Strain selection in Kluyveromyces marxianus var. lactis for galactose production
✍ Scribed by M.-Hélène Marin; Hélène Boze; G. Moulin; Prof. Dr. P. Galzy
- Publisher
- John Wiley and Sons
- Year
- 1987
- Tongue
- English
- Weight
- 390 KB
- Volume
- 27
- Category
- Article
- ISSN
- 0233-111X
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✦ Synopsis
After treatment with ethyl-methane sulfonate (EMS) mutants of Kluyveromyces marxianus var. lactis altered in galactose metabolism were obtained which were further improved by genetic recombination. Six types of mutants were detected. I n batch culture strains with double mutationseither in galactokinase and uridyltransferase, or in galactokinase and epimerase-and which also had a galactose permease transformed lactose to galactose and galactitol. Strains with the same double mutations but no galactose permease completely hydrolyzed lactose to galactose and galactitol under continuous culture.
De-proteinized whey and ultra-filtrates from the cheese industry are important sources of lactose. One of lactose applications involves the production of galactose via lactose hydrolysis. This can be accomplished by either enzymatic ( SHUKLA 1975, GREENBERG and MAIIONEY 1981) or microbial means. A number of microorganisms, e.g. Arthrobacter globiformis and Candida kefir, metabolize lactose and thereby liberate galactose into the medium (COOPER et al. 1978, MOULIN et al. 1976). However, the quantity of galactose excreted is small in comparison t o the amount of lactose consumed.
The objective of this work was t o find a method for the production of galactose via lactose fermentation by Kluyveromyces marxianus var. lactis.
Methods
Biological material : Parental strains were obtained from the Centraalbureau Voor Schimmelcultures as K . lactis C . B. S. 2359 (mating type a) and K . luctis C. B. S. 6315 (mating type a).
Culture conditions: Cultures were grown in ERLENMEYER flasks filled to a thenth of the total volume, incubated a t 28 "C and shaken (80 strokes/min). Culture media contained 6.7 g/l yeast nitrogen base (YNB DIBCO) with either 5 g/l glucose (YNBG), or 2 g/l and 20 g/l galactose (YNB gal), respectively, or 5 g/l lactose (YNB lact), or 5 g/l glycerol (YNB gly). Solid media contained YNB (DIFCO) 5 mg/l nicotinic acid (BARRERA and DOMINGWEZ 1980), and 30 g/l agar, p H 3.5. Cultures were incubated a t 28 "C and aerated (1.5 v/v/m).
Analytic methods: Growth was followed by optical density measurements a t 400nm by an ELVI colorimeter. An unit of OD corresponded to 2 mg/ml of dry weight. Sugar concentrations were measured by a WATERS High-Performance Liquid Chromatography apparatus (HPLC) equipped with a refractometer and a Bondapak-NH, column. Separation of galactitol from galactose was performed with an OMINEX HPX 87 H column. Protein concentrations were determined by the BIURET method according to STRICKLAND (1951).
Enzymatic assays: The activities of the different enzyme systems (lactose and galactose permeases, galactosidase, galactokinase, epimerase, transferase) in different strains were determined after growth on YNB glyc (10 g/l) and on YNB glyc (10 g/l) supplemented with galactose (10 g/l). Galactokinase (EC. 2.7.1.6.), epimerase (EC. 5.1.3.2.), and uridyltransferase (EC. 2.7.7.10.) activities were assayed by using enzymatic extracts prepared according to the ROBICHON-SZWL-MAJSTER method (1958). P-Galactosidase was assayed according to the method described by SHEETZ and DICKSON (1980).
Enzymatic extracts were obtained from crushed cells. Ten ml of a cellular suspension containing 60 g/l dry weight were homogeneized in the presence of glass beads (8 g) in an MKS BRAWN cell for 5 min. The mixture was then centrifuged a t 50,000 g for 25 min. The supernatant (Sb) was