Preparative Biochemistry & Biotechnology

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Improvement of enzymatic bioxylitol production

from sawdust hemicellulose: optimization of
parameters

Islam S. M. Rafiqul, Abdul Munaim Mimi Sakinah & Abdul Wahid Zularisam

To cite this article: Islam S. M. Rafiqul, Abdul Munaim Mimi Sakinah & Abdul Wahid Zularisam
(2021): Improvement of enzymatic bioxylitol production from sawdust hemicellulose: optimization of
parameters, Preparative Biochemistry & Biotechnology, DOI: 10.1080/10826068.2021.1897840

To link to this article: https://doi.org/10.1080/10826068.2021.1897840

Published online: 16 Mar 2021.

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PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY
https://doi.org/10.1080/10826068.2021.1897840

Improvement of enzymatic bioxylitol production from sawdust hemicellulose:

optimization of parameters
Islam S. M. Rafiqula , Abdul Munaim Mimi Sakinahb, and Abdul Wahid Zularisamc
a
Department of Genetic Engineering and Biotechnology, University of Chittagong, Chattogram, Bangladesh; bFaculty of Chemical and
Natural Resources Engineering, Universiti Malaysia Pahang, Kuantan, Pahang, Malaysia; cFaculty of Engineering Technology, Universiti
Malaysia Pahang, Kuantan, Pahang, Malaysia

ABSTRACT KEYWORDS
Enzymatic production of bioxylitol from lignocellulosic biomass (LCB) provides a promising alterna- Bioconversion; bioxylitol;
tive to both chemical and fermentative routes. This study aimed to assess the impacts of catalytic hemicellulosic hydrolysate;
variables on bioxylitol production from wood sawdust using xylose reductase (XR) enzyme and to optimization; xylose;
xylose reductase
optimize the bioprocess. Enzyme-based xylitol production was carried out in batch cultivation
under various experimental conditions to obtain maximum xylitol yield and productivity. The
response surface methodology (RSM) was followed to fine-tune the most significant variables such
as reaction time, temperature, and pH, which influence the synthesis of bioxylitol from sawdust
hydrolysate and to optimize them. The optimum time, temperature, and pH became were 12.25 h,
35
C, and 6.5, respectively, with initial xylose 18.8 g/L, NADPH 2.83 g/L, XR 0.027 U/mg, and agita-
tion 100 rpm. The maximum xylitol production was attained at 16.28 g/L with a yield and product-
ivity of 86.6% (w/w) and 1.33 g/Lh, respectively. Optimization of catalytic parameters using
sequential strategies resulted in 1.55-fold improvement in overall xylitol production. This study
explores a novel strategy for using sawdust hemicellulose in bioxylitol production by
enzyme technology.

Introduction
Wood sawdust, a waste of sawmill, is a lignocellulosic bio-
mass (LCB). Meranti wood sawdust (MWS) shows great
potential as a renewable raw material for producing several
value-added chemicals because of its low-cost, availability,
and high content of hemicellulose.[1,2] The hemicellulosic
part of MWS is selectively hydrolyzed by dilute acid to pro-
duce a xylose-rich hydrolysate,[1,3] which can be utilized as
an economic and potential starting substrate for the manu-
facture of various high-value bioproducts especially bioxyli-
tol. Xylitol, a functional sugar alcohol, is as sweet as sucrose.
It has 40% less calories than sucrose with a calorific value of
2.4 Cal/g.[4,5] Xylitol is one of the top 12 most valuable
chemicals that can be produced from LCBs released
by USDOE.[5]
Worldwide xylitol is industrially manufactured by a
chemical hydrogenation of pure xylose obtained from hard
woods and agri-residues.[4,6–8] However, the high production
cost of this method and the necessity to minimize the envir-
onmental impact, caused by the use of toxic nickel catalyst,
and high temperature and pressure, have led to extensive
exploration of alternative routes for producing xylitol.
Microbiological xylitol production from LCBs has been
extensively investigated as an alternative to the chemical
route.[9–12] The advantages of the fermentation process over
chemical ways are its cheaper production cost due to the
non-necessity of extensive xylose purification and low envir-
onmental impact.[4,10] The application of this process on an
industrial level is time-consuming, being associated with
some preparatory activities like sterilization and regular
inoculum development involving input of energy, labor, and
time, leading to decreased productivity.[4,11] The chemical
process has a yield of about 80% of the initial xylose,[6,7]
and for the microbial fermentation route, 65–85% of yield
has been reported.[4,7,13,14] However, the lower xylitol bio-
conversion rate and downstream processing problem are still
a challenge to establish a feasible, low-cost, and large-
scale technology.
The enzymatic approach of xylitol production from LCB
is a promising biotechnological alternative in both chemical
and microbial routes. There are a few reports on enzyme-
based xylitol production from commercial pure xylose by
xylose reductase (XR).[2,15–18] Neuhauser et al.[17] and
Nidetzky et al.[18] studied on enzymatic production of xylitol
from commercial xylose and reported a yield of 96% and
productivity of 3.33 g/Lh. Compared to the microbial pro-
cess, the cell free enzymatic system to xylitol synthesis is
expected to achieve a substantial increase in productivity as
mass transfer limitations are overcome in an enzyme
reactor. One significant advantage of enzymatic approach is
that it can afford an easy recovery of xylitol. This new

CONTACT Islam S. M. Rafiqul drsmrafiqcu@gmail.com Department of Genetic Engineering and Biotechnology, University of Chittagong, Chattogram-
4331, Bangladesh.
ß 2021 Taylor & Francis Group, LLC
2 S. M. RAFIQUL ET AL.
option would also be appropriate to current concepts and
interests of sustainability and ecosystem preservation due to
the use of LCBs and mild operating conditions. However, a
major and common shortcoming in the enzymatic approach
using lignocellulosic hydrolysate is that it contains not only
the sugar required for conversion but also inhibitors that
could slow down or prevent its bioconversion.[19,20] In
enzymatic bioxylitol synthesis, the obstacles related to reac-
tion inhibition can be overcome by treatment of the hydrol-
ysate prior to reaction or by the hydrolysis of LCM with
enzymes to generate an inhibitor-free hydrolysate.
XR is an intracellular and NADPH-dependent oxidore-
ductase enzyme usually found in yeast and filamentous
fungi. This enzyme has broad applications in the bioproduc-
tion of bioxylitol, biosorbitol, and bioethanol from
xylose.[15,21,22] XR is not commercially available and thus it
was produced in this study from adapted yeast Candida tro-
picalis. NADPH is a coenzyme that plays key role in reduc-
ing xylose to xylitol in organisms and an inadequate supply
of this reductant lowers the yield and productivity. In cell
free method, the added NADPH reduces xylose but high
cost of coenzyme makes the bioprocess challenging.[4,7] The
enzyme-based hydrogenation of xylose to xylitol is NADPH-
dependent, and thus, one mole of coenzyme is consumed
per mole of xylitol produced.[16,18] NADPH donates proton
(Hþ) and electron (2e–) to xylose in the XR-catalyzed pro-
duction of xylitol. Because enzyme and coenzyme are costly,
the XR-mediated hydrogenation of xylose requires recycling
of XR and regeneration of NADPH to make the enzymatic
route economic.
The goals of this study were to examine the impact of
catalytic variables reaction time, temperature, and pH on
bioxylitol production from MWS hemicellulosic hydrolysate
(MWSHH) by XR and to optimize the bioprocess. The
research was performed in two steps based on the prelimin-
ary information obtained from previous studies reported by
the authors.[2,23] Firstly, the central composite design (CCD)
was employed to establish the true optimum conditions to
improve xylitol yield and productivity. Secondly, the verifi-
cation process was followed to validate the CCD model.
Materials and methods
Preparation of MWS hydrolysate
Meranti wood sawdust (MWS) was collected from a local
sawmill and processed for hydrolysis under optimal condi-
tions based on the previous studies.[1] In brief, 3 g of MWS
(oven dried) was mixed with the required amount of sulfuric
acid solution (%, w/w) in screw capped conical flask
(250 mL), and the batch hydrolysis was performed in an
autoclave (Hiclave HVE-50, Japan) at 124
C with 3.26%
H2SO4 for 80 min using a liquid to solid ratio of 8 g/g. The
solid residue was removed by filtration (Whatman no. 1).
The resulting filtrate named MWS hemicellulosic hydrolys-
ate (MWSHH) was neutralized with CaO to pH 6.0 and
submitted to HPLC analysis. The MWSHH contained (g/L)
18.8 xylose, 4.64 glucose, 4.14 acetic acid, 2.55 arabinose,
1.55 lignin degradation products (LDPs), 0.55 furfural, and
0.08 hydroxymethylfurfural (HMF). It was stored at 4
C
and used in subsequent experiments.
Microorganism, media, and maintenance
The yeast strain, Candida tropicalis IFO 0618, was collected
from the ATCC, USA.
It was cultured at 30
C for 30 h on yeast-extract peptone
dextrose (YPD) agar medium with the composition (g/L) 20
glucose, 5 yeast extract, 5 peptone, 1 KH2PO4, 5
MgSO47H2O, and 20 agar in water and maintained at
4
C.[20,22] Synthetic growth medium (SGM) having the follow-
ing composition (g/L): 30 xylose, 3 yeast extract, 3 K2HPO4,
and 1 MgSO47H2O in water. The recipe of YP-hydrolysate
(YPH) agar medium was similar to that of the YPD agar
medium except that MWSHH (containing 18.8 g/L xylose) was
added instead of using glucose. Hydrolysate growth medium
(HGM) also prepared from MWSHH containing 18.8 g/L
xylose, and the remainder of the components was the same as
the SGM. The pH of the media was adjusted to 6.0. The sugar
solutions (e.g., glucose or xylose) and MWSHH were auto-
claved separately from other medium ingredients to avoid
undesired reactions. The sterilized carbon sources were then
mixed together with other components before use.
Preparation of adapted yeast and inoculum
To develop adapted C. tropicalis, six successive batch cul-
tures were performed with HGM at 30
C for 24 h at
150 rpm as outlined by the authors.[22] The adapted strain
was maintained on YPH agar plate. Fresh inoculum was
attained by transferring a single colony of adapted yeast
grown at 30
C for 36 h on YPH agar plate into an
Erlenmeyer flask containing 50 mL of HGM. The inoculated
flask was incubated at 30
C in a shaker incubator (Infors
HT Ecotron, Swizerland) for 24 h at 150 rpm. The inoculum
was used in subsequent experiments.
Preparation of XR and its activity assay
XR enzyme was prepared in the laboratory from adapted C.
tropicalis following the protocol described by Rafiqul and
Sakinah.[22] Briefly, a 10% inoculum of adapted yeast was
cultivated at 30
C and 150 rpm in a flask (1 L) containing
250 mL HGM. After 20 h cultivation, cells (4.87 g/L cell dry
weight) were harvested toward the end of the log growth
phase by centrifugation, rinsed the cell pellet with potassium
phosphate buffer (0.1 M; pH 7.0), resuspended the pellet in
buffer at a cell biomass to buffer ratio of 1:2 (w/v), and dis-
rupted the cell suspension by a cell homogenizer (Omni
Ruptor 4000, USA). Cell debris was discarded by centrifuga-
tion at 8,000g for 20 min at 4
C to attain a supernatant
solution. The supernatant was re-clarified by centrifugation
at 18,500g for 30 min and the refined supernatant was
used as crude XR enzyme.
XR activity was assayed spectrophotometrically at 340 nm
and 25
C by measuring the rate of NADPH oxidation at
1 min intervals for 5 min as also reported previously [22].
 PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 3

The XR reaction contained 0.2 mL potassium phosphate buf- Table 1. Experimental design and results of the CCD study for bioxyli-
tol production.
fer (0.1 M, pH 7.0), 0.2 mL mercaptoethanol (0.1 M), 0.1 mL
Coded variables Actual input variables Responses
crude XR, 0.1 mL NADPH (3.4 mM), and 1.2 mL sterile
water. The reaction was started by adding 0.2 mL D-xylose Time Temp. pH
Run X1 X2 X3 (X1; h) (X2;
C) (X3) Yp/s (%) Qp (g/Lh)
(0.5 M). The pre-boiled XR was added to the control instead
1 2 0 0 10 35 6.5 63.52 ± 2.34 1.19 ± 0.04
of fresh XR. One unit (U) of XR activity is defined as the 2 1 1 1 13 37 7.0 77.06 ± 2.77 1.11 ± 0.04
amount of enzyme required to oxidize 1 mmol NADPH per 3 0 0 0 12 35 6.5 84.73 ± 2.94 1.33 ± 0.06
min at pH 7.0. Specific activity is expressed as units of the 4 0 2 0 12 39 6.5 76.09 ± 3.43 1.19 ± 0.05
5 1 1 1 13 37 6.0 76.88 ± 3.44 1.11 ± 0.05
XR enzyme per milligram of protein and detonated as U/ 6 0 0 2 12 35 5.5 63.13 ± 2.34 0.99 ± 0.04
mg. The activity of crude XR was 11.16 U/mL with the spe- 7 1 1 1 11 37 7.0 78.88 ± 3.51 1.35 ± 0.06
1 1
cific activity of 0.91 U/mg. XR was stored at 80
C and uti- 8
9
1
1 1 1
13
11
33
33
6.0
7.0
76.21 ± 3.04
73.22 ± 2.40
1.10 ± 0.04
1.25 ± 0.04
lized in bioxylitol production. 10 0 0 0 12 35 6.5 85.27 ± 3.31 1.34 ± 0.05
11 0 0 0 12 35 6.5 85.53 ± 3.47 1.34 ± 0.05
12 1 1 1 11 37 6.0 67.73 ± 3.77 1.16 ± 0.06
Bioxylitol production 13 0 0 0 12 35 6.5 84.75 ± 3.75 1.33 ± 0.06
14 2 0 0 14 35 6.5 73.34 ± 3.82 0.98 ± 0.05
15 1 1 1 11 33 6.0 62.91 ± 2.42 1.08 ± 0.04
Bioxylitol was produced in batch mode from MWSHH by 16 0 0 0 12 35 6.5 84.11 ± 4.17 1.32 ± 0.07
isolated XR enzyme. The reaction medium contained phos- 17 1 1 1 13 33 7.0 72.87 ± 2.31 1.05 ± 0.03
phate buffer (0.1 M, pH 7.0), XR, and NADPH in a 50 mL 18 0 0 0 12 35 6.5 84.98 ± 4.88 1.33 ± 0.08
19 0 2 0 12 31 6.5 69.21 ± 1.95 1.08 ± 0.03
flask. Enzymatic reaction was initiated by adding MWSHH 20 0 0 2 12 35 7.5 72.16 ± 4.25 1.13 ± 0.07
as substrate. Preboiled XR was added to the control instead Yp/s: xylitol yield (% w/w); Qp: xylitol volumetric productivity (g/Lh)
of active XR. After thorough mixing, a 100 mL of reaction
mixture was withdrawn to use as a zero time reaction and Optimization procedure
inactivated by boiling, and then stored at 20
C until ana-
lysis. The remaining reaction mix was incubated at various Optimization study was a further continuation of previous
operating conditions in a shaker incubator. The residual XR studies based on the OFAT and FFD approaches to select the
activity was measured after diluting aliquots taken from the suitable range of factors and to identify the significant varia-
reaction mixture into the respective assay buffer. At the end bles, respectively, for xylitol production.[2,23] The three most
of reaction, the mixture was boiled to stop the enzyme important variables namely reaction time (X1), temperature
catalysis. The denatured protein in the reaction sample was (X2), and pH (X3) were chosen for further evaluation of their
impacts on bioxylitol production from MWSHH by XR using
eliminated by centrifugation. The resulting supernatant was
central composite design (CCD) in RSM. The CCD was
stored at 20
C and submitted to analysis. Initial xylose
adopted to illustrate the nature of response surface in the
concentration was maintained at 18.8 g/L in all xylitol pro-
experimental region and elucidate the optimum combinations
duction experiments to avoid the inhibitory impact of
of the factors used. The selected variables were studied at five
byproducts present in the MWSHH. Unless otherwise stated,
different levels (relatively low, low, basal, high, relatively high)
all the experiments were done in triplicate and mean values
coded (-2, 1, 0, þ1, þ2). Other four variables xylose concen-
were recorded. tration, agitation rate, NADPH, and enzyme load were set at
18.8 g/L, 100 rpm, 2.83 g/L, 0.027 U/mg (3% v/v of reaction vol-
Analytical determinations ume), respectively, based on the results of both the OFAT and
FFD studies. The Design ExpertV R software (Stat Ease Inc.,
Samples from MWS hydrolysis and bioxylitol production USA) was employed for the experimental matrix and analysis
experiments were analyzed by HPLC as reported else- of the observed data. According to the 23 CCD layout, a total
where.[23] Shortly, xylitol, xylose, glucose, acetic acid, and of 20 experiments including eight factorial points, six axial
arabinose titers were quantified by a chromatograph points (a¼ ±2), and six repetitions at the center point were
(Agilent 1200, Agilent, USA) with a RID and a Rezex RHM executed. Table 1 presents the CCD matrix of the experiment
Monosaccharide Hþ column (Phenomenex, USA) at 80
C with coded and actual input variables, including values of
using ultrapure water as mobile phase at a flow rate of responses Yp/s (% w/w) and QP (g/Lh). In this experimental
0.6 mL/min. Furfural and HMF concentrations were also design, Yp/s (xylitol yield) was defined by [(g of xylitol pro-
determined by HPLC, but with an UV-DAD set at 276 nm duced/g of xylose consumed at the end of each run)  100]
and a Zorbax eclipse XDB-C18 column (5 mm; Agilent, USA) and QP (volumetric productivity), defined by [xylitol concentra-
operated at 25
C. In this case, the mobile phase was aceto- tion (g/L)/overall reaction time (h)]. To predict the optimum
nitrile/water (1:8) with 1% (v/v) glacial acetic acid at a flow point, experimental results were adjusted to Eq. (1), a second
rate of 0.8 mL/min. The samples were diluted with ultrapure order polynomial model equation, as outlined by
water (1:5, v/v). Quantification of LDPs was performed spec- Montgomery.[25]
trophotometrically following the Prussian blue method[24] Y ¼ b0 þ b1 X1 þ b2 X2 þ b3 X3 þ b11 X12 þ b22 X22 þ b33 X32 þ b12 X1 X2
with tannic acid as standard. Enzyme activity was tested þ b13 X1 X3 þ b23 X2 X3
spectrophotometrically at 340 nm and cell dry weight was (1)
estimated by dry weight technique as described earlier.[22]
4 S. M. RAFIQUL ET AL.

Table 2. ANOVA for the quadratic model adjusted to xylitol yield (Yp/s) and productivity (QP).
Sum of squares Degree of freedom Mean square F-value Prob > F
Source Yp/s QP Yp/s QP Yp/s QP Yp/s QP Yp/s QP
Model 1127.56 0.30 9 9 125.28 0.034 511.80 496.60 <0.0001a <0.0001a
X1 99.60 0.050 1 1 99.60 0.050 406.88 731.58 <0.0001 <0.0001
X2 52.93 0.014 1 1 52.93 0.014 216.21 204.02 <0.0001 <0.0001
X3 82.63 0.022 1 1 82.63 0.022 337.54 321.50 <0.0001 <0.0001
X12 429.50 0.096 1 1 429.50 0.096 1754.54 1419.87 <0.0001 <0.0001
X22 238.22 0.061 1 1 238.22 0.061 973.14 903.71 <0.0001 <0.0001
X32 471.25 0.12 1 1 471.25 0.12 1925.11 1721.49 <0.0001 <0.0001
X1 X2 3.95 1.513E-003 1 1 3.95 1.513E-003 16.13 22.35 0.0025 0.0008
X1 X3 75.77 0.021 1 1 75.77 0.021 309.52 310.51 <0.0001 <0.0001
X2 X3 2.38 6.125E-004 1 1 2.38 6.125E-004 9.71 9.05 0.0110 0.0132
Residual 2.45 6.767E-004 10 10 0.24 6.767E-05
Lack of fit 1.23 3.934E-004 5 5 0.25 7.867E-05 1.01 1.39 0.4942b 0.3638b
Pure error 1.22 2.833E-004 5 5 0.24 5.667E-05
Cor total 1130.01 19 19
R2 0.9878 0.9867
R 0.9939 0.9933
Adj R2 0.9859 0.9854
Prob > F less than 0.05 indicate model terms are significant
a
Model is significant; bLack of fit is not significant

where Y is the predicted response variable; X1, X2, and X3
are the coded independent variables corresponding to reac-
tion time, temperature, and pH, respectively; b0 is the inter-
ception coefficient; b1, b2, and b3 represent the linear
coefficients; b11, b22, and b33 represent the quadratic coeffi-
cients and b12, b13, and b23 represent the second order inter-
action coefficients. The developed second order model was
statistically evaluated by analyzing the values of regression
coefficients, analysis of variance (ANOVA), F- and p-values.
The quality of fit of the model was expressed through the
determination coefficient (R2) and correlation coefficient
(R). For maximum xylitol biosynthesis, an optimum setting
of the variables was obtained by numerical analysis depend-
ing on the desirability criterion. Five sets of experiments
were performed at model recommended optimal conditions
to validate the CCD model. A final experiment was carried
out to confirm the model. To compare the responses, a con-
trol experiment was conducted using commercial pure
xylose under optimum conditions established by CCD.
Results and discussion
Statistical optimization of variables
The sequential optimization of process parameters following
classical OFAT and statistical approaches is very important
to improve xylitol production. A rough optimization of reac-
tion time, temperature, pH, NADPH concentration, enzyme
load, xylose concentration, and agitation by OFAT method
gave the values of 10 h, 30
C, 7.0, 3.66 g/L, 0.027 U/mg,
18.8 g/L, and 100 rpm, respectively. These conditions led to
56% (w/w) xylitol yield. The OFAT study explored that
among the seven factors examined, five factors namely time,
temperature, pH, NADPH, and enzyme load significantly
influenced the production of bioxylitol from MWSHH.[2]
The suitable conditions for the highest xylitol production
determined by FFD study were reaction time 12 h, tempera-
ture 35
C, pH 7.0, NADPH concentration 2.83 g/L, and
enzyme concentration 0.027 U/mg. Xylitol yield was 71% (w/
w) under these conditions.[23] It was emphasized that the
xylitol yield of 71% with conditions attained by the FFD,
which was 1.27-fold higher than the yield obtained by the
OFAT (56%). This result indicated that the XR efficiency for
xylitol production is synergistically influenced by time, tem-
perature, pH with low concentration of NADPH. In a previ-
ous study, the authors reported that xylitol yield increased
with increasing NADPH titer up to 3.66 g/L (with a yield of
53.83%) and then remained constant with further increase
in NADPH.[2] The presence of NADPH above the saturating
concentration could slow the conversion of xylose to xylitol.
Among the variables screened out through FFD, reaction
time, temperature and pH were identified as the most cru-
cial variables influencing xylitol bioconversion as reported
elsewhere by the authors.[23] Hence, these factors were fur-
ther optimized by RSM to develop a second order model,
which can predict the responses more accurately. The RSM
is a powerful mathematical and statistical tool useful for
modeling and analyzing the problems related to response of
interest influenced by different factors.[25,26] The influence
of the important input variables reaction time (X1), tempera-
ture (X2), and pH (X3) was fine-tuned using CCD in RSM
to determine the actual optimum conditions that will
enhance xylitol yield and productivity as output variables.
The outcomes of the RSM study on the production of bioxy-
litol are documented below.
Optimizing critical variables by RSM
The input and output variables were fitted to the second order
equation (Eq. (1)) and investigated in terms of the goodness
of fit of the model. Table 2 summarizes the ANOVA results
of quadratic models for xylitol yield (Yp/s) and productivity
(QP). The adequacy of the model was statistically tested by F-
and p-values, R2, and R. The larger F-value and the smaller
the P-value indicate that the corresponding model and the
individual coefficient are more significant.[25,26] The ANOVA
for xylitol yield (Table 2) demonstrated that the model was
quite significant, as was evident from the high F-value
(Fmodel¼511.80) and very small probability (p < 0.0001) value.

These outcomes ensured a satisfactory adjustment of the
model to the experimental data and implied that the model
terms have a significant impact on the response Yp/s.
Furthermore, the R2 was 0.9878, which indicates that 98.78%
of the variation in the response is attributed to the input vari-
ables. The value of R2 also indicates that only 1.22% of the
total variation was not explained by the model, which is
accounted as residual. The value of R for xylitol yield was
0.9939, implying a satisfactory correlation among the observed
and predicted results.
The quadratic effect of pH (X32), reaction time (X12), tem-
perature (X22), the linear effect of reaction time (X1), pH (X3),
and the interaction of reaction time and temperature (X1X3)
were the primary determining factors of the response in xylitol
yield (Yp/s) as they had the largest coefficient values (Eq. (2)).
Meanwhile, the linear effect of temperature (X2), and the inter-
action effect of reaction time and temperature (X1X2), tem-
perature and pH (X2X3) were the secondary determining
factors with medium coefficients. Among them, the linear
terms X1, X2 and X3, and the interaction term X2X3 had posi-
tive coefficient implying a favorable effect on xylitol yield. The
negative coefficient indicates an adverse impact on the
response. According to the magnitude of F-value, the order of
effects of model terms on xylitol yield is X32 > X12 > X23 >
X1 > X3 > X1 X3 > X2 > X1 X2 > X2 X3 :
Based on the results of ANOVA (Table 2), the F-value of
the model representing xylitol productivity (QP) was 496.60
with a very low P-value of <0.0001, indicated that the
model was highly significant. The value of R2 was 0.9867,
implying that only 1.33% of the variations are not inter-
preted by the model. The R value was calculated to be
0.9933, implying a better correlation between the actual and
predicted values of QP. Notably, the insignificant lack of fit
(p > 0.05) value was observed for both regression equations
and thus ensuring a satisfactory fitness of models to the
experimental data (Table 2). The adjusted R2 (adj R2) for
both Yp/s and QP were also found very high (0.9859 and
0.9854, respectively) indicating that the created models were
greatly significant and suitable for use in this experiment.
From the regression coefficient values (Eq. (3)), it can be
concluded that all the linear terms X1, X2, X3, their quad-
ratic and interaction terms except X2X3 had significant effect
on QP. The order of significant effects (based on F-value) of
model terms on xylitol productivity is found to be the same
as ranked for the xylitol yield. The second order equations
in terms of coded factors for xylitol yield (Eq. (2)) and
productivity (Eq. (3)) were derived from regression analysis
of data.
Yp=s ¼ þ84:86 þ 2:49X1 þ 1:82X2 þ 2:27X3  4:13X12  3:08X22
 4:33X32  0:70X1 X2  3:08X1 X3 þ 0:55X2 X3
(2)
Qp ¼ þ1:33  0:056X1 þ 0:029X2 þ 0:037X3  0:062X12  0:049X22
 0:068X32  0:014X1 X2  0:051X1 X3 þ 0:009X2 X3
(3)
The created empirical equations are mathematical correl-
ation models that can be used to navigate the design space
PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 5
to predict and optimize the responses by considering inter-
action effect of factors.
Interaction of variables
Estimation of xylitol yield and productivity over the critical
variables reaction time, temperature, and pH in the forms of
interaction and response surface plots are presented in
Figures 1 and 2, respectively. The response surface with an
elliptical or saddle nature of the contour diagram indicates
the significant interaction among the corresponding varia-
bles, whereas with circular contour diagram indicates the
negligible interaction.[25] The interaction of variables like
X1X2, X1X3, and X2X3 are common effects for Yp/s and QP,
which were found to remarkably affect the results of
responses. The contribution order of interaction effects
(based on coefficient- and F-values,) for xylitol yield and
productivity is identical and expressed asX1 X3 > X1 X2 >
X2 X3 : The mutual interactions of crucial variables during
optimization by CCD are detailed below:
Figure 1(a1, a2) shows the interaction effect of reaction
time and temperature on xylitol yield when pH was main-
tained at 6.5 as the center point. The interaction and
response surface graphs showed that the predicted xylitol
yield slowly increased with increase in reaction time and
reached to a maximum value (from 77.68% at 11 h to
81.26% at 13 h) while the temperature was kept at 37
C. A
similar pattern of curves was observed at low temperature
(33
C) where the yield enhanced rapidly from 72.63% at
11 h to 79.03% at 13 h (Figure 1(a1, a2)). The longer reaction
time gave maximum xylitol yield probably because of the
adequacy of the time required for the enzyme to bind as
well as to react with substrate (xylose). As reported by
Mussatto et al.[27] and Nidetzky et al.,[21] the enzymatic
reaction requires certain period for direct physical contact
and binding between enzyme and substrate in order to
product formation. These results highlighted that longer
reaction time will be favorable to attain the highest xylitol
yield (77.06%) that is resulted in run 2 (Table 1). The rela-
tively longer (14 h in run 14) or shorter (10 h in run 1) reac-
tion time led to reduction in xylitol yield to 73.34 and
63.52%, respectively.
The interaction relationship between reaction time and
pH for xylitol yield at a temperature of 35
C is illustrated
in Figure 1(b1, b2). A notable and constant improvement in
xylitol yield was observed for the increase of reaction time
(from 68.55% at 11 h to 79.70% at 13 h) while holding pH at
low level (6.0). At high pH condition (pH 7.0), a comple-
mentary result was also observed where the yield decreased
slightly from 79.25% at 11 h to 78.09% at 13 h of reaction.
These results indicate that a near-neutral pH facilitates the
ionization of XR functional groups and consequently enhan-
ces the production of xylitol. These findings led to the con-
clusion that the low initial pH is suitable for the enzymatic
bioconversion of xylose to xylitol that is evidenced by run 8
with a maximum yield of 76.21% (Table 1). However, the
high (pH 7.5 in run 20) and low (pH 5.5 in run 6) pH
decreased xylitol yield to 72.16 and 63.13%, respectively.
6 S. M. RAFIQUL ET AL.

Interaction Graph
85.53
X2: Temperature Actual Factor
X3: pH = 6.50
Temp = 37 ºC

85.44

Xylitol yield (%)

79.88
Xylitol yield (%)

82.24
79.04
74.22 Temp = 33 ºC 75.84
72.63
(a2)
68.56

(a1) 37.00
13.00
62.91
36.00
12.50
35.00 12.00
11.00 11.50 12.00 12.50 13.00 X2: Temperature 34.00 11.50 X : Time (h)
(ºC) 33.00 11.00
1

X1: Time (h)

Interaction Graph Actual Factor

85.53
X3: pH X2: Temp = 35.00 (ºC)
pH = 7.0

79.88 Xylitol yield (%) 85.35

Xylitol yield (%)

81.15
76.95
74.22 72.75
68.55
pH = 6.0 (b2)
68.56

(b1) 7.00
13.00
62.91 6.75
12.50
6.50 12.00
11.00 11.50 12.00 12.50 13.00
X3: pH 6.25 11.50 X : Time (h)
1
X1: Time (h) 6.00 11.00

Interaction Graph Actual Factor

X3: pH
85.53 X1: Time = 12.00 h

pH = 7.0

79.88 85.47
Xylitol yield (%)
Xylitol yield (%)

82.58
79.69
74.22 pH = 6.0 76.80
73.91
(c2)
68.56
(c1)
7.00
37.00
62.91 6.75
36.00
6.50 35.00
33.00 34.00 35.00 36.00 37.00
X3: pH 6.25 34.00
6.00 33.00 X2: Temperature
X2: Temperature (ºC)
(ºC)
Figure 1. Xylitol yield as affected by reaction time and temperature: (a1) interaction and (a2) response surface graph, reaction time and pH: (b1) interaction and (b2)
response surface graph, temperature and pH: (c1) interaction and (c2) response surface graph.

Figure 1(c1, c2) is the interaction and response surface
plots for the variation in xylitol yield as a function of tem-
perature and pH by setting the reaction time at 12 h as the
medium level. When the initial pH was fixed at a high value
(pH 7.0), the predicted xylitol yield gradually improved with
increasing temperature from 77.36% at 33
C to 82.09% at
37
C. Again, at a low pH (pH 6.0), the trend of xylitol yield
was similar in which the yield enhanced slightly from
73.91% at 33
C to 76.45% at 37
C (Figure 1(c1, c2)). The
maximum xylitol yield was obtained at high temperature
 PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 7

Interaction Graph
1.35
X2: Temperature
2 Actual Factor
3 X3: pH = 6.50

Productivity (g/L h)

Productivity (g/ L h)
1.30 Temp = 37 ºC
1.35
1.30
1.25
1.25
1.20
1.15 (a2)
Temp = 33 ºC
1.19

(a1)
37.00
1.14 13.00
36.00
12.50
11.00 11.50 12.00 12.50 13.00 35.00 12.00
X2: Temperature 34.00 11.50 X : Time (h)
X1: Time (h) (ºC) 33.00 11.00
1

Interaction Graph Actual Factor

1.36
X3: pH X2: Temp = 35.00 (ºC)
2
3

Productivity (g/ L h)
Productivity (g/L h)

1.30 pH = 7.0 1.36

1.30
1.25
1.24
(b1) 1.19
1.13 (b2)
pH = 6.0
1.18

7.00
13.00
1.12 6.75
12.50
6.50 12.00
11.00 11.50 12.00 12.50 13.00
X3: pH 6.25 11.50
X1: Time (h)
X1: Time (h) 6.00 11.00

Interaction Graph Actual Factor

1.34
X3:
2 pH X1: Time = 12.00 h
3
Productivity (g/ L h)
Productivity (g/L h)

1.34
1.29 pH = 7.0
1.30
1.25
(c1)
1.25
1.20
1.16
(c2)

1.20

pH = 6.0 7.00
37.00
6.75
1.15 36.00
6.50 35.00
33.00 34.00 35.00 36.00 37.00 X3: pH 6.25 34.00
6.00 33.00 X2: Temperature
X2: Temperature (ºC) (ºC)
Figure 2. Xylitol productivity as affected by reaction time and temperature: (a1) interaction and (a2) response surface graph, reaction time and pH: (b1) interaction
and (b2) response surface graph, temperature and pH: (c1) interaction and (c2) response surface graph.

possibly due to the availability of required activation energy
(36.2 kJ/mol) for XR. These outcomes suggested that high
temperature positively affected xylitol production and pro-
vided higher yield (78.88%) as determined by run 7 (Table
1). At a high temperature (at 39
C in run 4), xylitol yield
slightly decreased to 76.09% and at a relatively low
temperature (at 31
C in run 19) it markedly declined to
64.14%. The decrease in xylitol yield at high temperature is
due to a progressive loss in XR activity or inactivation of
NADPH, whereas at low temperature, the reaction rate
declines with temperature according to the Arrhenius equa-
tion, which are consistent with the previous report.[13,17,28]
8 S. M. RAFIQUL ET AL.

Table 3. Results of model validation and confirmation run for xylitol yield and productivity.
Variables Xylitol yield (Yp/s; %) Productivity (Qp; g/Lh)


Time (h) Temp ( C) pH Actual Predicted Residual Error Actual Predicted Residual Error
Run (X1) (X2) (X3) (%) (%)
1 12.25 35 6.5 86.57 85.36 11.21 1.40 1.33 1.31 10.02 1.40
2 12.25 35 6.6 86.41 85.34 11.07 1.24 1.33 1.31 10.02 1.24
3 12.25 35 6.4 83.18 85.36 2.18 2.62 1.28 1.31 0.03 2.62
4 12.25 35 6.7 81.81 85.34 3.53 4.31 1.26 1.31 0.05 4.31
5 12.25 35 6.3 86.54 85.35 11.19 1.38 1.33 1.31 10.02 1.38
Boldface values indicate validation and confirmation results under true optimal conditions with highest xylitol production at a minimum and acceptable percent-
age error.

The interaction effect of reaction time and temperature
on xylitol productivity at pH 6.5 is shown in Figure 2(a1,
a2). It was found that xylitol productivity improved with the
increase of reaction time to certain extent and then sharply
reduced with further increase of time. The elliptical diagram
presents that the productivity decreased from 1.32 g/Lh at
11 h to 1.18 g/Lh at 13 h while setting temperature at 37
C.
At low temperature (33
C) condition, a similar phenom-
enon was also found in which the maximum and minimum
productivities of 1.23 and 1.15 g/Lh were predicted at 11
and 13 h, respectively (Figure 2(a1, a2)). By analyzing these
results, it can be concluded that short reaction time will
offer higher xylitol productivity (1.35 g/Lh) as obtained by
run 7 (Table 1). At 14 h of reaction time (in run 14), the
productivity distinctly decreased to 0.98 g/Lh. However, at a
relatively short reaction time (10 h in run 1), the productiv-
ity slightly reduced to 1.19 g/Lh.
increase of temperature from 1.21 g/Lh at 33
C to 1.32 g/
Lh at 37
C when pH was maintained at 7.0. At low pH
(pH 6.0), a reciprocal phenomenon were observed where a
little improvement in productivity occurred (from 1.16 g/Lh
at 33
C to 1.19 g/Lh at 37
C). These outcomes indicated
that high temperature resulted in higher productivity
(1.35 g/Lh) as interpreted by run 7 (Table 1). However, xyli-
tol productivity decreased to 1.19 and 1.08 g/Lh at relatively
high (at 39
C in run 4) and low (at 31
C in run 19) tem-
peratures, respectively.
Validation of model
Model validation is an important step of optimization proce-
dures and is used to verify the acceptability, adequacy and
accuracy of the model [25,26]. Numerical optimization was
conducted using Design ExpertV program. Five proposed
R

In a previous work, the kinetic parameters (Km and
Vmax) were calculated during the investigation of the impact
of substrate concentrations (xylose or NADPH) on the rate
of xylitol formation by XR.[2] The authors reported that the
Km for the two substrates are interdependent. The values of
Km for xylose and NADPH were 11.89 g/L and 6.68 mg/L
with the corresponding Vmax of 27.78 and 1.91 mg/L.min,
respectively. These results indicated that lower Km and
higher Vmax of XR for xylose are associated with the greater
xylitol production titer. Additionally, the higher Vmax indi-
cated that this reaction would yield higher amount of xylitol.
As a result, high xylitol productivity as well as high product
titer was achieved.
Figure 2(b1, b2) depicts the influence of reaction time
and pH on xylitol productivity when temperature was held
constant at 35
C. At high pH (pH 7.0), increase in reaction
time led to a rapid decrease in productivity from 1.34 g/Lh
at 11 h to 1.13 g/Lh at 13 h. At a low pH (pH 6.0) condi-
tion, xylitol production rate increased with increase in time
up to its middle level (12 h), and then decreased back to the
initial value (1.16 g/Lh at 11 h) (Figure 2(b1, b2)). These
results pointed out that high pH condition facilitated the
XR-catalyzed xylitol bioconversion and contributed the high-
est productivity (1.25 g/Lh) as attained by run 9 (Table 1).
By using the highest pH value (pH 7.5 in run 20), the prod-
uctivity reduced to 1.13 g/Lh whereas at the lowest pH (at
pH 5.5 in run 6), it sharply reduced to 0.99 g/Lh.
The interactive effect of temperature and pH on xylitol
productivity while keeping reaction time at 12 h is presented
in Figure 2(c1, c2). Xylitol productivity increased with the
optimum conditions were chosen considering those levels of
variables that led to maximum Yp/s and Qp. Validation
experiments were performed at suggested optimum condi-
tions to justify these conditions. The proposed optimum
conditions with actual and predicted results are listed in
Table 3. The analyses of residual and percentage error were
done by comparing the actual values and its associated pre-
dicted values of responses from validation trails. The resid-
uals and percentage errors were computed based on Eqs. (4)
and (5).
Residual ¼ ðActual value  Predicted valueÞ (4)
% Error ¼ ðResidual =Actual valueÞ  100 (5)
The combination of parameter settings selected for max-
imum xylitol production were reaction time 12.25 h, tem-
perature 35
C, and pH 6.5. Under these conditions, 86.57%
of xylitol yield was obtained by experiments with a
productivity of 1.33 g/Lh, which are recorded in boldface in
Table 3. The percentage errors for both xylitol yield and
productivity are ranging from 1.24–4.31%, which implied
that the created models were accurate sufficiently because
the errors were well within acceptable value (5%). These
findings also implied that the models were to be accurate
and reliable for predicting xylitol yield and productivity
within 95% confidence interval (CI).
 PREPARATIVE BIOCHEMISTRY & BIOTECHNOLOGY 9

xylose Xylitol Glucose 1.33 g/Lh, respectively. This work revealed that enzymatic
(a1) Acetic acid Arabinose bioxylitol production can be improved by optimization of
20 5 process variables. From the sequential optimization studies,
Xylitol, Xylose, Glucose (g/L)

it was found that the xylitol yield of 86.57% with optimum

Arabinose, Acetic acid (g/L)

16 4 conditions obtained by the CCD, which was 1.55-fold and
1.22-fold higher than the yield achieved by the OFAT (56%)
12 3 and FFD (71%), respectively. These findings indicated the
significance and applicability of the sequential optimization
8 2 strategies for the improvement of xylitol production.
It is noted that 86.57% of xylose to xylitol bioconversion
4 1 achieved under optimized conditions. This outcome inter-
preted that the operating conditions employed led solely
0 0
xylose to xylitol biotransformation instead of being con-
0 2.25 4.25 6.25 8.25 10.25 12.25 verted to arabinitol by XR. This fact also interpreted that
Reaction time (h)
MWSHH did not cause undesired reaction with the enzyme,
although the enzymatic reaction was affected by the concen-
(a2) 2.0 0.085
tration of hydrolysate due to the increased amounts of toxic
compounds [15,20,21]. This study innovatively developed a
Furfural, LDPs (g/L)

1.6
1.2
Furfural LDPs
0.8
0.4
0.0
0 2.25 4.25 6.25 8.25
Reaction time (h)
Figure 3. Concentrations of (a1) xylitol, xylose, glucose, arabinose, acetic acid;
and (a2) furfural, HMF and LDPs in the reaction mixture. Bioxylitol production
from MWSHH was carried out for a period of 12.25 h at 35
C, pH 6.5 using
18.8 g/L crude xylose, 2.83 g/L NADPH, 0.027 U/mg XR, and 100 rpm agitation.
These optimal conditions led to a xylitol production of 16.28 g/L with a yield of
86.6% (w/w).
Model confirmation testing
Confirmation testing is the final step of optimization study
and is important to prove the developed model directly.
Based on validation results, optimum conditions for bioxyli-
tol production were chosen as reaction time 12.25 h, tem-
perature 35
C, and pH 6.5 (shown as boldface in Table 3).
The predicted xylitol yield and productivity under the afore-
mentioned optimum conditions were 85.36% and 1.31 g/Lh,
respectively, at 95% CI. These values of responses were con-
firmed by enzymatic reaction in triplicate sets of conform-
ation experiments. The values of Yp/s and Qp obtained were
86.57% (w/w) and 1.33 g/Lh, respectively, which were in
good agreement with the values predicted by the models.
These results proved that the model fitted to the observed
data. Thus, the selected optimum conditions were the most
suitable in practice for producing bioxylitol from lignocellu-
losic substrate.
The true optimum conditions for the production of bio-
xylitol were found as reaction time 2.25 h, temperature
35
C, pH 6.5, initial xylose concentration 18.8 g/L, NADPH
2.83 g/L, XR 0.027 U/mg and agitation 100 rpm. These con-
ditions resulted in the maximum xylitol production of
16.28 g/L with a yield and productivity of 86.57% (w/w) and
0.075 reaction medium utilizing crude MWSHH to produce bioxy-
HMF (g/L) litol with a yield of 86.57% which is close to the ones
HMF
0.065 obtained in industry.[7,13,17] However, one should keep in
mind that the xylitol yield and productivity recorded in the
current study might still be markedly enhanced by further
0.055
study on enzyme inhibitors and application of immobiliza-
tion process.
0.045
10.25 12.25
Final composition of reaction mixture
Bioxylitol production from MWSHH was conducted at opti-
mal conditions of reaction time 12.25 h, temperature 35
C,
pH 6.5, crude xylose 18.8 g/L, NADPH 2.83 g/L, XR 0.027 U/
mg, and agitation 100 rpm with a yield of 86.57%. To assess
the effect and potential of MWSHH as xylose source on
xylitol biosynthesis, fixed volume of MWSHH were applied
in the reaction media. A time course of changes in product
and reactant contents during enzymatic conversion under
optimized conditions is shown in Figure 3. During enzym-
atic reaction, a concomitant reduction in the concentration
of xylose was found in the course of xylitol formation. The
highest xylitol production (16.28 g/L) was achieved at a
xylose concentration of 18.8 g/L (Figure 3(a1)). This outcome
pointed out that no substrate inhibition occurred for a
xylose concentration of 18.8 g/L, and the conversion of
xylose to xylitol progressed at maximum speed. At concen-
trations above 18.8 g/L, xylitol production is less favored
because of the presence of various toxic compounds at high
levels.[2] At the end of the enzymatic conversion, concentra-
tion of residual xylose in the reaction mixture was 2.52 g/L
(13.4% of initial xylose). Glucose, arabinose, LDPs, furfural,
and HMF concentrations remained almost unchanged
throughout the reaction period (Figure 3(a1, a2)). Acetic acid
concentration slightly decreased with increasing reaction
time probably due to its partial evaporation during the reac-
tion. Hence, MWSHH can be utilized as a promising alter-
native source of xylose for producing bioxylitol.
10 S. M. RAFIQUL ET AL.
Table 4. Bioxylitol yield and productivity obtained from pure xylose and from MWSHH under optimized conditions.
Reaction Time
medium (h) Xylitol conc. (g/L)
Pure xylose 12.25 17.76
(18.8 g/L)
MWSHH 12.25 16.28
(18.8 g/L)
Control experiment
A control experiment was carried out using commercial
xylose in the optimum conditions and the responses (Yp/s
and Qp) were compared to reaction with MWSHH.
Xylitol yield and productivity obtained from pure xylose
and from MWSHH under optimized conditions is presented
in Table 4. According to Figure 3 and Table 4, xylose con-
sumption and xylitol production behaviors were somewhat
different for the control and MWSHH experiments. From
Table 4, it was found that there was a decrease in only
8.37% of Yp/s and 8.27% of Qp for the experiments using
MWSHH, compared to the control reaction with commer-
cial xylose. These findings highlighted that the enzymatic
route has potential as an alternative for the conventional
xylitol production ways.
The decrease in Yp/s and Qp might be attributed to the
synergistic inhibitory effect of different byproducts in the
MWSHH.[20] These results revealed that the MWSHH con-
tained xylose, glucose, acetic acid, arabinose, LDPs, furfural,
and HMF (in the concentrations of 18.8, 4.64, 4.14, 2.55,
1.55, 0.55, and 0.08 g/L, respectively) did not hinder the bio-
conversion courses. This phenomenon is noticeably advanta-
geous for the scale-up of this bioprocess as the use of
MWSHH presents a diminution of the process costs. Xylitol
can be purified and recovered from reaction mixture by
crystallization, which includes sequential steps of ethanol
precipitation, centrifugation, adsorption, evaporation, and
crystallization. Hence, MWSHH can be used as a promising
alternative source of xylose for producing bioxylitol.
Conclusion
This research reports a novel strategy to utilize lignocellulo-
sic hydrolysate as a source of xylose for enzymatic biocon-
version to xylitol, a specialty chemical. A 23 CCD in RSM
was adopted to fine-tune the impacts of critical variables on
bioxylitol production from sawdust hydrolysate by XR and
to develop models for attaining optimum modes with
respect to xylitol yield and productivity. Optimum condi-
tions found were reaction time 12.25 h, temperature 35
C,
and pH 6.5, which gave a xylitol yield and productivity of
86.6% (w/w) and 1.33 g/Lh, respectively. Optimizing process
variables with sequential strategies based on OFAT plan and
statistical program resulted in 1.55-fold improvement in
overall xylitol production. Utilization of lignocellulosic
hydrolysate as xylose source will discourage the use of high-
priced commercial xylose and also manufacture a value-
added product xylitol from low-cost MWS hydrolysate. This
is the first study trying to enhance enzymatic xylitol produc-
tion from hemicellulosic substrate by catalytic parameter
Yield
Remaining xylose (%) (Yp/s, %) Productivity (Qp, g/Lh)
5.53 94.47 1.45
13.40 86.57 1.33
optimization. Hence, this study will serve as a benchmark
for further work on the enzymatic production of bioxylitol
from LCB.
Disclosure statement
All the authors have consented for publication of the manuscript in
this journal. The manuscript has not been published elsewhere and
that it has not been submitted simultaneously for publication else-
where. No potential conflict of interest was reported by the author(s).
Funding
The authors would like to thank the University of Chittagong,
Bangladesh, Universiti Malaysia Pahang and Ministry of Higher
Education, Malaysia (MTUN-COE Research Grant No. RDU 121205)
for providing necessary facilities and funds in order to carry out
this study.
ORCID
Islam S. M. Rafiqul http://orcid.org/0000-0001-6901-4070
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