ß-galactosidase GALA from Bacillus circulans with high transgalactosylation activity.

ß-galactosidase catalyzes lactose hydrolysis and transfers reactions to produce prebiotics such as galacto-oligosaccharides (GOS) with potential applications in the food industry and pharmaceuticals. However, there is still a need for improved transgalactosylation activity of ß-galactosidases and reaction conditions of GOS production in order to maximize GOS output and reduce production costs. In this study, a ß-galactosidase gene, galA, from Bacillus circulans was expressed in Pichia pastoris, which not only hydrolyzed lactose but also had strong transgalactosylation activity to produce GOS. Response surface methodology was adopted to investigate the effects of temperature, enzyme concentration, pH, initial lactose concentration, and reaction time on the production of GOS and optimize the reaction conditions for GOS. The optimal pH for the enzyme was 6.0 and remained stable under neutral and basic conditions. Meanwhile, GALA showed most activity at 50°C and retained considerable activity at a lower temperature 30-40°C, indicating this enzyme could work under mild conditions. The enzyme concentration and temperature were found to be the critical parameters affecting the transgalactosylation activity. Response surface methodology showed that the optimal enzyme concentration, initial lactose concentration, temperature, pH, and reaction time were 3.03 U/mL, 500 g/L, 30°C, 5.08, and 4 h, respectively. Under such conditions, the maximum yield of GOS was 252.8 g/L, accounting for approximately 50.56% of the total sugar. This yield can be considered relatively high compared to those obtained from other sources of ß-galactosidases, implying a great potential for GALA in the industrial production and application of GOS.

Site-directed mutation of ß-galactosidase from Aspergillus candidus to reduce galactose inhibition in lactose hydrolysis.

ß-Galactosidase is widely used for hydrolysis of whey lactose. However, galactose inhibition has acted as a major constraint on the catalytic process. Thus, it is sensible to improve upon this defect in ß-galactosidase through protein modification. To reduce the galactose inhibition of Aspergillus candidus ß-galactosidase (LACB), four amino acid positions were selected for mutation based on their molecular bindings with galactose. Four mutant libraries (Tyr96, Asn140, Glu142, and Tyr364) of the LACB were constructed using site-directed mutagenesis. Among all of the mutants, Y364F was superior to the wild-type enzyme. The Y364F mutant has a galactose inhibition constant (K i) of 282 mM, 15.7-fold greater than that of the wild-type enzyme (K i = 18 mM). When 18 mg/ml galactose was added, the activity of the wild-type enzyme fell to 57% of its initial activity, whereas Y364F activity was maintained at over 90% of its initial activity. The wild-type enzyme hydrolyzed 78% of the initial lactose (240 mg/ml) after 48 h, while the Y364F mutant had a hydrolysis rate greater than 90%. The ß-galactosidase Y364F mutant with reduced galactose inhibition may have greater potential applications in whey treatment compared to wild-type LACB.

The use of Agrobacterium-mediated insertional mutagenesis sequencing to identify novel genes of Humicola insolens involved in cellulase production.

A transfer DNA (T-DNA)-tagged mutant library of Humicola insolens was screened for mutants with altered cellulase production using the plate-clearing zone assay. Three selected mutants (5-A7, 5-C6, and 13-B7) exhibited significantly depressed FPase, CMCase and xylanase activities compared with the wild-type strain upon shake-flask fermentation, while the pNPCase and pNPGase activities of the three mutants were relatively higher than those of the parental strain. Combined with the results of SDS-PAGE and mass spectrometry, we suggest that expression of the CMCases Cel6B, Cel7B, CMC3, and XynA/B/C was reduced in the mutant strains. Twelve putative T-DNA insertion sites were identified in the three mutants via Agrobacterium-mediated insertional mutagenesis sequencing (AIM-Seq). Bioinformatics analysis suggested that a putative dolichyl pyrophosphate phosphatase, two hypothetical proteins encoding genes of unknown function, and/or nine intergenic fragments may be involved in cellulase and hemicellulase production by H. insolens. This provides promising new candidate genes relevant to cellulase production by the fungus, which will be crucial not only for our understanding of the molecular mechanism underlying cellulase production, but also for strain improvement.

Enhanced Thermostability of Glucose Oxidase through Computer-Aided Molecular Design.

Glucose oxidase (GOD, EC.1.1.3.4) specifically catalyzes the reaction of ß-d-glucose to gluconic acid and hydrogen peroxide in the presence of oxygen, which has become widely used in the food industry, gluconic acid production and the feed industry. However, the poor thermostability of the current commercial GOD is a key limiting factor preventing its widespread application. In the present study, amino acids closely related to the thermostability of glucose oxidase from Penicillium notatum were predicted with a computer-aided molecular simulation analysis, and mutant libraries were established following a saturation mutagenesis strategy. Two mutants with significantly improved thermostabilities, S100A and D408W, were subsequently obtained. Their protein denaturing temperatures were enhanced by about 4.4 °C and 1.2 °C, respectively, compared with the wild-type enzyme. Treated at 55 °C for 3 h, the residual activities of the mutants were greater than 72%, while that of the wild-type enzyme was only 20%. The half-lives of S100A and D408W were 5.13- and 4.41-fold greater, respectively, than that of the wild-type enzyme at the same temperature. This work provides novel and efficient approaches for enhancing the thermostability of GOD by reducing the protein free unfolding energy or increasing the interaction of amino acids with the coenzyme.