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Preparation of lactose-free pasteurized milk with a recombinant thermostable β-glucosidase from Pyrococcus furiosus.

Li B, Wang Z, Li S, Donelan W, Wang X, Cui T, Tang D - BMC Biotechnol. (2013)

Bottom Line: The optimum temperature and pH for this β-glucosidase activity was 100°C and pH 6.0, respectively.The enzyme activity was not significantly inhibited by Ca2+.We tested the additive amount, hydrolysis time, and the influence of glucose on the enzyme during pasteurization and found that the enzyme possessed a high level of lactose hydrolysis in milk that was not obviously influenced by glucose.

View Article: PubMed Central - HTML - PubMed

Affiliation: Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012, China. Taixing.Cui@uscmed.sc.edu.

ABSTRACT

Background: Lactose intolerance is a common health concern causing gastrointestinal symptoms and avoidance of dairy products by afflicted individuals. Since milk is a primary source of calcium and vitamin D, lactose intolerant individuals often obtain insufficient amounts of these nutrients which may lead to adverse health outcomes. Production of lactose-free milk can provide a solution to this problem, although it requires use of lactase from microbial sources and increases potential for contamination. Use of thermostable lactase enzymes can overcome this issue by functioning under pasteurization conditions.

Results: A thermostable β-glucosidase gene from Pyrococcus furiosus was cloned in frame with the Saccharomyces cerecisiae a-factor secretory signal and expressed in Pichia pastoris strain X-33. The recombinant enzyme was purified by a one-step method of weak anion exchange chromatography. The optimum temperature and pH for this β-glucosidase activity was 100°C and pH 6.0, respectively. The enzyme activity was not significantly inhibited by Ca2+. We tested the additive amount, hydrolysis time, and the influence of glucose on the enzyme during pasteurization and found that the enzyme possessed a high level of lactose hydrolysis in milk that was not obviously influenced by glucose.

Conclusions: The thermostablity of this recombinant β-glucosidase, combined with its neutral pH activity and favorable temperature activity optima, suggest that this enzyme is an ideal candidate for the hydrolysis of lactose in milk, and it would be suitable for application in low-lactose milk production during pasteurization.

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Time course of recombinant enzyme properties and protein analysis during fermentation. A. Wet cell weight (square), total protein in supernatant (diamond), β-glucosidase activity (triangle), and β-galactosidase activity (circle) were assessed as a function of time for 120 h fermentation in a 5 L fermenter. The arrow shows the time point for addition of glucose. B. Total protein in culture supernatant was assessed by Coomassie Brilliant Blue R-250 staining following SDS-PAGE (10%) during high cell density fermentation in a 5 L fermenter. Arrow at approximately 120 kDa shows the recombinant β-glucosidase. Each lane shows culture supernatant at indicated time and M = protein size marker.
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Figure 1: Time course of recombinant enzyme properties and protein analysis during fermentation. A. Wet cell weight (square), total protein in supernatant (diamond), β-glucosidase activity (triangle), and β-galactosidase activity (circle) were assessed as a function of time for 120 h fermentation in a 5 L fermenter. The arrow shows the time point for addition of glucose. B. Total protein in culture supernatant was assessed by Coomassie Brilliant Blue R-250 staining following SDS-PAGE (10%) during high cell density fermentation in a 5 L fermenter. Arrow at approximately 120 kDa shows the recombinant β-glucosidase. Each lane shows culture supernatant at indicated time and M = protein size marker.

Mentions: Unlike the AOX1 promoter which requires methanol to initiate gene expression, the GAP promoter constitutively expresses genes in P. pastoris cells grown on many carbon sources [14-17]. Product formation appears to be growth associated in the GAP promoter constitutive expression system, therefore, a higher cell density would help to achieve higher product concentration. The fermentation was a fed-batch process carried out in 5 L fermenters. The X-33 cells were initially grown on basal salts medium and fed with 50% glucose after 48 h. Cell concentrations, total protein in supernatant, and enzyme activities during fermentation are shown in Figure 1A. The biomass accumulated at 120 h was 312 g/L, enzyme expression reached its peak with a yield of 740 mg/L, and the corresponding β-glucosidase activity was 27 1U/ml. We also tested the β-galactosidase activity of the enzyme and found it to be 94 U/ml, approximately 35% of the β-glucosidase activity. A protein corresponding to recombinant β-glucosidase, with a molecular mass of approximately 120 kDa, was detected upon performing SDS-PAGE analysis with Coomassie Brilliant Blue staining (Figure 1B). This band corresponds to the dimer unit of the enzyme. The native β-glucosidase is a tetramer, composed of four identical subunits, and each subunit has a molecular mass of 58 ± 2 kDa. The two monomer subunits dimerize by forming a disulfide bond and have a molecular mass of approximately 120 kDa. The enzyme dimers form a tetramer through non-covalent bonding, which is the functional native enzyme. It has been previously demonstrated that boiling in SDS is insufficient to denature the dimer which requires additional reagents, such as β-mercaptoethanol, to cleave the disulfide bonds.


Preparation of lactose-free pasteurized milk with a recombinant thermostable β-glucosidase from Pyrococcus furiosus.

Li B, Wang Z, Li S, Donelan W, Wang X, Cui T, Tang D - BMC Biotechnol. (2013)

Time course of recombinant enzyme properties and protein analysis during fermentation. A. Wet cell weight (square), total protein in supernatant (diamond), β-glucosidase activity (triangle), and β-galactosidase activity (circle) were assessed as a function of time for 120 h fermentation in a 5 L fermenter. The arrow shows the time point for addition of glucose. B. Total protein in culture supernatant was assessed by Coomassie Brilliant Blue R-250 staining following SDS-PAGE (10%) during high cell density fermentation in a 5 L fermenter. Arrow at approximately 120 kDa shows the recombinant β-glucosidase. Each lane shows culture supernatant at indicated time and M = protein size marker.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4016594&req=5

Figure 1: Time course of recombinant enzyme properties and protein analysis during fermentation. A. Wet cell weight (square), total protein in supernatant (diamond), β-glucosidase activity (triangle), and β-galactosidase activity (circle) were assessed as a function of time for 120 h fermentation in a 5 L fermenter. The arrow shows the time point for addition of glucose. B. Total protein in culture supernatant was assessed by Coomassie Brilliant Blue R-250 staining following SDS-PAGE (10%) during high cell density fermentation in a 5 L fermenter. Arrow at approximately 120 kDa shows the recombinant β-glucosidase. Each lane shows culture supernatant at indicated time and M = protein size marker.
Mentions: Unlike the AOX1 promoter which requires methanol to initiate gene expression, the GAP promoter constitutively expresses genes in P. pastoris cells grown on many carbon sources [14-17]. Product formation appears to be growth associated in the GAP promoter constitutive expression system, therefore, a higher cell density would help to achieve higher product concentration. The fermentation was a fed-batch process carried out in 5 L fermenters. The X-33 cells were initially grown on basal salts medium and fed with 50% glucose after 48 h. Cell concentrations, total protein in supernatant, and enzyme activities during fermentation are shown in Figure 1A. The biomass accumulated at 120 h was 312 g/L, enzyme expression reached its peak with a yield of 740 mg/L, and the corresponding β-glucosidase activity was 27 1U/ml. We also tested the β-galactosidase activity of the enzyme and found it to be 94 U/ml, approximately 35% of the β-glucosidase activity. A protein corresponding to recombinant β-glucosidase, with a molecular mass of approximately 120 kDa, was detected upon performing SDS-PAGE analysis with Coomassie Brilliant Blue staining (Figure 1B). This band corresponds to the dimer unit of the enzyme. The native β-glucosidase is a tetramer, composed of four identical subunits, and each subunit has a molecular mass of 58 ± 2 kDa. The two monomer subunits dimerize by forming a disulfide bond and have a molecular mass of approximately 120 kDa. The enzyme dimers form a tetramer through non-covalent bonding, which is the functional native enzyme. It has been previously demonstrated that boiling in SDS is insufficient to denature the dimer which requires additional reagents, such as β-mercaptoethanol, to cleave the disulfide bonds.

Bottom Line: The optimum temperature and pH for this β-glucosidase activity was 100°C and pH 6.0, respectively.The enzyme activity was not significantly inhibited by Ca2+.We tested the additive amount, hydrolysis time, and the influence of glucose on the enzyme during pasteurization and found that the enzyme possessed a high level of lactose hydrolysis in milk that was not obviously influenced by glucose.

View Article: PubMed Central - HTML - PubMed

Affiliation: Shandong University Qilu Hospital Research Center for Cell Therapy, Key Laboratory of Cardiovascular Remodeling and Function Research, Qilu Hospital of Shandong University, Jinan 250012, China. Taixing.Cui@uscmed.sc.edu.

ABSTRACT

Background: Lactose intolerance is a common health concern causing gastrointestinal symptoms and avoidance of dairy products by afflicted individuals. Since milk is a primary source of calcium and vitamin D, lactose intolerant individuals often obtain insufficient amounts of these nutrients which may lead to adverse health outcomes. Production of lactose-free milk can provide a solution to this problem, although it requires use of lactase from microbial sources and increases potential for contamination. Use of thermostable lactase enzymes can overcome this issue by functioning under pasteurization conditions.

Results: A thermostable β-glucosidase gene from Pyrococcus furiosus was cloned in frame with the Saccharomyces cerecisiae a-factor secretory signal and expressed in Pichia pastoris strain X-33. The recombinant enzyme was purified by a one-step method of weak anion exchange chromatography. The optimum temperature and pH for this β-glucosidase activity was 100°C and pH 6.0, respectively. The enzyme activity was not significantly inhibited by Ca2+. We tested the additive amount, hydrolysis time, and the influence of glucose on the enzyme during pasteurization and found that the enzyme possessed a high level of lactose hydrolysis in milk that was not obviously influenced by glucose.

Conclusions: The thermostablity of this recombinant β-glucosidase, combined with its neutral pH activity and favorable temperature activity optima, suggest that this enzyme is an ideal candidate for the hydrolysis of lactose in milk, and it would be suitable for application in low-lactose milk production during pasteurization.

Show MeSH
Related in: MedlinePlus