U.S. patent application number 15/442607 was filed with the patent office on 2018-03-08 for method for detecting products derived from glucuronide metabolites with the enzyme b-glucuronidase, and a reagent comprising said enzyme.
This patent application is currently assigned to LA PIEDRA BIOTECNOLOG A SPA. The applicant listed for this patent is LA PIEDRA BIOTECNOLOG A SPA. Invention is credited to Daniel E. ALMONACID CORONADO, Manuel ROZAS ANDREU.
Application Number | 20180067116 15/442607 |
Document ID | / |
Family ID | 57119795 |
Filed Date | 2018-03-08 |
United States Patent
Application |
20180067116 |
Kind Code |
A1 |
ROZAS ANDREU; Manuel ; et
al. |
March 8, 2018 |
METHOD FOR DETECTING PRODUCTS DERIVED FROM GLUCURONIDE METABOLITES
WITH THE ENZYME B-GLUCURONIDASE, AND A REAGENT COMPRISING SAID
ENZYME
Abstract
The present invention discloses a method and a highly efficient
reagent to detect products derived from glucuronide metabolites
presents in a sample, comprising the steps of adding to said sample
an enzyme with .beta.-glucuronidase activity originated from genus
Brachyspira or any variant or mutant derived thereof; incubating
the sample with the enzyme during a determined period of time; and
detecting the product derived from glucuronide metabolite by means
of a suitable technique. The reagent of the present invention
comprises an enzyme with .beta.-glucuronidase activity originated
from genus Brachyspira or any variant or mutant derived thereof;
and a suitable vehicle.
Inventors: |
ROZAS ANDREU; Manuel;
(Puerto Varas, CL) ; ALMONACID CORONADO; Daniel E.;
(Lafayette, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
LA PIEDRA BIOTECNOLOG A SPA |
Puerto Varas |
|
CL |
|
|
Assignee: |
LA PIEDRA BIOTECNOLOG A SPA
|
Family ID: |
57119795 |
Appl. No.: |
15/442607 |
Filed: |
February 24, 2017 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/948 20130101;
G01N 33/56916 20130101; G01N 2333/924 20130101; C12Q 1/34 20130101;
G01N 30/72 20130101; G01N 33/9486 20130101 |
International
Class: |
G01N 33/569 20060101
G01N033/569; C12Q 1/34 20060101 C12Q001/34 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 26, 2016 |
GB |
1614546.8 |
Claims
1. A method to detect products derived from glucuronide metabolites
in a sample, comprising the steps of: a) providing the sample in
which said product is to be detected, b) adding to said sample an
enzyme with .beta.-glucuronidase activity from genus Brachyspira or
any variant or mutant derived thereof, c) incubating the sample
with the enzyme for a determined period of time; and d) detecting
the product derived from said glucuronide metabolite through any
suitable technique.
2. The method of claim 1, wherein products derived from glucuronide
metabolites are from natural, semi-synthetic or synthetic
origin.
3. The method of claim 2, wherein products derived from glucuronide
metabolites are selected from the group consisting of an opioid, an
opiate, a cannabinoid, a benzodiazepine or any derivative
thereof.
4. The method of claim 1, wherein the sample is a biological
sample.
5. The method of claim 4, wherein the biological sample is selected
from the group consisting of saliva, whole blood, plasma, urine,
hair, skin, teeth, soft tissues, meconium and vitreous humor.
6. The method of claim 1, wherein the enzyme with
.beta.-glucuronidase activity originates from species Brachyspira
pilosicoh.
7. The method of claim 6, wherein the enzyme with
.beta.-glucuronidase activity is selected from the group consisting
of: a) an amino acid sequence comprising the amino acid sequence
shown in SEQ. ID. NO.1; b) an amino acid sequence sharing at least
a 70% of identity with the amino acid sequence defined in a).
8. The method of claim 1, wherein the incubation period of the
sample and the enzyme is between 1 and 30 minutes.
9. The method of claim 1, wherein incubation of sample and enzyme
is performed at a temperature range between 20 and 60 degrees
Celsius.
10. The method of claim 1, wherein incubation of sample and enzyme
is performed at a range of pH between 4.0 and 9.0.
11. The method of claim 1, wherein the suitable technique to detect
products derived from glucuronide metabolites is selected from the
group consisting of LC-MS, GC-MS, HPLC-MS, LC-HRMS, HPLC-HRMS,
LC-MS-MS, GC-MS-MS and HPLC-MS-MS.
12. A reagent to detect products derived from glucuronide
metabolites in a sample, comprising the steps of: a) an enzyme with
.beta.-glucuronidase activity originated from genus Brachyspira or
any variant or mutant derived thereof; and b) a suitable
vehicle.
13. The reagent of claim 12, wherein the enzyme with
.beta.-glucuronidase activity originates from species Brachyspira
pilosicoli.
14. The reagent of claim 13, wherein the enzyme with
.beta.-glucuronidase activity is selected from the group consisting
of: a) an amino acid sequence comprising the amino acid sequence
shown in SEQ. ID. NO.1; b) an amino acid sequence sharing at least
a 70% of identity with the amino acid sequence defined in a).
15. The reagent of claim 12, wherein the suitable vehicle is
selected from the group consisting of: water, salts, buffer
solutions, Tris, HEPES, DTT, citric acid, EDTA, glycerol, sugars,
amino acids or a combination thereof.
16. The reagent of claim 15, wherein the salts are selected from
the group consisting of: sodium phosphate, potassium phosphate,
sodium carbonate, sodium acetate, sodium citrate, sodium chloride
and potassium chloride.
17. The reagent of claim 15, wherein sugars are selected from the
group consisting of sorbitol, trehalose, sucrose, glucose, lactose,
mannitol and raffinose.
Description
TECHNICAL FIELD
[0001] The present invention is related with the technical field of
biotechnology, and particularly provides a method for detecting
glucuronide derivatives present in a sample, using for this purpose
a .beta.-glucuronidase enzyme from bacterial origin, as well as a
reagent including said enzyme.
BACKGROUND OF THE INVENTION
[0002] Conjugation of endogenous and exogenous compounds with
D-glucuronic acid is a metabolic pathway commonly known in animals
and humans, which is normally considered as a process for
detoxification of organisms. Through this process, glucuronide
conjugates or metabolites are produced, which are excreted through
urine or bile (Boppana V et al, J. Pharm. Sci. 1989. Vol
78(2):127-131). Compounds that are excreted in these ways are
mainly lipophilic, such as bilirubin, androgens, glucocorticoids,
among others, and xenobiotic substances such as opioids,
cannabinoids, benzodiazepines, among others.
[0003] Detection of these glucuronide metabolites in biological
fluids allows identification and quantitation of endogenous or
exogenous compounds present in the organism. Said detection can be
performed by means of chemical hydrolysis (acid hydrolysis) or by
means of enzymatic hydrolysis of said glucuronide metabolites.
Chemical hydrolysis is performed with an acid which hydrolyses most
of conjugates in a relative reduced period of time (from 60 to 90
minutes) and at a low cost. However, the acid conditions and
temperature required in this process degradate sensitive compounds,
for example, benzodiazepines. Moreover, working with acids in the
laboratory requires stricter security conditions, producing
corrosion of vulnerable equipment. For this reason, testing
laboratories for these purposes mainly employ enzymatic hydrolysis,
consisting in the use of .beta.-glucuronidases for hydrolyzation of
glucuronide metabolites in conditions more manageable than acid
hydrolysis.
[0004] Currently there exist diverse .beta.-glucuronidase enzymes
available from different origins, each one with optimum conditions
for hydrolysis of metabolites. Thus, parameters such as
concentration, pH, incubation period and temperature of reaction
vary depending on the enzyme used.
[0005] For example, there exist alternatives of enzymes obtained
from molluscs such as a .beta.-glucuronidase enzyme originated from
Patella vulgata described in document U.S. Pat. No. 4,473,640 of
Combie et al, which requires a temperature between 60 and 70
degrees Celsius, pH 5, and at least one hour to obtain only 60% of
hydrolysis of the glucuronide metabolite, and approximately three
hours as the optimum time to reach complete quantification of the
hydrolyzed metabolite. There also exist .beta.-glucuronidase
enzymes described from species Helix pomatia, Helix aspersa,
Haliotis rufescens, among others (WO 95/16050, Scheinmann et al;
Kemp et al, FAA. 2015. Report No. DOT/FAA/AM-15/6). Generally,
preparations of enzymes obtained from molluscs tend to have more
contaminant substances that may interfere with further analysis of
glucuronide metabolites.
[0006] On the other hand, there also exist alternatives to enzymes
from bacterial origin, such as .beta.-glucuronidase from
Escherichia coli. Comparative studies show that at an optimum pH
and depending on the type of glucuronide metabolite/substrate, it
is required an hour of incubation to obtain 40-80% of hydrolysis,
and between three to five hours to reach a complete hydrolysis
(Wakabashi et al, J Biol. Chem. 1961. Vol 236(4):996-1001). These
detection times are excessive for testing laboratories which
analyze drugs in biological samples, and the situation is even more
dramatic in cases such as analysis of natural opioids (codeine and
morphine) and semi-synthetic opioids (hydromorphone, oxycodone and
oxymorphone). The hydrolysis percentage of conjugated metabolites
of these opioids widely varies depending on the origin of the
enzyme used: after an incubation period of two hours of the
biological sample with .beta.-glucuronidase from Patella vulgata,
only a 21.degree. A of codeine and a 64% of morphine are recovered;
an 11.degree. A of codeine and a 35% of morphine are obtained when
.beta.-glucuronidase from Helix pomatia is used; and only a 9% of
codeine and morphine are recovered when .beta.-glucuronidase from
Escherichia coli is used (Wang et al., J Anal. Toxicol. 2006. Vol
30:570-575). For this reason, diverse researchers seek enzymes
capable of reduce the time needed to hydrolyze glucuronide
metabolites. The recently published patent application US
2016/0090582 discloses a recombinant .beta.-glucuronidase enzyme
from E. coli (IMCSzyme.TM.), which, according to the applicant,
detects the presence of drugs in biological samples in 30 minutes
or less, whose specific activity is three times higher than wild
type enzyme. However, in the examples section it is shown that 60
minutes are required to incubate the mutant enzyme with a sample
containing codeine-6-glucuronide at 55 degrees Celsius to recover
93.5% of codeine.
[0007] Therefore, new .beta.-glucuronidase enzymes are needed to
achieve quick hydrolysis of glucuronide metabolites and to obtain
high recovery percentages of their derivatives, in order to
implement a highly efficient method for detection and
quantification of the same.
SUMMARY OF INVENTION
[0008] The present invention discloses a method to detect products
derived from glucuronide metabolites in a sample, comprising the
steps of: [0009] a) providing the sample in which said product is
to be detected, [0010] b) adding to said sample an enzyme with
.beta.-glucuronidase activity from genus Brachyspira or any variant
or mutant derived thereof, [0011] c) incubating the sample with the
enzyme for a determined period of time; and [0012] d) detecting the
product derived from said glucuronide metabolite through any
suitable technique.
[0013] This method allows to detect any product derived from
glucuronide metabolites, whether from natural, semi-synthetic or
synthetic origin. In a preferred embodiment, said derived products
are selected from the group consisting of an opioid, an opiate, a
cannabinoid, a benzodiazepine or any derivative thereof.
[0014] In a preferred embodiment, the sample from which these
products derived from glucuronide metabolites are detected, is
selected from the group consisting of saliva, whole blood, plasma,
urine, hair, skin, teeth, soft tissues, meconium, vitreous humor,
water and food.
[0015] The enzyme with .beta.-glucuronidase activity of the present
invention, whose amino acid sequence is shown in SEQ. ID. NO.1,
preferably originates from species Brachyspira pilosicoli. The
invention also includes any derived enzyme or mutant thereof,
provided that it maintains its .beta.-glucuronidase activity.
Preferably, the mutant enzyme with .beta.-glucuronidase activity
shares at least an 80% of identity with the sequence defined in
SEQ. ID. NO.1.
[0016] In another preferred modality of the method of the present
invention, incubation period of sample with the enzyme is of at
least one minute, preferably between 2 and 120 minutes, and even
more preferably between 3 and 30 minutes. Incubation is performed
in a range of temperature between 20 and 60 degrees Celsius,
preferably between 50 and 55 degrees Celsius, and in a pH range
between 4.0 and 9.0, preferably between 6.5 and 7.5.
[0017] Detection of products derived from glucuronide metabolites
is performed through any suitable analytical technique, preferably
selected from the group consisting of liquid chromatography (LC) or
gas chromatography (GC), high performance liquid chromatography
(HPLC), followed by mass spectrometry (LC-MS, or GC-MS), or high
resolution mass spectrometry (LC-HRMS, HPLC-HRMS), or tandem mass
spectrometry (LC-MS-MS, GC-MS-MS, HPLC-MS-MS) or any other suitable
technique or combination of detection methods.
[0018] A second object of the present invention is a reagent for
detection of products derived from glucuronide metabolites in a
sample, which comprises an enzyme with .beta.-glucuronidase
activity originated from genus Brachyspira or any derived enzyme or
mutant thereof; and an appropriate vehicle. Preferably, the reagent
contains an enzyme with .beta.-glucuronidase activity originated
from species Brachyspira pilosicoli, whose amino acid sequence is
shown in SEQ. ID. NO.1, or any enzyme derived from said sequence
maintaining .beta.-glucuronidase activity. Preferably, the derived
or mutant enzyme with .beta.-glucuronidase activity shares at least
an 80% of identity with the sequence defined in SEQ. ID. NO.1.
[0019] In a preferred embodiment, the reagent contains the enzyme
in a suitable vehicle which is selected from the group consisting
of water, salts, buffer solutions, Tris, HEPES, DTT, citric acid,
EDTA, glycerol, sugars, amino acids or a combination thereof. Salts
are preferably selected from the group consisting of sodium
phosphate, potassium phosphate, sodium carbonate, sodium acetate,
sodium citrate, sodium chloride and potassium chloride. At the same
time, sugars are selected from the group consisting of sorbitol,
trehalose, sucrose, glucose, lactose, mannitol and raffinose.
BRIEF DESCRIPTION OF FIGURES
[0020] FIG. 1 shows vector pET-28a with the optimized nucleotide
sequence encoding .beta.-glucuronidase enzyme originated from B.
pilosicoli to be expressed in E. coli.
[0021] FIG. 2 shows an SDS-PAGE gel of .beta.-glucuronidase enzyme
purified with column Ni-NTA.
[0022] FIG. 3 shows recovery of codeine and morphine versus time,
using .beta.-glucuronidase from B. pilosicoli.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention relates to a method for detection of
products derived from glucuronide metabolites present in a sample,
comprising the use of a new .beta.-glucuronidase enzyme originated
from Brachyspira genus. Hydrolysis efficiency of said enzyme is
widely superior than all .beta.-glucuronidases currently known,
with a specific activity 6.6 times higher than mean specific
activity of a .beta.-glucuronidase obtained from E. coli. This
allows to detect products derived from glucuronide metabolites in
less than 15 minutes with a high recovery rate.
[0024] All the technical and scientific terms used to describe the
present invention have the same meaning understood by any person
with basic knowledge in the field under review. Nevertheless, to
define more clearly the scope of the invention, a list of
terminology used in this description is given below.
[0025] It should be understood that as used herein, the term
"glucuronide metabolite" refers to any compound conjugated with
glucuronic acid through a glycosidic bond. It is to be understood
that a glucuronide metabolite can be natural or synthetic, and
produced by chemical or enzymatic methods. The terms "conjugated
compound", "conjugated metabolite", "glucuronide compound", and
"glucuronide substrate" are indistinctly used to describe the
technical characteristics of the present invention.
[0026] The term "products derived from glucuronide metabolites" or
"recovered analytes" should be understood as the molecule or
original compound that is produced after hydrolysis or glycosidic
bond cleavage of a glucuronide metabolite. These derived products
or original compounds can be natural or synthetic, and produced by
chemical or enzymatic methods.
[0027] The term "enzyme" should be understood as a sequence of
amino acids which form a functional protein of interest for the
present invention. It is to be understood that the present
invention is not related with enzymes in their natural state, but
it relates to proteins in an isolated state, purified or partially
purified, or recombinant, obtained by any method of genetic
engineering known in the state of the art. Likewise, it is to be
understood that the term "mutant" or "mutant enzyme" refers to a
modified protein derived from the original amino acid sequence
maintaining its .beta.-glucuronidase enzymatic activity; and
sharing at least 70%, preferably 80% and more preferably 90% of
identity with the original sequence.
[0028] It is to be understood that the term "identity" among amino
acid sequences refers to the percent of identical amino acids that
the compared sequences share among them, in a particular sequence
alignment window. The percentage of identity can be calculated
using a sequence comparison algorithm or by manual alignment
together with visual inspection. For example, sequences and
percentages of identity can be obtained using computer resources
available on internet such as BLAST
(http://blast.ncbi.nlm.nih.gov/) or FastDB computer programs.
[0029] The term "Unit (U)" should be understood as the unit used to
measure the specific activity of an enzyme. One unit will produce
1.0 .mu.g of phenolphthalein from phenolphthalein glucuronide (PPG)
metabolite per hour at 37 degrees Celsius at pH 6.8.
[0030] The term "vehicle" should be understood as any substance
that is added to an active principle, in this case an enzyme, which
is useful as a support and allows to dilute or improve stability or
durability of the main compound.
[0031] As described before, an object of the present invention
relates to a method for detection of glucuronide metabolites in a
sample, which comprises the steps of adding an enzyme with
.beta.-glucuronidase activity from genus Brachyspira or any variant
or mutant derived thereof to the sample in which a product derived
from a glucuronide metabolite is to be detected, incubating the
sample with the enzyme for a determined period of time; and
detecting the product derived from said glucuronide metabolite
through any suitable technique.
[0032] This method is superior than others described in the state
of the art, since it includes the use of the new enzyme with
.beta.-glucuronidase activity originated from genus Brachyspira,
which has an improved affinity for glucuronide metabolites, a
specific activity 6.6 times higher than .beta.-glucuronidase
originated from E. coli, and therefore it is capable of hydrolyse
glucuronide metabolites to be detected in a period of time of at
least one minute, preferably between three and 30 minutes. This
enzyme is preferably originated from species Brachyspira
pilosicoli, whose amino acid sequence is shown in SEQ. ID. NO.1,
but it can be obtained from any species of genus Brachyspira.
Additionally, in the method of the present invention, any
derivative or mutant of this protein can be used, provided that it
maintains its .beta.-glucuronidase activity. Preferably, the
derived or mutant enzyme with .beta.-glucuronidase activity shares
at least a 70% of sequence identity, more preferably an 80% of
identity, and even more preferably a 90% of identity with the
sequence defined in SEQ. ID. NO.1.
[0033] The .beta.-glucuronidase enzyme originated from Brachyspira
can hydrolyse any glucuronide metabolite, whether from natural,
semi-synthetic or synthetic origin. Some examples of these
metabolites, without limitation, are: morphine-3-glucuronide,
morphine-6-glucuronide, codeine-6-glucuronide,
oximorphone-glucuronide, hydromorphone-glucuronide,
norbuprenorphine-glucuronide, buprenorphine-glucuronide,
oxazepam-glucuronide, temazepam-glucuronide, lorazepam-glucuronide,
alprazolam-glucuronide, midazolam-glucuronide,
nordiazepam-glucuronide, psilocin-glucuronide,
carboxy-tetrahydrocannabinol-glucuronide,
tetrahydrocannabinol-glucuronide, tetrahydrocannabinolic
acid-glucuronide, naloxone-3-glucuronide, tapentadol-glucuronide,
tricyclic antidepressant-glucuronide, cannabidiol-glucuronide,
among others. It is to be understood for the scope of the present
invention that the .beta.-glucuronidase enzyme originated from
Brachyspira can hydrolise any glucuronide metabolite and it is not
limited to the examples previously mentioned.
[0034] Therefore, this method allows to detect any product derived
from glucuronide metabolites, whether from natural, semi-synthetic
or synthetic origin. For example, the method can be used to analyze
drugs, toxic compounds derived from fungi, androgens, estrogens,
mineralocorticoids, glucocorticoids, fatty acid derivatives,
retinoids, among others.
[0035] In a preferred embodiment, products derived from glucuronide
metabolites are selected from the group consisting of opioids,
opiates, cannabinoids, benzodiazepines or any other derived
thereof. Preferably, the products derived from glucuronide
metabolites are selected from the group consisting of morphine,
codeine, oximorphone, hydromorphone, norbuprenorphine,
buprenorphine, oxazepam, temazepam, lorazepam, alprazolam,
midazolam, nordiazepam, psilocin,
11-nor-9-carboxy-tetrahydrocannabinol (THC-COOH),
tetrahydrocannabinol (THC), tetrahydrocannabinolic acid (THCa),
naloxone, tapentadol, tricyclic antidepressant, cannabidiol, among
others, without being limited to the examples described herein.
[0036] In a preferred embodiment of the present invention, the
method allows to detect any product derived from glucuronide
metabolites in any type of sample, which is preferably selected
from the group consisting of saliva, whole blood, plasma, urine,
hair, skin, teeth, soft tissues, water, food, meconium and vitreous
humor, without restriction to these types of samples. The following
biological samples can also be used: bile, earwax, phlegm, vomit,
aqueous humour, tears, amniotic fluid, vaginal secretion, semen,
pre-ejaculate fluid, mucus, sebum, sweat, excrement, or any other
suitable sample without limitation to the animal fluids previously
mentioned. Other suitable samples originate from a non-animal
source, such as food, water or chemical solutions from
laboratories.
[0037] In the method of the present invention, .beta.-glucuronidase
enzyme originated from genus Brachyspira is added to the sample and
the mixture is incubated for a determined period of time, in
suitable conditions. In a preferred embodiment, incubation period
of sample with the enzyme is of at least one minute, that is to
say, only one minute of reaction is enough to detect the presence
of a product derived of a glucuronide metabolite in a sample.
However, incubation period can be extended to 16 hours or even
more, provided that time is not an impediment to the technician
performing the method.
[0038] More preferably, incubation period is between 2 and 120
minutes, and even more preferably, incubation period is between 3
and 30 minutes, since obtaining the results efficiently and in the
shortest time is commonly desired. Additionally, adequate
incubation conditions refer to ideal conditions of temperature and
pH in order to allow the reaction to be produced in the time
periods mentioned. Regarding temperature, incubation is performed
preferably in a range between 20 and 60 degrees Celsius, and even
more preferably in a range of 50 to 55 degrees Celsius. Regarding
pH, incubation is performed in a range between 4.0 and 9.0 and
preferably between 6.5 and 7.5. As part of the elements routinely
used in this assays, incubation is performed in presence of buffer
solutions, standards of products derived from glucuronide
metabolites, polar or apolar solvents, among others.
[0039] After incubating the .beta.-glucuronidase enzyme with the
sample and before separating the products derived from glucuronide
metabolites through separation columns, an extraction step of said
metabolites can be optionally included to increase sensitivity of
analytical techniques. This extraction step can be performed by
means of liquid-liquid extraction (LLE), solid-liquid extraction
(SLE), extraction in solid phase (SPE), or any other suitable
technique. However, the step of extracting the products derived
from glucuronide metabolites adds a laborious step to the
analytical method and increases operational costs due to the use of
solvents and other materials.
[0040] To the method of the present invention, the latter step is
optional due to the availability of highly selective analytical
techniques that allow direct analysis by means of the technique
named "dilute and shoot", which consist of diluting the sample with
an adequate solution and injecting it directly into a separation
column. Moreover, the method does not require elimination of the
enzyme since the .beta.-glucuronidase of the present invention is
highly purified and has a high reactivity; and therefore a small
volume of the enzyme is required to react with glucuronide
metabolites. In a preferred embodiment the volume range of enzyme:
liquid sample used, for example, urine, is from 1:1 to 1:14.
Preferably, optimum concentration is between 0.02 and 0.25 mg of
enzyme per mL of total reaction. In this manner, the volume of
enzyme used does not affect or damage the separation column in case
it is directly injected to it. The adequate solvent can be for
example, water or a mixture of water with methanol, or any mixture
of solvents that can be used to dilute the sample.
[0041] Finally, detection of products derived from glucuronide
metabolites is performed through the use of separation columns and
analytical techniques which are selected from the group consisting
of: gas chromatography (GC), liquid chromatography (LC) or high
performance liquid chromatography (HPLC), solid phase
microextraction (SPME), with mass spectrometry detectors (GC-MS;
LC-MS; HPLC-MS; SPME-MS), high resolution mass spectrometry
(GC-HRMS; LC-HRMS; HPLC-HRMS), tandem mass spectrometry (GC-MS/MS;
LC-MS/MS; SPME-GC-MS/MS), time of flight mass spectrometry
(LC-TOF-MS), ultraviolet-visible spectrophotometry (GC-UV/Vis;
LC-UV/Vis; SPME-LC-UV/Vis), flame ionization detection (GC-FID;
LC-FID; SPME-GC-FID), photodiode array detection, (LC-DAD), or any
other suitable detection method, without limiting to the examples
previously mentioned.
[0042] A second object of the present invention is a reagent for
detection of products derived from glucuronide metabolites in a
sample, which comprises an enzyme with .beta.-glucuronidase
activity originated from genus Brachyspira or any derived enzyme or
mutant thereof; which is included in a suitable vehicle.
[0043] Preferably, the reagent contains an enzyme with
.beta.-glucuronidase activity originated from species Brachyspira
pilosicoli, or any derived enzyme or mutant thereof. Said enzyme
has an amino acid sequence shown in SEQ. ID. NO.1, but it can be
any enzyme mutant from said sequence and maintaining
.beta.-glucuronidase activity. Preferably, the derived or mutant
enzyme with .beta.-glucuronidase activity shares at least a 70% or
80% of identity with the sequence defined in SEQ. ID. NO.1, more
preferably a 90% of identity with the sequence defined in SEQ. ID.
NO.1.
[0044] In a preferred embodiment, the reagent contains between 0.1
and 2 mg/mL of enzyme, in which the same has a .beta.-glucuronidase
activity from 200,000 U/mL to 800.000 U/mL. In addition to
.beta.-glucuronidase enzyme, the reagent includes an appropriate
vehicle that varies depending on the product format and on the
function that the same performs. For example, the reagent including
the enzyme can be in a liquid format, which could require only
water as a vehicle.
[0045] To improve the stability of the enzyme, salts can be added
(at concentrations .mu.M or mM), or ethylenediamine tetraacetic
acid (EDTA), Tris, dithiothreitol (DTT),
hydroxyethylpiperazin-ylethanesulfonic acid (HEPES), citric acid,
among others, or a combination thereof. Preferably, salts are
selected from the group consisting of sodium phosphate, potassium
phosphate, sodium carbonate, sodium acetate, sodium citrate, sodium
chloride and potassium chloride, without limitation to the salts
previously described. In case that the reagent is frozen, the
vehicle to be used can include glycerol, preferably at a
concentration of 50%. On the other hand, if the reagent format is
powder, which can be produced by means of lyophilisation, spray
drying or any other pulverization method; vehicles able to protect
the enzyme from drying process are required such as sugars, amino
acids and some salts, which are present at a concentration range
between 0.001 to 2% based on liquid previous to the drying process.
In this case, sugars are preferably selected from the group
consisting of sorbitol, trehalose, sucrose, glucose, lactose,
mannitol and raffinose, without limitation to these described
examples. Therefore, the vehicle of the present invention fulfils
the purpose of being a diluting, preservating or stabilizing medium
for the enzyme.
[0046] The following examples are considered to illustrate the
invention and its preferred embodiments, but they must not be
considered under any circumstance as a restriction to the scope of
the invention, which is defined by the claims attached hereto.
EXAMPLES OF IMPLEMENTATION
Example 1
Synthesis and Expression of .beta.-glucuronidase Enzyme from B.
pilosicoli
[0047] To express the .beta.-glucuronidase enzyme, nucleotide
sequence from said protein was used, originated from Brachyspira
pilosicoli strain B2904 described in Genbank (access code
CP003490), which is shown in SEC. ID. NO.2, and codon optimization
was performed using the algorithm OptimumGene.TM. (GenScript) to
efficiently express said sequence in E. coli. To do this, codon
adaptation index (CAI), understood as distribution of frequency of
codon usage through the sequence, was adjusted from 0.66 to 0.88,
in which a CAI value of 1.0 is considered to be perfect in the
desired expression organism; and a CAI value greater than 0.8 is
regarded as good in terms of high gene expression levels.
Guanine-cytosine content was also optimized to extend mRNA
half-life and stem-loop structures were eliminated, since they
disrupt ribosome binding and mRNA stability. Optimized nucleotide
sequence obtained is shown in SEC. ID. NO.3. Amino acid sequence of
the enzyme is shown in SEC. ID. NO.1.
[0048] The optimized nucleotide sequence of the enzyme was cloned
into vector pET-28a, thus vector pET-28a-glucuronidase was
obtained, which is shown in FIG. 1. To express .beta.-glucuronidase
enzyme from B. pilosicoli in E. coli, the following protocol was
carried out: 5 .mu.L of competent E. coli strain BL21 cells were
incubated in ice with 10 ng of pET-28a-glucuronidase vector during
20 minutes. Then, bacterial cells were submitted to heatshock at 37
degrees Celsius during 3 minutes. Bacterial cells were then put in
ice for 30 seconds and later cells were recovered with 250 .mu.L of
LB io medium at 37 degrees Celsius and plated in LB-agar with 50
mg/L of kanamycin, and colonies were grown. Finally, a
.beta.-glucuronidase activity test was carried out on plates with
IPTG (isopropyl-.beta.-thiogalactoside) and X-Gal
(5-bromo-4-chloro-3 indolyl-beta-D-thiogalactopyranoside).
[0049] In order to purify the enzyme, E. coli BL21 transformed
cells were grown in 500 mL of LB medium supplemented with kanamycin
(50 mg/L), until obtention of an optical density of 0.3, measured
using a spectrophotometer at 600 nm (DO.sub.600).
[0050] Then, IPTG at a concentration of 1 mM was added to the
culture and then incubated at 37 degrees Celsius during 6 hours.
Cells were subjected to sonication during 3 minutes with an
amplitude of 60 (cycles of 15 seconds in ice). Later, the enzyme
was purified by means of a system for protein purification in
columns with Ni-NTA (Nickel-Nitriloacetic acid) resins with elution
of 2 mL of Tris-HCl 50 mM, pH 5.6 with imidazole 100 mM pH 5.9 or
with imidazole 500 mM pH 4.5.
[0051] FIG. 2 shows an SDS-PAGE gel of purified proteins in column
Ni-NTA. Each well contained proteins at a concentration of 15
pg/mL, except in crude extract (E. coli pET28a-glucuronidase
without purification in the column), in which 5 .mu.L were loaded.
The acronym Std corresponds to molecular weight standard in
kilodaltons (kD), Crude ex corresponds to crude extract, Glu E1
corresponds to elution with imidazole 100 mM pH 5.9 and Glu E2
corresponds to elution with imidazole 500 mM pH 4.5.
Example 2
Incubation Time of a Sample with .beta.-glucuronidase from B.
pilosicoli
[0052] The required time to hydrolyze morphine-3-glucuronide and
codeine-6-glucuronide with the enzyme .beta.-glucuronidase in
excess (named BGTurbo.TM.) from B. pilosicoli was evaluated with
the following protocol: 10 .mu.L of glucuronide metabolites at a
concentration of 100 .mu.g/mL, free standards (free codeine and
morphine calibrators at concentrations of 312.5, 625, 1,000, 1,250
and 1,875 ng/mL), and internal standards (40 .mu.L of deuterated
free drug at a concentration of 1 .mu.g/mL to each sample) were
added to a sample of 0.4 mL of blank urine and then submitted to
vortex agitation. Then, 360 .mu.L of sodium phosphate (pH 7; 140
mM) solution was added to each sample. Then, 240 .mu.L of
.beta.-glucuronidase (BGTurbo.TM.) were added to each sample and
then agitated by vortex. Samples were incubated at 55 degrees
Celsius in an oven during 0, 10, 20, 30, 40, 50 and 60 minutes. A
total of 21 samples were used: 7 points of measure at different
times, each one in triplicate. Calibrators were incubated during 30
minutes. The samples incubated at different times were removed from
oven and the reaction was stopped by adding 700 .mu.L of sodium
phosphate buffer solution 1, 5 M and agitated by vortex. Then, pH
8-9 was verified with pH paper, and 2 mL of chloroform: isopropanol
50:50 were added to samples, and they were agitated by vortex
during 2 minutes. Aqueous phase was discarded and organic phase was
transferred to 12.times.75 mm assay tubes and then evaporated under
nitrogen gas (in a water bath at 50 degrees Celsius) until
approximately 1 mL. Samples were then agitated by vortex previous
to evaporation until dryness. Samples were reconstituted in 1 mL of
HCl 0, 5 N in methanol for LC-MS/MS analysis.
[0053] Calculation of concentration of equivalents was performed as
follows:
Morphine:
[0054] Molecular weight of morphine: 285.34 g/mol [0055] Molecular
weight of morphine-3-glucuronide: 461.462 g/mol [0056] Proportion
of molecular weight of morphine/morphine-3-glucuronide:
[0057] Morphine equivalents:
0.6183 2 , 500 ng mL Morphine - 3 - glucuronide = 1 , 545.8 ng mL
Morphine ##EQU00001##
Codeine:
[0058] Molecular weight of codeine: 299.364 g/mol [0059] Molecular
weight of codeine-6-glucuronide: 475.49 g/mol [0060] Proportion of
molecular weight of codeine/codeine-6-glucuronide:
[0061] Codeine equivalents:
0.62959 2 , 500 ng mL Codeine - 6 - glucuronide = 1 , 574.0 ng mL
Codeine ##EQU00002##
[0062] Table 1 and FIG. 3 show the results obtained. Hydrolysis of
100% of codeine-6-glucuronide was obtained in less than 10 minutes.
In the case of morphin, approximately 65% of hydrolysis was
obtained within 10 minutes, but these values can be improved by
optimizing parameters of temperature, pH or the method to stop
reaction. Due to the use of internal controls for recovery of
products derived from glucuronide metabolites, values have a
standard deviation up to 20%.
TABLE-US-00001 TABLE 1 Tabulated results of FIG. 3. Time Codeine %
Yield Morphine % Yield (minutes) (ng/mL) Codeine (ng/mL) Morphine 0
30.0 1.9 195.8 12.7 10 1826.9 116.1 997.4 64.5 20 1867.5 118.6
969.2 62.7 30 1694.3 107.6 871.2 56.4 40 1821.0 115.7 811.5 52.5 50
1844.3 117.2 847.4 54.8 60 1819.5 115.6 823.4 53.3
Example 3
Calculation of Specific Activity of .beta.-glucuronidase from B.
pilosicoli
[0063] Specific activity of the .beta.-glucuronidase enzyme from B.
pilosicoli (BGTurbo.TM.) was calculated and compared with a mean
value of three calculations of the specific activity of
.beta.-glucuronidase from E. coli, using the following protocol:
350 .mu.L of sodium phosphate buffer solution (NaH.sub.2PO.sub.4)
0.1 M, pH 6.8 were mixed with 350 .mu.L of phenolphthalein
.beta.-D-glucuronide sodium salt (0.64 mg/mL) and the mixture was
incubated at 37 degrees Celsius. Then, 50 .mu.L of solution
containing the enzyme were added to the mixture and incubated at 37
degrees Celsius during 30 minutes. After io the first 15 minutes
the mixture was gently agitated and stopped at 30 minutes from the
beginning of the reaction with 2.5 mL of a glycine solution 0.2 M,
pH 10.4. Absorbance was read at 540 nm and the amount of enzyme
used was calculated (U/mL) when adjusting the values in a
calibration curve. Protein concentration was measured using
Bradford method, as widely known in literature. As shown in Table
2, results indicate that BGTurbo.TM. enzyme has a specific activity
6.6 times higher than mean specific activity of
.beta.-glucuronidase from E. coli.
TABLE-US-00002 TABLE 2 Enzymatic activity of .beta.-glucuronidase
from B. pilosicoli. Activity Proteins Specific Activity Enzyme
(U/mL) (mg/mL) (U/mg) BGTurbo .TM. 427,805 2.08 205,676 E. coli 1
68,779 2.28 30,166 E. coli 2 102,710 3.17 32,401 E. coli 3 85,084
2.89 29,441 E. coli mean value 30,669
Example 4
Comparison of .beta.-glucuronidase from B. pilosicoli with
.beta.-glucuronidases from Different Sources
[0064] To test the efficiency of .beta.-glucuronidase from B.
pilosicoli (BGTurbo.TM.), this enzyme was compared with two
.beta.-glucuronidases currently available in the market: DR2102
(Campbell Science) and BG100.TM. (KURA Biotec). To do this, 300 ng
of each of the glucuronide metabolites of buprenorphine, codeine,
hydromorphone, lorazepam, morphine, oxymorphone and
11-nor-9-carboxy-tetrahydrocannnabinol (THC-COON) were added to a
sample of blank urine, therefore obtaining spiked urine. Each
enzyme was used under the conditions proposed by each manufacturer.
Hydrolysis of 50 .mu.L of urine was performed, diluting it with 250
.mu.L of buffer solution at pH 6.8, 20 .mu.L of internal standards
(ISTD) and 10 .mu.L of DR2102 or BG100 enzymes, or 20 .mu.L of
BGTurbo.TM.. Incubation conditions are shown in Table 3.
TABLE-US-00003 TABLE 3 Conditions used for each enzyme. Conditions
DR2102 BG100 .TM. BGTurbo .TM. Urine (.mu.L) 50 50 50 ISTD (.mu.L)
20 20 20 Buffer (.mu.L) 25 25 25 Enzyme (.mu.L) 10 10 20
Temperature (.degree. C.) 62 68 50 Time (minutes) 60 30 15
[0065] After hydrolysis, samples were centrifuged at 24,000 g,
diluted with 450 .mu.L of a solution comprising 95% water and 5%
methanol, and charged to an HPLC column (Phenomenex). Protein
amount in samples was measured before and after centrifugation at
24,000 g using a NanoDrop spectrophotometer (Thermo Scientific). As
a result, the reactions performed were carried out with 1.787 mg/mL
of DR2102, 0.554 mg/mL of BG100.TM. and 0.103 mg/mL of
BGTurbo.TM..
[0066] Recovery yields of products derived from glucuronide
metabolites are shown in Table 4. As shown, mean recovery of
derived products was improved in a 15% when hydrolysis was
performed with BGTurbo.TM. in only 15 minutes and using 70% and 94%
less of protein amount in comparison to the other two enzymes. It
is interesting to mention that .beta.-glucuronidase enzyme from B.
pilosicoli is more reactive with codeine-6-glucuronide than the
other enzymes.
TABLE-US-00004 TABLE 4 Results of analites recovered from
hydrolysis of glucuronide metabolites with three
.beta.-glucuronidases enzymes from different origins DR2102 BG100
.TM. BGTurbo .TM. 60 minutes 30 minutes 15 minutes Recovered
Analyte (ng/mL) (ng/mL) (ng/mL) Buprenorphine 223.1 271.1 224.4
Codeine 159.4 173.8 243.2 Hydromorphone 176.8 196.0 196.2 Lorazepam
214.9 202.5 185.9 Morphine 174.5 190.9 198.1 Oximorphone 155.8
189.0 169.4 THC-COOH 75.5 146.0 140.6 Protein in sample 1.787
(mg/mL) 0.554 (mg/mL) 0.103 (mg/mL) pre-centrifugation Protein in
sample 0.725 (mg/mL) 0.280 (mg/mL) 0.085 (mg/mL)
post-centrifugation and injected on HPLC column
Example 5
Comparison of .beta.-glucuronidase from B. pilosicoli with Other
.beta.-glucuronidases from Bacterial Origin
[0067] In order to test enzyme speed of .beta.-glucuronidase from
B. pilosicoli (BGTurbo.TM.) in comparison with
.beta.-glucuronidases from bacterial origin, BGTurbo.TM. was
compared with EBG enzyme (recombinant .beta.-glucuronidase from E.
coli, KURA Biotec) and with IMCSzyme.TM. (Integrated
Micro-Chromatography Systems) at different incubation periods and
concentration. The method previously described was used, but
analyzing only hydrolysis of codeine-6-glucuronide and
morphine-3-glucuronide with the conditions described in Table
5.
TABLE-US-00005 TABLE 5 Conditions used for each enzyme. BGTurbo
.TM. EBG IMCSzyme .TM. Control Conditions A B C D F -- E Buffer
(.mu.L) 360 360 360 360 360 360 360 Water (.mu.L) 0 120 180 210 180
180 120 Spiked urine (.mu.L) 400 400 400 400 400 400 400 Enzyme
(.mu.L) 240 120 60 30 60 60 0 Internal Standard (.mu.L) 40 40 40 40
40 40 40 Storage buffer control (.mu.L) 0 0 0 0 0 0 120 Total
Volume (.mu.L) 1,040 1,040 1,040 1,040 1,040 1,040 1,040 Incubation
Time (minutes) 10 10 10 10 0, 10, 20 0, 10, 20 10 pH 7.0 7.0 7.0
7.0 7.0 7.0 7.0 Temperature (.degree. C.) 55 55 55 55 55 55 55
[0068] Each condition shown in Table 5 was assayed in duplicate,
and each analysis was measured in triplicate. Calculation of
concentration of equivalents was performed as described in Example
2.
[0069] Table 6 shows the results obtained from analysis of codeine,
and Table 7 shows results obtained from analysis of morphine. As
can be noticed, codeine-6-glucuronide was hydrolyzed in a mean
value of 109,59% within 10 minutes, using 240 .mu.L of BGTurbo.TM..
The value mentioned is greater than 100% because standards of
glucuronide metabolites used in the analysis are not completely
pure, and present a fraction of the original compound that is added
to the final result. On the other hand, when comparing hydrolysis
yield of codeine-6-glucuronide and morphine-3-glucuronide using the
same volume of the enzymes (60 .mu.L), it is shown that BGTurbo.TM.
is better than the other EBG and IMCSzyme.TM. enzymes within 10
minutes. Even after an incubation period of 20 minutes of the
samples with EBG and IMCSzyme.TM., these enzymes could not produce
the same hydrolysis yield that was obtained with BGTurbo.TM. within
10 minutes.
TABLE-US-00006 TABLE 6 Results of codeine analysis. Condition/
Codeine Codeine Average of Assay Sample (ng/mL) % Yield Assay Yield
(%) A/1 BGTurbo .TM. (10 min, 240 .mu.L) 1,740 110.55 109.59 A/2
BGTurbo .TM. (10 min, 240 .mu.L) 1,710 108.64 B/1 BGTurbo .TM. (10
min, 120 .mu.L) 1,270 80.69 78.14 B/2 BGTurbo .TM. (10 min, 120
.mu.L) 1,190 75.60 C/1 BGTurbo .TM. (10 min, 60 .mu.L) 879 55.84
54.19 C/2 BGTurbo .TM. (10 min, 60 .mu.L) 827 52.54 D/1 BGTurbo
.TM. (10 min, 30 .mu.L) 230 14.61 22.20 D/2 BGTurbo .TM. (10 min,
30 .mu.L) 469 29.80 F/1 EBG (0 min, 60 .mu.L) No Peak 0.00 0.00 F/2
EBG (0 min, 60 .mu.L) No Peak 0.00 F/1 EBG (10 min, 60 .mu.L) 154
9.78 10.48 F/2 EBG (10 min, 60 .mu.L) 176 11.18 F/1 EBG (20 min, 60
.mu.L) 396 25.16 27.10 F/2 EBG (20 min, 60 .mu.L) 457 29.03 1
IMCSzyme .TM. (0 min, 60 .mu.L) No Peak 0.00 0.00 2 IMCSzyme .TM.
(0 min, 60 .mu.L) No Peak 0.00 1 IMCSzyme .TM. (10 min, 60 .mu.L)
189 12.01 11.85 2 IMCSzyme .TM. (10 min, 60 .mu.L) 184 11.69 1
IMCSzyme .TM. (20 min, 60 .mu.L) 395 25.10 27.26 2 IMCSzyme .TM.
(20 min, 60 .mu.L) 463 29.42 E/1 Control (10 min) No Peak 0.00 0.00
E/2 Control (10 min) No Peak 0.00
TABLE-US-00007 TABLE 7 Results of the morphine analysis. Condition/
Morphine Morphine Average of Assay Sample (ng/mL) % Yield Assay
Yield (%) A/1 BGTurbo .TM. (10 min, 240 .mu.L) 796 51.49 51.49 A/2
BGTurbo .TM. (10 min, 240 .mu.L) 796 51.49 B/1 BGTurbo .TM. (10
min, 120 .mu.L) 717 46.38 46.90 B/2 BGTurbo .TM. (10 min, 120
.mu.L) 733 47.42 C/1 BGTurbo .TM. (10 min, 60 .mu.L) 687 44.44
43.70 C/2 BGTurbo .TM. (10 min, 60 .mu.L) 664 42.96 D/1 BGTurbo
.TM. (10 min, 30 .mu.L) 498 32.22 38.46 D/2 BGTurbo .TM. (10 min,
30 .mu.L) 691 44.70 F/1 EBG (0 min, 60 .mu.L) 179 11.58 12.71 F/2
EBG (0 min, 60 .mu.L) 214 13.84 F/1 EBG (10 min, 60 .mu.L) 609
39.40 39.62 F/2 EBG (10 min, 60 .mu.L) 616 39.85 F/1 EBG (20 min,
60 .mu.L) 629 40.69 43.47 F/2 EBG (20 min, 60 .mu.L) 715 46.25 1
IMCSzyme .TM. (0 min, 60 .mu.L) 142 9.19 10.19 2 IMCSzyme .TM. (0
min, 60 .mu.L) 173 11.19 1 IMCSzyme .TM. (10 min, 60 .mu.L) 603
39.01 39.20 2 IMCSzyme .TM. (10 min, 60 .mu.L) 609 39.40 1 IMCSzyme
.TM. (20 min, 60 .mu.L) 662 42.83 43.41 2 IMCSzyme .TM. (20 min, 60
.mu.L) 680 43.99 E/1 Control (10 min) No Peak 0.00 0.00 E/2 Control
(10 min) No Peak 0.00
Sequence CWU 1
1
31603PRTBrachyspira pilosicoli 1Met Val Asn Ser Met Leu Tyr Pro Arg
Glu Ser Arg Thr Arg Arg Val 1 5 10 15 Val Asp Ile Ser Gly Met Trp
Glu Phe Lys Ile Asp Ile Asn Asn Glu 20 25 30 Gly Arg Asn Ser Gly
Tyr Ala Asn Gly Leu Lys Asp Thr Thr Phe Ile 35 40 45 Pro Val Pro
Ser Ser Phe Asn Asp Leu Phe Thr Asp Lys Asn Ile Arg 50 55 60 Glu
His Ala Gly Asp Val Trp Tyr Glu Thr Ser Phe Tyr Leu Pro Leu 65 70
75 80 Glu Trp Lys Asp Lys Asp Val Asn Val Arg Phe Gly Cys Ala Thr
His 85 90 95 Glu Ala Thr Val Tyr Ile Asn Gly Lys Glu Val Cys Thr
His Val Gly 100 105 110 Gly Phe Met Pro Phe Asn Ala Pro Val Asn Glu
Ala Gly Ile Phe Gly 115 120 125 Glu Lys Asn Lys Leu Val Val Val Val
Asn Asn Glu Leu Ser Asn Thr 130 135 140 Thr Ile Pro Cys Gly His Thr
Glu Thr Lys Pro Ser Gly Lys Lys Tyr 145 150 155 160 Ile Lys Pro Ser
Phe Asp Phe Phe Asn Tyr Ala Gly Leu Asn Arg Pro 165 170 175 Val Lys
Ile Thr Val Thr Asn Lys Glu Tyr Ile His Asp Ile Asp Ile 180 185 190
Leu Ser Asp Val Asn Gly Ser Asp Gly Ile Val Asn Tyr Glu Val His 195
200 205 Thr Thr Gly Glu Asn Lys Val Tyr Ile Lys Ile Asn Asp Glu Glu
Gly 210 215 220 Lys Glu Val Ala Ser Cys Glu Gly Lys Ser Gly Lys Ile
Val Ile Lys 225 230 235 240 Asp Ala Lys Leu Trp Asn Pro Lys Ala Ala
Tyr Leu Tyr Lys Phe Ile 245 250 255 Ala Cys Ile Lys Asn Gly Asp Glu
Leu Ile Asp Glu Tyr Tyr Leu Asp 260 265 270 Phe Gly Ile Arg Thr Val
Lys Val Glu Gly Thr Lys Phe Leu Ile Asn 275 280 285 Gly Lys Pro Phe
Tyr Phe Thr Gly Phe Gly Lys His Glu Asp Ser Glu 290 295 300 Ile Ala
Gly Arg Gly Tyr Asn Pro Pro Val Ile Lys Arg Asp Phe Glu 305 310 315
320 Leu Ile Lys Trp Val Gly Ala Asn Ser Phe Arg Thr Ser His Tyr Pro
325 330 335 Tyr Ser Glu Glu Ile Met Gln Ala Ala Asp Arg Glu Gly Ile
Val Ile 340 345 350 Ile Asp Glu Val Ala Ala Val Gly Met Phe Asp Val
Gly Ser Val Leu 355 360 365 Asn Pro Ser Ala Ser Lys Thr Asp Tyr Phe
Ser Leu Asp Glu Val His 370 375 380 Ser Lys Thr Lys Glu Val His Lys
Lys Ala Val Glu Glu Leu Ile Lys 385 390 395 400 Arg Asp Lys Asn His
Pro Ser Val Val Met Trp Ser Leu Phe Asn Glu 405 410 415 Pro Asp Thr
Ser Lys Asp Glu Ala Val Pro Tyr Phe Glu Asp Ile Phe 420 425 430 Asn
Phe Ala Lys Ser Gln Asp Lys Gln Asn Leu Pro Lys Thr Phe Ala 435 440
445 Ala Ile Gln Ala Ser Ser Pro Gly Lys Cys Lys Cys Met His Leu Cys
450 455 460 Asp Val Ile Thr Leu Asn Arg Tyr Tyr Gly Trp Tyr Phe Leu
Gly Gly 465 470 475 480 Tyr Glu Ile Asp Met Ser Glu Glu Lys Phe Arg
Glu Glu Met Asn Leu 485 490 495 Tyr Ser Asn Met Asn Lys Pro Val Met
Phe Thr Glu Tyr Gly Ala Asp 500 505 510 Thr Tyr Ala Gly Val His Lys
Leu Pro Ser Val Met Trp Ser Glu Glu 515 520 525 Tyr Gln Cys Glu Tyr
Tyr Glu Met Asn Phe Lys Val Phe Asp Ser Tyr 530 535 540 Asp Phe Ile
Val Gly Glu Gln Leu Trp Asn Phe Ala Asp Phe Gln Thr 545 550 555 560
Thr Glu Gly Ile Phe Arg Val Asp Gly Asn Lys Lys Gly Ile Phe Thr 565
570 575 Arg Asn Arg Gln Pro Lys Ala Val Ala His Leu Ile Arg Ser Arg
Trp 580 585 590 Asn Lys Leu Pro Leu Asp Tyr Lys Ser Lys Lys 595 600
21809DNABrachyspira pilosicoli 2atggttaatt ctatgttata tcctagagag
agtaggacaa gaagagtagt agatatatct 60ggtatgtggg agtttaaaat tgatataaat
aatgaaggca gaaatagcgg atatgctaat 120gggcttaaag atactacatt
tatacctgtt ccatctagtt ttaatgattt atttacagat 180aaaaatataa
gagaacatgc tggtgatgta tggtatgaaa catcatttta tcttcctttg
240gaatggaaag acaaagatgt taatgtaaga tttggttgtg caactcatga
agctactgta 300tatattaatg gaaaagaagt atgtacacat gtaggtggat
ttatgccttt caatgctcct 360gtaaatgaag caggaatatt tggagaaaaa
aataaattag ttgtagttgt taataatgaa 420cttagcaata ctactatacc
ttgtggtcat acagaaacta aaccttcagg caaaaaatat 480attaaacctt
cttttgactt ttttaattat gcgggattaa acagacctgt aaaaattact
540gttacaaata aggaatatat acatgatata gatatattat cagatgtaaa
cggcagtgat 600ggaattgtaa attatgaagt acatactaca ggcgaaaaca
aagtatatat aaaaataaac 660gatgaagagg gaaaagaagt agcaagttgt
gaaggtaaaa gcggaaaaat agttattaaa 720gatgctaaat tatggaatcc
aaaggctgct tatttgtata agtttattgc atgcataaaa 780aatggcgatg
aactcataga tgaatattat ttagactttg gtataagaac tgttaaagtt
840gaaggaacaa aatttttaat taatggaaaa cctttctact ttacaggatt
tggtaaacat 900gaagacagtg aaatagcagg taggggttat aatcctcctg
taataaaaag agattttgag 960ctcattaaat gggtaggtgc aaattcattt
agaacatctc actatcctta tagtgaagaa 1020ataatgcagg cagctgacag
agaaggaatt gttattatag atgaagttgc tgctgtaggt 1080atgtttgatg
taggttctgt actaaatcct agtgcatcaa aaactgatta tttctcttta
1140gatgaagtac atagcaaaac taaagaagtt cataaaaaag ctgttgaaga
gcttattaaa 1200agagataaaa atcacccttc agttgttatg tggagtttat
tcaatgagcc tgatacttca 1260aaagacgaag ctgttccata ttttgaagat
atatttaatt ttgctaaaag tcaggataag 1320cagaatcttc ctaaaacttt
tgctgctatt caggcatctt ctcctggaaa atgtaagtgt 1380atgcatttat
gcgatgtaat tacattaaat agatactatg gatggtattt cttgggtggt
1440tatgaaattg atatgtcaga agaaaaattt agagaagaaa tgaatttgta
ttctaatatg 1500aataaaccag ttatgttcac agaatacgga gctgatactt
atgctggtgt tcataaactt 1560ccttccgtaa tgtggagcga agaatatcaa
tgcgaatact atgaaatgaa tttcaaagtt 1620tttgatagct atgactttat
agttggagaa caattatgga actttgctga ttttcaaact 1680actgaaggaa
tatttagggt agatggaaat aaaaaaggta tatttactag aaaccgtcag
1740cctaaagccg ttgctcatct tataagaagc agatggaata agttgccatt
agattataaa 1800tcaaaaaaa 180931823DNAArtificial SequenceOptimizado
para expresion en E. coli 3ccatgggcat ggtcaatagt atgctgtatc
cgcgtgaaag ccgcacccgc cgtgttgtgg 60acatctctgg catgtgggaa ttcaaaatcg
acatcaacaa tgaaggccgt aacagtggtt 120atgcgaatgg cctgaaagat
accacgttta ttccggtccc gagctctttt aacgacctgt 180ttaccgacaa
aaatatccgt gaacatgccg gtgatgtttg gtatgaaacg agcttctacc
240tgccgctgga atggaaagat aaagacgtga acgttcgctt tggttgcgcg
acccatgaag 300ccacggtgta tattaatggc aaagaagtct gtacccacgt
gggcggtttt atgccgttca 360acgcgccggt taatgaagcc ggcatcttcg
gcgagaaaaa caaactggtg gttgtcgtga 420acaatgaact gtccaatacc
acgattccgt gcggtcacac cgaaacgaaa ccgagcggca 480aaaaatacat
caaaccgtct tttgatttct ttaactacgc aggcctgaat cgtccggtta
540aaatcaccgt cacgaacaaa gaatacatcc atgatatcga catcctgtca
gatgtgaacg 600gctcggacgg tattgtgaat tatgaagttc acaccacggg
tgaaaacaaa gtttacatca 660aaatcaacga tgaagaaggc aaagaagtcg
cgagctgcga aggcaaatct ggtaaaattg 720tgatcaaaga tgccaaactg
tggaacccga aagcggccta cctgtacaaa ttcatcgcct 780gtatcaaaaa
cggtgatgaa ctgattgacg aatattacct ggattttggc atccgcaccg
840tcaaagtgga gggtacgaaa ttcctgatta acggcaaacc gttttatttc
accggctttg 900gtaaacatga agattctgaa atcgcaggcc gtggttacaa
cccgccggtt attaaacgcg 960actttgaact gatcaaatgg gtcggtgcta
atagcttccg tacctctcac tatccgtaca 1020gcgaagaaat tatgcaggca
gctgatcgcg aaggcatcgt gattatcgac gaagttgcgg 1080ccgtcggcat
gttcgatgtg ggctctgttc tgaatccgtc agcgtcgaaa accgattatt
1140tttcactgga cgaagttcat tcgaaaacga aagaagtcca caagaaagcg
gtggaagaac 1200tgattaaacg tgataaaaac catccgagtg ttgtcatgtg
gtccctgttc aatgaaccgg 1260atacctcaaa agacgaagca gtgccgtact
tcgaagatat cttcaacttc gctaaatcgc 1320aggacaaaca aaatctgccg
aaaacctttg cagctatcca ggcgagttcc ccgggtaaat 1380gcaaatgtat
gcatctgtgc gatgtgatta cgctgaaccg ttattacggc tggtatttcc
1440tgggcggtta cgaaatcgac atgagtgaag aaaaattccg cgaagaaatg
aacctgtact 1500ccaacatgaa caaaccggtt atgttcaccg aatatggtgc
agatacgtac gctggcgttc 1560acaaactgcc gagtgtcatg tggtccgaag
aataccagtg tgaatactac gaaatgaact 1620tcaaagtgtt cgatagctac
gactttattg ttggtgaaca gctgtggaat tttgcagatt 1680tccaaaccac
ggaaggcatt tttcgcgtgg acggtaacaa gaaaggcatc ttcacccgta
1740atcgccaacc gaaagctgtc gcacatctga ttcgctcccg ttggaacaaa
ctgccgctgg 1800actacaaaag taagaagaag ctt 1823
* * * * *
References