Enzymatic Synthesis of Lignin-Based Concrete Dispersing Agents.

Lignin is the most abundant aromatic biopolymer, functioning as an integral component of woody materials. In its unmodified form it shows limited water solubility and is relatively unreactive, so biotechnological lignin valorisation for high-performance applications is greatly underexploited. Lignin can be obtained from the pulp and paper industry as a by-product. To expand its application, a new synthesis route to new dispersing agents for use as concrete additives was developed. The route is based on lignin functionalisation by enzymatic transformation. Screening of lignin-modifying systems resulted in functionalised lignin polymers with improved solubility in aqueous systems. Through grafting of sulfanilic acid or p-aminobenzoic acid by fungal laccases, lignin became soluble in water at pH≤4 or pH≤7, respectively. Products were analysed and evaluated in miniaturised application tests in cement paste and mortar. Their dispersing properties match the performance criteria of commercially available lignosulfonates. The study provides examples of new perspectives for the use of lignin.

Engineered Bacillus pumilus laccase-like multi-copper oxidase for enhanced oxidation of the lignin model compound guaiacol.

Laccases and laccase-like multi-copper oxidases (LMCOs) are versatile and robust biocatalysts applied in a variety of oxidative processes, and various studies have attempted to improve their catalytic activity. Here we report the engineering of a bacterial LMCO for enhanced oxidation of the lignin-related compound guaiacol by a combination of structure-guided mutagenesis and DNA shuffling. Mutant L9 showed a 1.39 mM Km for guaiacol and a 2.5-fold increase in turnover rate (kcat/Km = 2.85·104 M-1s-1).

A novel enzymatic approach in the production of food with low purine content using Arxula adeninivorans endogenous and recombinant purine degradative enzymes.

The purine degradation pathway in humans ends with uric acid, which has low water solubility. When the production of uric acid is increased either by elevated purine intake or by impaired kidney function, uric acid will accumulate in the blood (hyperuricemia). This increases the risk of gout, a disease described in humans for at least 1000 years. Many lower organisms, such as the yeast Arxula adeninivorans, possess the enzyme, urate oxidase that converts uric acid to 5-hydroxyisourate, thus preventing uric acid accumulation. We have examined the complete purine degradation pathway in A. adeninivorans and analyzed enzymes involved. Recombinant adenine deaminase, guanine deaminase, urate oxidase and endogenous xanthine oxidoreductase have been investigated as potential additives to degrade purines in the food. Here, we review the current model of the purine degradation pathway of A. adeninivorans and present an overview of proposed enzyme system with perspectives for its further development.

The complete genome of Blastobotrys (Arxula) adeninivorans LS3 - a yeast of biotechnological interest.

BACKGROUND: The industrially important yeast Blastobotrys (Arxula) adeninivorans is an asexual hemiascomycete phylogenetically very distant from Saccharomyces cerevisiae. Its unusual metabolic flexibility allows it to use a wide range of carbon and nitrogen sources, while being thermotolerant, xerotolerant and osmotolerant. RESULTS: The sequencing of strain LS3 revealed that the nuclear genome of A. adeninivorans is 11.8 Mb long and consists of four chromosomes with regional centromeres. Its closest sequenced relative is Yarrowia lipolytica, although mean conservation of orthologs is low. With 914 introns within 6116 genes, A. adeninivorans is one of the most intron-rich hemiascomycetes sequenced to date. Several large species-specific families appear to result from multiple rounds of segmental duplications of tandem gene arrays, a novel mechanism not yet described in yeasts. An analysis of the genome and its transcriptome revealed enzymes with biotechnological potential, such as two extracellular tannases (Atan1p and Atan2p) of the tannic-acid catabolic route, and a new pathway for the assimilation of n-butanol via butyric aldehyde and butyric acid. CONCLUSIONS: The high-quality genome of this species that diverged early in Saccharomycotina will allow further fundamental studies on comparative genomics, evolution and phylogenetics. Protein components of different pathways for carbon and nitrogen source utilization were identified, which so far has remained unexplored in yeast, offering clues for further biotechnological developments. In the course of identifying alternative microorganisms for biotechnological interest, A. adeninivorans has already proved its strengthened competitiveness as a promising cell factory for many more applications.

Arxula adeninivorans recombinant guanine deaminase and its application in the production of food with low purine content.

Purines of exogenous and endogenous sources are degraded to uric acid in human beings. Concentrations >6.8 mg uric acid/dl serum cause hyperuricemia and its symptoms. Pharmaceuticals and the reduction of the intake of purine-rich food are used to control uric acid levels. A novel approach to the latter proposition is the enzymatic reduction of the purine content of food by purine-degrading enzymes. Here we describe the production of recombinant guanine deaminase by the yeast Arxula adeninivorans LS3 and its application in food. In media supplemented with nitrogen sources hypoxanthine or adenine, guanine deaminase (AGDA) gene expression is induced and intracellular accumulation of guanine deaminase (Agdap) protein occurs. The characteristics of the guanine deaminase isolated from wild-type strain LS3 and a transgenic strain expressing the AGDA gene under control of the strong constitutive TEF1 promoter were determined and compared. Both enzymes were dimeric and had temperature optima of 55°C with high substrate specificity for guanine and localisation in both the cytoplasm and vacuole of yeast. The enzyme was demonstrated to reduce levels of guanine in food. A mixture of guanine deaminase and other purine degradation enzymes will allow the reduction of purines in purine-rich foods.

Identification of uric acid as the redox molecule secreted by the yeast Arxula adeninivorans.

The yeast Arxula adeninivorans has been previously shown to secrete a large amount of an electro-active molecule. The molecule was produced by cells that had been cultivated in a rich medium, harvested, washed and then suspended in phosphate-buffered saline (PBS). The molecule was easily detectable after 60 min of incubation in PBS, and the cells continued to produce the molecule in these conditions for up to 3 days. The peak anodic potential of the oxidation peak was 0.42 V, and it was shown to be a solution species rather than a cell-attached species. We have optimised the production of the molecule, identified it by high-pressure liquid chromatography (HPLC) fractionation and high-resolution mass spectrometric analysis and determined the pathway involved in its synthesis. It has a mass/charge ratio that corresponds to uric acid, and this identification was supported by comparing UV spectra and cyclic voltammograms of the samples to those of uric acid. An A. adeninivorans xanthine oxidase gene disruption mutant failed to produce uric acid, which added further validity to this identification. It also demonstrated that the purine catabolism pathway is involved in its production. A transgenic A. adeninivorans strain with a switchable urate oxidase gene (AUOX) accumulated uric acid when the gene was switched off but did not when the gene was switched on. Cultivation of cells on amino acid and purine-free minimal media with an inorganic nitrogen source suggests that the cells synthesise purines from inorganic nitrogen and proceed to degrade them via the normal purine degradation pathway.

Arxula adeninivorans recombinant urate oxidase and its application in the production of food with low uric acid content.

Hyperuricemia and its symptoms are becoming increasingly common worldwide. Elevated serum uric acid levels are caused by increased uric acid synthesis from food constituents and reduced renal excretion. Treatment in most cases involves reducing alcohol intake and consumption of meat and fish or treatment with pharmaceuticals. Another approach could be to reduce uric acid level in food, either during production or consumption. This work reports the production of recombinant urate oxidase by Arxula adeninivorans and its application to reduce uric acid in a food product. The A. adeninivorans urate oxidase amino acid sequence was found to be similar to urate oxidases from other fungi (61-65% identity). In media supplemented with adenine, hypoxanthine or uric acid, induction of the urate oxidase (AUOX) gene and intracellular accumulation of urate oxidase (Auoxp) was observed. The enzyme characteristics were analyzed from isolates of the wild-type strain A. adeninivorans LS3, as well as from those of transgenic strains expressing the AUOX gene under control of the strong constitutive TEF1 promoter or the inducible AYNI1 promoter. The enzyme showed high substrate specificity for uric acid, a broad temperature and pH range, high thermostability and the ability to reduce uric acid content in food.