Polycistronic Expression System for Pichia pastoris Composed of Chitino- and Chitosanolytic Enzymes.

Chitin is one of the most abundant biopolymers. Due to its recalcitrant nature and insolubility in accessible solvents, it is often considered waste and not a bioresource. The products of chitin modification such as chitosan and chitooligosaccharides are highly sought, but their preparation is a challenging process, typically performed with thermochemical methods that lack specificities and generate hazardous waste. Enzymatic treatment is a promising alternative to these methods, but the preparation of multiple biocatalysts is costly. In this manuscript, we biochemically characterised chitin deacetylases of Mucor circinelloides IBT-83 and utilised one of them for the construction of the first eukaryotic, polycistronic expression system employing self-processing 2A sequences. The three chitin-processing enzymes; chitin deacetylase of M. circinelloides IBT-83, chitinase from Thermomyces lanuginosus, and chitosanase from Aspergillus fumigatus were expressed under the control of the same promoter in methylotrophic yeast Pichia pastoris and characterised for their synergistic action towards their respective substrates.

Preparative scale application of Mucor circinelloides ene-reductase and alcohol dehydrogenase activity for the asymmetric bioreduction of α,ß-unsaturated γ-ketophosphonates.

The fungus Mucor circinelloides exhibits high potential for green chemistry and technological applications. Recently M. circinelloides, which so far was considered mainly as a platform for biodiesel production, was found to exhibit high ene-reductase activity. In our current research we applied this promising microorganism to the biotransformation of a series of α,ß-unsaturated γ-ketophosphonates. The biotransformations were conducted using cheap corn steep liquor or minimal media. The products were obtained with excellent enantiomeric purity (>99% ee in most cases) and in good isolated yields, highlighting the great potential of this microorganism for asymmetric synthesis. Moreover, the products obtained may be further applied as chiral building blocks for the synthesis of biologically active compounds, such as glutamic acid or fosmidomycin derivatives.

Enzymatic Modifications of Chitin, Chitosan, and Chitooligosaccharides.

Chitin and its N-deacetylated derivative chitosan are two biological polymers that have found numerous applications in recent years, but their further deployment suffers from limitations in obtaining a defined structure of the polymers using traditional conversion methods. The disadvantages of the currently used industrial methods of chitosan manufacturing and the increasing demand for a broad range of novel chitosan oligosaccharides (COS) with a fully defined architecture increase interest in chitin and chitosan-modifying enzymes. Enzymes such as chitinases, chitosanases, chitin deacetylases, and recently discovered lytic polysaccharide monooxygenases had attracted considerable interest in recent years. These proteins are already useful tools toward the biotechnological transformation of chitin into chitosan and chitooligosaccharides, especially when a controlled non-degradative and well-defined process is required. This review describes traditional and novel enzymatic methods of modification of chitin and its derivatives. Recent advances in chitin processing, discovery of increasing number of new, well-characterized enzymes and development of genetic engineering methods result in rapid expansion of the field. Enzymatic modification of chitin and chitosan may soon become competitive to conventional conversion methods.

Oil accumulation and in situ trans/esterification by lipolytic fungal biomass.

The goal of this study was to increase the cost-effectiveness of oil production by an oleaginous and lipolytic strain M. circinelloides IBT-83, by optimizing both lipids accumulation in the mycelium containing intracellular lipases, and a one-step process coupling lipids extraction and enzymatic trans/esterification. In optimal conditions (culture medium composed of corn steep solids, plant oil, glucose and NO3-) over 50gd.w./dm3 of biomass containing over 60% of lipids was produced. The lipids extracted with acetone or petroleum ether contain free fatty acids and triacylglycerols. The supplementation of the second solvent with alcohol results in enzymatic trans/esterification of lipids with the yield of over 80% of esters in 1 h. To our knowledge, this is the first suggestion to convert fungal oils into esters during their extraction using intracellular lipases contained in the same fungus. What is important, it is possible to obtain a second product, lipase preparation, in this process.

Scale-up of PUF-immobilized fungal chitosanase-lipase preparation production.

Mucor circinelloides IBT-83 mycelium that exhibits both lipolytic (AL) and chitosanolytic (ACH) activities was immobilized into polyurethane foam in a 30 L laboratory fermenter. The process of immobilization was investigated in terms of the carrier porosity, its type, amount, and shape, location inside the fermenter, mixing, and aeration parameters during the culture, as well as downstream processing operations. The selected conditions allowed for immobilization of approximately 7 g of defatted and dried mycelium in 1 g of carrier, i.e., seven times more than achievable in 1 L shake-flasks. Enzymatic preparation obtained by this method exhibited both the chitosanolytic (ACH 432.5 ± 6.8 unit/g) and lipolytic (AL 150.0 ± 9.3 U/g) activities. The immobilized preparation was successfully used in chitosan hydrolysis to produce chitooligosaccharides and low molecular weight chitosan, as well as in waste fats degradation and in esters synthesis in nonaqueous media. It was found that the half-life of immobilized preparations stored at room temperature is on average of 200 days.

Immobilized preparation of cold-adapted and halotolerant Antarctic beta-galactosidase as a highly stable catalyst in lactose hydrolysis.

A cold-active beta-galactosidase of Antarctic marine bacterium Pseudoalteromonas sp. 22b was synthesized by an Escherichia coli transformant harboring its gene and immobilized on glutaraldehyde-treated chitosan beads. Unlike the soluble enzyme the immobilized preparation was not inhibited by glucose, its apparent optimum temperature for activity was 10 degrees C higher (50 vs. 40 degrees C, respectively), optimum pH range was wider (pH 6-9 and 6-8, respectively) and stability at 50 degrees C was increased whilst its pH-stability remained unchanged. Soluble and immobilized preparations of Antarctic beta-galactosidase were active and stable in a broad range of NaCl concentrations (up to 3 M) and affected neither by calcium ions nor by galactose. The activity of immobilized beta-galactosidase was maintained for at least 40 days of continuous lactose hydrolysis at 15 degrees C and its shelf life at 4 degrees C exceeded 12 months. Lactose content in milk was reduced by more than 90% over a temperature range of 4-30 degrees C in continuous and batch systems employing the immobilized enzyme.