CS271570B1 - Alpha amylace producing strain streptomyces liviidans as 28 - Google Patents

Abstract

Alpha-amylase enzyme present in Streptomyces lividans AS28 strain splits amylum to oligosaccharides that can be used as raw material in the food processing industry. Streptomyces lividans AS28 strain shows a high production of alpha-amylase that is lead by multiple-copy plasmide pAS28. The strain is stored in the Czechoslovak collection of micro-organisms at the University of J. E. Purkyne in Brne under CCM 4010 number.

Description

The invention relates to a strain of Streptomycez Lividans AS 28 producing alpha amylase.

This new production industrial bacterial strain is characterized by high production of the exocellular β-amylase enzyme.

Streptomyces produce a number of extracellular enzymes (Williame et al., Gen Gen Microbiol. 129: 1743, 1983). This means that, like the genus Bacillus, they have a highly efficient system of protein export from the cell. An effort has therefore been made to combine the long-term experience in the fermentation of streptomycetes obtained in the production of antibiotics by the production of some important enzymes of industrial interest. Gene manipulation was prepared by etreptomycetes secreting tyrosinase enzymes / Katz et al., ·. Gene. Microbiol. 129, 2703 (1983), endoglycosidesis H (Robbine et al., Vol. Biol. Chem., 256: 10640, 1981) and an agarase which hydrolyzes an agar or agarose polymer (Kendall and Cullum Gene 29: 315, 1984). In 1986 McKillop et al. / FEMS Microbiol. Lett. 36: 3, 1986 / cloned rapidly - Streptomyces amylase, hydroscopiue in Streptomaces lividane · Preparation of Streptomyces superproducing amylase is of great importance for the preparation of high maltose sugar syrup, which is a desired raw material for the food and fermentation industry.

A new strain of Streptomyces lividans AS28 was obtained by introducing the newly constructed multicopy plasmid pAS28, coding for the synthesis of / / - amylase, into the Streptomyces lividans TK24 strain. and multiplied by cloning in the multi-copy plasmid vector pI0622. The recombinant plasmid pSA28 is the subject of U.S. Pat. АО No. 269 939. Characteristic of the strain Streptomyces lividane AS28 It is the ability to grow on starchy substrates supplemented with organic and inorganic nitrogen while producing X-amylase. The end product of starch degradation Oligoeacharides · Production capacity of the strain remains fully maintained in both submerged batch and continuous cultivation · Submerged batch cultivation 8 2% starch As a substrate, the Streptomyces lividans AS28 strain produces 4,500 to 5,000 units-amylase (measured by Spofa) • amylase test) per ml medium ·

A process for the preparation of the monohocopium plasmid pAS28 which encodes the synthesis of β-amylase in etreptomycetes;

Chromosomal ONA was isolated from a strain of β-amylase of Streoptomycee limosus. Chromosome ONA was partially digested with Sau3AI or Mbol reetric endonuclease and the obtained reetric fragments were separated by molecular weight by sucrose gradient centrifugation. 2-4 kb fragments were used for ligation in vitro into the multicopy plasmid vector pI0622. By cloning at a single site of the plasmid vector for the Retric endonuclease BglII, the synthesis of the tyrosinase encoded by this plasmid was abolished and the plasmid vector containing the inserted restriction fragments of chOphreor DNA could be indentified in thioetrexines after transformation into the recipient Streptomycee lividans TK24 strain. plasmid pAS28 It is composed of the plasmid vector pI0622 (Kieser et al., MolGen. Genet. 185: 223, 1982) with an inert Sau3A1 non-spray fragment of the 3 kb fragment on which the gene for Z-amylase production is located. The restriction fragment also contains an intervention site for the retetric endonuclease -PvuII, SstI and EcoRI. The restriction endonucleases KpnI and EcoRI are located outside the coding region of amylase production. Sstl Retrieving Endonuclease Reaching Site It is located in the coding region of the β-amylase synthesis, and insertion into the Sst1 intervention site results in the loss of α-α-aylase production.

(a) Morphological observations

1 · Vegetative cells of Streptomyces lividans AS28 strain are Gram positive in multi-cell mycelium.

2. On liquid soils, S. lividans AS28 strain exhibits mycelial growth. Prices rarely grow in pairs.

CS 271 570 Bl

3. On solid soils, the mycelium is terminated by spores. Spores are thermosensitive ·

4. Growth on solid soils: colonies on agar are round, pale yellow or whitish. The colony profile is regular and flat. Colonies grow firmly into living agar. The surface of the colonies is mostly smooth, but can it be rough on drier agar with numerous scars? you.

b) Physiological conditions r

1. The strain grows strictly aerobically.

2. The temperature optimum of the batch is between 25 and 34 ° C. The temperature maximum is 37 to 45 ° C.

3. Active growth takes place at pH 5.5 to 8.0.

4. The starch forms a significant amount of sxocellular γ-amylase enzyme on starch substrates. Oligosaccharides are the final product of starch cleavage produced by β-amylase. The $ 6-amylase produced has a pH optimum of 8.0 and a temperature optimum of 37 ° C.

5. Glucose represses N-amylase formation. ·

c) Genetic characteristics

1. The strain has no detected restriction activity under conditions used for genetic transfer.

2. The Streptomyces lividans AS28 strain differs from the Streptomyces lividans T1 <24 strain in that it contains plasmid DNA pAS28 having a molecular weight of 921 < b which encodes thioetrepton resistance and β-amylase production.

Claims (1)

Streptomyces lividans strain AS28 CCM 4010 producing alpha amylase.