Adriano Gennari

Lactose Hydrolysis in Milk and Dairy Whey Using Microbial β-Galactosidases.

This work aimed at evaluating the influence of enzyme concentration, temperature, and reaction time in the lactose hydrolysis process in milk, cheese whey, and whey permeate, using two commercial β-galactosidases of microbial origins. We used Aspergillus oryzae (at temperatures of 10 and 55°C) and Kluyveromyces lactis (at temperatures of 10 and 37°C) β-galactosidases, both in 3, 6, and 9 U/mL concentrations. In the temperature of 10°C, the K. lactis β-galactosidase enzyme is more efficient in the milk, cheese whey, and whey permeate lactose hydrolysis when compared to A. oryzae. However, in the enzyme reaction time and concentration conditions evaluated, 100% lactose hydrolysis was not reached using the K. lactis β-galactosidase. The total lactose hydrolysis in whey and permeate was obtained with the A. oryzae enzyme, when using its optimum temperature (55°C), at the end of a 12 h reaction, regardless of the enzyme concentration used. For the lactose present in milk, this result occurred in the concentrations of 6 and 9 U/mL, with the same time and temperature conditions. The studied parameters in the lactose enzymatic hydrolysis are critical for enabling the application of β-galactosidases in the food industry.

Biocatalytic characterization of Aspergillus oryzae β-galactosidase immobilized on functionalized multi-walled carbon nanotubes

Abstract The objectives of this work were to immobilize commercial Aspergillus oryzae β-galactosidase on functionalized multi-walled carbon nanotubes (MWCNTs) using different treatments and to characterize the products. Treatments were performed with glutaraldehyde, ethylenediamine and a mixture of concentrated H2SO4:HNO3. The MWCNTs and their derivatives were characterized by thermogravimetric analysis. The immobilized enzymes were evaluated using inactivation kinetics, operating conditions, that is pH and temperature, kinetic parameters and lactose hydrolysis reusability. Immobilization yield and efficiency were significantly higher for β-galactosidase immobilized on MWCNTs functionalized by the acid mixture (Ac-Gal-MWCNTs). These values were 97% and 82%, respectively, after 3 h of immobilization. The activity of the Ac-Gal-MWCNTs was maintained at ∼51% of their initial activity after being stored for 90 days at 4 °C. The Ac-Gal-MWCNTs retained more than 90% of their initial activity up to the fourth recycle. As the acid functionalization was the most efficient method tested for immobilizing A. oryzae β-galactosidase on MWCNTs, this method shows promise for industrial applications.

Chelation by collagen in the immobilization of Aspergillus oryzae β-galactosidase: A potential biocatalyst to hydrolyze lactose by batch processes

This work is the first study of the immobilization of Aspergillus oryzae β-galactosidase (Gal) on powdered collagen (Col) that had formed a chelate with aluminum (Col-Al-Gal). Other collagen treatments, including those with acetic acid, glutaraldehyde, and a combination of aluminum and glutaraldehyde (Col-Al-Glu-Gal), were also tested. High-yield (superior to 80%) and high-efficiency (superior to 99%) immobilization was obtained for the derivatives Col-Al-Gal and Col-Al-Glu-Gal, even at high protein loads (500-1,000 mg g-1 of support). The storage stability of Gal immobilized on Col-Al and Col-Al-Glu resulted in Gal retaining approximately 60% of its initial activity after 90 days at 4 °C. The half-life values of derivatives Col-Al-Gal and Col-Al-Glu-Gal were higher than those of soluble enzyme at 65, 68, 70, and 73 °C. The derivatives Col-Al-Gal and Col-Al-Glu-Gal retained high enzyme activity in batch hydrolysis of lactose in permeate and lactose solutions for 50 and 60 cycles, respectively. Our results suggest that powdered collagen treated with aluminum, a low-cost support, is a promising support for the immobilization of β-galactosidase.

Modification of Immobead 150 support for protein immobilization: Effects on the properties of immobilized Aspergillus oryzae β-galactosidase

We studied the modification of Immobead 150 support by either introducing aldehyde groups using glutaraldehyde (Immobead‐Glu) or carboxyl groups through acid solution (Immobead‐Ac) for enzyme immobilization by covalent attachment or ion exchange, respectively. These two types of immobilization were compared with the use of epoxy groups that are now provided on a commercial support. We used Aspergillus oryzae β‐galactosidase (Gal) as a model protein, immobilizing it on unmodified (epoxy groups, Immobead‐Epx) and modified supports. Immobilization yield and efficiency were tested as a function of protein loading (10–500 mg g−1 support). Gal was efficiently immobilized on the Immobeads with an immobilization efficiency higher than 75% for almost all supports and protein loads. Immobilization yields significantly decreased when protein loadings were higher than 100 mg g−1 support. Gal immobilized on Immobead‐Glu and Immobead‐Ac retained approximately 60% of its initial activity after 90 days of storage at 4°C. The three immobilized Gal derivatives presented higher half‐lifes than the soluble enzyme, where the half‐lifes were twice higher than the free Gal at 73°C. All the preparations were moderately operationally stable when tested in lactose solution, whey permeate, cheese whey, and skim milk, and retained approximately 50% of their initial activity after 20 cycles of hydrolyzing lactose solution. The modification of the support with glutaraldehyde provided the most stable derivative during cycling in cheese whey hydrolysis. Our results suggest that the Immobead 150 is a promising support for Gal immobilization.