Enzyme or cell preparation with inulinase activity

Details Enzyme or cell preparation with inulinase activity Enzyme or cell preparation with inulinase activity

2001-11-09

2003-02-11

Achutamurthy, Ponnathapu (Department: 1652)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

Reexamination Certificate

active

06518047

ABSTRACT:

CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to European Application Serial No. EP 00870264.9, filed Nov. 9, 2000, the disclosure of which is herein incorporated by reference in its entirety.
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to enzyme or cell preparations with inulinase activities from
Penicillium restrictum,
to their obtention and to their use in the hydrolysis of fructan polymers.
2. Description of the Related Art
Inulin is a polydisperse composition made of oligo- and polysaccharides which are composed of fructose units linked together by &bgr;(2-1) linkages. Most molecules are terminated by a glucose unit. They can be hydrolyzed into monomers by acidic treatment (pH 1-2) for 1 to 2 hours at high temperature. However, undesirable secondary products may appear during this process, leading to a lower yield.
A known alternative to this process is the enzymatic hydrolysis. Enzymes preparations obtained from cultures of various micro-organisms have been described to hydrolyze inulin. Among these are the yeasts
Kluyveromyces marxianus,
Debaromyces,
Candida kefyr,
the molds
Aspergillus oryzae, Aspergillus ficuum, Fusarium oxysporum
and some bacterial species from the genus Actinomyces or Lactobacillus. There are also some plants that synthesize inulinases.
Some parameters are required for an economical and realistic industrial process of inulin hydrolysis:
The working temperature needs to be high (above 60° C.) (required to achieve an adequate level of inulin concentration in the solution to be treated, but minimizes also microbial infections).
The working pH should be slightly acidic (pH 4-6) to keep a good stability of the fructose reaction product.
The enzyme preparation should be stable in the working conditions.
The hydrolysis of the inulin should be as complete as possible, giving at least 98% of glucose and fructose yield on a dry weight basis.
The enzyme preparation should work in concentrated inulin solution (20% and higher).
It is therefore highly desirable to have an improved inulinase preparation and that preferably presents the following characteristics:
optimal temperature above 60° C., preferably at about 65° C.
optimal pH between about 4.0 and about 6.0.
high stability in working conditions.
achievement of a complete hydrolysis.
substrate concentration of about 20% or higher.
Another aim of the present invention is related to the use of said enzyme preparation, preferably in the absence of cells or cell debris, preferably in the form of extracellular medium comprising most of the enzymatic activity of said inulinase, for the enzymatic treatment of inulin and inulin containing materials or the synthesis of fructose oligomers.
SUMMARY OF THE INVENTION
The present invention is related to an isolated and purified enzyme with inulinolytic activity having more than 75% sequence identity with the amino acid sequence SEQ ID NO 12 to its encoding nucleotide sequence to a cell producing the enzyme and to the use for the degradation of inulin or inulin-containing plant material, especially for the production of fructose syrups and for the production of oligomers of fructose.
Some embodiments of the present invention are described in the following numbered paragraphs:
Paragraph 1: An isolated and purified enzyme with inulinolytic activity having more than 75% sequence identity with the amino acid sequence SEQ ID NO 12.
Paragraph 2: The enzyme according to paragraph 1, having more than 80% sequence identity with the amino acid sequence SEQ ID NO 12.
Paragraph 3: An isolated and purified enzyme amino acid sequence having the amino acid sequence of SEQ ID NO 12 or a portion thereof having an inulinolytic activity.
Paragraph 4: The enzyme according to paragraph 1, which presents an optimum enzymatic activity at a pH between about 4.0 and about 6.0 and at a temperature between about 60 and about 70° C.
Paragraph 5: An isolated and purified nucleotide sequence encoding the enzyme according to paragraph 1, 3 or 4.
Paragraph 6: An isolated and purified nucleotide sequence which encodes a polypeptide having a inulinolytic activity and has more than 75% sequence identity with the nucleotide sequence SEQ ID NO 9.
Paragraph 7: The isolated and purified nucleotide according to paragraph 6, which has more than 80% sequence identity with the nucleotide sequence SEQ ID NO 9.
Paragraph 8: An isolated and purified nucleotide sequence SEQ ID NO 9 or a portion thereof encoding a polypeptide having an inulinolytic activity.
Paragraph 9: A recombinant nucleotide sequence comprising, operably linked to the nucleotide sequence according to paragraph 5, one or more adjacent regulatory sequence(s), preferably originating from homologous microorganisms.
Paragraph 10: A vector comprising the nucleotide sequence according to paragraph 5.
Paragraph 11: The vector according to paragraph 10, being a plasmid incorporated in
Escherichia coli
and having the deposit number LMBP-4252.
Paragraph 12: A cell producing the enzyme according to paragraph 1.
Paragraph 13: The cell of paragraph 12, having a deposit number MUCL-42612.
Paragraph 14: The cell according to the paragraph 12 being a recombinant host cell transformed by the nucleotide sequence according to paragraph 5 or the vector according to paragraph 10 or 11.
Paragraph 15: The recombinant host cell according to paragraph 14, which is selected from the group consisting of bacteria or fungi, including yeast.
Paragraph 16: The cell according to any one of the preceding paragraphs 12 to 15, wherein the enzyme is extra-cellularly expressed by said cell.
Paragraph 17: The cell according to paragraph 12, wherein the enzyme is intra-cellularly expressed by said cell.
Paragraph 18: A solid support fixing an element selected from the group consisting of the cell according to paragraph 12, a cell extract of the cell according to paragraph 12 and/or the isolated and purified enzyme with inulinolytic activity according to paragraph 1.
Paragraph 19: A method for the degradation of inulin or inulin-containing plant material by the addition of the recombinant host cell according to paragraph 12 or the enzyme with inulinolytic activity according to paragraph 1.


REFERENCES:
patent: 0043169 (1983-10-01), None
patent: WO 9413821 (1994-06-01), None
Arand, M and Golubev AM et al, 2001 Exo-insulinase fromAspergillus awamorivar. 2250: enzymatic properties, sequence analysis and preliminary X-ray data. EMBL Acc# AJ315793.*
European Search Report from EP00870264 dated Apr. 26, 2001.
Onodera, et al., (1996)Molecular Cloning and Nucleotide Sequences of cDNA and Gene Encoding endo-Inulinase from Penicillium Purpurogenum. Biosci. Biotech.Biochem. 60(11)1780-1785.
Rehm, et al. (1998)Production of 1-Kestose in Transgenic Yeast Expressing a Fructosyltransferase from Aspergillus foetidus. J.Bacteriology. 180(5)1305-1310.
Onodera; EMBL/GENBANK/DDBJ databases;Penicillium Purpurogenum DNA for endo-inulinase Precursor, complete cds; 2 pgs.; Feb. 1/97 (created), Jan. 21/99 (Last updated).
Chapman, et al.; EMBL/GENBANK/DDBJ databases;Kluyveromyces Marxianus, Inulinase Preprotein; 2 pg.; Mar. 3/98 (Created), Mar. 11/98 (Last Updated).
Rehm; EMBL/GENBANK/DDBJ databases;Aspergillus Foetidus DNA for Sucrose: Sucrose 1-Fructosyltransferase Gene; 2 pgs; Mar. 3/98 (Created), Mar. 11/98 (Last updated).
Vandamme, et al. (1983)Microbial Inulinases: Fermentation Process, Properties, and Applications; Advances in Applied Microbiology, 29:139-177.
Boel, et al. (1984)Two Different Types of Intervening Sequences in the Glucoamylase Gene from Aspergillus niger, EMBO Journal 3(7):1581-1585.
Punt, et al. (1990)Functional Elements in the Promoter Region of the Aspergillus nidulans gpdA Gene Encoding Glyceraldehyde-3-phosphate dehydrogenase, Gene, 93:101-109.
Ettalibi, et al. (1990)Molecular and Kinetic Properties of Aspergillus ficuum Inulinases, Agric.Biol.Chem. 54(1)61-68.
Hynes, et al. (1983)Isolation of Genomic Clones Containing amdS Gene of Aspergillus nidulans and Their Use in the Analysis of Structural and Regula

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