Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 13, Num. 3, 1996, pp. 201

PROCEDURE FOR THE HIGH LEVEL RECOMBINANT DEXTRANASE PRODUCTION

Efrain Rodriguez, Kosara Sanchez, Hernan Roca, Bianca Garcia, Jose Cremata and Julio Delgado

Center of Genetic Engeneering and Biotechnology, P.O. Box 6162, Havana, Cuba.

Code Number:BA96079
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Introduction

The production of industrial enzymes using gene technology and its correspondent new production technologies offers several benefits including the utilization of safe, well studied host micro-organism and very strong promoters in short scale and large capacity production plants at reduced costs (1). The methylotrophic yeast Pichia pastoris is able to produce large quantities of heterologous proteins under regulation of the alcohol oxidase promoter during growth on methanol (2, 3). We present here a procedure for recombinant dextranase production using the yeast P. pastoris which has integrated to its chromosome this gene under the alcohol oxidise promoter. The amount of secreted enzyme to the medium reaches 3.5 g/L.

Materials and Methods

The powerful transformant of recombinant yeast P. pastoris, producer of fungal dextranase, was selected in shake flask culture and then it was studied in 5 L fermenter using a minimal salts medium and molasses as carbon source at initial growth phase. The scale up process was carried out in a 75 and 300 L fermenters. The stability of secreted to the supernatant enzyme was studied, and established its final formulation. The efficiency of the product was probed in a Cuban sugar factory. A cost estimate for a 250 tons production plant was done.

Results and Discussion

On shake flask study 16 different clones were analyzed during 120 h in 1 % of methanol and the best producer reached 100 EUA/mL (approx. 0.1 g of enzyme/litre). The 5 L fermentation process was developed maintaining the oxygen dissolved tendency to decrease by the way of change of the methanol flow in the second production phase. The methanol flow was started at 2 g/Lh and once the tendency showed to increase then the flow was increased in 0.5 g/Lh up to 4 g/Lh. The amount of secreted dextranase to the culture broth reached 3 500 EUA/mL after 110 h (Figure 1). The level of purity of the enzyme in the supernatant is shown in Figure 2. The scale up process was carried out by the same strategy and showed a similar level of secreted enzyme at the same time in both 75 and 300 L fermenters (Figure 1). The stability of the enzyme in the supernatant was checked at 4 and 30 C with the addition of 0.05 % sodium benzoate.

Figure 1. 5 and 75 L fermentation.
Wet biomass (5L), Wet biomass (75L), Enz. Unit Act. (5L), Enz. Unit Act. (75L)

Figure 2. 75 L fermentation (PAGE 10%) (D- Dextranase).
A) Dextranase from P. minioluteum.
B, C, D, E) Culture supernatant of 26, 38, 62 and 110 h of fermentation.
F, G, H, I) Biomass of 26, 38, 62, and 110 h of fermentation

References

1 EA Falch. Industrial Enzymes Development in Production and Application. Biotechnology Advances 1991; 9(4): 643-658.

2 RS Siegel, RA Brierley. Methylotrophic yeast P. pastoris Produced in High-Cell-Density Fermentations with High Cell Yields as Vehicle for Recombinant Protein Production. Biotechnology and Bioengineering 1989;34:403- 404.

3 JM Cregg, TS Vedvick, WC Raschke. Recent Advances in the Expression of Foreign Genes in P. pastoris. Bio/Technology 1993; 11:905-910.

Copyright 1996 Elfos Scientiae

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