Notes
One-step Removal of Proteases in a Commercial Lactase Preparation from Kluyveromyces lactis Using an Anion-exchange Membrane
2020 年 26 巻 1 号 p. 65-68
詳細
2020 年 26 巻 1 号 p. 65-68
To reduce proteases in a commercial lactase preparation from K. lactis, enzyme preparations were diluted by a buffer with KCl and the dilutions were flushed through a weak anion-exchange membrane. For KCl concentration in the range of 0.20 to 0.25 M, more than 90% of lactase was recovered and protease level was decreased to less than 20%. Purified and pre-purified lactase solutions added in excess to milk and incubated for 3 months at 30 °C showed degraded milk protein bands on SDS-PAGE by treatment with the pre-purified lactase but not purified lactase. A one-step purification method for protease removal from the K. lactis lactase preparation was performed only by flushing the solution through an anion-exchange membrane.
Most industrial enzyme preparations include not only the main target enzyme but also other contaminating ones. When these preparations are used for food processing, proteases from microbes often lead to contamination (Fia et al., 2014, Hollá et al., 2019). Lactase (β-galactosidase, EC 3.2.1.23) is a glycoside hydrolase enzyme that catalyzes the hydrolysis of lactose to glucose and galactose. The dairy industry worldwide produces lactose-free or -low milk in which lactose has been decomposed for lactose-intolerant consumers using a commercial lactase prepared from Kluyveromyces lactis (Mlichová and Rosenberg, 2006). Lactase preparations from K. lactis also include a small amount of proteases since these enzymes are localized in the yeast cell. Milk proteins in lactase-treated milk are reported to be degraded by incubation with contaminating proteases for a long time (Tossavainen and Kallioinen, 2007). Additionally, excessive contaminating proteases accelerate the development of off-flavor in milk (Mittal et al., 1991). Therefore, removal of proteases from lactase preparations is very important when manufacturing lactose-hydrolyzed products.
Multiple techniques, such as column chromatography, salt precipitation and others, are generally adopted to purify lactase from K. lactis (Dickson et al., 1979, Husssein et al., 1988, Lima et al., 2016). A simpler purification method, however, is required for commercial applications. Recently, an anion exchange hollow fiber membrane was reported to be an effective tool in biopharmaceutical production (Shirataki et al., 2011, Chou et al., 2015, Kao et al., 2016). The deployment barrier of the membrane may be less than that of an industrial column chromatography because ultrafiltration and microfiltration membranes are reasonably used for concentration and sterilization in the enzyme industry, respectively. In this study, we attempted to establish a simple process using the anion-exchange membrane for the removal of proteases from the lactase preparation from K. lactis.
Enzyme assays
a) Lactase activity Lactase activity was determined by measuring the absorbance at 420 nm of o-nitrophenol liberated from o-nitrophenyl-β-D-galactopyranoside (ONPG, Sigma-Aldrich, St. Louis, USA). The enzyme solution (500 µL) and 500 µL of 0.1 M KH2PO4-NaOH with 0.1 mM MnCl2 buffer (pH 6.5) were incubated for 3 min at 37 °C, and then, the reaction was initiated by adding 1 mL of 0.1% (w/v) ONPG solution. After incubation for 1 min at 37 °C, the reaction was stopped by adding 2 mL of 0.2 M Na2CO3 solution. A blank was prepared by adding the stop solution before the substrate solution and was incubated in the same manner. One unit (U) of the lactase activity was defined as the amount of enzyme that increased the absorbance by 1 AU per 10 min in the condition.
b) Protease activity Protease activity was determined by measuring the absorbance at 275 nm of tyrosine liberated from casein, bovine milk, carbohydrate, and fatty-acid free (Calbiochem, Merck Millipore, Darmstadt, Germany). The enzyme solution (500 µL) was incubated for 3 min at 30 °C, and the reaction was initiated by adding 2.5 mL of 0.6% (w/v) casein solution in 0.1 M KH2PO4-NaOH with 0.1 mM MnCl2 buffer (pH 6.5). Following incubation for 60 min at 30 °C, the reaction was stopped by adding 2.5 mL of stop solution including 0.11 M trichloroacetic acid, 0.22 M sodium acetate, and 0.33 M acetic acid. Thereafter, it was filtered with a filter paper No. 4A (ADVANTEC, Tokyo, Japan), and the absorbance of the filtrate was measured. A blank was prepared by adding the stop solution before the substrate solution and was incubated in the same manner. Standard tyrosine was prepared as 10 mg/mL by adding 1 mL of 1 M HCl, and that was diluted to 1 mg/mL by 0.1 M KH2PO4-NaOH buffer (pH 6.5). This reaction required vigorous mixing. One unit of the protease activity (PU) was defined as the amount of enzyme that released 1 µg of tyrosine per 10 min in the condition.
Removal of proteases from the lactase preparation by anion-exchange membrane GODO-YNL (Lot 23020, GODO SHUSEI, Chiba, Japan) was used as the commercial lactase preparation and was diluted to 1:10 by 20 mM KH2PO4-NaOH buffer (pH 6.5) with 0 to 0.40 M KCl. An aliquot of lactase dilution (10 mL) was flushed through QyuSpeed D (0.6 mL, Asahi Kasei Medical, Tokyo, Japan) or Mustang Q (25 mmφ, Pall, Port Washington, USA) equilibrated with the same buffer. All permeates were fractionated. QyuSpeed D has a diethylamino group as a weak anion-exchange membrane, and Mustang Q has a quaternary amine group as a strong anion-exchange membrane.
SDS-PAGE analysis for milk proteins treated by the lactase preparation The diluted lactase preparations before and after flushing the anion-exchange membrane were designated as a pre-purified lactase and a purified lactase, respectively. Each of the lactase samples was sterilized with a 0.2 µm filter, and then, the enzyme solutions were aseptically added to commercial milk, which had been sterilized by UHT conditions (140 °C, 2 s). These milk samples were incubated at 30 °C for 1 and 3 months and were analyzed by SDS-PAGE with a 15% gel.
Effect of KCl concentration on the anion-exchange membrane step The lactase preparation was diluted by buffer with 0 to 0.40 M KCl. Lactase and protease activities in the enzyme dilution were 5 700 U/mL and 2.5 PU/mL, respectively. The dilution was flushed through the weak anion-exchange membrane. The lactase yields in permeates with 0 to 0.10 M KCl were less than 30% of the total activity in the pre-purified lactase. Those with more than 0.30 M of KCl gave higher yields, but the protease activity decrease was not sufficient. The most effective purification conditions were KCl concentrations from 0.20 to 0.25 M. Lactase yield and the protease reduction were more than 90% and less than 20%, respectively (Table 1). The lactase yield was 98%, but the protease activity only decreased to 56% when the strong anion-exchange membrane was used with the 0.20 M KCl condition. Therefore, it was found that use of the weak anion-exchange membrane was important for effective reduction of proteases in lactase preparations from K. lactis.
| KCl concentration (M) |
Lactase yield (%) |
Protease yield (%) |
|---|---|---|
| Pre-purified | 100 | 100 |
| 0 | 30 | 0 |
| 0.10 | 30 | 0 |
| 0.15 | 63 | 3 |
| 0.20 | 90 | 20 |
| 0.25 | 95 | 18 |
| 0.30 | 93 | 35 |
| 0.35 | 90 | 60 |
| 0.40 | 91 | 65 |
A representative result from two independent experiments is shown. A nearly identical result was confirmed when it was retested.
The subunit of lactase in K. lactis is about 120 kDa, and its oligomeric structure is a dimer or tetramer (Poch et al., 1992, Pereira-Rodríguez et al., 2012). Generally, molecular masses of proteases are less than 100 kDa. These findings suggest that the proteases were not removed by molecular size sieving through the membrane pore. In contrast, the isoelectric point of lactase was estimated to be 5.41 from the amino acid sequence (ExPASy compute pI, https://web.expasy.org/compute_pi/), and those of two aminopeptidases from K. lactis have been reported to be 4.82 and 5.1 (Flores et al., 1999). Thus, separation may be achieved by a small difference of adsorption on the weak anion-exchange membrane under this condition. This separation should also be performed with several chromatography columns (Dickson et al., 1979, Hussein et al., 1988, Lima et al., 2016). However, the use of this membrane is thought to give a higher throughput than columns because of the flow rate and flow pressure. This method is industrially useful as a one-step purification technique that avoids the usual two steps of adsorption and elution of the target, and it may be able to share the equipment of ultrafiltration or microfiltration, which is often used in the enzyme industry.
Degradation of milk proteins by proteases in the lactase preparation The sample with 0.20 M KCl that was treated by the weak anion-exchange membrane was used as the typical purified lactase. Each of purified and pre-purified lactase was added at 5 and 50 U/mL in commercial milk. These milk samples were incubated at 30 °C for 1 and 3 months. As shown in Fig. 1, protein bands in the milk with 5 U/mL of lactase activity were hardly different between purified and pre-purified lactase solutions regardless of the incubation conditions. In contrast, the milk proteins with 50 U/mL of pre-purified lactase showed degradation on SDS-PAGE at incubation times of more than 1 month, whereas those with the purified lactase were not affected, even if the solution was added in excess. There was no difference in lactose degradation between these lactase solutions. Purified lactase with 0.25 M KCl should not degrade milk protein because the protease activity was less. Although lactase is almost always used in amounts less than 5 U/mL in milk, large contamination of protease activity in the enzyme preparation could make quality control difficult. K. lactis produces various proteases inside the cell (Flores et al., 1999). It is expected that the lactase preparation includes several proteases since lactase is also an intracellular enzyme. Acquiring deletion variants for each protease is complicated and is not realistic. The easy one-step method to decrease contaminating proteases in the commercial lactase preparation from K. lactis was established. The anion-exchange membrane can be repeatedly used with regeneration and was used more than 10 times in this study. We propose that this technique can contribute to the development of the dairy industry, although a decision of equipment size and a breakthrough test for the membrane are needed as a scale-up step.
SDS-PAGE of the milk proteins incubated with the lactase preparation for 1 mont. (A) and 3 months (B). The values (5 and 50 U/mL) are shown as the final concentrations of the lactase activity. The enzyme solutions that were and were not flushed through the weak anion-exchange membrane with 0.20 M KCl in Table 1 were used as the purified lactase and the pre-purified lactase, respectively. Milk proteins were identified in comparison with the report by Tossavainen and Kallioinen (2007). A representative result from two independent experiments is shown. A nearly identical result was confirmed when it was retested with the other purified lactase.
In this study, we proposed a one-step purification method for protease removal in lactase preparations from K. lactis only by flushing the solution through an anion-exchange membrane, and purified lactase with less than 20% of protease did not degrade milk proteins such as casein.
Acknowledgements We would like to thank Asahi Kasei Corporation for providing the anion-exchange membrane (QyuSpeed D).
