Alpha/beta hydrolase-fold enzymes

Details Alpha/beta hydrolase-fold enzymes Alpha/beta hydrolase-fold enzymes

1999-07-20

2001-11-13

Nashed, Nashaat T. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S222000, C435S252310, C435S320100, C435S212000, C510S392000, C008S401000

Reexamination Certificate

active

06316241

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to alpha/beta hydrolase-fold enzymes derived from gram-positive microorganisms. The present invention provides the nucleic acid and amino acid sequences for the hydrolases and methods for their use.
BACKGROUND OF THE INVENTION
The alpha/beta hydrolase fold common to several hydrolytic enzymes of differing phylogenetic origin and catalytic function was described by Ollis et al. (1992, Protein Eng. 5(3):197-211). The core of each enzyme in this family was described as being similar: an alpha/beta sheet of eight beta-sheets connected by alpha-helices with conserved arrangement of catalytic residues. Members of this family were found to have a catalytic triad which is borne on the conserved loop structure found in the fold. In the five members discussed in Ollis et al., the catalytic residues always occur in the same order in the primary sequence: nucleophile, acid, histidine. Furthermore, the catalytic triad residues of the members had similar topological and three dimensional positions.
Members of the hydrolase family include a hydroxylyase (Wajant, et al., 1996, J. Biol. Chem. 271(42):25830-25834) which comprises the active site motif Gly-X-Ser-X-Gly/Ala and the residues Serine 80, Aspartic 208, and Histidine 236 which are critical for enzyme activity; 2-hydroxymuconic semialdehyde hydrolase, XylF (Diaz E., 1995, J. Biol. Chem. 270(11):6403-6411), which comprises the residues Ser107, Asp228 and His 256; non-heme haloperoxidases comprises oxidases, which perform halogenation and which are related to esterases (Pelletier et al., 1995, Biochim Biophys Acta, 1995,1250(2):149-157) which comprises the residues Serine 97, Aspartic acid 229, Histidine 258; and dipeptidyl-peptidase IV (David et al., 1993, J. Biol. Chem. 268(23):17247-17252), which comprises the residues Ser624Asp702, His734).
SUMMARY OF THE INVENTION
The present invention relates to the identification of gram-positive microorganism members of the family of alpha/beta hydrolase fold enzymes which are characterized by structural relatedness and which comprises conserved catalytic triads. These newly identified members of this family can be used in industrial applications, e.g., the textile industry, in cleaning compositions, such as detergents, and in animal feeds.
Accordingly, the present invention provides compositions comprising a hydrolase selected from the group consisting of YUXL, YTMA, YITV, YQKD, YCLE, YTAP, YDEN, YBFK, YFHM, YDJP, YVFQ, YVAM, YQJL, SRFAD, YCGS, YTPA, YBAC, YUII, YODD, YJCH, YODH which can be used in detergent compositions, compositions for the treatment of textiles; and animal feeds, for example. The present invention also provides commercial applications of the compositions, e.g., their use in methods for treating textiles and methods for cleaning.
The present invention provides amino acid sequences for hydrolases obtainable from B. subtilis (B. subtilis hydrolases). Due to the degeneracy of the genetic code, the present invention encompasses any nucleic acid sequence that encodes the specific hydrolase amino acid sequence shown in the Sequences.
The present invention provides methods for detecting gram positive microorganism homologs of the B. subtilis hydrolases that comprise hybridizing part or all of the nucleic acid encoding the hydrolase with nucleic acid derived from gram-positive organisms, either of genomic or cDNA origin. In one embodiment, the gram-positive microorganism includes
B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus and Bacillus thuringiensis.
The invention further provides for a hydrolase that has at least 80%, at least 85%, at least 90%, and at least 95% homology with the specific amino acid sequences shown in the sequences. The invention also provides for a nucleic acid which encodes a hydrolase that has at least 80%, at least 85%, at least 90% and at least 95% homology with the naturally occurring nucleic acid sequence found in Bacillus subtilis.
In a preferred embodiment, the present invention provides the naturally occurring hydrolase nucleic acid molecule having the sequence found in Bacillus subtilis 1-168 strain (Bacillus Genetic Stock Center, accession number 1A1, Columbus, Ohio) as disclosed infra.
The present invention provides expression vectors and host cells comprising a nucleic acid encoding a gram positive hydrolase. The present invention also provides methods of making the gram positive hydrolase.
The present invention encompasses novel amino acid variations of hydrolase amino acid sequences from gram positive microorganisms disclosed herein that have hydrolytic activity. Naturally occurring hydrolases derived from gram positive microorganisms disclosed herein as well as proteolytically active amino acid variations or derivatives thereof, have application in the textile industry, in cleaning compositions and methods and in animal feed compositions.
In another embodiment, a host cell is engineered to produce a hydrolase of the present invention. In a further aspect of the present invention, a hydrolase from a gram positive microorganism is produced on an industrial fermentation scale in a host expression system. The host cell may be a gram-negative or gram-positive microorganism, a fungal organism, or higher Eucaryotes. Gram negative microorganisms include but are not limited to members of Enterobacteriaceae; gram positive microorganisms include but are not limited to members of Bacillus and Pseudomonase; fungal organisms include but are not limited to Aspergillus and Tricoderma; and higher eucaryotes include mammalian cells.
The gram positive microorganism host cell may be normally sporulating or non-sporulating and may be modified in other ways to facilitate expression of the hydrolase. In a preferred embodiment, the gram positive host cell is a Bacillus. In another embodiment, the Bacillus includes
B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus and B. thuringiensis
. In a further preferred embodiment, the Bacillus host cell is Bacillus subtilis.
DETAILED DESCRIPTION
Definitions
The present invention relates to a newly characterized hydrolases from gram positive organisms. In a preferred embodiment, the gram positive organisms is a Bacillus. In another is preferred embodiment, the Bacillus includes
B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus and B. thuringiensis.
In another preferred embodiment, the gram positive organism is Bacillus subtilis and the hydrolases have the amino acid sequence disclosed in the Sequences. In a preferred embodiment, the hydrolase is encoded by the naturally occurring nucleic acid that is found at the respective positions of B. subtilis detailed infra.
As used herein, “nucleic acid” refers to a nucleotide or polynucleotide sequence, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin which may be double-stranded or single-stranded, whether representing the sense or antisense strand. As used herein “amino acid” refers to peptide or protein sequences or portions thereof. A “polynucleotide homologue” as used herein refers to a gram positive microorganism polynucleotide that has at least 80%, at least 85%, at least 90% and more preferably at least 95% identity to
B.subtilis
hydrolase, or which is capable of hybridizing to
B.subtilis
hydrolase under conditions of high stringency and which encodes an amino acid sequence having hydrolase activity.
The terms “isolated” or “purified” as used herein refer to a nucleic acid or peptide or protein that is removed from at least one component with which it is naturally associated. As used herein, isolated nucleic acid can include a vector comprising the nucleic acid.
As used herein, the term “overexpressing” when referring to the production of a protein in a host cell means that the protein is produ

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