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.

Biosurfactant from endophytic Bacillus pumilus 2A: physicochemical characterization, production and optimization and potential for plant growth promotion.

BACKGROUND: Microbial surfactants called biosurfactants, thanks to their high biodegradability, low toxicity and stability can be used not only in bioremediation and oil processing, but also in the food and cosmetic industries, and even in medicine. However, the high production costs of microbial surfactants and low efficiency limit their large-scale production. This requires optimization of management conditions, including the possibility of using waste as a carbon source, such as food processing by-products. This papers describes the production and characterization of the biosurfactant obtained from the endophytic bacterial strain Bacillus pumilus 2A grown on various by-products of food processing and its potential applications in supporting plant growth. Four different carbon and nitrogen sources, pH, inoculum concentration and temperature were optimized within Taguchi method. RESULTS: Optimization of bioprocess within Taguchi method and experimental analysis revealed that the optimal conditions for biosurfactant production were brewer's spent grain (5% w/v), ammonium nitrate (1% w/v), pH of 6, 5% of inoculum, and temperature at 30 °C, leading to 6.8 g/L of biosurfactant. Based on gas chromatography-mass spectrometry and Fourier transform infrared spectroscopy analysis produced biosurfactant was determined as glycolipid. Obtained biosurfactant has shown high and long term thermostability, surface tension of 47.7 mN/m, oil displacement of 8 cm and the emulsion index of 69.11%. The examined glycolipid, used in a concentration of 0.2% significantly enhanced growth of Phaseolus vulgaris L. (bean), Raphanus L. (radish), Beta vulgaris L. (beetroot). CONCLUSIONS: The endophytic Bacillus pumilus 2A produce glycolipid biosurfactant with high and long tem thermostability, what makes it useful for many purposes including food processing. The use of brewer's spent grain as the sole carbon source makes the production of biosurfactants profitable, and from an environmental point of view, it is an environmentally friendly way to remove food processing by products. Glycolipid produced by endophytic Bacillus pumilus 2A significantly improve growth of Phaseolus vulgaris L. (bean), Raphanus L. (radish), Beta vulgaris L. (beetroot). Obtained results provide new insight to the possible use of glycolipids as plant growth promoting agents.

Biosurfactant production and hydrocarbon degradation activity of endophytic bacteria isolated from Chelidonium majus L.

BACKGROUND: The process of plant growth in the contaminated environment is often inhibited and entails the neutralization of harmful compounds. To reduce the negative impact of harmful compounds microorganisms produce unique compounds called biosurfactants. This paper describes the potential of culturable endophytic microorganisms from synanthropic plant-Chelidonium majus L. for the production of biosurfactants, as indirect plant promoting factors as well as their degradation activity. Emulsifying activity and degradation potential of tested strains were assessed by cultivation of isolates in the presence of diesel oil and waste engine oil. RESULTS: Ten bacterial strain were isolated. Analysis of emulsifying activity revealed that all isolates possessed the ability for biosurfactant production. However, one of the isolated endophytes-2A, identified as Bacillus pumilus, exhibited the highest emulsifying activity (OD500 1.96). The same strain has shown very high degradation potential, both for diesel oil and waste engine oil hydrocarbons. Results obtained with the Phytotoxkit tests revealed that the addition of biosurfactant isolated from B. pumilus 2A strain resulted in stimulation of seed germination in soil contaminated with diesel oil (137%) and waste engine oil (120%). Positive impact of the biosurfactant produced by B. pumilus 2A on the growth of Sinapis alba in hydrocarbons contaminated soil was demonstrated. CONCLUSIONS: The endophytic strain identified as Bacillus pumilus 2A produce biosurfactant that is able to act as plant-growth promoting agent. Endophytic bacteria isolated from Chelidonium majus L. exhibit potential for hydrocarbons degradation and biosurfactant production. These properties provide promising perspectives for application of biosurfactants as potential agents for bioremediation of environment contaminated with hydrocarbons.

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.