Development of a Hybrid Bioinorganic Nanobiocatalyst: Remarkable Impact of the Immobilization Conditions on Activity and Stability of ß-Galactosidase.

Hybrid bioinorganic biocatalysts have received much attention due to their simple synthesis, high efficiency, and structural features that favor enzyme activity and stability. The present work introduces a biomineralization strategy for the formation of hybrid nanocrystals from ß-galactosidase. The effects of the immobilization conditions were studied, identifying the important effect of metal ions and pH on the immobilization yield and the recovered activity. For a deeper understanding of the biomineralization process, an in silico study was carried out to identify the ion binding sites at the different conditions. The selected ß-galactosidase nanocrystals showed high specific activity (35,000 IU/g biocatalyst) and remarkable thermal stability with a half-life 11 times higher than the soluble enzyme. The nanobiocatalyst was successfully tested for the synthesis of galacto-oligosaccharides, achieving an outstanding performance, showing no signs of diffusional limitations. Thus, a new, simple, biocompatible and inexpensive nanobiocatalyst was produced with high enzyme recovery (82%), exhibiting high specific activity and high stability, with promising industrial applications.

Biocatalysis in the winemaking industry: Challenges and opportunities for immobilized enzymes.

Enzymes are powerful catalysts already being used in a large number of industrial processes. Impressive advantages in enzyme catalysts improvement have occurred in recent years aiming to improve their performance under harsh operation conditions far away from those of their cellular habitat. Production levels of the winemaking industry have experienced a remarkable increase, and technological innovations have been introduced for increasing the efficiency at different process steps or for improving wine quality, which is a key issue in this industry. Enzymes, such as pectinases and proteases, have been traditionally used, and others, such as glycosidases, have been more recently introduced in the modern wine industry, and many dedicated studies refer to the improvement of enzyme performance under winemaking conditions. Within this framework, a thorough review on the role of enzymes in winemaking is presented, with special emphasis on the use of immobilized enzymes as a significant strategy for catalyst improvement within an industry in which enzymes play important roles that are to be reinforced paralleling innovation.

Synthesis and purification of galacto-oligosaccharides: state of the art.

Lactose-derived non-digestible oligosaccharides are prominent components of functional foods. Among them, galacto-oligosaccharides (GOS) outstand for being prebiotics whose health-promoting effects are supported on strong scientific evidences, having unique properties as substitutes of human milk oligosaccharides in formulas for newborns and infants. GOS are currently produced enzymatically in a kinetically-controlled reaction of lactose transgalactosylation catalyzed by ß-galactosidases from different microbial strains. The enzymatic synthesis of GOS, although being an established technology, still offers many technological challenges and opportunities for further development that has to be considered within the framework of functional foods which is the most rapidly expanding market within the food sector. This paper presents the current technological status of GOS production, its main achievements and challenges. Most of the problems yet to be solved refer to the rather low GOS yields attainable that rarely exceed 40 %, corresponding to lactose conversions around 60 %. This means that the product or reaction (raw GOS) contains significant amounts of residual lactose and monosaccharides (glucose and galactose). Efforts to increase such yields have been for the most part unsuccessful, even though improvements by genetic and protein engineering strategies are to be expected in the near future. Low yields impose a burden on downstream processing to obtain a GOS product of the required purity. Different strategies for raw GOS purification are reviewed and their technological significance is appraised.

Ultrasound-assisted enzymatic synthesis of galacto-oligosaccharides using native whey with two commercial ß-galactosidases: Aspergillus oryzae and Kluyveromyces var lactis.

Native whey obtained during casein micelle microfiltration was used as a novel source to produce galacto-oligosaccharides (GOS). Since the presence of macromolecules and other interferers reduces biocatalyst performance, this work evaluated the effect of different ultrasound processing conditions on GOS synthesis using concentrated native whey. Ultrasonic intensities (UI) below 11 W/cm2 tended to increase the activity in the enzyme from Aspergillus oryzae for several minutes but accelerated the inactivation in that from Kluyveromyces lactis. At 40 °C, 40 % w/w native whey, 70 % wave amplitude, and 0.6 s/s duty-cycle, a UI of 30 W/cm2 was achieved, and the increased specific enzyme productivity was similar to the values obtained with pure lactose (∼0.136 g GOS/h/mgE). This strategy allows for obtaining a product containing prebiotics with the healthy and functional properties of whey proteins, avoiding the required purification steps used in the production of food-grade lactose.

Enzymatic production of lactulose by fed-batch and repeated fed-batch reactor.

The effects of the most significant operational variables on reactor performance of fed-batch and repeated fed-batch were evaluated in the lactulose production by enzymatic transgalactosylation. Feed flowrate in the fed stage (F) and fructose to lactose molar ratio (Fr/L) were the variables that mostly affected the values ​​of lactulose yield (YLu), lactulose productivity (πLu) and selectivity of transgalactosylation (SLu/TOS). Maximum YLu of 0.21 g lactulose per g lactose was obtained at 50% w/w inlet carbohydrates concentration (IC) of, 50 °C, Fr/L 8, F 1 mL⋅min-1, 200 IU∙gLactose-1 reactor enzyme load and pH 4.5. At these conditions the selectivity was 7.4, productivity was 0.71 gLu∙g-1∙h-1and lactose conversion was 0.66. The operation by repeated fed batch increases the efficiency of use of the biocatalysts (EB) and the accumulated productivity compared to batch and fed batch operation with the same biocatalyst. EB obtained was 4.13 gLu∙mgbiocatalyst protein-1, 10.6 times higher than in fed-batch.

Conventional and non-conventional applications of ß-galactosidases.

ß-Galactosidase is one of the most important industrial enzymes, that has been used for many decades in the dairy industry. The main application of ß-galactosidase is related to the production of low-lactose and lactose-free milk and dairy products, which are now common consumer goods in supermarket shelves. This is a well-established market that is expected to keep on growing as these products become more accessible to mid-income people worldwide. However, a fresh air has come into the ß-galactosidase business as non-conventional applications arose in recent decades based on its transgalactosylation activity. This capacity is certainly a major asset for a commodity enzyme that can be used now as a catalyst for the upgrading of readily available and cheap lactose into high added-value glycosides in processes of organic synthesis in tune with green chemistry principles within the framework of sustainability. This is a reflection of a paradigm shift, where enzymes are now being considered as apt catalysts for the synthesis of valuable organic compounds. This article reviews the main applications of ß-galactosidase, going from its conventional use related to its hydrolytic activity to the ongoing non-conventional applications in the synthesis of high added-value oligosaccharides based on its transgalactosylation activity.

Improvements in the production of Aspergillus oryzae ß-galactosidase crosslinked aggregates and their use in repeated-batch synthesis of lactulose.

Aspergillus oryzae ß-galactosidase was immobilized by aggregation and crosslinking, obtaining catalysts (CLAGs) well-endowed for lactulose synthesis. Type and concentration of the precipitating agent were determinants of immobilization yield, specific activity and thermal stability. CLAGs with specific activities of 64,007, 48,374 and 44,560 IUH g-1 were obtained using 50% v/v methanol, ethanol and propanol as precipitating agents respectively, with immobilization yields over 90%. Lactulose synthesis was conducted at 50 °C, pH 4.5, 50% w/w total sugars, 200 IUH g-1 of enzyme and fructose/lactose molar ratio of 8 in batch and repeated-batch operation. Lactulose yields were 0.19 g g-1 and 0.24 g g-1 for fructose to lactose molar ratios of 4 mol mol-1 and 8 mol mol-1 while selectivities were 3.3 mol mol-1 and 6.6 mol mol-1 respectively for CLAGs obtained by ethanol and propanol precipitation. Based on these results, both CLAGs were selected for the synthesis in repeated-batch mode. The cumulative mass of lactulose in repeated-batch was higher with CLAGs produced by ethanol and propanol precipitation than with the free enzyme. 86 and 93 repeated-batches could have been respectively performed with those CLAGs considering a catalyst replacement criterion of 50% of residual activity, as determined by simulation.

Continuous enzymatic synthesis of lactulose in packed-bed reactor with immobilized Aspergillus oryzae ß-galactosidase.

Lactulose synthesis from fructose and lactose in continuous packed-bed reactor operation with glyoxyl-agarose immobilized Aspergillus oryzae ß-galactosidase is reported for the first time. Alternative strategies to conventional batch synthesis have been scarcely explored for lactulose synthesis. The effect of flow rate, substrates ratio and biocatalyst-inert packing material mass ratio (MB/MIM) were studied on reactor performance. Increase in any of these variables produced an increase in lactulose yield (YLu) being higher than obtained in batch synthesis at comparable conditions. Maximum YLu of 0.6 g·g-1 was obtained at 50 °C, pH 4.5, 50% w/w total sugars, 15 mL·min-1, fructose/lactose molar ratio of 12 and MB/MIM of 1/8 g·g-1; at such conditions yield of transgalactosylated oligosaccharides (YTOS) was 0.16 g·g-1, selectivity (lactulose/TOS molar ratio) was 5.4 and lactose conversion (XLactose) was 28%. Reactor operation with recycle had no significant effect on yield, producing only some decrease in productivity.

ß-Galactosidase from Exiguobacterium acetylicum: Cloning, expression, purification and characterization.

The main goal of this work was to evaluate the performance of ß-galactosidase from Exiguobacterium acetylicum MF03 in both hydrolysis and transgalactosylation reactions from different substrates. The enzyme gene was expressed in Escherichia coli BL21 (DE3), sequenced, and subjected to bioinformatic and kinetic assessment. Results showed that the enzyme was able to hydrolyze lactulose and o-nitrophenyl-ß-d-galactopyranoside, but unable to hydrolyze lactose, o-nitrophenyl-ß-d-glucopyranoside, butyl- and pentyl-ß-d-galactosides. This unique and novel substrate specificity converts the E. acetylicum MF03 ß-galactosidase into an ideal catalyst for the formulation of an enzymatic kit for lactulose quantification in thermally processed milk. This is because costly steps to eliminate glucose (resulting from hydrolysis of lactose when a customary ß-galactosidase is used) can be avoided.

Co-immobilized ß-galactosidase and Saccharomyces cerevisiae cells for the simultaneous synthesis and purification of galacto-oligosaccharides.

Simultaneous synthesis and purification (SSP) of galacto-oligosaccharides (GOS) from lactose was conducted using a combi-biocatalyst formed by crosslinked enzyme aggregates of Aspergillus oryzae ß-galactosidase and Saccharomyces cerevisiae cells co-immobilized by entrapment in calcium alginate gel particles. Product yield obtained with the combi-biocatalyst was similar than obtained with the soluble enzyme (23.3%), having a final purity of 25.7%. During the simultaneous process, ethyl-ß-galactoside was produced from the ethanol generated as a metabolic product of yeast cells, but ethyl-ß-galactoside was considerably decreased at high aeration (4 vvm). The combi-biocatalyst can be recovered and reused but its performance is limited by the reduction of the metabolic capacity of the cells. In this way, a process was developed for the SSP of GOS from lactose, obtaining a comparable product yield and higher specific productivity than in a conventional synthesis.

Study of the ichthyotoxic microalga Heterosigma akashiwo by transcriptional activation of sublethal marker Hsp70b in Transwell co-culture assays.

Despite the advance of knowledge about the factors and potential mechanisms triggering the ichthyotoxicity in microalgae, these remain unclear or are controversial for several species (e.g. Heterosigma). Neither typical toxicity tests carried out with cell extracts nor direct exposure to harmful species were proved suitable to unravel the mechanism of harm. Ichthyotoxic species show a complex harmful effect on fish, which is mediated through various mechanisms depending on the species. In this work, we present a method to study sub-lethal effects triggered by reactive oxygen species of a population of harmful algae in vivo over a fish cell line. To that end, Transwell co-cultures in which causative and target species are separated by a 0.4 µm pore membrane were carried out. This allowed the evaluation of the effect of the released molecules by cells in a rapid and compact test. In our method, the harmful effect was sensed through the transcriptional activation of sub-lethal marker Hsp70b in the CHSE214 salmon cell line. The method was tested with the raphidophyte Heterosigma akashiwo and Dunaliella tertiolecta (as negative control). It was shown that superoxide intracellular content and its release are not linked in these species. The methodology allowed proving that reactive oxygen species produced by H. akashiwo are able to induce the transcriptional activation of sub-lethal marker Hsp70b. However, neither loss of viability nor apoptosis was observed in CHSE214 salmon cell line except when exposed to direct contact with the raphidophyte cells (or their extract). Consequently, ROS was not concluded to be the main cause of ichthyotoxicity in H. akashiwo.