Selection of method for obtaining an active lactase preparation from Penicillium notatum
✍ Scribed by Janusz Szczodrak; Adrian Wiater
- Publisher
- John Wiley and Sons
- Year
- 1998
- Tongue
- English
- Weight
- 65 KB
- Volume
- 38
- Category
- Article
- ISSN
- 0233-111X
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✦ Synopsis
Crude lactase (β-galactosidase) preparations of Penicillium notatum 1 were obtained from the culture supernate by means of concentration under reduced pressure, freeze-drying, fractional precipitation with alcohols (methanol, ethanol, propanol, isopropanol) and acetone, ammonium sulfate salting out, and ultrafiltration. The last three methods of enzyme recovery from the post-culture fluid yielded the best results in regard to specific and overall activities of the enzymatic preparations.
The enzyme β-D-galactoside-galactohydrolase (EC 3.2.1.23), known as lactase or β-galactosidase, hydrolyzes lactose to glucose and galactose, that are sweeter, better digestible and soluble than lactose. Commercial lactase is mainly produced from Aspergillus niger as well as from the yeast Kluyveromyces lactis. The yeast enzyme functions best at neutral pH and is used to treat milk or sweet whey, whereas lactases from filamentous fungi have a lower pH optimum of 3 -5 and are used primarily to process acid wheys ZACCHI 1990, HOLSINGER and.
Crude lactase preparations have been obtained by fractional precipitation with ammonium sulfate or organic solvents, and ultrafiltration of the post-culture fluids . However, data concerning loss of enzymatic activity during application of any particular methods for enzyme recovery from the culture medium have been scarcely presented. On the other hand, the choice of an appropriate method of enzyme precipitation in industry depends on its economical viability.
Recently, the fungus Penicillium notatum 1 was selected characterizing a relatively high activity of extracellular lactase . The objective of this investigation was to evaluate the utility of various methods of precipitating and concentrating enzymes for obtaining an active lactase preparation.
Methods
Fungal strain, media and growth conditions: Penicillium notatum 1 was maintained on 2% malt agar slants. The medium for lactase production (pH 3.0) consisted of (g ⋅ l -1 ): lactose 10.0, (NH 4 ) 2 HPO 4 5.0, K 2 HPO 4 1.0, MgSO 4 ⋅ 7 H 2 O 1.0, CaCl 2 1.0, peptone 1.5, and yeast extract 1.0. Shake cultures were conducted in 500-ml conical flasks containing 100 ml of the sterile medium. The flasks were inoculated with 2.0 ml of conidial suspensions (about 2 ⋅ 10 7 conidia ⋅ ml -1 ) and placed on a rotary shaker at 220 rev ⋅ min -1 and 28 ºC for 4 d.
Enzyme recovery:
The mycelium was removed from the culture broth by centrifugation at 6000 rev ⋅ min -1 for 10 min. From clear supernate fluid the enzyme was recovered: a) by precipitation with methanol, ethanol, propanol, isopropanol and acetone; the chilled solvent (-20 ºC) was mixed with enzyme solution (pH 4.0) at volume ratios 1 : 1, 1 : 2, 1 : 3 or 1 : 4 and the mixture was allowed to stand for 2 h in the cold. The precipitate was collected by centrifugation (14 000 rev ⋅ min -1 , 10 min) and dissolved in 50 ml of 0.1 M citrate-phosphate (MCILVAINE) buffer (pH 4.0). The residual solvent