Mirosława Szczęsna-Antczak

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

ABSTRACT 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.

Engineering of lipase-catalyzed transesterification reaction media using water and diethylamine

Abstract The objective of presented study was to maximize yields of 2-methylbutyl esters, derived by transesterification reactions mediated by sn-1,3-specific lipases, through engineering of reaction medium. Effects of water and diethylamine (DEA) concentrations on the efficiency of plant oils 2-methylbutanolysis, catalyzed by either mycelium-bound Mucor circinelloides lipase (powder) or commercial immobilized lipase Lipozyme TL IM, were determined. Water content monitoring in reaction mixtures enabled to optimize the initial water content in terms of preventing the dehydration of enzyme’s microenvironment and increasing 2-methylbutyl esters yields. These yields were found to be increased by addition of either suitable amounts of water (0.5–1.5%) or diethylamine (10–30 mM) to the mixture of substrates. The presented results suggest that at low concentrations, diethylamine molecules contribute to retaining water in the microenvironment of enzyme that gives rise to increased transesterification yields and significantly reduced amounts of residual mono- and 1,2-diacyl-glycerols.

Cold-Active Yeast Lipases: Recent Issues and Future Prospects

Microbial lipases are, besides proteases, enzymes of the highest biotechnological potential, catalyzing not only hydrolytic reactions but also – in media of low water activity – synthesis reactions. These enzymes are used in pharmaceutical, food, and household chemicals industries and for the treatment of environmental pollution. Production of lipases is constantly increasing and now accounts for more than one-fifth of the global enzyme market. This review is dedicated to cold-active lipases of yeasts, of which lipases A and B of Pseudozyma (formerly Candida) antarctica have been the most thoroughly investigated. This chapter covers distinctive structural features and specificity of these enzymes in comparison with selected mesophilic lipases as well as modifications (together with diverse immobilization techniques on various supports) directed to improving catalytic properties and stability of these proteins. The application potential of cold-active yeast lipases is discussed; the most important applications include enantio- and regioselective biotransformations, production of biofuels, detergents, food additives, structured triacylglycerols, etc. Some lipases from mesophilic yeasts (e.g., non-conventional yeast Yarrowia lipolytica) show characteristic features of cold-active enzymes, and examples of their use are also considered.

ISOLATION, MOLECULAR CLONING AND CHARACTERISATION OF TWO GENES CODING CHITIN DEACETYLASE FROM MUCOR CIRCINELLOIDES IBT-83

Chitosan is a linear N-deacetylated derivative of chitin, soluble in acetic solutions. The deacetylation of chitin can be achieved enzymatically using chitin deacetylase (ChDa) (EC 3.5.1.41), which hydrolyses the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin and chitosan. Complementary DNA (cDNA), which encodes ChDa, was isolated from M. rouxii s well as other fungi. Chitin deacetylase activity was detected in partially purified and concentrated crude extract of the protein from Mucor circinelloides IBT-83. Additionally, two open reading frames (ORF), putatively encoding ChDa, were identified and amplified from cDNA of this strain. Each ORF was molecularly cloned and sequenced. Amino acid sequences of ChDaI and ChDaII were predicted, using nucleotide sequences of these cDNA clones, and analysed by means of bioinformatics tools.

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.