BRENDA - Enzyme Database show
show all sequences of 3.1.1.41

Efficient production of peracetic acid in aqueous solution with cephalosporin-deacetylating acetyl xylan esterase from Bacillus subtilis

Tao, W.; Xu, Q.; Huang, H.; Li, S.; Process Biochem. 50, 2121-2127 (2015)
No PubMed abstract available

Data extracted from this reference:

Cloned(Commentary)
Commentary
Organism
expression in Escherichia coli
Bacillus subtilis
Engineering
Amino acid exchange
Commentary
Organism
synthesis
synthesis of peracetic acid. Recombinant enzyme can be efficiently produced in a low-cost autoinduction medium with an activity of 6800 U/ml. Optimal synthesis of peracetic is achieved with 0.30 mg/ml crude enzyme, 300 mM glycerol triacetate, and 1 M hydrogen peroxide, pH 8.0, and 2°C, which produces approximately 150 mM of peracetic acid within 5 min. When immobilized on an acrylate amino resin, the activity of the immobilized enzyme is 383.7 U/g
Bacillus subtilis
Inhibitors
Inhibitors
Commentary
Organism
Structure
ethanol
product inhibition
Bacillus subtilis
KM Value [mM]
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1
-
H2O2
free enzyme, pH 6.5, 25°C
Bacillus subtilis
1.15
-
H2O2
immobilized enzyme, pH 6.5, 25°C
Bacillus subtilis
140.7
-
glycerol triacetate
free enzyme, pH 6.5, 25°C
Bacillus subtilis
315.3
-
glycerol triacetate
immobilized enzyme, pH 6.5, 25°C
Bacillus subtilis
409.1
-
ethyl acetate
free enzyme, pH 6.5, 25°C
Bacillus subtilis
Organism
Organism
Primary Accession No. (UniProt)
Commentary
Textmining
Bacillus subtilis
M1JUH6
-
-
Substrates and Products (Substrate)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ethyl acetate + H2O2
-
752071
Bacillus subtilis
ethanol + peracetic acid
-
-
-
?
glycerol triacetate + H2O2
-
752071
Bacillus subtilis
glycerol diacetate + peracetic acid
-
-
-
?
Subunits
Subunits
Commentary
Organism
?
x * 35600, SDS-PAGE
Bacillus subtilis
Cloned(Commentary) (protein specific)
Commentary
Organism
expression in Escherichia coli
Bacillus subtilis
Engineering (protein specific)
Amino acid exchange
Commentary
Organism
synthesis
synthesis of peracetic acid. Recombinant enzyme can be efficiently produced in a low-cost autoinduction medium with an activity of 6800 U/ml. Optimal synthesis of peracetic is achieved with 0.30 mg/ml crude enzyme, 300 mM glycerol triacetate, and 1 M hydrogen peroxide, pH 8.0, and 2°C, which produces approximately 150 mM of peracetic acid within 5 min. When immobilized on an acrylate amino resin, the activity of the immobilized enzyme is 383.7 U/g
Bacillus subtilis
Inhibitors (protein specific)
Inhibitors
Commentary
Organism
Structure
ethanol
product inhibition
Bacillus subtilis
KM Value [mM] (protein specific)
KM Value [mM]
KM Value Maximum [mM]
Substrate
Commentary
Organism
Structure
1.1
-
H2O2
free enzyme, pH 6.5, 25°C
Bacillus subtilis
1.15
-
H2O2
immobilized enzyme, pH 6.5, 25°C
Bacillus subtilis
140.7
-
glycerol triacetate
free enzyme, pH 6.5, 25°C
Bacillus subtilis
315.3
-
glycerol triacetate
immobilized enzyme, pH 6.5, 25°C
Bacillus subtilis
409.1
-
ethyl acetate
free enzyme, pH 6.5, 25°C
Bacillus subtilis
Substrates and Products (Substrate) (protein specific)
Substrates
Commentary Substrates
Literature (Substrates)
Organism
Products
Commentary (Products)
Literature (Products)
Organism (Products)
Reversibility
ethyl acetate + H2O2
-
752071
Bacillus subtilis
ethanol + peracetic acid
-
-
-
?
glycerol triacetate + H2O2
-
752071
Bacillus subtilis
glycerol diacetate + peracetic acid
-
-
-
?
Subunits (protein specific)
Subunits
Commentary
Organism
?
x * 35600, SDS-PAGE
Bacillus subtilis
KCat/KM [mM/s]
kcat/KM Value [1/mMs-1]
kcat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.089
-
ethyl acetate
free enzyme, pH 6.5, 25°C
Bacillus subtilis
0.4
-
glycerol triacetate
immobilized enzyme, pH 6.5, 25°C
Bacillus subtilis
1.09
-
H2O2
immobilized enzyme, pH 6.5, 25°C
Bacillus subtilis
2.53
-
glycerol triacetate
free enzyme, pH 6.5, 25°C
Bacillus subtilis
3.3
-
H2O2
free enzyme, pH 6.5, 25°C
Bacillus subtilis
KCat/KM [mM/s] (protein specific)
KCat/KM Value [1/mMs-1]
KCat/KM Value Maximum [1/mMs-1]
Substrate
Commentary
Organism
Structure
0.089
-
ethyl acetate
free enzyme, pH 6.5, 25°C
Bacillus subtilis
0.4
-
glycerol triacetate
immobilized enzyme, pH 6.5, 25°C
Bacillus subtilis
1.09
-
H2O2
immobilized enzyme, pH 6.5, 25°C
Bacillus subtilis
2.53
-
glycerol triacetate
free enzyme, pH 6.5, 25°C
Bacillus subtilis
3.3
-
H2O2
free enzyme, pH 6.5, 25°C
Bacillus subtilis
Other publictions for EC 3.1.1.41
No.
1st author
Pub Med
title
organims
journal
volume
pages
year
Activating Compound
Application
Cloned(Commentary)
Crystallization (Commentary)
Engineering
General Stability
Inhibitors
KM Value [mM]
Localization
Metals/Ions
Molecular Weight [Da]
Natural Substrates/ Products (Substrates)
Organic Solvent Stability
Organism
Oxidation Stability
Posttranslational Modification
Purification (Commentary)
Reaction
Renatured (Commentary)
Source Tissue
Specific Activity [micromol/min/mg]
Storage Stability
Substrates and Products (Substrate)
Subunits
Temperature Optimum [°C]
Temperature Range [°C]
Temperature Stability [°C]
Turnover Number [1/s]
pH Optimum
pH Range
pH Stability
Cofactor
Ki Value [mM]
pI Value
IC50 Value
Activating Compound (protein specific)
Application (protein specific)
Cloned(Commentary) (protein specific)
Cofactor (protein specific)
Crystallization (Commentary) (protein specific)
Engineering (protein specific)
General Stability (protein specific)
IC50 Value (protein specific)
Inhibitors (protein specific)
Ki Value [mM] (protein specific)
KM Value [mM] (protein specific)
Localization (protein specific)
Metals/Ions (protein specific)
Molecular Weight [Da] (protein specific)
Natural Substrates/ Products (Substrates) (protein specific)
Organic Solvent Stability (protein specific)
Oxidation Stability (protein specific)
Posttranslational Modification (protein specific)
Purification (Commentary) (protein specific)
Renatured (Commentary) (protein specific)
Source Tissue (protein specific)
Specific Activity [micromol/min/mg] (protein specific)
Storage Stability (protein specific)
Substrates and Products (Substrate) (protein specific)
Subunits (protein specific)
Temperature Optimum [°C] (protein specific)
Temperature Range [°C] (protein specific)
Temperature Stability [°C] (protein specific)
Turnover Number [1/s] (protein specific)
pH Optimum (protein specific)
pH Range (protein specific)
pH Stability (protein specific)
pI Value (protein specific)
Expression
General Information
General Information (protein specific)
Expression (protein specific)
KCat/KM [mM/s]
KCat/KM [mM/s] (protein specific)
749261
Singh
Structural role of a conserve ...
Thermotoga maritima, Thermotoga maritima ATCC 43589, Thermotoga maritima DSM 3109
Proteins
85
694-708
2017
-
-
1
1
2
-
-
18
-
-
2
-
-
6
-
-
1
-
-
-
13
-
23
-
3
-
1
20
1
1
-
-
-
-
-
-
-
1
-
1
2
-
-
-
-
18
-
-
2
-
-
-
-
1
-
-
13
-
23
-
3
-
1
20
1
1
-
-
-
-
-
-
19
19
746975
Singh
Crystal structure of Thermoto ...
Thermotoga maritima, Thermotoga maritima ATCC 43589, Thermotoga maritima DSM 3109
Biochem. Biophys. Res. Commun.
476
63-68
2016
-
-
-
1
-
-
-
-
-
-
-
-
-
8
-
-
1
-
-
-
-
-
16
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
16
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
749834
Ma
-
High-level expression of Ceph ...
Bacillus subtilis, Bacillus subtilis SIL3
Biochem. Eng. J.
114
183-190
2016
-
1
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
749837
Ma
-
One-pot enzymatic production ...
Pseudomonas sp. SE83
Biochem. Eng. J.
95
1-8
2015
-
1
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
752071
Tao
-
Efficient production of perac ...
Bacillus subtilis
Process Biochem.
50
2121-2127
2015
-
-
1
-
1
-
1
5
-
-
-
-
-
1
-
-
-
-
-
-
-
-
2
1
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
1
-
-
1
-
5
-
-
-
-
-
-
-
-
-
-
-
-
2
1
-
-
-
-
-
-
-
-
-
-
-
-
5
5
729073
Tian
A novel cephalosporin deacetyl ...
Bacillus subtilis, Bacillus subtilis CICC 20034
Appl. Microbiol. Biotechnol.
98
2081-2089
2014
-
-
1
-
-
-
15
-
-
-
2
2
-
2
-
-
1
-
-
-
6
-
13
1
1
1
3
-
1
1
1
-
-
-
-
-
-
1
-
-
-
-
-
15
-
-
-
-
2
2
-
-
-
1
-
-
6
-
13
1
1
1
3
-
1
1
1
-
-
1
1
-
-
-
730072
Wang
Double knockout of beta-lactam ...
Escherichia coli, Escherichia coli JM105
J. Biosci. Bioeng.
113
737-741
2012
-
-
-
-
1
-
-
-
-
-
-
2
-
7
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
730907
Levisson
Functional and structural char ...
Thermotoga maritima
Proteins
80
1545-1559
2012
-
-
1
1
-
-
2
-
1
2
-
2
-
1
-
-
1
1
-
-
-
-
4
1
1
-
-
-
1
-
-
-
1
-
-
-
-
1
-
1
-
-
-
2
1
-
1
2
-
2
-
-
-
1
-
-
-
-
4
1
1
-
-
-
1
-
-
-
-
2
2
-
-
-
747164
Hedge
The structural basis for the ...
Thermotoga maritima, Thermotoga maritima DSM 3109
Biochim. Biophys. Acta
1824
1024-1030
2012
-
-
1
-
1
-
-
10
-
-
-
-
-
4
-
-
1
-
-
-
-
-
15
-
1
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
1
-
-
-
-
10
-
-
-
-
-
-
-
1
-
-
-
-
15
-
1
-
-
-
1
-
-
-
-
-
-
-
10
10
677538
Martinez-Martinez
A colorimetric assay for the d ...
Bacillus pumilus, Bacillus pumilus CECT 5072
Anal. Biochem.
369
210-217
2007
-
1
1
-
-
-
-
2
-
-
-
-
-
4
-
-
1
-
-
-
-
-
4
-
1
-
-
-
1
-
-
-
-
-
-
-
1
1
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
1
-
-
-
-
4
-
1
-
-
-
1
-
-
-
-
-
-
-
-
-
671502
Takimoto
Batch production of deacetyl 7 ...
Bacillus subtilis, Bacillus subtilis SHS0133
Appl. Microbiol. Biotechnol.
65
263-267
2004
-
1
1
-
-
1
1
-
-
-
-
2
-
5
-
-
1
-
-
-
-
1
4
-
-
-
1
-
-
-
2
-
-
-
-
-
1
1
-
-
-
1
-
1
-
-
-
-
-
2
-
-
-
1
-
-
-
1
4
-
-
-
1
-
-
-
2
-
-
-
-
-
-
-
675216
Basch
Expression of a cephalosporin ...
Rhodotorula toruloides
J. Ind. Microbiol. Biotechnol.
31
531-539
2004
-
1
1
-
-
-
-
-
-
-
-
1
-
4
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
1
1
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
652917
Vincent
Multifunctional xylooligosacch ...
Bacillus subtilis
J. Mol. Biol.
330
593-606
2003
-
-
-
1
-
-
-
-
-
-
-
-
-
3
-
-
-
1
-
-
-
-
5
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
5
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
649401
Velasco
Cloning and characterization o ...
Acremonium chrysogenum, Acremonium chrysogenum C10 / ATCC 48272
Appl. Microbiol. Biotechnol.
57
350-356
2001
-
-
-
-
-
-
2
1
1
-
2
-
-
7
-
1
1
-
-
-
-
-
4
-
1
1
-
-
1
1
-
-
-
1
-
-
-
-
-
-
-
-
-
2
-
1
1
-
2
-
-
-
1
1
-
-
-
-
4
-
1
1
-
-
1
1
-
1
-
-
-
-
-
-
651823
Lee
-
Isolation, analysis, and expre ...
Staphylococcus sp.
J. Biochem. Mol. Biol.
34
274-277
2001
-
-
1
-
-
-
-
-
-
-
1
-
-
1
-
1
1
-
-
-
-
-
2
-
1
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
1
-
-
-
1
1
-
-
-
-
2
-
1
-
-
-
1
-
-
-
-
-
-
-
-
-
649397
Cardoza
A cephalosporin C acetylhydrol ...
Amycolatopsis lactamdurans, Amycolatopsis lactamdurans MA4213
Appl. Microbiol. Biotechnol.
54
406-412
2000
-
-
-
-
-
-
5
2
1
-
1
-
-
4
-
-
1
-
-
1
1
-
8
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
5
-
2
1
-
1
-
-
-
-
1
-
1
1
-
8
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
170677
Takimoto
High-level expression, purific ...
Bacillus subtilis, Bacillus subtilis SHS 0133
J. Biosci. Bioeng.
87
456-462
1999
-
-
1
-
-
-
-
5
-
-
1
-
-
7
-
-
1
-
-
-
1
1
11
1
1
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
5
-
-
1
-
-
-
-
1
-
-
1
1
11
1
1
-
-
-
1
-
-
-
-
-
-
-
-
-
170672
Sakai
-
Purification and properties of ...
Rhodotorula glutinis, Rhodotorula glutinis 38B1
J. Ferment. Bioeng.
85
53-57
1998
-
-
-
-
-
-
6
4
1
-
2
-
-
2
-
1
1
-
-
-
1
-
8
1
1
1
1
4
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
6
-
4
1
-
2
-
-
-
1
1
-
-
1
-
8
1
1
1
1
4
1
1
-
-
-
-
-
-
-
-
170675
Politino
Purification and characterizat ...
Bacillus subtilis, Rhodotorula toruloides
Appl. Environ. Microbiol.
63
4807-4811
1997
-
-
-
-
-
1
3
1
-
-
2
-
-
2
-
1
1
-
-
-
1
-
7
1
1
-
2
-
-
1
-
-
-
-
-
-
-
-
-
-
-
1
-
3
-
1
-
-
2
-
-
-
1
1
-
-
1
-
7
1
1
-
2
-
-
1
-
-
-
-
-
-
-
-
170676
Sakai
Bioconversion of 7-aminocephal ...
Rhodotorula glutinis
Appl. Environ. Microbiol.
62
2669-2672
1996
-
1
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
170674
Mitsushima
Gene cloning, nucleotide seque ...
Bacillus subtilis, Bacillus subtilis SHS 0133
Appl. Environ. Microbiol.
61
2224-2229
1995
-
-
1
-
-
-
-
-
-
-
-
-
-
8
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
170673
Takimoto
-
Purification, characterization ...
Bacillus subtilis, Bacillus subtilis ATCC 6633, Bacillus subtilis SHS 0133
J. Ferment. Bioeng.
77
17-22
1994
-
-
-
-
-
-
7
7
-
-
3
-
-
6
-
-
1
-
-
-
1
-
16
2
1
-
4
2
2
-
1
-
-
-
-
-
-
-
-
-
-
-
-
7
-
7
-
-
3
-
-
-
-
1
-
-
1
-
16
2
1
-
4
2
2
-
1
-
-
-
-
-
-
-
660484
Verweij
-
Industrial transformation of p ...
Escherichia coli, Pseudomonas sp., Pseudomonas sp. GK-16
Rec. Trav. Chim. Pays-Bas
112
66-81
1993
-
2
-
-
-
-
-
-
-
-
-
-
-
3
-
-
-
-
-
-
-
-
3
-
2
-
-
-
2
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3
-
2
-
-
-
2
-
-
-
-
-
-
-
-
-
170663
Singh
-
Cephalosporin acetylesterase a ...
Fusarium oxysporum, Fusarium oxysporum AY F-298
Eur. J. Appl. Microbiol. Biotechnol.
9
15-18
1980
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
-
-
-
-
-
-
-
6
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
6
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
170667
Abbott
Cephalosporin acetylesterase ( ...
Bacillus subtilis, Bacillus subtilis WRRL-B-558
Methods Enzymol.
43
731-734
1978
-
-
-
-
-
-
2
3
-
-
-
-
-
2
-
-
-
-
-
-
-
1
12
-
1
-
1
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
3
-
-
-
-
-
-
-
-
-
-
-
1
12
-
1
-
1
-
1
-
-
-
-
-
-
-
-
-
170664
Abbott
Immobilization of a cephalospo ...
Bacillus subtilis
Biotechnol. Bioeng.
18
1033-1042
1976
-
-
-
-
-
1
2
1
-
-
-
-
-
2
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
2
-
1
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
170665
Hinnem
Enzymatic hydrolysis of cephal ...
Acremonium chrysogenum
Antimicrob. Agents Chemother.
9
824-830
1976
-
-
-
-
-
-
2
1
1
-
1
1
-
1
-
-
1
-
-
1
1
-
2
-
1
1
1
-
1
1
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
1
1
-
1
1
-
-
-
1
-
1
1
-
2
-
1
1
1
-
1
1
-
-
-
-
-
-
-
-
170670
Nuesch
-
A biochemical and genetical ap ...
Acremonium chrysogenum
Proc. Int. Symp. Genet. Ind. Microorg. (McDonald, K. D. , ed. )
451-472
1976
-
-
-
-
-
-
1
-
-
-
1
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
1
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
170666
Abbott
Physical properties and kineti ...
Bacillus subtilis
Appl. Microbiol.
30
413-419
1975
-
-
-
-
-
-
2
2
-
-
1
-
-
2
-
-
1
-
-
-
-
1
2
-
1
1
2
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2
-
2
-
-
1
-
-
-
-
1
-
-
-
1
2
-
1
1
2
-
1
-
-
-
-
-
-
-
-
-
170668
Abraham
Cephalosporin acetylesterase ( ...
Citrus sp.
Methods Enzymol.
43
728-731
1975
-
-
-
-
-
1
-
3
-
-
-
-
-
1
-
-
-
-
-
1
-
-
4
-
-
-
1
-
1
1
2
-
-
-
-
-
-
-
-
-
-
1
-
-
-
3
-
-
-
-
-
-
-
-
-
1
-
-
4
-
-
-
1
-
1
1
2
-
-
-
-
-
-
-
170669
Fujisawa
-
Deacetylcephalosporin C format ...
Acremonium chrysogenum
Agric. Biol. Chem.
39
1303-1309
1975
-
-
-
-
-
-
4
-
-
-
-
-
-
1
-
-
1
-
-
1
1
-
1
-
1
-
1
-
2
1
-
-
-
-
-
-
-
-
-
-
-
-
-
4
-
-
-
-
-
-
-
-
-
1
-
1
1
-
1
-
1
-
1
-
2
1
-
-
-
-
-
-
-
-
170671
Fujisawa
New findings on cephalosporin ...
Acremonium chrysogenum
Nature New Biol.
246
154-155
1973
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
1
-
-
-
-
-
1
-
1
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
1
-
1
-
-
-
-
-
-
-