Purified Beta-Galactosidase for the Treatment of Lactose Intolerance

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Purified Beta-Galactosidase for the Treatment of Lactose Intolerance

²-galactosidase is a bacterial enzyme. Study of this enzyme allowed researchers to develop operon model and determine the role of the enzyme in regulating gene expression. Besides this historic significance, ²-galactosidase also have essential enzymatic functions, including to hydrolyze lactose into glucose and galactose, to synthesize allolactose by transgalactosylation of lactose, and to produce monosaccharides by cleaving allolactose . Ability of ²-galactosidase to hydrolyze lactose is vital for energy production. Resulting products of lactose hydrolyzes enter glycolysis and are converted to ATP, energy source used by organisms. This hydrolyze process is turned on when there is lack of glucose and presence of lactose in the environment. In the absence of glucose, ²-galactosidase is essential for energy production and thus survival of the organisms. In addition to enzymatic functions, ²-galactosidase is also used significantly in molecular biology. It acts as a reporter marker to monitor gene expression and is use to distinguish recombinant plasmid through blue and white screening (Juers et al., 2012).

²-galactosidase can be purified by first introducing ²-galactosidase gene into plasmid, which is then introduced into Escherichia coli culture. This will allow for increase in concentration of the enzyme. Then, the cells are lysed to break the cell membrane and release cell content, including ²-galactosidase. After, the cell content must go through various separation steps to purify ²-galactosidase from other proteins. This could be achieved by (NH4)SO4 fractionation, size exclusion chromatography using Sepharose G-75, and DEAE chromatography (Hu et al., 1959).

During (NH4) SO4 fractionation, addition of salt results in salting-in, where solubility of protein increases. With addition of more salt salting-out occurs, where solubility of protein decreases and precipitation occurs. The precipitation, containing ²-galactosidase, can then be separated by chromatography. ²-galactosidase is a tetramer with molecular weight of 540 kDa. Since, Sepharose G-75 has exclusion limit of 80 kDa, ²-galactosidase will elute out of the column without entering resin pores, along with other protein of similar size. Thus, the sample must further be filtered to separate ²-galactosidase from other proteins. The final separation occurs through DEAE chromatography. In this step, the elution from size exclusion chromatography is added to the column, resulting in binding of ²-galactosidase to the resin. All the unbound protein are wash off while, bound protein remains, which can be eluted by increasing ionic strength of NTM elution buffer (Hu et al., 1959). The purity and identity of each separation steps can then be analysed using SDS-PAGE and Western Blot analysis.

In humans, lactase, small intestine enzymes, are responsible for digesting lactose. However, when there is low concentration of lactases, lactose begins to buildup. They eventually move from small intestine to large intestine. In large intestine, these lactose are digested by microorganisms, resulting in production of gaseous by-products. These gaseous by-products cause the symptoms of lactose intolerance including bloating, cramps and accumulation of water in the large intestine. Since lactose intolerance affects ~70% of the worlds adult population, modifying ²-galactosidase for safe and more efficient use is essential (Lehman and Wolyniak, 2015).

Currently, treatment of lactose intolerance includes adding ²-galactosidase to lactose-containing products and ²-galactosidase supplements. ²-galactosidase is added to products containing lactose, including yogurt, sour cream, milk and some cheese, during production (Saqib et al., 2017). The result is creation of lactose-hydrolysed products with low concentration of lactose (Saqib et al., 2017). Although, lactose-hydrolysed products are effective, recent study shows that lactose are not hydrolyed 100% and there is still low concentration of lactose in products, even after treatment with ²-galactosidase (Rosolen et al., 2015). Thus, studying the structure of ²-galactosidase can provide solution for successfully achieving 100% hydrolysis of lactose in lactose products.

Another treatment of lactose intolerance involves ingesting ²-galactosidase capsules before consuming lactose-containing products (Lehman and Wolyniak, 2015). Supplement have shown to prevented symptoms of lactose intolerance (Lehman and Wolyniak, 2015). However, research shows supplements being degraded due to the low pH level of the stomach (OConnell and Walsh, 2006). OConnell and Walsh studied four main commercial lactases used as treatment for lactose intolerance (2006). They found that all four lactases functioned at maximum activity of 55 to 65% at 37°C and at pH level of 3.0 to 6.5 (OConnell and Walsh, 2006). But all lactases significantly lost their activity in in vitro digestive environment, and only 0 to 65% of their original activities was retained (OConnell and Walsh, 2006). They also found that the most effective lactase out of the four, cleaved only 2.7 g of lactose per tablet (OConnell and Walsh, 2006). Thus, lactases not only loss significant amount of their activity but many lactase tablets is required for effective treatment.

Furthermore, a study on mutant strain of E. coli revealed ²-galactosidase with higher activity than normal (Martinez-Bilbao et al., 1991). This mutant was discovered to have its Gly-794 replaced with Asp (Martinez-Bilbao et al., 1991). Due to this substitution, activity of ²-galactosidase increased significantly, especially with lactose as a substrate. This study by Martinez-Bilbao and team also revealed this ²-galactosidase to be heat-sensitive in comparison to normal ²-galactosidase (1991). Based on the result of this study, structure of the purified enzyme can be examined and ways of replacing Gly-794 with Asp, while maintaining the heat-tolerance of normal enzyme, can be determined.

Purifying ²-galactosidase will allow for analysis of the structure and specific parts of the structure that are involved in the process of cleaving lactose into glucose and galactose. The structural information can be used to increase efficiency and effectiveness of current treatment for lactose intolerant.

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