Expression of Bacillus amyloliquefaciens transglutaminase in recombinant E. coli under the control of a bicistronic plasmid system in DO-stat fed-batch bioreactor cultivations.

We studied the expression of Bacillus amyloliquefaciens transglutaminase cloned in Escherichia coli BL21(DE3)pLysS harboring the plasmid pBAD/3C/bTGase, a bicistronic expression system, in bioreactor cultivation. Batch and fed-batch controlled as DO-stat strategies were employed for the production of the recombinant enzyme. In 30 h-batch cultivations using Terrific broth (TB), 6 g/L of biomass and 3.12 U/mgprotein of transglutaminase activity were obtained. DO-stat fed-batch cultivations under the control of oxygen concentration (DO-stat) using TB as medium but fed with glucose allowed the increment in biomass formation (17.5 g/L) and enzyme activity (6.43 U/mgprotein). DO-stat fed-batch using mineral medium (M9) and fed with glucose under the same conditions produced even higher enzymatic activity (9.14 U/mgprotein). The pH effect was investigated, and the best enzymatic activity could be observed at pH 8. In all cultivations, the bicistronic system remained stable, with 100% of plasmid-bearing cells. These results show that E. coli bearing bicistronic plasmid constructs to express recombinant TGase could be cultivated in bioreactors under DO-stat fed-batch using mineral medium and it is a promising strategy in future optimizations to produce this important enzyme.

Production of volatile compounds by yeasts using hydrolysed grape seed oil obtained by immobilized lipases in continuous packed-bed reactors.

Lipases CAL-B, TLL, and RML were used in the synthesis of free fatty acids of grape seed oil as heterogeneous substrate. The best enzyme was used to optimize the reaction variables temperature, enzyme content, and molar ratio of water:oil in batch reactions using experimental planning. The ideal conditions to produce free fatty acids using pure RML were 45 °C, 12:1 substrate molar ratio, and 15% enzyme, resulting in 66% of oil hydrolysis and a productivity of 0.54 mol L-1 min-1 in 4 h of reaction at 180 rpm. Repeated batches of reaction were performed testing the operational stability of RML, results showing that this enzyme could be used for at least 20 cycles keeping more than 80% of its initial activity, suggesting its potential use in industrial processes. The synthesis of free fatty acids was then evaluated in continuous reactions using packed-bed reactor (PBR). The highest productivity in the continuous process was 6.85 mol L-1 min-1, using only RML, showing an operational stability higher than 80% of its initial conversion capacity after 11 days of operation, at a flow rate of 0.13 mL min-1 at 45 °C. We evaluated the use of this hydrolyzed oil as substrate for lactone bioproduction using Galactomyces geotrichum UFMG-CM-Y3276, G. geotrichum UFMG-CM-Y3558, and Geotrichum klebahnii UFMG-CM-Y3014 screened for their oil-hydrolysis ability. Volatile compounds were qualitatively identified in GC-MS as γ-octalactone and γ-nonalactone.

Transglutaminases: part I-origins, sources, and biotechnological characteristics.

The transglutaminases form a large family of intracellular and extracellular enzymes that catalyze cross-links between protein molecules. Transglutaminases crosslinking properties are widely applied to various industrial processes, to improve the firmness, viscosity, elasticity, and water-holding capacity of products in the food and pharmaceutical industries. However, the extremely high costs of obtaining transglutaminases from animal sources have prompted scientists to search for new sources of these enzymes. Therefore, research has been focused on producing transglutaminases by microorganisms, which may present wider scope of use, based on enzyme-specific characteristics. In this review, we present an overview of the literature addressing the origins, types, reactions, and general characterizations of this important enzyme family. A second review will deal with transglutaminases applications in the area of food industry, medicine, pharmaceuticals and biomaterials, as well as applications in the textile and leather industries.

Review transglutaminases: part II-industrial applications in food, biotechnology, textiles and leather products.

Because of their protein cross-linking properties, transglutaminases are widely used in several industrial processes, including the food and pharmaceutical industries. Transglutaminases obtained from animal tissues and organs, the first sources of this enzyme, are being replaced by microbial sources, which are cheaper and easier to produce and purify. Since the discovery of microbial transglutaminase (mTGase), the enzyme has been produced for industrial applications by traditional fermentation process using the bacterium Streptomyces mobaraensis. Several studies have been carried out in this field to increase the enzyme industrial productivity. Researches on gene expression encoding transglutaminase biosynthesis were performed in Streptomyces lividans, Escherichia coli, Corynebacterium glutamicum, Yarrowia lipolytica, and Pichia pastoris. In the first part of this review, we presented an overview of the literature on the origins, types, mediated reactions, and general characterizations of these important enzymes, as well as the studies on recombinant microbial transglutaminases. In this second part, we focus on the application versatility of mTGase in three broad areas: food, pharmacological, and biotechnological industries. The use of mTGase is presented for several food groups, showing possibilities of applications and challenges to further improve the quality of the end-products. Some applications in the textile and leather industries are also reviewed, as well as special applications in the PEGylation reaction, in the production of antibody drug conjugates, and in regenerative medicine.

Enzymatic synthesis of ethyl esters from waste oil using mixtures of lipases in a plug-flow packed-bed continuous reactor.

This work describes the continuous synthesis of ethyl esters via enzymatic catalysis on a packed-bed continuous reactor, using mixtures of immobilized lipases (combi-lipases) of Candida antarctica (CALB), Thermomyces lanuginosus (TLL), and Rhizomucor miehei (RML). The influence of the addition of glass beads to the reactor bed, evaluation of the use of different solvents, and flow rate on reaction conditions was studied. All experiments were conducted using the best combination of lipases according to the fatty acid composition of the waste oil (combi-lipase composition: 40% of TLL, 35% of CALB, and 25% of RML) and soybean oil (combi-lipase composition: 22.5% of TLL, 50% of CALB, and 27.5% of RML). The best general reaction conditions were found to be using tert-butanol as solvent, and the flow rate of 0.08 mL min-1 . The combi-lipase reactors operating at steady state for over 30 days (720 h), kept conversion yields of ∼50%, with average productivity of 1.94 gethyl estersgsubstrate-1 h-1 , regardless of the type of oil in use. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:952-959, 2018.

Transesterification of Waste Frying Oil and Soybean Oil by Combi-lipases Under Ultrasound-Assisted Reactions.

This work describes the use of an ultrasound system for the enzymatic transesterification of oils using combi-lipases as biocatalyst. The reactions were carried out evaluating the individual use of waste oil and fresh soybean oil, and the immobilized lipases CALB, TLL, and RML were used as biocatalysts. It was performed in a mixture design of three factors to obtain the ideal mixture of lipases according to the composition of fatty acids present in each oil, and the main reaction variables were optimized. After 18 h of reaction, ultrasound provided a biodiesel yield of about 90% when using soybean oil and 70% using the waste oil. The results showed that ultrasound technology, in combination with the application of enzyme mixtures, known as combi-lipases, and the use of waste oil, could be a promising route to reduce the overall process costs of enzymatic production of biodiesel.

Effects of immobilization, pH and reaction time in the modulation of α-, ß- or γ-cyclodextrins production by cyclodextrin glycosyltransferase: Batch and continuous process.

This study reports the immobilization of a ß-CGTase on glutaraldehyde pre-activated silica and its use to production of cyclodextrins in batch and continuous reactions. We were able to modulate the cyclodextrin production (α-, ß- and γ-CD) by immobilization and changing the reaction conditions. In batch reactions, the immobilized enzyme reached to maximum productions of 4.9mgmL-1 of α-CD, 3.6mgmL-1 of ß-CD and 3.5mgmL-1 of γ-CD at different conditions of temperature, pH and reaction time. In continuous reactor, varying the residence time and pH it was possible to produce at pH 4.0 and 141min of residence time preferentially γ-CD (0.75 and 3.36mgmL-1 of α- and γ-CD, respectively), or at pH 8.0 and 4.81min α- and ß-CDs (3.44 and 3.51mgmL-1).

Immobilization of Thermomyces lanuginosus lipase by different techniques on Immobead 150 support: characterization and applications.

Thermomyces lanuginosus lipase (TLL) was immobilized on native and modified Immobead 150, with epoxy groups removed by hydrolysis and oxidized to add aldehyde on its surface. Immobilizations on both supports were performed by adsorption, adsorption and cross-linking, covalent attachment, multipoint covalent attachment, and, for the modified support, multipoint covalent attachment using ethylenediamine. Biocatalysts were evaluated for thermal and solvent stabilities, and the best biocatalyst was also tested after incubation in ionic liquids and used in the synthesis of butyl butyrate and isoamyl butyrate. Multipoint covalent immobilized TLL on the native Immobead 150 (Emulti) showed a half-life of 5.32 h at 70 °C, being approximately 30 times more stable than its soluble form; it showed high stability in acetone, hexane, and isooctane. Its enzymatic activity was up to 40% when incubated in ionic liquids. Ester synthesis produced yields of esterification above 60% in 24 h. Of all immobilization protocols, the Emulti performed best concerning the thermal, solvent, and ionic liquids stabilities. Emulti was successfully applied to the synthesis of butyl butyrate and isoamyl butyrate, which are very important products for the food and beverage industries.