Thermoresistant, recombinant β-galactosidase from Thermotoga maritima was purified and immobilized on the surface of epoxy-coated magnetic beads. The enzyme, which has hexameric quaternary structure as shown by gel filtration chromatography, attaches to the resin through multiple covalent linkages that involve different subunits. The bound enzyme shows higher stability than the free form. The immobilized enzyme showed to be efficient for the hydrolysis of lactose and the biosynthesis of galactooligosaccharides (GOS). The chemical structure of synthesized GOS has been determined by NMR revealing that the main product was β-3′-galactosyl lactose. Although β-galactosidases from different sources have been used for the same purposes, the distinct advantage of the methodology described in this communication is that the enzyme can be easily produced, purified and immobilized in large quantities.

Glycoside hydrolases, specifically β-galactosidases, can be used to synthesize galacto-oligosaccharides (GOS) due to the transglycosylating (secondary) activity of these enzymes. Site-directed mutagenesis of a thermoresistant β-galactosidase from Thermotoga maritima has been carried out to study the structural basis of transgalactosylation and to obtain enzymatic variants with better performance for GOS biosynthesis. Rational design of mutations was based on homologous sequence analysis and structural modeling. Analysis of mutant enzymes indicated that residue W959, or an alternative aromatic residue at this position, is critical for the synthesis of β-3'-galactosyl-lactose, the major GOS obtained with the wild-type enzyme. Mutants W959A and W959C, but not W959F, showed an 80% reduced synthesis of this GOS. Other substitutions, N574S, N574A, and F571L, increased the synthesis of β-3'-galactosyl-lactose about 40%. Double mutants F571L/N574S and F571L/N574A showed an increase of about 2-fold.

In this work we report a detailed analysis of the topology and phylogenetics of family 2 glycoside hydrolases (GH2). We distinguish five topologies or domain architectures based on the presence and distribution of protein domains defined in Pfam and Interpro databases. All of them share a central TIM barrel (catalytic module) with two β-sandwich domains (non-catalytic) at the N-terminal end, but differ in the occurrence and nature of additional non-catalytic modules at the C-terminal region. Phylogenetic analysis was based on the sequence of the Pfam Glyco_hydro_2_C catalytic module present in most GH2 proteins. Our results led us to propose a model in which evolutionary diversity of GH2 enzymes is driven by the addition of different non-catalytic domains at the C-terminal region. This model accounts for the divergence of β-galactosidases from β-glucuronidases, the diversification of β-galactosidases with different transglycosylation specificities, and the emergence of bicistronic β-galactosidases. This study also allows the identification of groups of functionally uncharacterized protein sequences with potential biotechnological interest.

Glucose oxidase is one of the most conspicuous commercial enzymes due to its many different applications in diverse industries such as food, chemical, energy and textile. Among these applications, the most remarkable is the manufacture of glucose biosensors and in particular sensor strips used to measure glucose levels in serum. The generation of ameliorated versions of glucose oxidase is therefore a significant biotechnological objective. We have used a strategy that combined random and rational approaches to isolate uncharacterized mutations of Aspergillus niger glucose oxidase with improved properties. As a result, we have identified two changes that increase significantly the enzyme's thermal stability. One (T554M) generates a sulfur-pi interaction and the other (Q90R/Y509E) introduces a new salt bridge near the interphase of the dimeric protein structure. An additional double substitution (Q124R/L569E) has no significant effect on stability but causes a twofold increase of the enzyme's specific activity. Our results disclose structural motifs of the protein which are critical for its stability. The combination of mutations in the Q90R/Y509E/T554M triple mutant yielded a version of A. niger glucose oxidase with higher stability than those previously described.

Enzymatically-active bacterial cellulose (BC) was prepared by non-covalent immobilization of a hybrid enzyme composed by a β-galactosidase from Thermotoga maritima (TmLac) and a carbohydrate binding module (CBM2) from Pyrococcus furiosus. TmLac-CBM2 protein was bound to BC, with higher affinity at pH 6.5 than at pH 8.5 and with high specificity compared to the non-engineered enzyme. Both hydrated (HBC) and freeze-dried (DBC) bacterial cellulose showed equivalent enzyme binding efficiencies. Initial reaction rate of HBC-bound enzyme was higher than DBC-bound and both of them were lower than the free enzyme. However, enzyme performance was similar in all three cases for the hydrolysis of 5% lactose to a high extent. Reuse of the immobilized enzyme was limited by the stability of the β-galactosidase module, whereas the CBM2 module provided stable attachment of the hybrid enzyme to the BC support, after long incubation periods (3 h) at 75 °C.

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We describe a simple, efficient process for the production of 6-kestose, a trisaccharide with well-documented prebiotic properties. A key factor is the use of a yeast transformant expressing an engineered version of Saccharomyces invertase with enhanced transfructosylating activity. When the yeast transformant was grown with 30 % sucrose as the carbon source, 6-kestose accumulated up to ca. 100 g/L in the culture medium. The 6-kestose yield was significantly enhanced (up to 200 g/L) using a two-stage process carried out in the same flask. In the first stage, the culture was grown in 30 % sucrose at physiological temperature (30 °C) to allow overexpression of the invertase. In the second stage, sucrose was added to the culture at high concentration (60 %) and the temperature shifted to 50 °C. In both cases, 6-kestose was synthesized with high specificity, representing more than 95 % of total FOS.

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