polyphenols bioaccessibility, NS and BS were either digested in water (WT) or incorporated into home-made biscuits (HB), crisp-bread (CB) and full-fat milk (FM).

Emergence of terpene cyclization in Artemisia annua.

The emergence of terpene cyclization was critical to the evolutionary expansion of chemical diversity yet remains unexplored. Here we report the first discovery of an epistatic network of residues that controls the onset of terpene cyclization in Artemisia annua. We begin with amorpha-4,11-diene synthase (ADS) and (E)-β-farnesene synthase (BFS), a pair of terpene synthases that produce cyclic or linear terpenes, respectively.

Comparative analysis and validation of the malachite green assay for the high throughput biochemical characterization of terpene synthases.

Terpenes are the largest group of natural products with important and diverse biological roles, while of tremendous economic value as fragrances, flavours and pharmaceutical agents. Class-I terpene synthases (TPSs), the dominant type of TPS enzymes, catalyze the conversion of prenyl diphosphates to often structurally diverse bioactive terpene hydrocarbons, and inorganic pyrophosphate (PPi). To measure their kinetic properties, current bio-analytical methods typically rely on the direct detection of hydrocarbon products by radioactivity measurements or gas chromatography-mass spectrometry (GC-MS).

Expression of active recombinant human gastric lipase in Nicotiana benthamiana using the CPMV-HT transient expression system.

Recombinant human gastric lipase (hGL) was transiently expressed in Nicotiana benthamiana leaves using the CPMV-HT expression system. Expression levels of up to 0.5mg recombinant hGL per gram of infiltrated leaf tissue were obtained.

Evaluation of the prebiotic properties of wheat arabinoxylan fractions and induction of hydrolase activity in gut microflora.

Dietary supplementation with prebiotics may result in the stimulation of the growth of beneficial bacteria such as lactobacilli and bifidobacteria in the human gastrointestinal tract. The effect of water-unextractable arabinoxylans (WU-AX) derived from wheat on the modulation of gut bacterial composition was investigated using a mixed culture fermentation system. A prebiotic index (PI) score of 2.

Understanding the structural basis for substrate and inhibitor recognition in eukaryotic GH11 xylanases.

Endo-beta1,4-xylanases (xylanases) hydrolyse the beta1,4 glycosidic bonds in the backbone of xylan. Although xylanases from glycoside hydrolase family 11 (GH11) have been extensively studied, several issues remain unresolved. Thus, the mechanism by which these enzymes hydrolyse decorated xylans is unclear and the structural basis for the variation in catalytic activity within this family is unknown.

In vitro three-stage continuous fermentation of wheat arabinoxylan fractions and induction of hydrolase activity by the gut microflora.

In vitro fermentations were carried out by using a model of the human colon to stimulate microbial activities of gut bacteria. The model consisted of a three-stage culture system. Bacterial populations were evaluated under the effect of three types of arabinoxylan, a nonstarch polysaccharide derived from wheat, the water-unextractable arabinoxylan fraction (WU-AX), WU-AX pretreated with exogenous xylanase and the soluble water-extractable arabinoxylan fraction (WE-AX).

Probing the structural basis for the difference in thermostability displayed by family 10 xylanases.

Thermostability is an important property of industrially significant hydrolytic enzymes: understanding the structural basis for this attribute will underpin the future biotechnological exploitation of these biocatalysts. The Cellvibrio family 10 (GH10) xylanases display considerable sequence identity but exhibit significant differences in thermostability; thus, these enzymes represent excellent models to examine the structural basis for the variation in stability displayed by these glycoside hydrolases. Here, we have subjected the intracellular Cellvibrio mixtus xylanase CmXyn10B to forced protein evolution.

A family 10 Thermoascus aurantiacus xylanase utilizes arabinose decorations of xylan as significant substrate specificity determinants.

Xylan, which is a key component of the plant cell wall, consists of a backbone of beta-1,4-linked xylose residues that are decorated with arabinofuranose, acetyl, 4-O-methyl d-glucuronic acid and ferulate. The backbone of xylan is hydrolysed by endo-beta1,4-xylanases (xylanases); however, it is unclear whether the various side-chains of the polysaccharide are utilized by these enzymes as significant substrate specificity determinants. To address this question we have determined the crystal structure of a family 10 xylanase from Thermoascus aurantiacus, in complex with xylobiose containing an arabinofuranosyl-ferulate side-chain.

Tailored catalysts for plant cell-wall degradation: redesigning the exo/endo preference of Cellvibrio japonicus arabinanase 43A.

Enzymes acting on polymeric substrates are frequently classified as exo or endo, reflecting their preference for, or ignorance of, polymer chain ends. Most biotechnological applications, especially in the field of polysaccharide degradation, require either endo- or exo-acting hydrolases, or they harness the essential synergy between these two modes of action. Here, we have used genomic data in tandem with structure to modify, radically, the chain-end specificity of the Cellvibrio japonicus exo-arabinanase CjArb43A.

Enzymic production of a feruloylated oligosaccharide with antioxidant activity from wheat flour arabinoxylan.

Background: Main cereals such as rice, wheat, barley, and corn belong to the family Gramineae and have similar cell-wall composition. Since cereal cell walls are a good source of dietary fibre, meeting one-half of the daily requirement of 30 g of dietary fibre can be achieved by the regular consumption of cereals. Many studies have dealt with the isolation of feruloylated oligosaccharides from Gramineae by treatment with polysaccharide hydrolysing enzymes.