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Metagenomic approach for the isolation of a thermostable β-galactosidase with high tolerance of galactose and glucose from soil samples of Turpan Basin.

Zhang X, Li H, Li CJ, Ma T, Li G, Liu YH - BMC Microbiol. (2013)

Bottom Line: Furthermore, Gal308 displayed a very high tolerance of galactose and glucose, with the highest inhibition constant K(i,gal) (238 mM) and K(i,glu) (1725 mM) among β-galactosidases.This study will enrich the source of β-galactosidases, and attract some attentions to β-galactosidases from extreme habitats and metagenomic library.These properties make it a good candidate in the production of low-lactose milk and dairy products after further study.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of life sciences, Sun Yat-sen University, Guangzhou 510275, People's Republic of China. lsslig@mail.sysu.edu.cn.

ABSTRACT

Background: β-Galactosidases can be used to produce low-lactose milk and dairy products for lactose intolerant people. Although commercial β-galactosidases have outstanding lactose hydrolysis ability, their thermostability is low, and reaction products have strong inhibition to these enzymes. In addition, the β-galactosidases possessing simultaneously high thermostability and tolerance of galactose and glucose are still seldom reported until now. Therefore, identification of novel β-galactosidases with high thermostability and tolerance to reaction products from unculturable microorganisms accounting for over 99% of microorganisms in the environment via metagenomic strategy is still urgently in demand.

Results: In the present study, a novel β-galactosidase (Gal308) consisting of 658 amino acids was identified from a metagenomic library from soil samples of Turpan Basin in China by functional screening. After being overexpressed in Escherichia coli and purified to homogeneity, the enzymatic properties of Gal308 with N-terminal fusion tag were investigated. The recombinant enzyme displayed a pH optimum of 6.8 and a temperature optimum of 78 °C, and was considerably stable in the temperature range of 40 °C - 70 °C with almost unchangeable activity after incubation for 60 min. Furthermore, Gal308 displayed a very high tolerance of galactose and glucose, with the highest inhibition constant K(i,gal) (238 mM) and K(i,glu) (1725 mM) among β-galactosidases. In addition, Gal308 also exhibited high enzymatic activity for its synthetic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG, 185 U/mg) and natural substrate lactose (47.6 U/mg).

Conclusion: This study will enrich the source of β-galactosidases, and attract some attentions to β-galactosidases from extreme habitats and metagenomic library. Furthermore, the recombinant Gal308 fused with 156 amino acids exhibits many novel properties including high activity and thermostability at high temperatures, the pH optimum of 6.8, high enzyme activity for lactose, as well as high tolerance of galactose and glucose. These properties make it a good candidate in the production of low-lactose milk and dairy products after further study.

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Effect of pH (A) and temperature (B) on activity (square) and stability(circle) of Gal308 using lactose as substrate. Data points are the average of triplicate measurements; error bars represent ±1 SD.
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Figure 3: Effect of pH (A) and temperature (B) on activity (square) and stability(circle) of Gal308 using lactose as substrate. Data points are the average of triplicate measurements; error bars represent ±1 SD.

Mentions: The optimal pH of recombinant Gal308 was investigated by measuring the enzymatic activity towards lactose at various pH values (pH 2.0-10.0) and 78°C. Gal308 displayed the highest activity at pH 6.8. Even at pH 4.0 and pH 10.0, recombinant enzyme still exhibited 31.6% and 18.9% of the maximum activity, respectively (Figure 3A). Moreover, the enzyme was found to be stable in the pH range of 5.0 - 8.0, and more than 70% of the maximum activity was remained (Figure 3A). Thus, the pH properties of Gal308 are suitable in lactose hydrolysis of natural milk (pH 6.7-6.8). The optimal temperature for the enzyme was 78°C (Figure 3B). The thermostability of Gal308 was drastically decreased when the temperature was more than 80°C, and the enzyme was almost completely inactivated at 90°C (Figure 3B). However, the enzyme was fairly stable for a temperature range of 40°C - 70°C, and its activity almost kept unchangeable after incubation for 60 min. Therefore, Gal308 is especially suitable for hydrolysis of lactose during milk pasteurization (62.8°C - 65.6°C for 30 min) when compared with a commercially available β-galactosidase from Kluyveromyces lactis (the optimal temperature is approximately 50°C).


Metagenomic approach for the isolation of a thermostable β-galactosidase with high tolerance of galactose and glucose from soil samples of Turpan Basin.

Zhang X, Li H, Li CJ, Ma T, Li G, Liu YH - BMC Microbiol. (2013)

Effect of pH (A) and temperature (B) on activity (square) and stability(circle) of Gal308 using lactose as substrate. Data points are the average of triplicate measurements; error bars represent ±1 SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016535&req=5

Figure 3: Effect of pH (A) and temperature (B) on activity (square) and stability(circle) of Gal308 using lactose as substrate. Data points are the average of triplicate measurements; error bars represent ±1 SD.
Mentions: The optimal pH of recombinant Gal308 was investigated by measuring the enzymatic activity towards lactose at various pH values (pH 2.0-10.0) and 78°C. Gal308 displayed the highest activity at pH 6.8. Even at pH 4.0 and pH 10.0, recombinant enzyme still exhibited 31.6% and 18.9% of the maximum activity, respectively (Figure 3A). Moreover, the enzyme was found to be stable in the pH range of 5.0 - 8.0, and more than 70% of the maximum activity was remained (Figure 3A). Thus, the pH properties of Gal308 are suitable in lactose hydrolysis of natural milk (pH 6.7-6.8). The optimal temperature for the enzyme was 78°C (Figure 3B). The thermostability of Gal308 was drastically decreased when the temperature was more than 80°C, and the enzyme was almost completely inactivated at 90°C (Figure 3B). However, the enzyme was fairly stable for a temperature range of 40°C - 70°C, and its activity almost kept unchangeable after incubation for 60 min. Therefore, Gal308 is especially suitable for hydrolysis of lactose during milk pasteurization (62.8°C - 65.6°C for 30 min) when compared with a commercially available β-galactosidase from Kluyveromyces lactis (the optimal temperature is approximately 50°C).

Bottom Line: Furthermore, Gal308 displayed a very high tolerance of galactose and glucose, with the highest inhibition constant K(i,gal) (238 mM) and K(i,glu) (1725 mM) among β-galactosidases.This study will enrich the source of β-galactosidases, and attract some attentions to β-galactosidases from extreme habitats and metagenomic library.These properties make it a good candidate in the production of low-lactose milk and dairy products after further study.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of life sciences, Sun Yat-sen University, Guangzhou 510275, People's Republic of China. lsslig@mail.sysu.edu.cn.

ABSTRACT

Background: β-Galactosidases can be used to produce low-lactose milk and dairy products for lactose intolerant people. Although commercial β-galactosidases have outstanding lactose hydrolysis ability, their thermostability is low, and reaction products have strong inhibition to these enzymes. In addition, the β-galactosidases possessing simultaneously high thermostability and tolerance of galactose and glucose are still seldom reported until now. Therefore, identification of novel β-galactosidases with high thermostability and tolerance to reaction products from unculturable microorganisms accounting for over 99% of microorganisms in the environment via metagenomic strategy is still urgently in demand.

Results: In the present study, a novel β-galactosidase (Gal308) consisting of 658 amino acids was identified from a metagenomic library from soil samples of Turpan Basin in China by functional screening. After being overexpressed in Escherichia coli and purified to homogeneity, the enzymatic properties of Gal308 with N-terminal fusion tag were investigated. The recombinant enzyme displayed a pH optimum of 6.8 and a temperature optimum of 78 °C, and was considerably stable in the temperature range of 40 °C - 70 °C with almost unchangeable activity after incubation for 60 min. Furthermore, Gal308 displayed a very high tolerance of galactose and glucose, with the highest inhibition constant K(i,gal) (238 mM) and K(i,glu) (1725 mM) among β-galactosidases. In addition, Gal308 also exhibited high enzymatic activity for its synthetic substrate o-nitrophenyl-β-D-galactopyranoside (ONPG, 185 U/mg) and natural substrate lactose (47.6 U/mg).

Conclusion: This study will enrich the source of β-galactosidases, and attract some attentions to β-galactosidases from extreme habitats and metagenomic library. Furthermore, the recombinant Gal308 fused with 156 amino acids exhibits many novel properties including high activity and thermostability at high temperatures, the pH optimum of 6.8, high enzyme activity for lactose, as well as high tolerance of galactose and glucose. These properties make it a good candidate in the production of low-lactose milk and dairy products after further study.

Show MeSH
Related in: MedlinePlus