Proposal Synthesis Matrix Analysis of Literature

Last Name: _Weinberg_________________ First Name: _David_______________ Period: _4__

Overarching question: _Between 3 enzymes, 2 E. coli-based and 1 abalone-based beta-glucuronidases, which combination of
enzyme source, activity units, temperature, and incubation time in sample preparation by enzyme hydrolysis of codeine-6-
glucuronide yields the most accurate results for percent recovery of analytes upon analysis?___

Key Terms (list and define in the space below)

Alphabetize your Identify as Key Technical Definition According to Field of Study
key words Word or Concept
Acid/Base Hydrolysis Concept Acid hydrolysis use protons from acids as catalysts in substitution reactions. Base hydrolysis involves
the hydroxyl (OH-) group of the base acting as the substitution in a reaction (S. Vander Wielen,
personal communication, October 11, 2018).
Aliquot Key Word A known amount of a substance, generally a liquid, that is usually a fraction of a whole (McNaught,
Wilkinson, & Jenkins, 1997).
Analyte Key Word A part of a substance that is being analyzed (McNaught, Wilkinson, & Jenkins, 1997).
Beta-glucuronidase Key Word Beta-glucuronidase (GUS) is an enzyme that is used in the hydrolysis of glucuronides from bodily fluids
(GUS) prior to analysis (“β-Glucuronidase (beta-Glucuronidase)”, n. d.).
Enzyme Activity Unit Concept The amount of an enzyme that catalyzes the conversion of a specific amount of a substance per
minute under optimal conditions for the enzyme (Nomenclature Committee of the International Union
of Biochemistry (NC-IUB), 1979).
Enzyme Hydrolysis Concept A reaction in which an enzyme facilitates the breaking of bonds in molecules in the presence of water
(Yang, Dai, Ding, & Wyman, 2011).
Hydrolysis Concept Solvolysis by water, solvolysis being the reaction with a solvent involving the breaking of bonds in the
solute (McNaught, Wilkinson, & Jenkins, 1997).
Incubation Key Word The facilitation, or catalyzation, of a reaction by controlling environmental parameters around the
reaction (R. Godwin, personal communication, September 14, 2018).
Internal Standard Key Word “A compound added to a sample in known concentration to facilitate the qualitative identification
and/or quantitative determination of the sample components,” (McNaught, Wilkinson, & Jenkins,
1997).
Liquid Chromatography Key Word Liquid chromatography is a method where molecules in a mixture are applied to a solid, moved along
that solid by a liquid solution, and separated from each by various interactions between the individual
molecules and the solid (Coskun, 2016).
Mass Spectrometry Key Word Mass spectrometry is a method of measuring the characteristics of molecules by ionizing a sample,
separating them by mass and charge using electromagnetic fields, and detecting them and creating a
 graph based on the results (Reusch, 2013). Tandem mass spectrometry uses multiple mass separators
to obtain a higher resolution of ions (“Tandem Mass Spectrometry (MS/MS)”, 2015).
Metabolite Key Word “A substance made or used when the body breaks down food, drugs or chemicals, or its own tissue,”
(“Metabolite”, n. d.).
Quality Control Concept Quality control is an aspect of laboratory management taken to assure the validity of results and to
improve analysis associated with measurement in the lab (Howanitz & Howanitz, 1983).

Synthesis Matrix Analysis of Literature

Foundational Sub Problem 1: What are the differences between acid/base hydrolysis and enzyme hydrolysis, and what are their respective
advantages?
APA Purpose Framework Sample Design Variables/ Results Controversies, Assumptions, Implications
format instruments disagreements Limitations for practice,
reference Overarching Hypothesis/ with other and research,
Question Objective How the Validity How the authors Delimitations theory
data was and hypothesis was
collected? Reliability supported/rejected You will add a
list of authors
Conclusion and referenced in
further studies this section on
a separate
page
Peciulyte “The “An “Six “An “The “It serves to This “Different “Something
enzymatic interdiscipl differe interdisci enzymatic emphasize that for conclusion fibril models does happen
, A., hydrolysis inary nt plinary hydrolysab efficient from the have been with the
of cellulose approach cellulo approach ility, enzymatic article is proposed in LFD as the
Karlstro is generally was sic was measured hydrolysis, the actually in the result of
considered adopted in substra adopted as the surface of disagreement literature… acid and
m, K., to be a the current tes in the conversion cellulosic with one of In the other, enzymatic
sustainable study… in were current yield of substrates need to the authors’ the fibril hydrolysis,
Larsson, means of order to used in study… cellulosic be accessible to previous is composed though we
obtaining characteriz this in order substrates the enzymes, studies: “This of 36 cannot
P. T., & monosaccha e the study to to glucose implying that the is opposite to elementary explain the
rides that supramolec (Table characteri after 2 typical fibre wall the trends chains, in mechanism.
can be ular 3). ze the days, pore sizes need to observed for which it is ” (p. 6)
Olsson, converted structure of Avicel supramol was quite be larger than the protein assumed that
into a cellulosic ® PH- ecular different typical sizes of the production the fibril “It would be
L. number of substrates 101 structure for the six enzyme molecules among our aggregates useful to
products via during en (Fluka of substrates which are around samples and have a evaluate the
(2015). microbial zymatic BioCh cellulosic investigate 10 nm [26].” (p. suggests that hexagonal changes in
fermentation hydrolysis. emika, substrates d (Table 3) cellulosic cross-section NMR
Impact [1].” (p. 1) ” (p. 2) Cork, during en 2).” (p. 4) substrates that [23].” (p. 2) spectra
Ireland zymatic “Although the are more resulting
of the “However, “We ) has hydrolysi “The experimental recalcitrant to “It should be from
the believe that been s.” (p. 2) solubilized conditions were enzymatic noted that the changes in
supramol enzymatic investigatio used in monosacch identical, the hydrolysis FSP method the
hydrolysis ns of many “The arides were conversion yield may result in used is not supramolec
ecular of cellulose cellulose studies characteri quantified of the cellulosic higher applicable to ular
is often structures and stics of using high- substrates ranged enzyme substrates structure of
structure incomplete with therefo the performanc from 2% to 91%. production lacking fibre cellulose
[3] and we nanometre re Avicel, e This was when they are cell wall that may
of do not yet resolution served OPHS- anion interpreted as an used as morphology, occur
have a full will as a ND, exchange indication that the carbon and such as the during the
cellulose understandi provide a referen OPHS-D chromatogr structural energy NCC-OPHS- time course
ng of the more ce.” and NCC- aphy with a properties of sources for ND of
on the process.” precise (p. 9) OPHS- Dionex cellulose may fungi.” substrate.” enzymatic
(p. 1) picture of ND ISC-5000 affect enzymatic (p. 5) (p. 7) hydrolysis.”
efficienc the process substrates system hydrolysability.” (p. 9)
as both were coupled to (p. 4) “It should be
y of enzymes measured a CarboPac noted that “Other
and the before PA1 (250 “Only weak average pore mechanisms
enzymati building and after mm× 4 correlations were sizes were may also
blocks of enzymatic mm i.d.) found between used in this alter the
c cellulose hydrolysi column initial SSA and study. Pore supramolec
have these s.” (Dionex, enzymatic size ular
hydrolysi dimensions (p. 6) Stockholm, conversion yield distributions structure of
.” (p. 3) Sweden) of the cellulose and the cellulose I.
“Stone and a substrates.” (p. 4) detailed Several
s. and pulsed am structural mechanisms
Scallan perometric “This observation characteristic may be
Biotechn have detector led us to s of the pore working
developed (HPAEC- hypothesize that system, such simultaneou
ology for a method PAD).” there may exist a as sly,
based on (p. 10) relation between bottlenecks enhancing
Biofuels, solute the structural and dead-end or
exclusion “Average properties of the pores may counteractin
8(56), 1- that can pore sizes substrate used for considerably g the
be used to were the fungal growth reduce the expected
13. estimate computed and the capacity SSA spectral
the total by of the produced accessible to changes,
https://do water combining enzymes to the enzymes. making it
content of estimates hydrolyse the This means difficult to
i.org/10. the fibre of the SSA same substrate.” that the cor relate the
wall pore from NMR (p. 5) relation observed
1186/s13 system spectra between spectral
when in a with results “It appears that average pore changes to
068-015- water- from FSP the observed size and specific
swollen measureme increase in LFD in conversion mechanisms
0236-9 state, nts.” (p. this case is yield may .” (p. 9)
which 11) independent of not be
they call whether acid or perfect but,
the FSP enzymatic as the data in
[41].” hydrolysis Figure 4
(p. 7) is used.” (p. 6) suggests,
the impact of
“All “The structural such pore
samples changes induced system
were by acid hydrolysis characteristic
wetted may make it s on the
with easier for the conversion
deionized enzymes to split yield is
water in the fibril probably
the range aggregates apart most
of 40 to during enzymatic pronounced
60% hydrolysis, in substrates
water possibly due to where the
content fibril or fibril average pore
and aggregate size is in the
packed shortening which order of, or
uniformly might occur smaller than,
in a during the acid the typical
zirconium hydrolysis step.” size of
oxide (p. 6) enzymes
rotor. molecules.”
Recordin “Investigating the (p. 8)
g spectra relationship
from wet between the
rather average pore size
than dry and the
samples hydrolysability of
give a the substrates
higher showed that
apparent conversion yield
resolution increased with
.” (p. 10) increasing pore
size of the
“FSP substrate (Figure
measurem 4).” (p. 8)
ents were
conducted “Larger pore sizes
according in the cellulosic
to Stone substrates resulted
and in higher
Scallan conversion yield.”
 [41].” (p. 9)
(p. 11)
“Both acid and
enzymatic
hydrolysis can
increase the LFD,
but no plausible
mechanism
leading to the
observed increase
in LFD could be
given.” (p. 9)
Greenwo “Hemicellul “In this “Exper “A “Within “The maximum “Yan et al. “To define “Calculatio
ose forms a work, we imental discrete the fibre, xylose yields also use a the model ns exist in
od, A. monolayer propose a ly, acid populatio the volume obtained for each similar ratio domain, it is the
coating fibre scale hydrol n balance fraction of temperature to describe an assumed that literature to
A., around model that ysis approach the solid ranged from unreactive the distinguish
cellulose marries the was is used to biomass is 63.2% at 110°C component of hydrolysis of the yield
Farrell, and thus chain conduc account denoted by after 360 min to cellulose in a each due to the
inhibits the length ted by for the es and the 92.1% obtained at model of individual preheating
T. W., enzymatic dependenc loading chain volume 155°C after 20 cellulosic acid bagasse fibre time from
saccharificat y of 5 g of degradati fraction of min.” (p. 6) hydrolysis; in the cell is the true
Zhang, ion process population dried on the acid however, independent experimenta
[3,4]. Acid balance sugarc kinetics filled pores “Therefore, even the of any other. l yield;
Z., & pretreatment equations ane by is labelled though the model interpretation Hence, the however,
is a method with “hard- bagass formulati ev. The looked capable of this simplifying these
O'Hara, of removing to- e into a ng chain hydrolysate of reproducing the parameter is assumption calculations
hemicellulos hydrolyse” 66-mL scission contains xylose curve, it not the same is made that are typically
I. M. e from kinetics.” Dioniu as a series only the can be seen that in this context the based on
bagasse by (p. 2) m™ of acid under a more [13].” (p. 3) hydrolysis of simpler
(2015). hydrolysing cell, polymer solution stringent fitting all the kinetics
the linkages “By which degradati and hence regime, the model materials in [30]. An
between the comparing was on in this does not the cell can investigatio
A novel monomeric the model subseq equations region of accurately capture be n into such
units of the results to uently [9].” (p. the domain the chemical approximate calculations
populatio hemicellulos oligomer filled 3) ev=1 and/or physical d by a model for non-
e polymers.” profiles in with throughout. processes that are of the linear
n (p. 1) addition to 0.5 “The ” (p. 2) occurring during hydrolysis of population
the wt% temperatu dilute acid a single balance
balance “Ensuring monomer sulfuri re used “The hydrolysis of bagasse kinetics
the (xylose) c for acid parameters bagasse fibre, and fibre.” (p. 2) may
model efficiency of yield curve, acid.” hydrolysi ˆF and ea hence, its provide an
acid the (p. 2) s was in a represent usefulness as a “We assume interesting
for the pretreatment variability range of the fixed predictive tool reaction and future
improves associated “Sugar 110°C to volume would seem to be one- course of
dilute the overall with the cane 170°C in fractions of questionable.” dimensional enquiry.”
cost- model bagass increment lignocellul (p. 7) transport (p. 6)
acid effectivenes parameters e was s of ose and across two
s of is collect 15°C.” unreactive “It is observed distinct “Further
hydrolysi bioethanol restricted. ed (p. 10) hemicellul that the inclusion regions of experimenta
production Hence, it is from ose, of the hard-to- the domain, tion is
s of from second hypothesise Raceco respectivel hydrolyse the fibre (0 < required to
generation d that the urse y.” (p. 4) parameter, a, r< determine
hemicell feedstocks resultant Sugar results in an Ri) and the an
[5].” (p. 1) parameters Mill “These improved fit hydrolysate expanded
ulose. carry more (Mack experiment between the (Ri < r < temperature
weight than ay al results model and the Ro).” (p. 2) profile for
Biotechn those Sugar were used experimental a(T), and
which have Limite to first data over a broad “In the without
ology for not been d) in calibrate range of oligomer literature, the this
subjected Macka and then profiles, where the existence of information,
Biofuels, to an y, validate the sum of the a fast and it is difficult
equivalentl Austral model squared residuals slow to assume
8(26), 1- y stringent ia.” presented was reduced to hydrolysing the
fitting (p. 10) in the Φ=59.01 for hemicellulos functional
process.” Model non-zero a from e fraction is form of
13. (p. 2) Developme Φ=1182 when readily temperature
nt section.” a=0.” (p. 7) observed [7]; dependence
https://do (p. 6) however, the at
“It is observed population temperature
i.org/10. “The that at both balance s outside
validity of temperatures, the framework the scope of
1186/s13 a as a model predictions does not the
useful compare readily allow experimenta
068-015- fitting tool reasonably well to for l work
has been the experimental Kobayashi conducted
0211-5 demonstrat results.” (p. 9) and Sakai’s here (110°C
ed…” separate to 170°C).”
(p. 7) “The results in “hard-to (p. 8)
Figure 9 suggest hydrolyse”
“Thus, that the model and “easy-to- “The model
rather than described… is a hydrolyse” presented
using the useful tool for classes to be here has
yield data accurately incorporated reproduced
measured predicting the into the laboratory
at 155°C yield of model [8]. In scale
and 170°C hemicellulose order to best experimenta
to calibrate obtained from the approximate l results. To
our model, acid pretreatment this apply such a
this of sugarcane phenomenon model to an
informa bagasse.” (p. 9) while industrial
tion maintaining “reactor”
was used to full chain scale would
validate the length be largely
model.” dependence, beneficial
(p. 9) it is assumed in reducing
that the rate the number
of hydrolysis of resource
of the slow intensive
component hydrolysis
of experiments
hemicellulos required to
e is determine
effectively optimal
zero on the reactor con
timescale of ditions.
the fast Further
hydrolysis investigatio
reaction.” n is needed
(p. 3) to
determine
“It is the
assumed that applicability
only one of this
mole of model on
hydrogen such a
ions is scale.”
liberated (p. 10)
from one
mole of
sulfuric acid
[14,15].”
(p. 4)

“It was
assumed that
the porosity
of the fibre
was initially
25.4% (v/v)
based on the
porosity
measurement
of rice hulls
[18].”
(p. 4)

“In addition
to those
already
outlined,
there are five
further
assumptions
implicit in
the model
equations…
Finally, it is
assumed that
the domain is
isothermal
and
isobaric.”
(p. 5)

“As
discussed
previously, it
is difficult to
experimental
ly determine
rate
parameters in
lignocellulosi
c materials
 at high
temperatures
due to the
short
timescales
involved and
the
limitations of
the
experimental
setup.”
(p. 9)

“Careful
consideration
must be
given to the
interpretation
of parameters
obtained
from model
fitting when
only a single
set of data
(for example,
xylose yield)
is used to
constrain the
fit.” (p. 9)
Boison, “Phenylbuta “After “Incurr “This “A Waters “The results show This article “OXPBZ–D9 “The
zone (PBZ) reviewing ed paper Acquity that in the absence mentions no (C19H11D9 recovery of
J., has been the results equine describes UPLC of enzymatic controversies N2O3; MW= PBZ and
used for a from that tissue the results interfaced hydrolysis, liver or 333.43) was OXPBZ
Dowling, long time in study, it was of a to a Waters tissue obtained disagreements obtained as residues are
treating racewas generat comparati Micromass from the horse with other internal improved
T., horses for decided to ed by ve study Quattro sacrificed 6 days authors. standard for with the
musculoskel investigate dosing that was Premier post dose the addition of
Johnson, etal pain and if including a 10- conducted mass contained the quantificatio a ß-
inflammatio an year- in our spectromet highest n of OXPBZ glucuronida
R., & n but it is enzymatic old laboratory er with a concentration of but, unlike se enzyme
not hydrolysis female for the Z-spray PBZ followed by PBZ-D10 hydrolysis
Kinar, J. approved step to the horse analysis interface kidney and which step. It is,
for use in sample with of PBZ controlled muscle. With the showed the therefore,
(2016). horses preparation PBZ at and by additional same strongly
destined for procedure 8.8mg/ OXPBZ MassLynx enzymatic recovery recommend
Analysis human would have kg with and version 4.1 hydrolysis step in efficiency as ed that
consumptio an impact once a without software the sample PBZ, methods
of n.” (p.1) on the day for hydrolysi operated in preparation OXPBZ-D9 developed
recovery 3 days s.” (p. 1) the positive procedure, the showed for the
phenylbu “Since more and the and electrospra recovery of PBZ recovery analysis of
and more analysis of sacrific “Collision y were elevated efficiencies PBZ and its
tazone race horses PBZ and ed energies, ionization by about a factor that were OXPBZ
are finding OXPBZ within precursor, mode was of 1.3 in liver, 1.4 highly metabolite
residues their way residues 6 days. and used for in kidney, and 4.7 variable consider the
into the food from Liver, product the liquid times in muscle compared inclusion of
in horse chain, it is equine kidney, ions used chromatogr tissues.” (p. 3) to OXPBZ this enzyme
imperative tissue and for the aphy- itself, and so hydrolysis
tissues that residues samples.” muscle quantitati tandem “With enzyme it was step.” (p. 3)
of PBZ and (p. 1) tissue ve and mass hydrolysis, decided not
with and its active sample confirmat spectromet however, the to include it
metabolite, s ory ry (LC- recovery of in the
without oxyphenbut obtaine analysis MS/MS) OXPBZ in equine quantitative
azone d from of PBZ analysis.” liver was elevated analysis
enzyme (OXPBZ), this and (p. 2) by a factor of 5.7, procedure for
be horse OXPBZ followed by OXPBZ.”
monitored were residues kidney by 1.4.” (p. 2)
hydrolysi in sacrificed homog in the (p. 3)
horses to enized samples The article
s by LC- ensure that prior to using mentions no
consumers extracti PBZ-D10 assumptions.
MS/MS. are not on.” as
exposed to (p. 1) internal The article is
Drug any standard also
unwanted are shown delimited to
Testing drug in Table horse tissues.
residues 3.” (p. 2)
and resulting
from PBZ
Analysis, administrati
on.” (p. 1)
8(5-6),

535-538.

doi:10.1

002/dta.2

020

Liu, L., “[Rice bran] “One “Indivi “Complex “All “Meanwhile, “In addition, This article “As shown
has gained possible dual enzyme solvents complex enzyme Schmidt, mentions no in Fig. 1, an
Wen, increasing method of phenoli hydrolysi used in treatment Gonçalves, assumptions unknown
attention degrading c s was chromatogr significantly Prietto, or peak
W., world wide the cell standar then aphy were increased the Hackbart, and limitations. It followed by
due to its wall to ds performe of HPLC contents of free, Furlong is delimited gallic acid
Zhang, many further were d in grade and soluble conjugate, (2014) also to rice bran present as
beneficial release purcha triplicate, other and total found that but makes soluble
nutritional phenolics sed where chemicals phenolics by 2.17- fermentation note of conjugate in
R., Wei, and is complex from this were of , 1.33-, and 1.46- with Rizhopus similar liquefaction
biological enzyme Aladdi mixture analytical fold more than oryzae findings in group was
Z., Deng, effects. hydrolysis n was reagent gelatinization (p < increased the studies on higher than
However, it using Reagen incubated grade.” 0.05).” (p. 3) amount of grape seed, that in
Y., Xiao, is presently protease, ts with a (p. 2) free phenolics grape gelatinizatio
underutilize cellulase (Shang complex “Compared to in rice bran. pomace, and n group,
J., & d due to its and hai, of “The total gelatinization, However, it is cranberry and then
poor flavor glucoamyla China). enzymes phenolic liquefaction difficult to seed. almost
Zhang, and se. The ” (p. 2) consisting content in slightly, but compare the disappeared
solubility, work of 0.5% both the significantly, value from in complex
M. and its presented “The glucoamy free and increased the this study enzyme
primary here fresh lase, soluble amounts of free, with that from hydrolysis
(2017). current use focuses on rice 1.5% conjugate soluble conjugate, our study due group.
is in the this method bran protease fractions and total to different Thus, the
Complex production of used in and 1.5% was flavonoids by extraction and further
of fertilizer enzymatic this cellulase, measured 8.94%, 5.64% and quantification studies are
enzyme and animal release of study based on by the 7.72%, methods.” required to
feed. phenolics had the Folin– respectively (p < (p. 6) identify the
hydrolysi Therefore, from rice been weight of Ciocalteu 0.05), while unknown
how to best bran.” defatte rice bran, colorimetri complex enzyme compound.”
s utilize rice (p. 2) d using for 190 c method treatment (p. 5)
bran supercr min at (Singleton, increased the
releases has become itical 57.5 C°. Orthofer, amounts by “Along
an intense carbon Samples & 69.3%, 96.1% and those lines,
antioxida focus of dioxide from each Lamuela- 79.1%, another
research.” (Rice of the Raventos, respectively.” mechanism
tive (p. 1) Resear three 1999).” (p. 3) by which
ch stages (p. 2) complex
phenolic “One Institut were “Generally, the enzyme
benefit of e of collected “The total total amount of treatment
s from rice bran is Guang and flavonoid each phenolic may
that it is a dong phenolics content compound increase the
source of Acade were was significantly antioxidant
rice bran. bioactive my of extracted determined increased activity of
phenolics. Agricu and according following rice bran
Food These ltural quantified to the enzymatic extract may
phenolic Scienc .” (p. 2) colorimetri processing (p < be by
Chemistr compounds es, c method 0.05) with the significantly
have potent China) “The with minor exception of increasing
y, 214, 1- antioxidant and mobile modificatio syringic acid, gallic acid
and free was phase was n (Min, which did not levels, a
8. radical made a 0.4% Gu, change phenotype
scavenging up of aqueous McClung, significantly noted in our
https://do properties, 52.87 solution Bergman, (p > 0.05).” (p. 4) study.”
which % of acetic & Chen, (p. 7)
i.org/10. prevent carboh acid 2012).” “Complex
chronic ydrate, (solution (p. 2) enzyme treatment “The results
1016/j.fo diseases, 29.66 A) and further increased of this study
such as % acetonitril the free, soluble provide
odchem. cancer, starch, e conjugate and useful
diabetes, 16.72 (solution total FRAP values information
2016.07. obesity and % B), by 2.53-, 2.64- for
cardiovascul protein using the and 2.59-fold, processing
038 ar diseases , following respectively, rice bran
(Lai, Chen, 23.21 gradient compared to into
Chen, % program: following functional
Chang, & crude 0–40 min, gelatinization (p < beverage
Cheng, fiber solution 0.05).” (p. 5) rich in
2012; and B 5–25%; phenolics
Okarter & 12.35 40–45 “Complex enzyme and
Liu, 2010; % ash min, treatment flavonoids
Verschoyle based solution significantly and
et al., on dry B 25– increased the free, amplified
2007).” weight 35%; 45– soluble conjugate antioxidant
(p. 1) (DW). 50 min, and total ORAC activity.”
” (p. 2) solution values, which (p. 7)
“These B 35– were 1.83, 1.30,
reports 50%. and 1.42 times
suggest that Other higher,
the soluble chromato respectively,
conjugate graphic compared to
form is an condition values after
important s included gelatinization (p <
source of an 0.05).” (p. 5)
phenolic injection
compounds volume of “However,
in cereals. 20 lL, a treatment by
However, flow rate gelatinization and
little of 1.0 liquefaction
information mL/min, destroy the
is available a run time crystalline
on soluble of 50 structure of starch,
conjugate min, the main
phenolics column component of rice
and their temperatu bran, allowing
antioxidant re of 30 C enzymes to attach
activity in and to their substrates
rice bran.” detection (Blazek & Gilbert,
(p. 2) wavelengt 2010; Li et al.,
h of 280 2015).” (p. 5)
nm.” (p.
3) “Treatment with
the enzymes
“All glucoamylase,
experime cellulase and
nts were protease caused
repeated hydrolysis of
3 times starch,
and data polysaccharides,
are protein and fiber
expressed in the rice bran
as mean ± and disrupted the
standard interactions
deviation between the
(SD). phenolics and cell
Data were wall components,
analyzed thus the
by one- corresponding
way bound phenolics
ANOVA were released into
followed free or soluble
by the conjugates
SNK-q (Tables 1 and 2).”
test using (p. 6)
SPSS13.0
software “Generally,
(SPSS liquefaction and
Inc. complex enzyme
Chicago, treatment together
IL, USA). increased the total
The level soluble amount of
of each phenolic
significan compound studied
ce with the exception
was set at of syringic acid
p < 0.05.” (Table 3).” (p. 6)
(p. 3)
“In summary, the
manner in which
each specific
phenolic was
released differed
based on their
attachments to cell
wall components,
as well as the
specificity of the
enzymes.” (p. 6)

“This present
study
demonstrated that
complex enzyme
treatment
significantly
increased the free
FRAP and ORAC
antioxidant
activity in rice
bran extract.”
(p. 6)

“Taken together,
our results
indicate that
complex enzyme
treatment
increases the total
soluble
antioxidant
activity of rice
bran extract by
releasing bound
antioxidants.”
(p. 7)
Hilpman “Waste “In the “In the “The “The “Results for xylan The article “The factor “Applicatio
biomass, present present experime carbohydra hydrolysis as a mentions no 0.85 n of the
n, G., mostly of work, work, nts were te and the function of controversies describes, heterogeneo
plant origin, hydrolysis the carried monomer temperature are or that the used us catalyst
Becher, is a main of xylan utilized out in a content of given in Fig. 3. disagreements xylan Smopex-
renewable into hemice jacketed the All three with other consists of 101
N., feedstock monosacch llulose glass samples experiments were authors. ca. 85% indicated
for both arides was was batch reac were conducted with xylose that a higher
Pahner, biofuels and studied in extract tor. The analyzed HCl at pH 1. As monomers, conversion
basic or the ed temperatu by GC- expected, xylan which are of xylan and
F.-A., specialty temperatur from re was FID, which hydrolysis rate is hence xylose
chemicals. e range 70– the kept was a strongly enhanced assumed to yields were
Kusema, Despite 90 °C hardwo constant PerkinElm with increasing determine the reached in
ongoing using a od using a er Clarus temperature.” maximum comparison
B., attempts to range of species heating 500 (p. 3) possible to HCl at
establish homogeneo silver jacket instrument. xylose the same
Mäki- biorefinery us birch connected ” (p. 2) “Obviously the amount after reaction
concepts, catalysts. (Betula to a higher reaction complete conditions
Arvela, there is still For pendul water- “The temperature xylan (T = 90 °C,
a lack of comparison a).” (p. filled oligomer influences the hydrolysis.” H+ = 0.01
P., fundamental , the 1) thermosta content hydrolysis rate (p. 3) mol/l in the
knowledge heterogene t. was and selectivity, reaction
Lange, in some ous catalyst Atmosphe determined resulting in better solution).
areas of Smopex- ric with a conversion, at However,
R., . . . biomass 101 was pressure PerkinElm the same time catalyst
conversion. also during er above a certain deactivation
Salmi, T. One of these utilized.” heating Autosyste temperature level due to
gaps are (p. 2) was m XL GC leading to thermal leaching
(2016). hemicellulos maintaine with FI- degradation of of acid sites
es, being “The aim d via a detector.” monomers and was
Acid part of of the standard (p. 2) formation of or observed.”
lignocellulo experiment glass unwanted (p. 5)
se which is s was to condenser “Degradati by-products. High
hydrolysi the most investigate fitted on yields of xylose
abundant the acid to the top products can already be
s of biomass catalyzed of the were obtained at
representing hydrolysis reactor, identified moderate
xylan. almost 70% of xylan to which via a GC– temperatures
of the total xylose at was MS (T<100 °C) as
Catalysis plant moderate independe equipped visible from Fig. 3
biomass.” reaction ntly with a in agreement with
Today, (p. 1) conditions cooled MSD 5973 literature data
with with cold HP [20,21,23].
259(2), common water. detector.” Contrary to
mineral Mixing (p. 2) glucose and
376-380. and organic was mannose, xylose
acids, as realized “The molar is much less stable
https://do well as by a mass [24,25].” (p. 3)
solid lateral average
i.org/10. catalyst stirrer weight “As visible from
Smopex- made of (MW) and Fig. 3 hydrolysis
1016/j.ca 101.” (p. 2) PTFE, the molar rate at 70 °C was
running at mass very slow and
ttod.201 minimum distribution within 24 h a
500 rpm.” (MWD) xylose yield of
5.04.044 (p. 2) were only 5% was
analyzed obtained. With
“The with high elevating
following pressure temperature the
main size rate as well as the
reaction exclusion xylose yield
parameter chromatogr increased with
s were aphy the latter reaching
varied (HPLC- 66% and 86% at
during the SEC). This 80 and 90 °C after
experime is in an 24 h, respectively.
nts (Table online Thus it can be
1): (a) combinatio concluded that
type of n with a efficient
the acid multiangle hydrolysis of
catalyst laser light xylan within a
(HCl, scattering reasonable
H2SO4, (MALLS) reaction time
trifluoroa instrument requires reaction
cetic and a temperature
acid/TFA, refractive higher than 80 °C,
oxalic index (RI) but still lower
acid, detector.” than 100 °C.” (p.
Smopex- (p. 3) 4)
101), (b)
catalyst “A xylose yield of
loading, 100% was
influencin achieved at pH 0.5
g proton (H+ 0.316), 86%
concentra at pH 1 (H+ 0.1)
tion (pH and 10 % at pH 2
0.5–3; H+ H+ 0.01),
0.361– respectively
0.001 within 24 h. In the
mol/l), case of pH equal
(c) to 3 (proton
temperatu concentration
re (70–90 0.001 mol/l), the
°C), (d) reaction rate was
xylan very slow and
concentra hardly any xylose
tion (0.5– was observed
5 wt.%).” in the reaction
(p. 2) solution. Such
 strong dependence
“To between the
eliminate reaction rate and
the proton
influence concentration is in
of agreement with
different the literature
catalyst [22,23]. Efficient
loadings, hydrolysis can be
xylan achieved at proton
hydrolysi concentration
s was corresponding to
conducted pH-values
separately significantly
with HCl lower than pH 2.
and However, too high
Smopex- proton
101 at concentration (or
both 90 pH below ca. 0.5)
°C and may lead to the
equal undesired by-
proton products in xylan
(acid) hydrolysis.”
[H+] (p. 4)
concentra
tion of “The assumption
0.01 mol/l that the starting
in the xylan
reaction concentration
medium could have an
(Fig. 6).” effect on the
(p. 4) reaction rate was
investigated
during a set of
experiments
with higher initial
xylan
concentrations
(Fig. 5). The
results indicated
rather similar
hydrolysis
behavior at least
in the range 0.5–5
wt.%.” (p. 4)

“Interestingly, a
higher xylose
yield was obtained
with Smopex-101
at the end of the
experiment in
comparison with
HCl. It should be
emphasized that
degradation
products, typically
formed under
more severe
conditions [26],
were not
determined during
the reaction.” (p.
5)
 Synthesis Matrix Analysis of Literature
Foundational Sub Problem 2: What does beta-glucuronidase do and what are some of its sources?
APA Purpose Framework Sample Design Variables/ Results Controversies, Assumptions, Implications
format instrument disagreements Limitations for practice,
reference Overarching Hypothesis/ s How the with other and research,
Question Objective How the hypothesis was authors Delimitations theory
data was Validity supported/rejected
collected? and You will add a
Reliability Conclusion and list of authors
further studies referenced in
this section on
a separate
page
Liang, “Interestingl “In light of “Daph “A “All “M-1 and M-2 This article “In “Severe
y, with such the netin Shimadzu incubation were detected in mentions no particular, underestima
S.-C., poor deficiency (97%).. LC/MS- s were the present of controversies because tions of the
pharmacoki of current . and 2010EV performed UDPGA. They or UGTs are clearance of
Xia, Y.- netic profile, knowledge D- mass in 3 were further disagreement. located propofol
daphnetin on sacchar spectrome independe identified, within the (56.28 vs.
L., Hou, still exerts a daphnetin ic acid ter nt respectively, to be lumen of the 0.16 l/h),
strong anti metabolism 1,4- (Kyoto, experiment 7-O and 8-O endoplasmic naloxone
J., Ge, inflammator and the lactone Japan) s in mono- reticulum, it (76.72 vs.
y activity potential were with an duplicate.” glucuronides of is not uncom 1.20 l/h),
G.-B., effect in influence purcha ESI (p. 3) daphnetin by mon for in and
vivo. of sed interface comparing the vitro morphine
Zhang, [6,9,10] daphnetin from was used “The HPLC retention assessments (44.40 vs.
This could metabolism Sigma- to identity of times and mass of 4.92 l/h) by
J.-W., be explained on its Aldric identify daphnetin spectra with those glucuronidati .27
by assuming pharmacolo h (St. daphnetin and its of authentic on to glucuronida
that gical Louis, and its metabolite chemical underestimat tion have
He, Y.- daphnetin is effects, the MO).” metabolit peaks standards which e been
converted present (p. 2) es, formed in were markedly the observed by
Q., . . . into active study is operating incubation biosynthesized extent to using in
metabolite(s designed to in both samples previously 14.” which the vitro data
Yang, L. ) after (1) negative was (p. 4) process Thus,
metabolism. characteriz and confirmed occurs in further
(2016). ” (p. 1) e the in positive by “In the presence vivo.” studies on
vitro ion reference of NADPH gen (p. 8) daphnetin
Methylat metabolic modes to eration system, metabolism,
profile of from m/z authentic neither substrate particularly
ion, daphnetin 100 to standards.” depletion nor new studies
in different 1000.” (p. 4) metabolite peaks performed
glucuron human (p. 2) were detected in vivo, are
liver tissue (data not shown), still
idation, preparation “All suggesting that needed.”
s, (2) assays xCYP enzymes (p. 8)
and compare were are not involved in
the conducted daphnetin
sulfonati contributio at 37C for metabolism.”
ns of 60 min (p. 4)
on of different with the
metabolic final “Of the 3
daphneti pathways protein conjugation
to concentra pathways, the
n in daphnetin tion of Vmax/Km value
clearance 0.2 for methylation
human …” (p. 2) mg/mL.” was highest (470
(p. 3) mL/min/mg
hepatic protein), followed
“Glucuro by
preparati nidation glucuronidation
was (425 mL/min/mg
investigat protein) and
ons in ed by sulfonation (165.9
incubatin mL/min/mg
vitro: g HLS9 protein; Table 2).”
(0.2 mg 7 (p. 4)
Metaboli protein/m
L) at 37C “Disappearance of
c for 10 daphnetin corre
min in a lated well with its
profiling, medium metabolites
(200 mL) formation (Fig. 4).
pathway containin After 120-min
g 5 mM incubation… the
comparis MgCl2, glucuronoconjugat
25 es (M-1, 19.1%;
on, and mg/mL M-2, 15.2%)...
alamethic contributed to
bioactivit in, 10- 34.3%... of total
mM D- metabolism of
y saccharic daphnetin.”
acid 1,4- (p. 4)
analysis. lactone,
1-600 “Of the 3
Journal mM of metabolic
daphnetin pathways,
of , and 50- methylation might
mM Tris- be a main pathway
Pharmac HCl responsible for the
buffer hepatic clearance
eutical (pH 7.4).” of daphnetin…
(p. 3) However, the
Sciences, contribution of
glucuronidation to
daphnetin
105(2), metabolism could
be equally
808-816. important.”
(p. 8)
https://do

i.org/10.

1016/j.x

phs.2015

.10.010

Krahulec “Bacterial “In this “Potent “A “Bootstrap “Detailed analyses This article This article “We
beta- study, ial 594 multiple ping of the enzymes mentions no mentions no conclude
, J., glucuronida recently aa alignment analysis of revealed a small controversies limitations to that the
se has been identified large was trees was number of highly or the study or shown data
Szemes, considered new beta- protein created used to conserved disagreements any strongly
for many glucuronid of using determine residues with other assumptions. support the
T., & years to be ase 1,785 neighbor- the tree responsible for authors. The study is hypothesis
exclusive from S. bp joining topology catalytic action of delimited to of a cloned
Krahulco for equi subsp. large method, significanc the enzymes. Site characterizin beta-
Escherichia zooepidemi open and e.” (p. 2) directed mutation g a new beta- glucuronida
vá, J. coli cus has reading distance analysis of E. coli glucuronidas se gene.” (p.
and closely been frame tree was beta-galactosidase e clone. 9)
(2010). related characteriz was formed demonstrated
Enterobacte ed by subject based on significance of
Bioinfor riaceae [1]. several ed to the Glu-461 residue
However, bioinformat blastp- alignment for the enzymatic
matics recent ics tools.” protein with reaction and other
studies (p. 2) s bootstrap experiments
confirmed similar ping assigned this
character that beta- ity analysis residue the acid
glucuronida search [14].” base catalyst
ization se activity [11]. A (p. 2) action, and an
can also be set of additional
of found in a 10 residue Glu-537 a
limited protein role of
potential number of s nucleophile in the
other was active site [27,
new bacteria, selecte 28].” (p. 3)
particularly d from
beta- gram- the “The calculated
positive protein distances propose
glucuron inhabitants similar greater relatedness
of ity of the analyzed
idase gastrointesti search protein to the
nal tract [2, result beta-
from 3].” (p. 1) and glucuronidases,
was including those of
streptoco used human and mouse,
for compared to
ccus equi analysi proteins with beta-
s in galactosidase
subsp. Clustal activity.” (p. 6)
X
zooepide progra “A sugar-binding
m domain of
micus. [13].” glycosyl
(p. 2) hydrolases family
Molecula 2 proteins was
identified near to
r N terminus of the
studied protein.”
(p. 6)
Biotechn
“Interestingly,
ology, along with highly
similar sequences
44(3), found in closely
related
232-41. streptococci, a
high degree of
doi:10.1 similarity was
detected for
007/s120 Haemophilus
somnus beta-
33-009- glucuronidas e.”
(p. 7)
9234-0

Hassan, “Human “Our “The “To “Data were “The GUS This article “The r.m.s. “It would be
beta- analysis of highest obtain a collected polypeptide mentions no differences interesting
M. I., glucuronida the high - high using the contains 629 controversies after to test the
se (GUS) resolution produc resolution APS residues and a 22 or superposition importance
Waheed, acts as an structure ing crystal BEAM- residue long disagreements of the four of these
exoglycosid provides clone structure LINE-17- signal sequence with other subunits four
A., ase in new insight of a of human ID of [5]. The sequence authors. between residues to
lysosomes for better stably- GUS we synchrotro alignment of coordinates lysosomal
Grubb, J. and is understandi transfe purified n source, at human GUS of Ca was targeting of
involved in ng of the cted human a with other lower than GUS
H., Klei, stepwise function of CHO GUS and wavelengt mammalian GUSs 0.2 and, experimenta
degradation GUS and cell produced h of λ = [34,35,36,37] and therefore, all lly by
H. E., of lysosomal line a high 0.98 on E. coli GUS [38] of the deletion
glucuronic targeting.” (CHO- quality Bruker is shown in Fig. 1, following analysis.”
Korolev, acid- (p. 2) K1), crystal, AXIOM which indicates discussion (p. 6)
containing was which 200 CCD that sequences are will be based
glycosamino scaled diffracted detector. highly on a single “Our
S., & glycans up, and up to 1.7 Data were conserved.” monomer.” structure
(GAGs) secrete A processed (p. 3) (p. 3) analysis,
Sly, W. including d resolution with combined
heparan enzym .” (p. 2) AUTOMA “The overall with an
S. sulfate, e was R and structure of GUS extensive
dermatan collect SCALEPA contains four list of
(2013). sulfate, and ed for CK from chains held mutations
chondroitin the HKL together by non- causing
High sulfate purific package covalent GUS
[1,2].” ation [57]. The interactions (Fig. deficiency
resolutio (p. 1) of overall 2).” (p. 3) in human
native completen and other
n crystal “This GUS ess of the “The sequence of site-directed
enzyme is of using data was human GUS mutagenesis
structure great an 88% at 1.7 contains four studies
importance immun resolution. potential provide
of because it oaffinit ” (p. 9) glycosylation sites a better
hydrolyzes y (Fig. 1).” (p. 4) understandi
human GAGs, and chroma ng
its tograp “Importantly, the of the
β- deficiency hy high-resolution mechanisms
causes proced structure revealed of
glucuron mucopoly ure glycosylation of lysosomal
saccharidosi describ a second residue, targeting.”
idase s type VII ed in Asn272, which (p. 9)
(MPSVII) detail was not
reveals [11], also elsewh previously
known as ere observed.” (p. 4)
structural Sly [55].”
syndrome (p. 9) “Mutation of
basis of [12].” different
(p. 1) “The combinations of
highest glycosylation sites
lysosome - led to reduction in
produc enzyme activity,
targeting ing possibly because
clone unglycosylated
. PLoS of a protein is unable
stably- to form soluble
One, transfe homotetramers
cted [10].” (p. 5)
8(11), CHO
cell “This analysis
e79687. line suggests that
(CHO- despite the
https://do K1), remarkable
was differences in the
i.org/10. scaled overall structure
up, and of GUS and
1371/jou secrete cathepsin D, a
d similarity in
rnal.pone enzym lysosomal
e was targeting motif
.0079687 collect make both
ed for enzymes a
the substrate for
purific phosphotransferas
ation e, which targets
of them to the
native lysosomes (Fig.
GUS 4C).” (p. 5)
using
an “We compared the
immun refined structure
oaffinit of human GUS to
y bacterial GUS.
chroma Both structures
tograp are
hy superimposable
proced with an r.m.s
ure deviation of 1.06
describ for Ca atoms…
ed in despite a
detail relatively low
elsewh sequence
ere similarity (45%).”
[55].” (p. 6)
(p. 9)
“However, a
remarkable
difference was
observed in the
loop (Ser360 to
Glu378) near the
catalytic site of
bacterial GUS.
Such a loop is
completely absent
in human GUS
(Fig. 6B). This
loop is formed
by 17 extra
residues in
bacterial GUS as
is evident from the
sequence
alignment (Fig.
1)... Absence of
these 17 residues
in mammalian
GUS explains
why a potent
inhibitor of
bacterial GUS
does not inhibit
human GUS,
despite the overall
structural
similarity of the
enzymes.” (p. 6)

“The active sites

of both of these
proteins are
structurally
similar, a
significant
difference is that
E. coli beta -
galactosidase is a
metalloenzyme
and requires M
for the catalysis
whereas GUS has
no such
requirement [54].”
(p. 7)

“These findings
suggest that all
glycosyl
hydrolases
 evolved from a
common ancestor
and acquired extra
residues that
confer substrate
specificity and
allow hydrolases
to perform
different functions
in different sub-
cellular
localizations.”
(p. 8)
Gagez, “In addition, “The “A “All “All “CDCA and TFP “UGT1A3- “A limitation “No
drug current pool of incubatio reagents were metabolized mediated of this study selective
A.-L., glucuronidat work mixed ns were were of exclusively by glucuronidati is that we did substrate
ion exhibits aimed at men done analytical UGT1A3 and - on of CDCA not include has been
Rouguie substantial establishin and under grade.” 1A4, respectively. was best- in this assay identified so
interindivid g an ‘‘n-in- women linear (p. 2) AZT, ETO, and fitted by selective far for
g-Malki, ual one’’ assay HLMs, condition MEF were the substrate substrates for UGT2B17,
variability to as well s with “Best-fit selectively inhibition UGT2B17 and apart
K., that can phenotype as respect models metabolized by model, (expressed in from its
result in six major micros to time were their enzymes whereas the human major role
Sauvage, altered drug human omes and selected (UGT2B7, -1A1, Trottier et al. prostate but in androgen
pharmacolo UGTs prepare protein among and -1A9, reported also in the metabolism
F.-L., gical (UGT1A1, d from concentra Michaelis– respectively) with Michaelis– liver), for [24], its
profiles -1A3, - baculo tion. The Menten, minor Menten UGT2B4, contribution
Marquet, [10]. Thus, 1A4, -1A6, virus- incubatio substrate involvement kinetics for and (more to drug
it becomes -1A infecte n buffer inhibition, (<10%) of other this pathway regrettably) metabolism
P., & increasingly 9, and - d consisted and UGTs (Fig. 2). [19]. The for remains to
apparent 2B7) in insect of 0.1 M substrate The range of UGT2B15.” be
Picard, that the human cells Tris–HCl autoactivat glucuronidation of CDCA (p. 6) ascertained.
study of liver express (final pH ion (Hill) SER was concentration Similarly,
N. drug microsome ing the 7.4) models on catalyzed by s tested here “Here we did UGT2B4,
glucuronidat s (HLMs) human containin the basis UGT1A6 and - was higher not evaluate reported as
(2012). ion needs to using UGT1 g 10 mM of the 2B17, but the than that used functional the most
be included incubations A1, - MgCl2.” Akaike activity of in this capabilities abundant
Simultan in the of selective 1A3, - (p. 2) informatio UGT1A6 was previous of UGTs at UGT in the
process of substrates 1A4, - n criterion more than 5-fold publication saturating liver,
eous enzyme followed 1A6, - “Substrat (AIC).” greater than the (10–750 vs. concentration appears to
reaction by a single 1A9, - es and (p. 2) activity observed 1–250 𝜇M), s.” (p. 7) exhibit low
evaluatio phenotyping LC– 2B4, - microsom with UGT2B17.” which activity
during drug MS/MS 2B7, - es were “The (p. 4) probably “In addition, toward
n of six developmen determina 2B15, preincuba chromatog explains why ETO drugs and
t, but this tion of their and - ted for 5 raphic “ETO the authors glucuronidati has no
human requires glucuronid 2B17 min at 37 system glucuronidation did not on was selective
qualified es.” (p. 1) (Super C° in a consisted was substantially observe significantly substrate
glucuron biological somes) shaking of a binary enhanced by SER substrate inhibited by identified
systems and a water pump (median +21% inhibition.” CDCA, TFP, [25]. Its
idation (e.g., liver control bath, and (Shimadzu [minimum +17, (p. 7) and AZT, high hepatic
microsomes, prepara the LC-20AD) maximum +40]), although expression
activities hepatocytes) tion, reaction and an whereas it tended “The none of these may be due
with known was was autosample to be decreased by glucuronidati substrates to its crucial
in liver enzyme purcha initiated r CDCA (20% [+2, on of MEF was role in the
contents or sed by the (Shimadzu -34]), AZT (-18% was best metabolized glucuronida
microso activities.” from addition SIL-20AC) [-12,-34]), and described by UGT1A1. tion of
(p. 1) BD of the equipped TFP (-22% [-2,- using the A possible potentially
mes Bioscie cosubstrat with a 100- 27]). On the other Michaelis– explanation hepatotoxic
nces e.” (p. 2) [micron] hand, ETO Menten for this could bile acids
using Gentes sample inhibited CDCA model, be that [10]. In
t “Potential loop.” (p. glucuronidation (- which is only noncompetiti contrast to
liquid (Wobu interactio 3) 23% [-12, -42]). partly in ve inhibition the two
rn, ns TFP also accordance occurred, above-
chromato MA, between “The markedly with the which has mentioned
USA). UGT detection inhibited the literature; been UGTs,
” (p. 2) probes was glucuronidation of Gaganis previously UGT2B15
graphy- were performed CDCA (-28% [- et al. reported described for is thought
“Huma examined using an 31, -17]) and AZT Michaelis– this enzyme to be an
tandem n liver by Applied (-42% [-29, Menten [35]. The important
tissue incubatin Biosystem -47]), whereas its kinetics using mechanism contributor
mass sample g pooled s 4000 own human kidney of such to drug
s HLMs QTRAP glucuronidation microsomes inhibition metabolism
spectrom derived with each mass was substantially and reported is not fully [25].
from substrate spectromet decreased by Hill kinetics understood… Further
etry. 44 alone or er CDCA (-31% [- using In this study, work needs
surgica in equipped 25, -41]). No rUGT1A9. In we did not to be done
Analytic l combina with a change greater addition, investigate in to establish
specim tion with Turbo V than 15% of the Km much detail whether S-
al ens each Ionspray control metabolic estimated here the oxazepam
were other source and rates was (164 𝜇M mechanism (a probe
Biochemi obtaine substrate controlled observed for other using HLMs involved in recently
d from following by Analyst substrate and 11 𝜇M these described
stry, Biopre the 1.5 combinations.” using rUG different for this
dic general software.” (p. 4) T1A9) were interactions enzyme),
427(1), Interna incubatio (p. 3) slightly because it can be
tional n “The relative different from was far included in
52-59. (Renne procedure “Intraassay standard those reported beyond the the current
s, described precision deviations (RSDs) by Gaganis et scope of the procedure
https://do France above (n was of the results al. (23 𝜇M study.” (p. 7) [26].” (p. 6)
).” (p. =2 studied by obtained with the using human
i.org/10. 2) experime preparing n-in-one kidney “A limitation “In
nts and procedure, microsomes of our conclusion,
1016/j.ab performe analyzing evaluated by and 449 𝜇M method is the current
d in five phenotyping the using that the assay can be
.2012.04. duplicate) independe same HLM recombinant glucuronides used
.” (p. 2) nt preparation in five UGT1A9) were effectively
031 replicates independent [29].” (p. 7) determined to rapidly
of quality experiments, relative to assess
controls ranged from 4.3 to the glucuronida
prepared as 26.7%.” (p. 5) calibration tion
described curves of activities of
above at “The method their six major
different showed good respective UGTs in
concentrati intraassay parent probe. HLMs. It
ons (40, precisions for all However, offers the
80, 200, compounds, with this has no potential of
400, 800, mean relative consequence being
2000, and errors (MREs) for the automated
4000 lg/L) less than 16.5% relative on 96-well
on a given and RSDs always comparison plates to
day. less than 16.0%.” of enzyme further
Interassay (p. 5) activities increase the
precision (i.e., analytical
and “Only a 2-fold comparison throughput.
linearity difference was of enzyme This
were observed between relative procedure
evaluated the lowest and activities may also
from the highest UGT1A9 with and be useful
analysis of activities. All without for the
a other UGTs had competitive evaluation
calibration higher activity inhibitor or of UGT
set each ranges, with 8- to for a given activities
day for 5 16-fold variation genotype).” using other
days.” (p. between the (p. 7) models
3) minimal and (e.g.,
maximal values.” hepatocytes,
“Statistical (p. 5) liver slices)
analysis to as well as
compare “Accordingly, for the
glucuronid UGT1A1, -1A3, - screening of
ation rates 1A4, and -2B7 potential
obtained were the only UGT
using the UGTs of the nine inhibitors.”
n-in-one tested here able to (p. 7)
strategy conjugate ETO,
and CDCA, TFP, and
individual AZT, respectively,
incubation confirming that
s of each these substrates
probe and are excellent
to evaluate markers for these
genotype– UGTs.” (pp. 5-6)
phenotype
relationshi “Despite the
ps was probes being
carried selectively
out using metabolized by
the their respective
nonparame enzymes,
tric Mann– significant
Whitney interactions were
and observed when
Kruskal– coincubations
Wallis were performed.”
tests, (p. 7)
respectivel
y. Two-
tailed P <
0.05 was
considered
as
statistically
significant.
” (p. 4)
Sakuram “Herbal “In this “L. “All “All other “The purification This article This article “The GUSs
medicines study, we brevis native chemicals increased the mentions no mentions no from
a, H., including aimed to subsp. enzyme used in this specific activity controversies limitations or Lactobacill
baicalin are identify coagul purificati study were 1,800-fold, and or assumptions us species
Kishino, used for and ans on of the purification disagreements made. The are thought
antibacterial characteriz FERM procedure analytical yield was found to with other study is to be useful
S., (Kubo et al. e the BP- s were grade and be only 0.47 %.” authors. delimited to for the
1981), anti- enzyme 4693 carried commercia (p. 5) examining an construction
Uchibori inflammator hydrolyzin was out at 0–4 lly enzyme from of a safe
y g baicalin used in °C, and available.” “A BLAST search L. brevis bioconversi
, Y., (Shen et al. from L. the 20 mM (p. 3) (Altschul et al. subsp. on system
2003), and brevis study.” potassium 1997) on this coagulans of prodrugs
Yonejim anticancer subsp. (p. 3) phosphate “The sequence and made such as
activities coagulans buffer subunit indicated that this from baicalin,
a, Y., (Liu et al. (designated (KPB) molecular protein shows recombinant because
2003; as (pH 6.5) mass of the 100% sequence DNA in E. Lactobacill
Ashida, Ikemoto et LcGUS30). was used purified homology with an coli. Effects us species
al. 2000). ” (p. 2) as a enzyme O-glycosyl on and are
H., Kita, Baicalein, standard was hydrolase from L. compatibiliti generally
the aglycone “In buffer.” estimated brevis ATCC 367 es in humans recognized
K., . . . of addition, (p. 3) by (ABJ65276.1) can only be as safe
baicalin, is we compariso (LVIS_2226), hypothesized (GRAS)
Ogawa, absorbed revealed “Sodium n of the molecular weight . microorgani
more the dodecyl mobility of 56 kDa, and sms. In this
J. quickly and physicoche sulfate– with that length of 510 study, we
shows more mical polyacryl of the amino acids.” (p. succeeded
(2014). effective properties amide gel standard 6) in the
properties and electroph protein.” identificatio
β- than substrate oresis (p. 3) “Surprisingly, n and
baicalin specificity (SDS- domain analysis cloning of
Glucuron (Zhao et al. of PAGE) “Protein using Pfam (Finn the enzyme
2006; Xing LcGUS30. was concentrati et al. 2010) converting
et al. 2005; ” performe ons were indicated that this baicalin to
idase Lai et al. (p. 2) d in a determined enzyme belongs to baicalein
2003). 10% by a Bio- GH family 30, from L.
from However, it concentra Rad which contains brevis
is not easy tion of Protein glucosylceramidas subsp.
Lactobac to polyacryl Assay Kit e… but does not coagulans,
obtain amide gel (Bio-Rad contain GUS LcGUS30.”
illus baicalein using the Co. Ltd., (Cantarel et al. (p. 8)
directly tris(hydro CA, USA). 2009; St John et
brevis from S. xymethyl) Bovine al. 2010).” (p. 6) “As
baicalensis amino serum mentioned
useful because of methane– albumin “The optimum pH above,
its glycine was used was 4.5 in citrate GUSs are
for low content buffer as the buffer (Fig. 4a). classified
(ca. 0.2 %). system.” protein This enzyme into GH
baicalin Therefore, (p. 3) standard.” showed below 50 families 1,
the (p. 3) % of its maximal 2, and 79,
hydrolysi bioconversi “The activity at pH and
on of purified “The above 6.0.” (p. 7) therefore,
s belongs baicalin to protein physicoche the
baicalein was mical “The optimum discovery of
to (Fig. 1a) is extensivel properties temperature was LcGUS30 is
an attractive y of the at 55 °C (Fig. the first
glycosid method, dialyzed recombina 4b).” finding of
because against 20 nt enzyme (p. 7) GUS
e baicalin is mM KPB were belonging
present in (pH 6.5) determined “The activity of to GH
hydrolas higher and used by using 1 LcGUS30 was family 30.”
content in S. for mM pNP- stable in the pH (p. 8)
e family baicalensis further GlcA. The range between 4.5
(ca. 6–10 characteri optimal and 9.0 (Fig. 4c)
30. %).” (p. 2) zation.” temperatur and was stable
(p. 4) e was below 60 °C for
“ß- examined 30 min (Fig. 4d).”
Applied Glucuronida “The by (p. 7)
ses (EC activity incubating
Microbio 3.2.1.31, measurem the “LcGUS30 could
GUSs) can ents of reaction hydrolyze only the
logy and hydrolyze ß- the mixture substrates
D- enzyme containing conjugated with
Biotechn glucuronic were 100 mM glucuronic acid…
bonds via a performe citrate This indicated that
ology, retaining d using buffer (pH LcGUS30 is a
mechanism baicalin, 4.5) at GUS that has
98(9), and are wogonosi various strict glycon
often used de, temperatur specificity. In
4021- to convert estrone- es for 1 contrast,
baicalin to GlcA, min to LcGUS30 enzyme
4032. baicalein.” phenolpht prevent did not show strict
(p. 2) halein- damage to substrate
https://do GlcA, the specificity toward
“L. brevis MU- enzyme. aglycones.” (p. 7)
i.org/10. subsp. GlcA, and The
coagulans is pNP- optimal pH
1007/s00 known for linked value was
its potential monosacc determined
253-013- benefits to harides at 37 °C...”
human including (p. 5)
5325-8 health pNP-
(Kishi et al. GlcA as “The
1996), such substrates temperatur
that it is . The e and pH
worth standard stabilities
analyzing assays were
the function were determined
of L. brevis performe at 37 °C by
subsp. d in 100 measuring
coagulans mM the
in flavonoid citrate residual
metabolism. buffer activities
” (p. 2) (pH 5.0) after
at 37 °C incubating
“In this for 5 min the enzyme
study, we (for in 100mM
aimed to phenolpht citrate
identify and halein- buffer
characterize GlcA, (pH 4.5)
the enzyme MU- for 30 min
hydrolyzing GlcA, and at various
baicalin pNP- temperatur
from L. linked es and
brevis monosacc after
subsp. harides dialyzing
coagulans hydrolysi the enzyme
(designated s) or 30 overnight
as min (for against
LcGUS30).” baicalin, various
(p. 2) wogonosi 100mM
de, and buffers at 4
estron- °C,
GlcA respectivel
hydrolysi y.” (p. 5)
s).” (p. 4)

“The
enzyme
convertin
g baicalin
to
baicalein
 was
purified
from L.
brevis
subsp.
coagulans
through
three
successiv
e steps of
separation
by
monitorin
g the
baicalin
hydrolytic
activity.”
(p. 5)
Taylor, “Alternative “Rather “Sampl “1,600 “After “Through the This article “The use of a “Resorufin
ly, an than using es of units of extraction addition of makes no single well area
L. L., enzymatic a synthet the ß- and resorufin- mention of an control for counts (an
hydrolysis single ic Glucuroni enzymatic glucuronide and controversies monitoring indication
Flint, N. step using control to urine dase hydrolysis, subsequent or glucuronidas of
ß provide an (7) or enzyme urine monitoring of disagreements e activity to successful
A., Ma, - indication patient (IMCS) samples resorufin, the IHI, with other indicate enzymatic
Glucuronida of enzyme urine was were we were able to authors. adequate activity)
V., Hill, se addition as were added to analyzed identify sufficient performance were
may be well as obtaine the by a glucuronidase for all observed
B. M., implemente enzyme d and reaction previously activity by the patients to vary
d to increase function 200 mixture validated ß-Glucuronidase could lead widely from
Clark, C. the for an µLs of and qualitative enzyme on a to false patient to
sensitivity entire batch sample incubated LC-TOF- sample-by-sample negative patient
of benzos by of samples, was at room MS basis.” (p. 2) results due to indicating a
J., & hydrolyzing we transfe temperatu method enzyme potential,
ß-D- investigate rred re for 15 (8).” (p. 2) “An optimal inhibition previously
Strathma glucuronic d the into a min. hydrolysis time and/or lack unaddressed
acid from use of a 96 After “Retention was achieved of addition.” need to
nn, F. G. benzo- known deep incubatio times must around 15 min (p. 4) monitor
glucuronide glucuronid well n, 180 be within with 1,600 units enzymatic
(2017). conjugates ated plate.” µLs of 0.05 min of enzyme used, activity in
in urine substrate as (p. 2) sodium of the as each
Internal samples.” an IHI bicarbona expected shown in Figure sample.” (p.
(p. 2) (resorufi te (0.1 M, retention 1A.” (p. 2) 3)
hydrolysi ß-D pH 9.0) time to be
glucuronid extraction considered “There
s e) added to buffer present for was a minor
each was each addition in
indicator sample as a mixed analyte. completion time
mechanism into the The mass of ~20–30 min
for for reaction.” must be due to aliquoting
monitoring (p. 2) within and incubating
sample enzymatic 10 ppm buffer and enzyme
activity “The and the in each sample
specific from assay was compound using a liquid
patient to calibrated score must handler.” (p. 2)
monitori patient.” with a be [greater
(p. 2) single than or “Of the 668
ng of β- point cali equal to] patient samples
“By bration 70 for an tested, 284
glucuron monitoring at the analyte to samples showed a
the forma cutoff be benzodiazepine to
idase tion of the concentra considered be present using
non- tion for positive. an enzymatic
activity. glucuronid all Additionall hydrolysis
ated analytes. ya step that was not
product in This was minimum previously
Journal each verified area count observed when
sample, we with two threshold tested without a
of were able quality must be hydrolysis step (a
to verify controls, met for positivity rate
Analytic enzyme one each increase of
addition as placed at analyte 42.5%).” (p. 2)
al well as a 50% of (varies
mechanism the cutoffamongst “Benzodiazepine
Toxicolo to establish concen analytes) concentrations
minimum trations and the increased
gy, 41, enzyme and one peaks must approximately 3-
activity for placed at be near fold with the
407-411. use on a 150% of Gaussian addition of the
sample-by- the cutoffshape with hydrolysis step.
https://do sample concentra no co- Resorufin was not
basis.” (p. tions. eluting detected in the
i.org/10. 2) Each compound patient samples
reportables without an
1093/jat/ analyte or tailing. enzymatic
was Furthermor hydrolysis step;
bkx027 representee, the however, samples
d in all deuterated that included
quality markers an enzymatic
controls from the IS hydrolysis step
and the added showed the
calibrator.
during presence of the
” (p. 2) extraction resorufin analyte.”
must be (p. 3)
“Enzyme present
activity with good “The R2 values
was qualitative ranged from 0.01
monitored criteria.” to 0.0973,
by LC- (p. 4) demonstrating a
 TOF-MS lack of strong
detection correlation
of the and indicating the
non- resorufin
glucuroni variability was
dated likely independent
product.” of extraction
(p. 2) inefficiency as
monitored by the
“Enzyme included ISs.” (p.
concentra 3)
tions
were
tested at
3,200
units,
1,600
units and
800 units
with
incubatio
n times of
5, 10, 15,
30 and 60
min.” (p.
2)
Morris, “Enzyme “The “b- “Aliquots “The “At 0 min (no This article “Transformat “Further
hydrolysis application Glucur of drug- hydrolysis incubation time mentions no ion of studies with
A. A., can be of a novel onidas free urine efficiency but 10 min delay controversies oxazepam to a larger
costly and recombinan e from were was for injection on or nordiazepam sample pool
Chester, time- t b- abalon fortified assessed instrument), mean disagreements under are needed
consuming, glucuronid e separately in triplicate analyte recovery with other several to confirm
with ase for (catalo with with .91% from authors. hydrolysis the extent of
S. A., reported rapid g# glucuroni individual hydrolyzed conditions oxazepam
incubation hydrolysis DR210 des of glucuronid oxazepam and has been transformati
Stricklan times in 2, Lot oxazepam e controls lorazepam reported and on in
ranging benzodiaze # , for glucuronide variability of addition to
d, E. C., from 0.5 to pine 40109- lorazepa oxazepam, controls and mean nordiazepam nordiazepa
20 h (5–7), urinalysis 02) m and lorazepam analyte recovery conjugates m reactivity
& depending was was temazepa and .80% from due to the with this
on the investigate purcha m at temazepam hydrolyzed less stable enzyme.”
McIntire, enzyme d. The sed 2,500 at the temazepam amine (p. 5)
source.” (p. effectivene from ng/mL recommen glucuronide glucuronide
G. L. 1) ss of the Campb for use in ded controls were linkage has “The
recombinan ell hydrolysi optimum observed at RT been maximum
(2014). t enzyme Scienc s temperatur (Figure 2).” (p. 3) suggested (8, number of
relative to e optimizati e of 55C 10).” (p. 5) analytical
Rapid b- (Rockf on (9) at “Maximum column
glucuronid ord, studies.” incubation hydrolysis of the “Also, the injections of
enzymati ase from IL, (p. 2) times of 5, glucuronide time between recombinant
abalone USA) 15, 30 and controls was dosing and enzyme-
c was as a “Multi- 60 min and observed at 45 sample treated
assessed.” solutio point at room min at 55C collection samples is
hydrolysi (p. 2) n calibratio temperatur (mean analyte and/or varied expected to
contain n curves e (RT) at recovery 94%) patient exceed that
s using a ing were incubation and at 5 min at metabolic seen with
minim prepared times of 0, RT (mean analyte profiles may abalone-
novel um in normal 5, 10 and recovery 94% for also have had treated
100,00 human 15 min.” oxazepam and an impact.” samples and
recombin 0 urine with (p. 2) lorazepam and (p. 5) early
Fishma the 80% for anecdotal
ant b- n following “Target temazepam).” (p. “Total evidence
units/ calibrator concentrati 3) lorazepam supports
glucuron mL s in ons for the concentration this
b- establishe authentic “The maximum s had an approach. A
glucur d linear specimens hydrolysis of overall detailed
idase in onidas range for were oxazepam and negative bias evaluation
e each derived lorazepam and a to confirm
benzodia and analyte from glucuronide relatively this is
8,000 (20–5,000 sample controls at RT high standard underway.”
zepine Fishma ng/mL): processing equivalent to deviation, (p. 5)
n 20, 50, involving measurements at but there was
urinalysi units/ 75, 250, reference 55C provided no “Work
mL 500, hydrolysis convenience with statistically applying
s. sulfata 1,000, of sample the elimination of significant this enzyme
se.” (p.2,500 and with b- heat activation difference to other
Journal 1) 5,000 glucuronid and was between analyses is
ng/mL. ase from considerably the sample underway.”
of “Reco The abalone faster than the sets. The (p. 5)
mbinan calibratio diluted 5- optimized high standard
Analytic t b- n curves fold in incubation time of deviation
glucur underwen water 30 min for the may have
al onidas t the same under abalone b- resulted
e dilution previously glucuronidase.” from patient
Toxicolo (catalo and validated (p. 3) to patient
g # 04- sample optimal matrix
gy, 38, 01F- preparatio conditions “Mean analyte variability,
050K, n (Table I).”recovery for all the effect of
610-614. Lot condition (p. 3) glucuronide which could
# s as speci controls did not not be
https://do E13- mens.” “For increase at longer minimized in
1210, (p. 2) benzodiaze incubation times the absence
i.org/10. E13- pine at both of a
1224, “Randoml confirmati temperatures.” (p. matching
1093/jat/ E14- y selected on using a 3) deuterated
0101, authentic 20 ng/mL internal
bku083 E14- benzodiaz reporting “The unique standard.”
0124, epine cutoff, potential of the (p. 5)
E13- urine spec urine IMCSzymeTM
1203 imens specimens recombinant
and that were were b-glucuronidase
E13- previousl hydrolyzed was demonstrated
0924) y and with fast
was confirmed analyzed benzodiazepine
obtaine positive on a TLX- hydrolysis which
d from for… 4 is complete at RT
Integra were Multiplexe before the samples
ted sequester d HPLC can be injected
Micro- ed and with onto the HPLC–
Chrom stored at Agilent MS-MS
atograp 48C until 1,200 instrument. The
hy analysis.” Series use of this enzyme
System (p. 2) Binary will decrease
s Pumps processing time
(Colu “IMCSzy ScientificT due to an absence
mbia, meTM UltraTM of incubation
SC, recombin coupled time, requiring no
USA) ant b- to a heat activation
as a glucuroni Thermo and thus removing
solutio dase was TSQ a
n buffered Quantum processing step
contain to the Triple- from the analysis
ing recomme Stage of
50,000 nded Quadrupol benzodiazepines
units/ optimum e Mass in urine.” (p. 5)
mL b- (pH 6.8) Spectromet
glucur (9) and er using a
onidas the previously
e.” (p. hydrolysi validated
1) s condi multiplexe
tions d method.”
“Certif were (p. 3)
ied evaluated
drug using “In the
negativ glucuroni HPLC–
e de MS-MS
normal controls confirmati
human and on method,
urine authentic the limit of
was urine detection
obtaine specimen (LOD) was
d from s (Table defined as
UTAK I).” (p. 2) the lowest
Labora concentrati
tories, on at
Inc. which the
(Valen identity of
cia, the analyte
CA, could be
USA). accurately
” (p. 2) established
in terms of
retention
time and
ion ratio.
The limit
of
quantitatio
n (LOQ)
was
defined as
the lowest
concentrati
on at
which both
the
identity
and
concentrati
on of the
analyte
could be
accurately
established
. LODs
and LOQs
were 20
ng/mL for
all
analytes.”
(p. 3)

“The
hydrolysis
efficiency
was
calculated
by dividing
the
resulting
analyte
concentrati
on by the
target
concentrati
on of the
test
compound
 in the
sample and
multiplyin
g by
100%.” (p.
3)

References (Both from FSP 1, FSP 2, FSP 3 etc.; and references from the controversies, disagreements with other authors’
column)
*Note: Always in APA format on a separate page.

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