Introduction

The enzyme lactase or Beta-galactosidase is released by the intestinal villi and it hydrolyzes the lactose into galactose and glucose. Lactase is known to be responsible for the digestion of bovine milk consisting of 4.8% of lactose, on average. Lactose fermentation and malabsorption of lactose in the gut results from the deficiency of the gene responsible for this protein. Intolerance to lactose will develop when the affected individuals suffer from diarrhea, flatulence, abdominal pain, bloating, and other gastrointestinal symptoms after the consumption of lactose.

The lactose intolerant people will avoid taking dairy products which might lead to calcium and vitamin D deficiencies. This deficiency might lead to fractures and decreased bone mineral density. Consuming lactose free milk by adding lactase enzyme or Beta-glucosidase to the milk will maintain good health and will avoid the lactose intolerance symptoms.

Bacteria can effectively produce lactase enzyme, but they cannot be used in food preparation due to the microbial contamination risk. The lactase enzymes constitute mesophilic enzymes from Aspergillus sp. and Kluyveromyces sp. The fungal enzymes can hydrolyze lactose in Whey at acidic pH, while yeast can effectively hydrolyze milk lactose at neutral pH. Pyrococcus furiosus has several hydrolytic enzymes like Beta-glucosidase that has a half-life of 85 hours at 100 degree C. The enzyme Beta-glucosidase was cloned and functionally characterized in Saccharomyces cerevisiae, while the expression in batch culture could yield very low amounts. This enzyme is mostly used for lactose hydrolysis as it is highly stable and has elevated catalytic activity in lactose presence.

In the current study, Beta-glucosidase gene from Pyrococcus furiosus was cloned into pGAPZaA vector and the vector was transformed into Pichia pastoris X-33 strain for the large scale production of heterologous proteins. The promoter used was glyceraldehyde-3-phosphate dehydrogenase (GAP), which helps in constitutive expression of several heterologous proteins. The analysis of low lactose pasteurized milk production was done by establishing lactose hydrolysis through Beta-glucosidase in pasteurization conditions. The researchers here provide a method of large scale production of "food grade" lactase enzyme that is thermostable and is suitable for use in the pasteurization process.

Results of the study

The GAP promoter could express the genes constitutively in Pichia pastoris cells that were grown on several carbon sources. The higher cell density helps in achieving higher concentration of the product. The fermentation process carried out was fed batch type and it was done in 5 liter volume fermenters. The X-33 cells were grown on basal salt medium and the medium is supplemented with 50 percent glucose. The biomass of the organism that was deposited after 120 hours was 312g/liter and the expression of the enzyme has attained its peak value of 740 mg/liter and the Beta-glucosidase activity was determined as 27 IU/ml.

The SDS-PAGE analysis has revealed that a protein similar to the recombinant Beta-glucosidase having the molecular mass of 120k Da was identified. This band indicated the dimer form of the enzyme. The native enzyme Beta-glucosidase is a tetramer consisting of four subunits and each of the subunit having a molecular mass of 58k Da. Tetramer form of the enzyme is its functional form.

Purification of recombinant Beta-glucosidase

The crude samples of the enzyme Beta-glucosidase were subjected to one-step weak anion exchange chromatography to elute the target recombinant protein. The supernatant was loaded in SDS-PAGE. This method could result in 1.9 times of purified enzyme and 80.8 percent recovered from the supernatant.

Enzyme characteristics

The optimum temperature at which the enzyme remains stable and active is 100 degree C. The enzyme activity was decreased to approximately 7 percent of the maximum activity. The enzyme Beta-glucosidase could maintain stability and 80 percent of its maximum activity between 30 degree C and 120 degree C. This enzyme could maintain optimum activity at pH 6 and was found inactive at pH range of 4 to 9. The maximum activity of the enzyme was retained by 80 percent at pH range of 5 to 8 and when the enzyme was incubated for one hour at 37 degree C.

The relative activity of the enzyme Beta-glucosidase was decreased to 78 percent by the metal ion Cu2+. The activity of the recombinant enzyme was less or not influenced by other metal ions. It was found that calcium ion did not influence the enzyme activity significantly.

Hydrolysis of lactose in milk

The Beta-glucosidase enzyme was able to hydrolyze milk lactose at 65 degree C for about half an hour and this was examined through HPLC analysis. The analysis results showed that 50 percent of hydrolysis of lactose in milk were achieved by 17 U/ml of recombinant enzyme. Enhancing the recombinant Beta-glucosidase enzyme levels to 498 U/ml led to 90 percent of hydrolysis.

The efficiency of enzyme hydrolysis was tested at 311 U/ml concentration of the enzyme Beta-glucosidase in 5 to 30 minutes. It was found that the hydrolysis efficiency after 30 mints. was same as that of after 20 mints. So, the activity of the enzyme was enough to remove lactose from the milk in the pasteurization conditions. The hydrolysis of lactose were found to be least influenced by high concentrations of glucose indicating the tolerance capacity of the enzyme against glucose.

Reference:

Bin Li, Zemin Wang, Shiwu Li, William Donelan, Xingli Wang, Taixing Cui and Dongqi Tang. Preparation of lactose-free pasteurized milk with a recombinant thermostable Beta-glucosidase from Pyrococcus furiosus. BMC Biotechnology 2013, 13:73.

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