Accepted Manuscript Title: Production optimization of invertase by Lactobacillus brevis Mm-6 and its immobilization on alginate beads Authors: Ghada E.A. Awad, Hassan Amer, Eman W. El-Gammal, Wafaa A. Helmy, Magdy M.M. Elnashar PII: DOI: Reference:
S0144-8617(12)01262-3 doi:10.1016/j.carbpol.2012.12.039 CARP 7306
To appear in: Received date: Accepted date:
27-11-2012 13-12-2012
Please cite this article as: Awad, G. E. A., Amer, H., El-Gammal, E. W., & Elnashar, M. M. M., Production optimization of invertase by Lactobacillus brevis Mm-6 and its immobilization on alginate beads, Carbohydrate Polymers (2010), doi:10.1016/j.carbpol.2012.12.039 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Production optimization of invertase by Lactobacillus brevis Mm1 6 and its immobilization on alginate beads
2 3
Ghada E.A. Awada*, Hassan Amera, Eman W. El-Gammala, Wafaa 4 A.Helmya, Magdy M. M. Elnasharb
ip t
a
5
Dep. of Chemistry of Microbial and Natural Products; National
Research Center, Giza-Cairo-Egypt.
7
cr
b
6
Centre of Scientific Excellences, Polymers Department, Biopolymers 8
us
& Nanobiotechnology Group, National Research Center, Giza, Egypt9 10
[email protected]
11
an
Corresponding author: *Ghada E.A. Awad; E-mail:
Fax:( +202) 33370931 12
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ed
M
Phone : (+202) 33335933
13 14 15 16
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17 18 19 20 21 22 23 24
Abstract
25
1 Page 1 of 37
A sequential optimization strategy, based on statistical experimental 26 designs, was employed to enhance the production of invertase 27 by lactobacillus brevis Mm-6 isolated from breast milk. First, a 2-level 28 Plackett-Burman design was applied to screen the bioprocess 29
ip t
parameters that significantly influence the invertase production.30The second optimization step was performed using fractional factorial 31
cr
design in order to optimize the amounts of variables have the highest 32
us
positive significant effect on the invertase production. A maximal 33 enzyme activity of 1399 U/ml was more than five folds the activity 34
grafted
alginate
beads
to
an
obtained using the basal medium. Invertase was immobilized 35 onto improve
the
enzyme's
stability. 36
M
Immobilization process increased the operational temperature from 37 30 to 60 °C compared to the free enzyme. The reusability test proved 38the
ed
durability of the grafted alginate beads for 15 cycles with retention 39 of 100 % of the immobilized enzyme activity to be more convenient 40for
pt
industrial uses.
41
Ac ce
42
Keywords: Breast milk, Invertase, Immobilization, Lactobacillus 43 brevis, Central composite design, Grafted alginate beads
44 45 46 47 48
1.
Introduction
49
2 Page 2 of 37
Breast milk is widely acknowledged as the most complete 50 form of nutrition for infants, with a range of benefits for infants' health, 51 growth, immunity and development. Many studies were done52 on Lactobacillus and Bifidobacterium spp. isolated from breast milk53and
ip t
their role as probiotic. However, there are limited studies on using 54 these bacteria in the production of important enzymes with 55 high biotechnological approaches.
cr
56
us
Invertase (β-D-fructofuranosid fructohydrolase, EC 3.2.1.26) 57 catalyses the hydrolysis of sucrose into an equimolar mixture 58 of
an
glucose and fructose Danisman et al. (2004). Inverted sugar has a lower 59 crystallinity than sucrose, which is important in food industry 60 to
M
ensuring that the products remain fresh and soft for a long 61 time. Invertase is being widely applied in food industry, particularly in62the
ed
production of the chocolate with liquid center Tomotani & Vitolo 63 (2007). At high concentration of sucrose this enzyme shows 64
pt
transglycosidase activity and thus can be used in the production of 65the
Ac ce
fructooligosaccharides (FOS) – sugars with probiotic properties Ning 66 et al. (2010). It is worth to mention that invertase is also used in67the production of artificial honey and is widely used in production of high68 fructose syrup (HFS) which is largely employed as a sweetener in 69 food and pharmaceutical industries as well as the source for attaining 70 crystalline fructose. It has more desirable functional properties such 71 as high osmotic pressure, high solubility, a source of instant energy 72 as well as preventing crystallization of sugar in food products Kurup 73 et al.
3 Page 3 of 37
(2005). Moreover, invertase is used as plasticizing agents in cosmetics, 74 medicines and paper industry.
75 76
Optimization of fermentation has long been used in enhancing 77
ip t
the yield of many bioprocesses. Optimization studies involving a 78 onefactor-at-a-time approach is tedious and tend to overlook the effects 79 of
cr
interacting factors but might lead to misinterpretations of results Zhang 80
us
et al. (1996). Response surface methodology (RSM), which has 81 been extensively applied in optimization of medium composition, conditions 82 is83the
an
of enzymatic hydrolysis, and fermentation Cui et al. (2006)
collection of mathematical and statistical techniques for experiment 84
conditions
of
different
M
design, model development, evaluation factors, and optimum 85 biotechnological
bioprocess.
Statistical 86
ed
optimization not only allows quick screening of large experimental 87 domain, but also reflects the role of each of the components. 88
pt
Optimization through factorial design and the application of response 89
Ac ce
surface methodology is a common practice in biotechnology90for optimizing the media components and process parameters Rao et 91 al. 1993; Chen, (1996).
92
Due to the vast use of invertase in the food industries, several 93
attempts have already been performed to immobilize it on different 94 supports in order to extend its stability, providing re-usage possibility 95 and preventing its loss upon processing. Many different supports 96 have been investigated (Arica & Bayramoglu 2006; Altinok et al. 2008).97The immobilization process has been mostly realized by covalent bonding 98
4 Page 4 of 37
of the amino-groups of the protein to the proper functional groups 99 of the support Azodi et al. (2011).
100
According to our knowledge, we are reporting for the first 101time, the ability of Lactobacillus brevis Mm- 6 isolated from breast 102 milk to invertase
which
can
be
safely
used
in
different 103
ip t
produce
biotechnological approaches. A sequential optimization strategy 104 for
cr
identified the enzyme production parameters in basal production 105
us
medium as an effective tool for medium engineering. First, Plackett– 106 Burman screening design was applied to address the most significant 107
an
factors affecting enzyme production. Second, central composite108 design (CCD) was applied to determine the optimum level of each109 of the
M
significant parameters that brings maximum invertase production. 110 In the second part of our study, we studied the immobilization of invertase 111
ed
onto grafted alginate beads to improve the enzyme's stability112 to be convenient for different industrial applications. We optimized 113 the
pt
enzyme loading capacity as well as examining the temperature stability 114
Ac ce
and the reusability of the immobilized enzyme. 2. Materials and methods
115 116
2.1 Microorganism
117
2.1.1. Samples collection
118
Breast milk samples were collected from different mothers during 119 the first three months of breastfed. Milk samples are collected in sterile containers 120 and store in refrigerator at 4°C until reached to the laboratory. One ml121 of the sample was inoculated in tubes containing 9 ml of MRS broth medium, 122 the inoculated tubes were incubated at 37°C for 48 h. One milliliter of the 123 culture 5 Page 5 of 37
was plated onto MRS agar medium, incubation was done under anaerobic 124 condition. Successive purification was done for isolates till reaching to a125 single colony Solis et al. (2010). Bacterial isolates were routinely grown on126 MRS 127
2.1.2. Identification of invertase producing bacteria
128
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broth medium at 37ºC and preserved at -80ºC in 50 % glycerol.
The most potent isolate have the ability to produce invertase 129 had
cr
been examined by cell morphology, gram staining and spore production 130
us
properties. Identification of bacterial isolate was kindly carried131 out City for Scientific Research, Alexandria, Egypt by using 16S rRNA tequnic. 132 133
an
Enzyme assay
Invertase activity was measured by the release of reducing 134
M
sugars according to Miller (1959). The reaction mixture comprised 135 250 µl of 0.1 M sucrose as a substrate, 200 µl of 0.1 M acetate buffer 136 at pH
ed
value 5.0 and 50 µl of diluted enzymatic extract. Incubation was 137 set at 37 °C for 30 min and then the reaction was stopped by adding 138 1 ml of
pt
Di–Nitro Salicylic acid (DNS), the mixture boiled for 5 min. 139 to
Ac ce
complete color development. One invertase unit (U) was defined140 as the amount of enzyme releasing 1 µmol of reducing sugars per 141 minute
2.2.
under assay conditions.
142
Enzyme production conditions
143
Cultures were allowed to grow at 37 °C in 250 ml conical144 flasks
6 containing 50 ml of MRS broth medium. Then 0.5 ml (1.2 145×10
CFU/ml) of the overnight culture was used as inoculum 146 for the designed production medium of the following composition (g/l):147 Yeast extract, 2; Sucrose, 100. inoculated with 2 % of (1.2×106 CFU/ml) 148
6 Page 6 of 37
inoculum and incubated at 37 °C for 3 days. Cultures were centrifuged 149 at 5000 rpm at 4 °C for 15 min. The culture supernatant was used150 as the crude enzyme. Results reported were the average values with standard 151 152
Statistical designs
153
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2.3.
deviations.
2.3.1. Plackett–Burman design
154
cr
For multivariable processes such as biochemical systems, 155 in
us
which numerous potentially influential factors are involved, 156it is necessary to analyze the process with an initial screening design 157prior
an
to optimization Box et al. (1978). Plackett–Burman experimental 158 design Plackett & Burman (1946) was used to evaluate the 159 relative
M
importance of various nutrients for the production of invertase160 by L.
ed
brevis Mm-6 in submerged fermentation. Eleven components 161were selected for the study, each variable represented at two levels, 162high
pt
concentration (1) and low concentration (−1) in 12 trials as shown 163 in Table 1. pH, glucose, sucrose, malt extract, peptone, yeast 164 extract,
Ac ce
NaCl, CaCl2, MgSO4, CuSO4 , and FeSO4. Each row represents165 a trial run and each column represents an independent variable concentrations. 166 Plackett–Burman experimental design is based on the first order167 linear model:
168
Y= B0 +Σ Bi Xi
Eq. 1 169
Where Y is the response (invertase production) , B0 is the model 170 intercept and Bi is the variables estimates. The effect of each variable 171 was determined by the following equation,
172
7 Page 7 of 37
E(Xi ) = 2(Σ M i+ −M i−)/N
Eq.173 2
Where E(Xi) is the effect of the tested variable. Mi
+
and Mi− represent 174
invertase production from the trials where the variable (Xi) measured 175 was present at high and low concentrations, respectively and N176 is the
ip t
number of trials in Eq. 2. The standard error (SE) of the concentration 177 effect was the square root of the variance of an effect, and 178 the
cr
significance level (p-value) of each concentration effect 179 was determined using student’s t-test t(Xi) = E(Xi)/SE
us
180
Eq.181 3
an
Where E(Xi) is the effect of variable Xi.
183
M
2.3.2. Central composite design
182
After the identification of components affecting the production 184
ed
by Plackett–Burman design three variables (malt extract, CuSO 185 4, and FeSO4 concentrations) were selected for response surface methodology 186
pt
of central composite design (CCD). CCD proposed by (Box et al. 1871987
Ac ce
; Adinarayana et al. 2003) was selected for this study, a 23 factorial 188 design with six star points and six replicates at the central points 189were employed to fit the second-order polynomial model, the experimental 190 design consisted of 20 runs and the independent variables were 191 studied at five different levels. The experimental design used for the study 192 is shown in Table 3. All the experiments were done in triplicate 193 and the average of invertase production obtained was taken as the dependent 194 variable or response (Y). The second-order polynomial coefficients 195 were calculated and analyzed using the ‘SPSS’ software (Version 196 16.0)
8 Page 8 of 37
Second degree polynomials, Eq.(4), which includes all interaction 197 terms, were used to calculate the predicted response:
198
2 YActivity =ß0 + ß 1 X 1 + ß 2X2 + ß 3X3 + ß 11X12 + ß 22X22 + ß 33C199 3
+ ß12X1X2 + ß 13X1X3 + ß 23X2X3
Eq. 4 200
ip t
201
cr
Where YActivity was the predicted production of invertase (U/ml), 202 and X1, X2 and X3 were the independent variables corresponding203 to the
us
concentration of malt extract , CuSO4 and FeSO4 respectively; 204 ß 0 was
quadratic coefficients,
an
the intercept, ß 1, ß 2 , ß 3 were linear coefficients, ß 11, ß 22, ß205 33 are ß 12, ß 13, ß 23 are cross product coefficients. 206
M
Statistical analysis of the model was performed to evaluate the analysis 207 of variance (ANOVA). Statistical significance of the model equation 208
ed
was determined by Fisher’s test value, and the proportion of variance 209 explained by the model was given by the multiple coefficient 210 of
pt
determination for each variable, the quadratic models were represented 211
Ac ce
as contour plots (3D) and response surface curves were generated 212 by using STATISTICA (0.6).
2.4.
213
Immobilization of Invertase onto grafted alginate beads
2.4.1. Preparation of grafted alginate beads
214 215
Alginate beads were made by using the Encapsulator,216 model IR50 purchased from Innotech Encapsulator (Switzerland). Alginate 217 was dissolved in distilled water to give a final concentration of 218 1.5% (w/v) and dropped through 300 μm nozzle in a hardening solution 219
9 Page 9 of 37
using the Two-Step Method Danial et al. (2010), the beads were220 treated by 2% (w/v) CaCl2 (Ca2+) for 2 h then dropped in a solution221 of 2% (w/v) CaCl2 dissolved in 4% (v/v) polyethylenimine at pH 8 for 2222 h. The treated gels were then soaked in a solution of 2.5%223(v/v)
ip t
glutaraldehyde (GA) for 2 h to incorporate the new functionality, 224 aldehyde group.
225
227
us
2.5.2. Immobilization of invertase onto grafted alginate beads
cr
226
The immobilization process is done by soaking 0.5 g of228 the gel
an
beads in 5 ml of invertase (appropriate dilution was done in229 0.1 M
M
acetate-phosphate buffer at pH 5 for 24 h). The gel beads were 230 washed twice thoroughly for 30 min with acetate buffer to get rid 231 of any
ed
unbound enzyme. The beads containing the immobilized enzyme 232were stored at 4 °C for further measurements. For determination of 233 activity
pt
of the immobilized enzyme, an equal amount of 0.5 g gel of gel234 beads were incubated into 1.25 ml of 0.1 M sucrose and 1 ml of buffer235 for 30
Ac ce
min at 37 °C, an aliquot from the reaction mixture was assayed 236 for fructose determination as described before in material and methods. 237
2.5.3. Optimum temperature
238
The optimum temperature for the free and immobilized 239
invertase was examined by incubating the reaction mixture for 240both free and immobilized enzymes at different temperatures ranging 241from 25 to 90 °C for 30 min. The optimum temperature has been taken 242 as
10 Page 10 of 37
100 % activity and the relative activity at each temperature 243 was expressed as a percentage of the 100 % activity.
244
2.5.4. Operational stability
245
ip t
The reusability of immobilized invertase on alginate beads 246 was studied by incubation of 0.5 g of loaded gel beads with 1.25 ml247 of 0.1
cr
M sucrose and 1ml of buffer for 30 min at 60 °C. Fructose was assayed 248 as described before. The same gel beads were then washed with249 acetate
us
buffer and re-incubated with another substrate solution; this procedure 250
an
was repeated 20 times, and the initial activity was considered as 251 100 %. The relative activity was expressed as a percentage of the 252 starting 253
M
operational activity.
254
3.1. Identification of the invertase producing isolate
255
Among
ed
Results and discussion
30 Lactobacillus spp. isolated from breast 256milk
pt
samples, isolate No. 6. give a highest yield of invertase was 257 chosen for further investigation .
258
Ac ce
3.
Isolate No. 6 was characterized as Gram-positive, nonspore 259
forming rods and catalase-negative. The invertase producing bacterial 260 isolate was identified by using 16S rRNA tequnic in City for Scientific 261 Research, Alexandria, Egypt as Lactobacillus brevis and designated 262 as Lactobacillus
brevis
Mm-6.
Lactobacillus
spp.
lactobacilli, 263
staphylococci, streptococci, micrococci, and enterococci are 264 the bacterial strains which commonly isolated from breast milk,265 which should be considered as components of the natural microflora266 rather 11 Page 11 of 37
than as mere contaminant bacteria Wold & Adlerbreth. (2000) 267 and Martín et al. (2005).
268
3.5. Optimization of invertase production by multi-factorial experiments269 A sequential optimization approaches were applied 270 in this
ip t
study. The first approach deals with screening for nutritional 271 factors affecting growth of L. brevis Mm-6 with respect to invertase 272
cr
production. The second approach is to optimize the factors that 273 control the enzyme production process.
us
3.5.1.
274
Evaluation of the factors affecting invertase productivity
275
an
In the first approach, the Plackett–Burman design was 276 applied to reflect the relative importance of various medium components. 277
M
Eleven different factors (variables) including medium constitution 278 and initial pH were chosen to perform this optimization process. 279 The
ed
averages of invertase activity for the different trials were given in 280U/ml (Table 1). The main effect of each variable upon invertase activity 281 was
pt
estimated as the difference between both averages of measurements 282
Ac ce
made at the high level (+1) and at the low level (-1) of that factor. 283 The data in Table 1 showed a wide variation from 233 to 1200 U/ml 284 of invertase activity. This variation reflects the importance of medium 285 optimization to attain higher productivity. The analysis of the data 286from the Plackett–Burman experiments involved a first order (main 287 effects) model. The main effects of the examined factors on the enzyme 288 activity were calculated and presented graphically (Figure 1) offers the 289view for
the ranking of factor estimates obtained by Plackett–Burman 290
design.
291
12 Page 12 of 37
292
Table 2. shows the regression coefficients, calculated t- test 293 , Pvalues
of the tested variables. Malt extract, CuSO4, FeSO4, sucrose, 294
peptone, culture pH and CaCl2, showed positive effect on invertase 295
ip t
activity. Glucose, yeast extract, NaCl and MgSO4were contributed 296 negatively.
297
cr
The first order linear model describing the correlation between 298 the eleven factors and the Invertase activity could be presented 299 as
us
follows:
an
Y activity = 686.617+ 44.250X1+104.583 X2 ‒ 6.467 X3+94.400
300 301
X4+44.733 X5 ‒17.783 X6 ‒12.933 X7 ‒36.950 X8 + 40.433 X9 + 130.433 302 Eq.5
303
M
X10 + 68.917 X11
Based on the calculated t -test and p-values (Table 2), it was 304 evident
ed
that the medium components malt extract, CuSO4 and FeSO305 4 were
pt
found to be the most significant variables affecting invertase activity. 306 Effect of malt extract on invertase production was referred to307malt
Ac ce
extract represent a complex of nitrogen source. It is reported 308 that nitrogen source is essential for microbial growth of Lactobacillus 309spp. (Kwon et al. 2000). Copper is an essential trace element in310 living systems, where it serves as a cofactor in enzymes that function 311 in energy generation, oxygen transport, signal transduction and312 many other processes (Kim et al. 2008; Claus, 2010; Schut et al.313 2011). Other variables with less significant effect were not included in the 314next optimization experiment, but instead were used in all trials at their 315level (+1) level, for the positively contributing variables. According to 316these 13 Page 13 of 37
results, a medium of the following composition (g/l), sucrose, 317100; peptone, 2; CaCl2, 1 and pH 8 was used as a plain medium for318 further investigations. 3.5.2.
319
Optimization of the culture conditions by central composite design 320
ip t
In order to search for the optimum concentration of the 321most significant medium components (malt extract, CuSO4 and 322 FeSO4)
cr
showing confidence level 99% and above in the Plackett–Burman 323
us
design for invertase production, experiments were performed according 324 to the CCD experimental. The coded and uncoded levels of the 325three
an
independent variables investigated are listed in Table 3. The table 326also shows the CCD experimental plan, the observed and predicted 327
M
invertase production. Multiple regression analysis of the experimental 328 data gave the following second order polynomial equation (6). 329
ed
Yactivity= ‒1513.029 + 695.457X1+ 142561 X2+193847 X3 ‒75.334X12 ‒330 4.269
332 333
Ac ce
Eq.6
pt
X22 ‒ 4.306 X32 ‒149.125 X1X2 -18896 X1X3 ‒2.906331X2X3
Where Yactivit is the response (invertase production) and334 X1, X2
and X3 are the coded values of the test variables (malt 335 extract, CuSO4and FeSO4) respectively. The three-dimensional response 336 surface and the two-dimensional contour plots are the graphical 337 representations of the regression equation. They are helpful 338 in understanding both the main and the interaction effects of the factors 339 on the response value. Figure 2. A–C showe the response surface 340 and contour plots of malt extract and CuSO4, malt extract and FeSO 3414and
14 Page 14 of 37
CuSO4and FeSO4on invertase
production respectively, keeping 342 the
other component at the fixed zero level.
343
Table 4 shows the regression results from the data of 344 central composite designed experiments. The larger the magnitude of345 the t-
coefficient (Aravindan & Viruthagiri
ip t
value and smaller the p-value, the more significant is the corresponding 346 2007) This implies that 347 the
cr
variable with the largest effect was the linear effect of the 348 FeSO4
us
concentration and the squared term of the FeSO4concentration. 349This importance of FeSO4 as a medium component in the invertase 350
an
production was not clear in the literatures. Furthermore, quadratic 351 effect of the FeSO4 and CuSO4 are more significant than the 352malt
M
extract.
353
ed
The results obtained by (ANOVA) analysis showed 354 a significant F-value ( 11.591) which implied the model to be significant. 355
pt
Model terms having values of Prob > F ( 0.0001 ) less than 3560.05, considered significant.
2 The determination of coefficient (R357 ) was
Ac ce
calculated as 0.913 for invrtase activity (a value of R2 > 0.75 indicated 358 the aptness of the model) which indicates the statistical model 359 can explain 91.3% of variability in the response. The goodness of the360 model can be checked by the determination of coefficient (R2) and correlation 361 coefficient (R). The R2 value is always between 0 and 1. The closer 362 the R2 to 1, the stronger the model and the better in predicted response 363 Munk et al. (1963). The value of R (0.955) for (Eq. 6) being close 364 to 1 indicated a close agreement between the experimental results and 365 the theoretical values predicted by the model equation. An overall366 5.14 15 Page 15 of 37
fold increase in invertase was being achieved after application 367 of RSM. This reflects the necessity and value of optimization process. 368 There is no report available on optimization of invertase by369 using RSM production of invertase. All reports available so far 370 are on of invertase by one variable at a time by371 using
ip t
optimization
Saccharomyces cerevisiae Andjelkovic et al. (2010) But in 372 general
cr
different studies were done for optimization of enzymes production 373
3.5.3.
us
using RSM Abdel-Fattah et al. (2005) and Awad et al. (2011 a,b). 374 Validation of the model
375
an
The validation was carried out under optimum conditions of the376 media predicted by the polynomial model. The experimental invertase 377
production of 1411 U/ml after 3 days of fermentation 379
ed
invertase
M
production of 1399 U/ml was obtained which is closer to the predicted 378
validating the proposed model. The production of invertase 380 by
pt
Lactobacillus brevis Mm-6 at the optimized conditions is much381 higher than that of invertase produced
by Yarrowia lipolytica 382 SUC+
Ac ce
transformantsand reported by Zbigniew et al. (2011). The combination 383 of Plackett–Burman design and central composite design was shown 384 to be effective and reliable in selecting the statistically significant385 factors and finding the optimal concentrations of those factors for invertase 386 production by Lactobacillus brevis Mm-6 in submerged fermentation. 387 A second order polynomial model was established using 388 central composite design to identify the relationship between the three389 factors and the invertase yield. The final concentrations of the medium 390 components optimized with RSM were (g/l) malt extract, 2; peptone, 391 2; 16 Page 16 of 37
sucrose, 100; CaCl2, 1; CuSO4, 0.01 and FeSO4, 0.01. The initial 392 pH was adjusted to 8 as a plane medium for further investigations. 393 Immobilization of invertase onto grafted alginate beads
394
3.6.1.
Optimization of the enzyme loading capacity
395
ip t
3.6.
Optimization of enzyme loading capacity (ELC) was 396 carried
cr
out by soaking 0.5 g of the gel beads in 5 mL of original enzyme 397
us
solution and diluted enzyme 1:20, 1:15, 1:10, 1:05 and 1:02 in398 0.1 M acetate phosphate buffer at pH 5 for 24 h. The ELC increased gradually 399
an
by increasing the enzyme concentration, to reach ELC of 825 U/g 400 gel beads at 1:05 dilution, after which
any increase of the enzyme 401
M
concentration has slight effect on the ELC to reachits maximum 402 ELC. of 850 U/g gel beads using the original crude enzyme (data not shown). 403 most of aldehyde groups have 404been
ed
That could be explained that
pt
engaged with the enzymes at 1: 05 dilution and any more enzymes 405have less chances to find free aldehyde groups to bind with Elnashar 406et al.
Ac ce
(2009) This result is in agreement with the results obtained 407 in our previous study on the same polymer by Elnashar &Yassin (2009a) 408 and Danial et al. (2010)
3.6.2.
409
Optimization of temperature of immobilized invertase on grafted410 alginate
411
One of the main goals of this article was to improve 412 the enzyme’s operational temperature to be suitable for industrial use. 413 The operational temperature of the enzyme covalently immobilized414 to the 17 Page 17 of 37
grafted gel revealed higher temperature stability over the free enzyme, 415 as shown in Fig. 3.where the optimum temperature of the free enzyme 416 was at 30 °C, and that for the immobilized enzyme was at 60 °C. 417 The shift of the optimum temperature towards higher temperatures when 418 the
ip t
biocatalyst is immobilized indicates that the enzyme structure 419 is strengthened by the immobilization process. This could be regarded 420 to
cr
the formation of a molecular cage around the protein molecule 421
solution Tor et al. (1989) and Elnashar &Yassin (2009 b).
423
Operational stability of immobilized invertase
424
an
3.6.3.
us
(enzyme) to protect the enzyme from the high temperature of the 422bulk
The main advantage of immobilization of enzyme is the 425 easy separation and
M
reusability of the enzyme. The results indicated that the immobilized 426 invertase using the grafted alginate retained 100 % of its activity after 15427 reuses and then it
ed
started to decline to reach around 80 % after 20 cycles ( Figure 4428 ). These results are for the favor of grafted alginate as it uses the covalent technique. 429In another word,
pt
the immobilized invertase covalently immobilized to the grafted 430alginate could be
Ac ce
reused more times than other techniques using physical bonds. For 431example, Tanaka et al. (1984) used Polyethylene amine to entrap the glucoamylase 432into alginate, and they retained only 60 % of the enzyme activity by the 8th use, whereas 433 we retained 100 % of the immobilized enzyme activity for 15th use. These 434results could be attributed to the loss of enzyme from the alginate pores by reuses435 as they were using the non covalent technique (entrapment), while we were using 436 the covalent technique. That's mean the covalent technique is more economic 437as it saves time, carriers, and enzymes. However, the slight decrease in enzyme 438 activity by the 20th
18 Page 18 of 37
use using the grafted gel might be attributed to inactivation 439 of enzyme due to continuous use Nakane et al. (2001).
440 441
4.
Conclusions
442
ip t
In this report we studied for the first time the production 443 of
invertase enzyme from Lactobacillus brevis Mm-6 isolated from444 breast
cr
milk as a source of safe and important biotechnologically enzymes. 445
us
The optimization of the enzyme production by using statistical 446 experimental design was done.
A highly significant quadratic 447
an
polynomial equation obtained by the central composite design448 was very useful for determining the optimal concentrations of constituents 449
M
that have significant effects on invertase production. A high similarity 450
ed
was observed between the predicted and experimental results,451 which reflected the accuracy and applicability of RSM to optimize the 452 process
pt
for invertase production. The immobilization process revealed453 a high enzyme loading capacity of 850 U/g gel beads with an optimum 454
Ac ce
thermal stability at 60°C compared to the free enzyme, which was 455at 30 °
C. Moreover, the immobilized enzyme has been reused for 15456 times
without any loss of its activity. As a result, the new immobilized 457 enzyme has proven to be a good candidate in the industries.
5. Acknowledgements
458 459
This work was supported by National Research Center,
460
Chemistry of natural and microbial products department and the 461 Center
19 Page 19 of 37
of Excellence for Advanced Sciences, group of biopolymers & 462 Nanobiotechnology in Egypt.
463 464
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ip t
584 585
Ligand:
ed
M
an
us
cr
586 587 588 589 590 591 592
pt
Fig. 1. Effect of medium composition factors on the enzyme activity 593 (U/ml) produced by Lactobacillus brevis Mm-6
Ac ce
594
Fig. 2. A. Response surface plot of invertase production by Lactobacillus 595 brevis Mm-6
showing the Interactive effects of different concentrations of malt extract596 and CuSO4 at X3= 0 Fig. 2. B. Response surface plot of invertase production by Lactobacillus 597 brevis Mm-6
showing the Interactive effects of different concentrations of malt extract598 and FeSO4 at X2= 0 Fig. 2. C. Response surface plot of invertase production by Lactobacillus 599 brevis Mm-6 showing the Interactive effects of different concentrations of CuSO4 and 600 FeSO4 at X1=0 Fig. 3. Temperature-activity profile for immobilized and free invertase 601 25 Page 25 of 37
Fig. 4 . Operational stability of immobilized invertase on and grafted 602 alginate 603 604 605
ip t
606 607
Ac ce
pt
ed
M
an
us
cr
608
26 Page 26 of 37
Ac ce
pt
ed
M
an
us
cr
ip t
608 High light: 609 610 611 612 We studied the productionof invertase from Lactobacillus sp. from breast 613milk 614 A sequential optimization strategy for identified the enzyme production 615 parameters 616 Immobilization of invertase onto grafted alginate beads. 617 618 Immobilized invertase reuse for 15 cycles with retention of 100% of its619 activity 620
27 Page 27 of 37
ip t cr
Table 1. Randomized Plackett–Burman experimental design showed coded and598 uncoded variables levels for evaluating its influencing on invertase 599
us
production from Lactobacillus brevis Mm-6
600
Malt extract
Peptone
Yeast extract
Sucrose X5 -1(50)
X1 1(6)
X2 1(2)
X3 -1(0.5)
X4 1(2)
2
1(8)
1(2)
-1(0.5)
-1(0.5)
3
1(8)
1(2)
1(2)
-1(0.5)
4
1(8)
-1(0.5)
1(2)
5
1(6)
1(2)
1(2)
6
1(8)
1(2)
7
1(8)
8 9
11
12
NaCl
CaCl2
MgSO4
CuSO4
FeSO4
Invertase activity
X7 -1(0.1)
X8 1(1.0)
X9 -1(0.01)
X10 1(0.01)
X11 -1(0.001)
(U/ml) 670.1± 0.74
-1(50)
1(100)
-1(0.1)
-1(0.1)
-1(0.01)
1(0.01)
1(0.01)
860.5± 0.32
-1(50)
-1(50)
-1(0.1)
-1(0.1)
1(0.1)
1(0.01)
1(0.01)
1200± 0.74
-1(0.5)
1(100)
1(100)
1(1.0)
-1(0.1)
1(0.1)
-1(0.001)
1(0.01)
466.8± 1.2
1(2)
1(100)
1(100)
1(1.0)
1(1.0)
1(0.1)
1(0.01)
-1(0.001)
628.2±1.14
ep te -1(0.5)
1(2)
1(100)
-1(50)
1(1.0)
1(1.0)
-1(0.01)
1(0.01)
1(0.01)
709.9± 0.22
-1(0.5)
1(2)
1(2)
-1(50)
1(100)
-1(0.1)
1(1.0)
1(0.1)
-1(0.001)
1(0.01)
639.9± 0.21
-1(6)
1(2)
1(2)
-1(0.5)
1(100)
-1(50)
1(1.0)
-1(0.1)
1(0.1)
1(0.01)
-1(0.001)
833.6± 0.74
1(8)
-1(0.5)
-1(0.5)
1(2)
1(100)
-1(50)
1(1.0)
1(1.0)
-1(0.01)
-1(0.001)
1(0.01)
656.1±1.12
Ac c
10
Glucose
X6 1(100)
d
1
an
pH
M
Run No.
-1(6)
-1(0.5)
1(2)
1(2)
-1(50)
-1(50)
-1(0.1)
1(1.0)
1(0.1)
-1(0.001)
-1(0.001)
593.8±1.14
-1(6)
-1(0.5)
-1(0.5)
-1(0.5)
1(100)
1(100)
1(1.0)
-1(0.1)
-1(0.01)
-1(0.001)
-1(0.001)
747.5± 0.72
-1(6)
-1(0.5)
-1(0.5)
-1(0.5)
-1(50)
-1(50)
-1(0.1)
-1(0.1)
-1(0.01)
-1(0.001)
-1(0.001)
233.0± 0.55
Numbers between parakeet represent actual concentrations of tested variables (g/l); R2= 99.99 % 601
26 Page 28 of 37
Table 2. Statistical analysis of Plackett- Burman design showing coefficient values, t-and P- values for each variable on invertase activity P-value
Confidence level (%)
686.617
pH
44.250
1.317
0.108
89.14
Malt extract
261.33
3.912
0.001
99.85
Peptone
-6.467
-1.329
0.137
89.54
Yeast extract
94.400
-0.733
0.240
76.00
Sucrose
44.733
2.065
Glucose
-17.783
-0.528
NaCl
-12.933
-1.152
CaCl2
-36.950
1.212
MgSO4
40.433
CuSO4
130.433
Fe2SO4
68.917
cr
Intercept
ip t
t-statistics
0.032
96.71
us
Coefficient
0.304
69.58
0.137
86.18
0.126
87.34
-1.329
0.137
86.21
3.138
0.005
99.47
2.829
0.008
99.18
an
Variables
M
602 603
607 608 609 610 611 612
pt
606
Ac ce
605
ed
604
613 614 615 616 617 618 27
Page 29 of 37
619 620 621
Table 3. Central composite design (CCD) consisting of 20 experiments for three experimental factors in coded and actual values for the production of invertase by Lactobacillus brevis Mm-6
622 Factor levels Malt extract
CuSO4
FeSO4
No.
(X1)
(X2)
(X3)
coded
Actual
(g/L)
(g/l)
-1
1
-1
0.005
2a
+1
4
-1
0.005
3a
-1
1
+1
0.02
4a
+1
4
+1
5a
-1
1
-1
6a
+1
4
-1
7a
-1
1
8a
+1
9b
-2
10b
+2
11b
0
12b
Actual
Observed
Predicted
(g/l)
-1
0.005
250±1.3
406
-1
0.005
1036±1.4
1077
-1
0.005
959±0.74
701
-1
0.005
1300±1.2
1374
0.005
+1
0.02
1359±1.2
1212
0.005
M
+1
0.02
907±3.2
1032
+1
0.02
+1
0.02
881±3.4
861
4
+1
0.02
+1
0.02
803±0.76
675
0.5
0
0.01
0
0.01
725±0.99
936
6
0
0.01
0
0.01
1062±1.3
1020
2
-2
0.001
0
0.01
1026±3.4
819
0
2
+2
0.04
0
0.01
120±2.2
230
13b
0
2
0
0.01
-2
0.001
767±1.4
691
14b
0
2
0
0.01
+2
0.04
480±1.4
436
15c
0
2
0
0.01
0
0.01
1399±3.5
1411
16c
0
2
0
0.01
0
0.01
1392±1.3
1411
17c
0
2
0
0.01
0
0.01
1396±0.72
1411
18c
0
2
0
0.01
0
0.01
1398±1.1
1411
19c
0
2
0
0.01
0
0.01
1399±2.4
1411
20c
0
2
0
0.01
0
0.01
1394±3.2
1411
pt
0.02
ed
an
1a
Ac ce 623
Coded
(U/ml)
cr
Actual
us
Coded
Invertase activity
ip t
Trial
a Fractional 23 factorial design, b star points, c central points
28
Page 30 of 37
624 625 626
Table 4. Model coefficients estimated by multiples linear regression (significance of regression coefficients)
627
630
-1513
357.820
-4.228
X1
695
146.675
4.741
X2
142561
27180.033
5.245
X3
193847
27180.033
7.132
X12
-75.334
17.441
X22
- 4.269
644166.361
X32
- 4.306
644166.361
X1X2
-149.125
4899.989
X1X3
-18896
X2X3
-2.906
ip t
Intercept
P-value
0.002 0.001
cr
t- test
0.000 0.000 0.002
-6.684
0.000
-6.628
0.000
-0.030
0.976
4899.989
-3.856
0.003
975943.688
-2.978
0.014
ed
M
-4.319
an
us
Standard error
F value =11.591; P>F= 0.0001 ; R2= 0.913; R = 0.955; Adjusted R2= .8340
pt
629
Regression coefficient
Ac ce
628
Term
29
Page 31 of 37
Fig. 1. Effect of medium composition factors on the invertase) production by Lactobacillus 634 635
Ac ce
pt
ed
M
an
us
cr
ip t
brevis Mm-6
636 637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664
30 Page 32 of 37
Fig. 2. A. Response surface plot of invertase production by Lactobacillus brevis634 Mm-6 showing the Interactive effects of different concentrations of malt extract635 and CuSO4 at 636
pt
ed
M
an
us
cr
ip t
X3= 0
637 638 639 640
Ac ce
641 642 643 644 645 646 647 648 31 Page 33 of 37
Fig. 2. B. Response surface plot of invertase production by Lactobacillus brevis634 Mm-6 showing the Interactive effects of different concentrations of malt extract635 and FeSO4 at 636
pt
ed
M
an
us
cr
ip t
X2= 0
637 638 639 640
Ac ce
641 642 643 644 645 646 647 32 Page 34 of 37
Fig. 2. C. Response surface plot of invertase production by Lactobacillus brevis634 Mm-6 showing the Interactive effects of different concentrations of CuSO4 and 635 FeSO4 at 636
X1=0
pt
ed
M
an
us
cr
ip t
637
638 639 640 641
Ac ce
642 643 644 645 646 647 648 649 650 651 652 653 654 655 33 Page 35 of 37
634
Fig. 3. Temperature-activity profile for immobilized and free invertase
635
Ac ce
pt
ed
M
an
us
cr
ip t
636
637 638 639 640 641 642 643 644 645 646 647 648 649 650 651 652 653 654 655 656 657 658 659 660 661 662 663 664 665 666 667 668 669 670
34 Page 36 of 37
Fig. 4 . Operational stability of immobilized invertase on and grafted alginate 634 635
639
Ac ce
pt
ed
M
an
us
cr
ip t
636 637 638
35 Page 37 of 37
