Improvements in the production of Aspergillus oryzae β-galactosidase crosslinked aggregates and their use in repeated-batch synthesis of lactulose

Journal Pre-proofs Improvements in the production of Aspergillus oryzae β-galactosidase crosslinked aggregates and their use in repeated-batch synthesis of lactulose Cecilia Guerrero, Carla Aburto, Sebastián Súarez, Carlos Vera, Andrés Illanes PII: DOI: Reference:

S0141-8130(19)35123-2 https://doi.org/10.1016/j.ijbiomac.2019.09.117 BIOMAC 13362

To appear in:

International Journal of Biological Macromolecules

Received Date: Revised Date: Accepted Date:

4 July 2019 16 August 2019 16 September 2019

Please cite this article as: C. Guerrero, C. Aburto, S. Súarez, C. Vera, A. Illanes, Improvements in the production of Aspergillus oryzae β-galactosidase crosslinked aggregates and their use in repeated-batch synthesis of lactulose, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac. 2019.09.117

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Improvements in the production of Aspergillus oryzae β-galactosidase crosslinked aggregates and their use in repeated-batch synthesis of lactulose. Cecilia Guerrero1*, Carla Aburto1, Sebastián Súarez1, Carlos Vera2, Andrés Illanes1. 1. School of Biochemical Engineering, Pontificia Universidad Católica de Valparaíso (PUCV), Valparaíso, Chile. 2. Department of Biology, Faculty of Chemistry and Biology, Universidad de Santiago de Chile (USACH), Santiago, Chile. *: corresponding author. Tel. 56 32- 2272035; E-mail address: [email protected]

1

Abstract Aspergillus oryzae β-galactosidase was immobilized by aggregation and crosslinking, obtaining catalysts (CLAGs) well-endowed for lactulose synthesis. Type and concentration of the precipitating agent were determinants of immobilization yield, specific activity and -1 thermal stability. CLAGs with specific activities of 64,007, 48,374 and 44,560 IUH˜g were

obtained using 50% v/v methanol, ethanol and propanol as precipitating agents respectively, with immobilization yields over 90%. Lactulose synthesis was conducted at 50°C, pH 4.5, 50 % w/w total sugars, 200 IUH˜g of enzyme and fructose/lactose molar ratio -1

of 8 in batch and repeated-batch operation. Lactulose yields were 0.19 g˜g-1 and 0.24 g˜g-1 for fructose to lactose molar ratios of 4 mol˜mol-1 and 8 mol˜mol-1 while selectivities were 3.3 mol˜mol-1 and 6.6 mol˜mol-1respectively for CLAGs obtained by ethanol and propanol precipitation. Based on these results, both CLAGs were selected for the synthesis in repeated-batch mode. The cumulative mass of lactulose in repeated-batch was higher with CLAGs produced by ethanol and propanol precipitation than with the free enzyme. 86 and 93 repeated-batches could have been respectively performed with those CLAGs considering a catalyst replacement criterion of 50% of residual activity, as determined by simulation.

Keywords: β-galactosidase; crosslinked aggregates; CLEAs.

2

1. Introduction Chemical industry is experiencing a sustained transition to greener processes with reduced generation of residues and elimination of toxic or hazardous materials, this being particularly apparent for the production of fine chemicals and pharmaceuticals1-2. Within this scenario, enzyme biocatalysis has find a place in industries of organic synthesis with no tradition in using bioactive materials. Enzymes are effective catalysts under mild operation conditions that are used in conventional reactors with considerable savings in terms of investment and operation costs, well in compliance with the principles of sustainable chemistry3-4. Main drawback of enzymes as process catalysts is their poor operational stability which is usually much lower than chemical catalysts and several strategies have been implemented to overcome this limitation, including protein engineering and immobilization engineering1,5-6. Enzyme immobilization stands out having several advantages that allow an increase in stability, better operational control, flexibility in reactor design and operation, easy recovery of product, catalyst recovery and reutilization5,7-8, and even catalyst reactivation9-10. Better control and flexibility in the design and operation of the reactor are keys aspects for the kinetically controlled synthesis of lactulose from lactose and fructose catalyzed by β-galactosidase. β-Galactosidase is poorly specific with respect to the galactose acceptor, so the galactosyl-enzyme complex formed can react either with a fructose molecule to synthesize lactulose or with other molecules of lactose forming galacto-oligosaccharides (GOS), these reactions being in competence11-13. Therefore, new strategies of operation for conducting such reaction may allow increasing yield, productivity and selectivity of lactulose synthesis. Among them, repeated-batch operation with immobilized enzymes has proved to be a sound strategy for increasing the mass of lactulose and transgalactosylated oligosaccharides (TOS) produced per unit mass of catalyst. However, the characteristics of the catalyst will be determinant for the reaction performance13-14. Several strategies have been used for Aspergillus oryzae β-galactosidase immobilization, namely entrapment, adsorption, covalent binding, crosslinking or combinations thereof 14-19. Each of them has advantages and disadvantages, and selection of the best one should take into consideration the enzyme characteristics, type of support, reaction conditions and type of reactor where the catalyst will be used

5,19-20

.

Immobilization of β-galactosidase by aggregation and crosslinking has been described, the 3

resulting catalyst representing a new generation of carrier-free biocatalysts, which are easy to prepare and comparatively cheaper since no inert support is required

2,21-22

. No pure

enzymes are required and specific activity can be considerably higher than in conventional carrier-bound enzymes. Crosslinked enzyme aggregates have acceptable mechanical properties, thermostability and resistance to organic solvents, which favor their recovery and reutilization

4, 23

; however, alteration of the enzyme structure by formation of new

chemical bonds leading to enzyme inactivation is a significant drawback. Despite this, such modifications may be purposely used for modulating the selectivity of the enzyme for a particular substrate or generating particular byproducts1,24. Lack of support may be adverse in terms of mechanical stability limiting their use under harsh reaction conditions, but this may be circumvented by encapsulation25 at the expense of reducing their specific activity. Another potential advantages of crosslinked enzyme aggregates is the possibility of coimmobilizing enzymes (combi-catalysts)2, stabilizing multimeric enzymes by precluding subunit dissociation26 and co-precipitating enzymes with ionic polymers to provide a hydrophilic microenvironment27.

Preparation of crosslinked enzyme aggregates is done in two steps: firstly, the enzymes are physically aggregated by non-denaturing precipitation and then crosslinked by a bifunctional reagent 4,13,15. Crosslinking reagent reacts with the amino groups in the surface of the protein molecule leading to the formation of insoluble, active and stable enzyme aggregates: Best precipitating agent depends on biochemical and structural characteristics of the enzyme. Most used crosslinking reagents are glutaraldehyde, ethylene polymers and polyaldehyde dextran

4,23-24

. The catalytic behavior of crosslinked enzyme aggregates is

highly dependent on the properties of the precipitating agent used28. So, the main purpose of this work is the evaluation of the type and concentration of the precipitating agent, and also the concentration of crosslinking agent and time of crosslinking on the immobilization yield and the specific activity and stability of crosslinked aggregates of A. oryzae βgalactosidase (CLAGs), aiming to determine the operational conditions maximizing these parameters. Produced CLAGs were tested as catalysts for the kinetically controlled synthesis of lactulose from lactose and fructose, evaluating the effect of the precipitating agent used on CLAGs performance in batch and repeated-batch operation. Both modes of 4

operation were compared in terms of the cumulative mass of product per unit mass of enzyme protein and in the cumulative productivity of lactulose. Repeated-batch operation allowed determining the stability and efficiency of the produced CLAGs under reaction conditions.

2. Materials and Methods 2.1. Materials Lactose monohydrate, fructose, glucose, galactose, sucrose, o-nitrophenol (o-NP), onitrophenyl-β-D-galactopyranoside (o-NPG) and galacto-oligosaccharides (GOS) standards were supplied by Sigma (St Louis, MO, USA). Lactulose was supplied by Discovery Fine Chemicals (Wimborne, UK). The precipitating agents: methanol, ethanol, 1-propanol, isopropanol, 1-butanol, 2-butanol, tert-butanol, isobutanol, 1-pentanol, 2-pentanol, 3pentanol, 4-methyl-2-pentanol, tert-pentanol, hexane, heptanol, acetone, acetonitrile, ethyl acetate and polyethylene glycol 400 were supplied by Merck (Darmstadt, Germany). All other reagents were of the highest purity available as supplied by Sigma or Merck (Darmstadt, Germany). The enzyme used was Enzeco™ Fungal Lactase Concentrate, a commercial preparation of A. oryzae β-galactosidase kindly donated by Enzyme Development Corporation, New York, USA.

2.2. HPLC analysis of the reaction products Substrates (lactose and fructose) and products (lactulose, GOS, galactose and glucose) of the reaction of lactulose synthesis were analyzed in a Jasco RI 2031 HPLC delivery system, provided with refractive index detector, isocratic pump (Jasco PU2080) and autosampler (Jasco AS 2055). BP-100 Ca++ columns (300 mm x 7.8 mm) for carbohydrate analysis (Benson Polymeric, Reno, USA) were used. Samples were eluted with milli-Q water at a flow rate of 0.5 mLwmin-1. Column and detector temperatures were 80 and 40 °C respectively. ChromPass software was used for integrating the chromatograms. Composition of samples was determined assuming the proportionality of the area of each peak to the weight percentage of the respective sugar 29.

5

Standards of galactose, lactose, glucose, fructose, lactulose, 4β-galactobiose and 3α-4β-3α galactotetraose were used to determine their retention times checking that measurements were in the linear range (0 - 50 gwL-1).

2.3. Determination of enzyme activity Hydrolysis rate was determined by measuring o-NP formation calculated from absorbance measurements at 420 nm in a Jenway 6715 spectrophotometer and the corresponding calibration curve. One international unit of hydrolytic activity (IUH) was defined as the amount of E-galactosidase that hydrolyzes 1 μmol of o-NPG per minute at 45 mM o-NPG, pH 4.5 and 40 °C 30. The free enzyme preparation had a specific activity of 186,000 IUH˜g-1 at such assay conditions 14.

2.4. Preparation and characterization of β-galactosidase immobilized by aggregation and crosslinking (CLAGs).

β-Galactosidase immobilized by aggregation and crosslinking (CLAG) was produced by precipitating A. oryzae β-galactosidase under non-denaturing conditions with different precipitating agents and then crosslinking the precipitated protein with the bifunctional reagent glutaraldehyde as previously described by Guerrero et al.13. Best conditions for CLAGs production were determined evaluating the type and concentration of the precipitating agents, the crosslinker concentration (glutaraldehyde to protein mass ratio) and time of crosslinking as variables and immobilization yield (percentage of contacted activity expressed in the CLAG, YIA) and specific activity of the biocatalysts (IU˜gbiocatalyst1

, asp) as response parameters. Nineteen solvents (methanol, ethanol, 1-propanol,

isopropanol, 1-butanol, 2-butanol, tert-butanol, isobutanol, 1-pentanol, 2-pentanol, 3pentanol, 4-methyl-2-pentanol, tert-pentanol, hexane, heptanol, acetone, acetonitrile, ethyl acetate and polyethylene glycol 400) were used as precipitating agents at concentrations ranging from 10 to 90 % v/v.

2.4.1. Effect of the precipitating agent on CLAGs formation. 6

In the first step, precipitating agents were selected in terms of YIA and asp using 85% v/v, 5.5 g of glutaraldehyde per gram of protein, and 3 and 5 h of crosslinking.

2.4.2. Effect of concentration of precipitating agent on CLAGs formation. Once the best precipitating agents were selected, the effect of their concentration was evaluated at 10, 20, 30, 40, 50, 60, 65, 70, 75, 80, 85 and 90% v/v, maintaining the crosslinker concentration and crosslinking time at 5.5 g of glutaraldehyde per gram of protein, and 3 and 5 h respectively, always using YIA and asp as response parameters. 2.4.3. Effect of crosslinker concentration on CLAGs formation The effect of crosslinker concentration at 0.5, 1.5, 3.5, 5.5 and 1 g of glutaraldehyde per gram of protein was then evaluated with the selected precipitating agents at their best concentrations, always using YIA and asp as response parameters.

2.4.4. Effect of crosslinking time on CLAGs formation Using the selected precipitating agents at their best concentration and at the best crosslinker concentration, the effect of crosslinking time was evaluated at 1, 3, 5, 12 and 24 h, always using YIA and asp as response parameters. All the experiments made on CLAGs formation were performed in duplicate and the measurements of specific activity and immobilization performance were also done in duplicate, with standard deviations never exceeding 5%.

2.5. Thermal stability of CLAGs. Thermal stability of all CLAGs was evaluated at 50°C under non-reactive conditions, to assess their potential stabilization and reutilization. Inactivation profiles for all CLAGs were adequately described by a model based on a two-stage series mechanism with no residual activity (Eq. 1), as proposed by Henley & Sadana31: 7

e eo

= ቂ1+A∙ k

k1 2 -k1

ቃ ∙exp൫-k1 ∙t൯- ቂA∙ k

k1 2 -k1

ቃ ∙exp൫-k2 ∙t൯

(Eq.1)

where e0 and e are the initial and residual enzyme activity after time t respectively, k1 and k2 are the transition rate constants from the native to the intermediate and from the intermediate to the final enzyme species, respectively, and A is the specific activity ratio of the intermediate and initial enzyme species. Stabilization factor (SF) is defined as the immobilized enzyme to the free enzyme half-life ratio, half-life (t1/2) being the time when the enzyme activity has been reduced to 50% of its initial value. The assays were carried out in duplicate, with standard deviations never exceeding 5%.

2.6. Synthesis of lactulose with CLAGs in batch operation. CLAGs were evaluated as catalysts in the kinetically controlled synthesis of lactulose from lactose and fructose. The reaction was conducted at 50°C, pH 4.5, 50% w/w total sugars concentration and fructose/lactose molar ratios (RF/L) of 4 and 8. Reaction was monitored during 7 h until reaching maximum lactulose yield, where the reaction parameters were determined according to Guerrero et al.14:

- Lactulose yield (YLu), representing the mass of lactulose synthesized (MLu) per unit mass of limiting substrate (lactose) (MLac), evaluated at the maximum lactulose concentration attained during synthesis: YLu =

MLu MLac

(Eq.2)

- Transgalactosylated oligosaccharides (TOS) yield (YTOS), defined as the ratio of TOS mass (MTOS) to the initial mass of lactose, evaluated at the maximum lactulose concentration obtained during the synthesis:

8

YT =

MT MLac

(Eq.3)

where TOS denotes transgalactosylated oligosaccharides which are inevitably synthesized along with lactulose. - Specific productivity of lactulose synthesis (πLu), representing the mass of lactulose produced per unit mass of protein in the enzyme preparation (MP) and unit time (t), evaluated at the maximum lactulose concentration attained during synthesis: ɎLu =

MLu  M ή–

(Eq.4)

- Selectivity of lactulose synthesis (SLu), representing the molar ratio of lactulose to TOS in the reaction medium, evaluated at the maximum lactulose concentration attained during synthesis: Lu =

Lu 

(Eq.5)

where NLu and NTOS represent the moles of lactulose and TOS respectively. Product distribution was then determined by analysing the amounts of lactulose and TOS synthesized. The assays were carried out in duplicate, with standard deviations never exceeding 5%. Quantification of carbohydrates was carried out as described in section 2.2.

2.7. Synthesis of lactulose with CLAGs in repeated-batch operation. CLAGs with the highest asp, and thermal stability were evaluated in the synthesis of lactulose in repeated-batch operation at 50 °C, pH 4.5 and 200 IUH·g-1 of sugars at RF/L of 8; each batch was stopped at the maximum lactulose concentration and the time of reaction was adjusted to the point of maximum lactulose yield. The parameters for the evaluation of lactulose synthesis: cumulative mass of lactulose (RC,Lu) and TOS (RC,TOS) per unit mass of contacted protein in CLAGs formation, and cumulative specific productivity of lactulose (πRB,Lu) and TOS (πRB,TOS), were defined according to Guerrero et al.13,32. 9

- Cumulative lactulose mass (RC,Lu), defined as the mass of lactulose accumulated (MLu) per unit mass of enzyme protein (Mp), evaluated at the maximum lactulose concentration obtained in each batch during synthesis: RC,Lu =

MLu MP

(Eq. 6)

- Cumulative TOS mass (RC,TOS), defined as the mass of lactulose accumulated (MLu) per unit mass of enzyme protein (Mp), evaluated at the maximum lactulose concentration obtained in each batch during synthesis: RC,TOS=

MTOS MP

(Eq. 7)

- Cumulative lactulose specific productivity (πC,Lu), defined as the mass of lactulose accumulated (MLu) per unit mass of enzyme protein (Mp) and unit of time (t), evaluated at the maximum lactulose concentration obtained in each batch during synthesis: πC,Lu =

MLu  MP ∙t

(Eq. 8)

- Cumulative TOS specific productivity TOS (πC,TOS), defined as the mass of TOS accumulated (MLu) per unit mass of enzyme protein (Mp) and unit of time (t), evaluated at the maximum lactulose concentration obtained in each batch during synthesis: πC,TOS =

MTOS  MP ∙t

(Eq. 9)

Product distribution was then determined by analysing the amounts of lactulose and TOS synthesized. The assays were carried out in duplicate, with standard deviations never exceeding 5%. Quantification of carbohydrates was done described in section 2.2.

10

3. Results and Discussion 3.1. Effect of type and concentration of precipitating agent on CLAGs formation. The precipitating agent plays an important role in the formation of crosslinked aggregates being the main responsible for the adoption of a certain enzyme configuration and eventual loss of activity

21,33-34

. Figure 1 shows the effect of the 19 precipitating agents on YIA and

asp of CLAGs obtained at 85 %v/v precipitating agent and 5.5 g of glutaraldehyde per gram of protein at three different crosslinking times. As seen, high asp of CLAGs was obtained after 1 h of crosslinking, but YIA was lower than obtained at 3 and 5h, which is due to the lower recovery of CLAGs mass at the former condition. From the 19 precipitating agents tested, propanol, isopropanol and tert-butanol allowed obtaining CLAGs with asp over 20,000 IUH˜g at YIA over 70 %, which is twice the asp reported with ammonium sulfate for -1

the same β-galactosidase13. Figure 1 Only few of the precipitating agents produced CLAGs with high asp at high YIA, which may be due to the potential deleterious effect of the high concentration used (85 % v/v). To test this, the precipitating agents were used at different concentrations in the range from 10 to 90% v/v. Experiments were conducted at 5.5 g of glutaraldehyde per gram of protein and 3 and 5 h of crosslinking time. Results are presented in Figures 2 and 3 in terms of asp and YIA respectively, showing that the maximum values of asp and YIA were strongly dependent on the precipitating agent used and its concentration. Significant differences were also obtained between the values obtained at 3 and 5 h of crosslinking; however, their maximum values were obtained at the same precipitating agent concentration for both crosslinking times. In the case of acetone, higher asp of CLAGs was obtained at 65% v/v, corresponding to 25,220 IUH˜g-1 and 68 % YIA for 5 h of crosslinking time (Figures 2a and 3a). In the case of methanol higher asp and YIA were obtained at 50 % v/v and 3 h of crosslinking time, being 64,007 IUH˜g-1 and 100 %, respectively (Figures 2b and 3b). In the case of ethanol (Figures 2c and 3c) higher values of asp and YIA were 49,173 IUH˜g and 90%, obtained at -1

the same conditions as in methanol. In the case of propanol (Figures 2d and 3d) higher asp -1 and YIA were obtained at 50 % v/v, being 44,560 IUH˜g and 90%, respectively. In the case

11

of isopropanol (Figures 2e and 3e), higher asp and YIA were obtained at 60 % v/v, being -1 30,852 IUH˜g and 74%, respectively. In the case of tert-butanol (Figures 2f and 3f), higher -1 asp and YIA were obtained at 65 % v/v, being 42,154 IUH˜g and 80%, respectively. In the

case of methanol and ethanol, highest values were obtained at 3 h of crosslinking; for the other solvents, highest values were obtained at 5 h of crosslinking. Then, concentration and crosslinking time yielding the highest values of asp and YIA were selected for each precipitating agent used. Figure 2 Figure 3 The effect of the type of precipitating agent on asp and YIA (Figure 2 and Figure 3) agreed well with results reported for crosslinked enzyme aggregates of Geobacillus thermodenitrificans X1 xylanase, where YIA over 90 % were obtained when using ethanol, acetone, isopropanol and propanol as precipitating agents33. Amaral-Fonseca et al.21 reported higher values of asp Aspergillus niger amyloglucosidase precipitated with acetone, ethanol and isopropanol than obtained with ammonium sulfate and similar results were reported for β-mannanase 21,35. Values of asp and YIA in Figures 2 and 3 are significantly higher than previously reported for crosslinked aggregates of the same enzyme. Gaur et al. (2006) reported a YIA of only 13,5 %, which is about 20 % of the lower value obtained in this work, while Guerrero et al.13 reported values of asp of 15,000 IUH˜g and YIA of 30% for CLAGs obtained by -1

precipitation with ammonium sulfate, which are much lower than all values here reported. Differences in YIA among precipitating agents may be due to the different mechanisms of aggregation they exert as a consequence of changes in the hydration of the enzyme molecules or in the dielectric constant of the solution21. Values of asp and YIA shown in Figures 2 and 3 are very much higher than reported by A. oryzae β-galactosidase immobilization in carrier-bound systems. Guerrero et al. (2017), evaluated the immobilization in functionalized agarose matrices, namely, monofunctional glyoxylagarose and heterofunctional amino-glyxoxyl-, carboxy-glyoxyl- and chelate-glyoxylagarose; highest asp and YIA values were obtained by immobilization in amino-glyxoxyl12

agarose, being 3676 IUH˜g and 30%, respectively14. Similar results were reported by -1

immobilization on heterofunctional

chitosan, obtaining 2951

IUH˜g

-1

and 30%

respectively17. Values of asp are clearly much lower (more than one order of magnitude) than in CLAGs because in carrier-bound systems a significant portion of the catalyst mass is inert support material. In this way, highly active and stable catalysts were obtained by aggregation and crosslinking of A. oryzae β-galactosidase using different type and concentrations of precipitating agents, establishing that the biocatalyst characteristics are highly dependent on them.

3.2. Effect of crosslinker concentration on CLAGs formation. CLAGs were prepared without co-feeders or any other aid and were formed using glutaraldehyde as crosslinking agent. Table 1 shows the effect of crosslinker concentration (g of glutaraldehyde per g of protein) at 3 h of crosslinking on asp and YIA of CLAGs produced by the six precipitating agents selected and at 3 h of crosslinking time. Table 1 shows that there is no crosslinked aggregates formed with all six precipitating agents at 0.5 and 1.5 g of glutaraldehyde per gram of protein, except for the case of acetone precipitation in which CLAGs are formed at 1.5 g of glutaraldehyde per gram of protein. Table 1 In the case of methanol, ethanol, propanol and tert-butanol precipitation, the high glutaraldehyde concentration (11 g per gram of protein) caused a reduction in expressed enzyme activity and immobilization yield, which is due to excessive interaction between the crosslinker and the enzyme protein, altering the structural conformation of the enzyme molecules. This leads to the formation rigid and big CLAGs, so that diffusional restrictions increase and asp decreases 33,36. For the CLAGs obtained by methanol, ethanol, propanol, isopropanol or tert-butanol precipitation, maximum values of asp and YIA were obtained at a glutaraldehyde concentration of 5.5 g per gram of protein. In the case of acetone precipitation, maximum 13

asp was obtained at 11 g of glutaraldehyde per gram of protein, but value was only slightly higher than obtained at 5.5 g per gram of protein Table 1). Since high glutaraldehyde concentration may cause excessive modification of the enzyme protein36, the latter was selected for CLAGs production with the six selected precipitating agents. This concentration is the same reported for CLAGs produced by ammonium sulfate precipitation13,15 and is within the ranges reported by other crosslinked aggregates of other enzymes33-34,37-38.

3.3. Effect of crosslinking time on CLAGs formation. Crosslinking time is a key parameter in the formation of CLAGs. If too short, structural integrity will be poor and aggregates will be soft and hard to recover, leading to low asp and YIA. If too long, excessive modification of protein structure will produce stiff particles increasing diffusional restrictions and therefore reducing asp and YIA33,36. Table 2 shows the effect of crosslinking time on asp and YIA, in the range from 1 to 24 h at 5.5 g of glutaraldehyde per g of protein and the concentration of precipitating agents previously selected (Table 1). CLAGs obtained by methanol precipitation presented maximum asp and YIA at 1 hour of crosslinking, while at 3 h for CLAGs obtained by ethanol precipitation and 5 h for CLAGs obtained by acetone, propanol, isopropanol and tert-butanol precipitation. At 12 and 24 h of crosslinking activity of all CLAGs was reduced, being the effect more pronounced in the case of CLAGs obtained by tert-butanol and propanol precipitation, which is in agreement with previous reports on crosslinked aggregates of other enzymes 3334,36-37

. Table 2

3.4. Thermal stability of CLAGs. Figure 4 shows the thermal inactivation profiles at 50°C under non-reactive conditions of CLAGs produced with the six selected precipitating agents. Figure 4 As seen, all seven CLAGs have inactivation patterns quite similar than the one of the free enzyme, which is in agreement with previous reports for this type of immobilization13. 14

However, significant increase in thermal stability has been reported for the same enzyme immobilized in glyoxyl-agarose and amino-glyoxyl-agarose14 where the inert support contributes to a more robust catalyst structure. Having no scaffold, stability of enzymes is carrier-frees systems is usually be similar than the stability of the soluble counterpart13. Table 3 shows the values of the inactivation parameters of CLAGs obtained by non-linear fitting of the experimental data to a two-stage series mechanism with no residual activity31. Table 3 Half-life (t1/2) of all biocatalysts was also determined. Those of acetone and methanol precipitated CLAGs were similar than reported for the free enzyme and ammonium sulfate precipitated CLAGs13, while those precipitated with propanol and isopropanol were more stable, with t1/2 2.34 and 1.84 times the one of the free enzyme, respectively. CLAGs obtained by ethanol and tert-butanol precipitation, had a t1/2 1.58 times higher than reported for the free enzyme and ammonium sulfate precipitated CLAGs13. In this way, more stable catalysts have been obtained with CLAGs obtained by ethanol, propanol, isopropanol and tert-butanol precipitation (Table 3), which is important in terms of process economics. Values of t1/2 here obtained are similar than those reported for the immobilization of A. oryzae β-galactosidase by covalent linkage to mono- and heterofunctional agarose supports14. Type and concentration of the precipitating agent were crucial in the production of CLAGs, having a strong effect on asp and YIA, as well as in the catalysts’ thermal stability.

3.5. Synthesis of lactulose with CLAGs in batch operation. CLAGs produced with the six selected precipitating agents were used as catalysts for the kinetically controlled reaction of lactulose synthesis from lactose and fructose, with the purpose of determining their effect on the catalytic performance of CLAGs. Values of the reaction parameters of lactulose synthesis: yield (YLu), selectivity (SLu/TOS) and specific productivity (πLu), are presented in Figure 5. Figure 5 15

Values of YLu, YTOS and SLu/TOS were similar for all CLAGs, irrespective of the precipitating agent used. However, πLu varied according to the precipitating agent used, being higher for CLAGS produced by ethanol, propanol, methanol and tert-butanol precipitation than by isopropanol and acetone precipitation, at both substrates ratios evaluated. Values of YLu, YTOS and SLu/TOS are similar than reported for ammonium sulfate precipitated CLAGs13, but lower than obtained by carrier-bound immobilized A. oryzae βgalactosidase, which might be a consequence of more severe diffusional restrictions in crosslinked enzyme aggregates. On the other hand, πLu obtained with CLAGs are higher than obtained with the enzyme bound to heterofunctional amino-glyoxyl-agarose and chelate-glyoxyl-agarose supports14. Since YLu, YTOS and SLu/TOS were similar for all CLAGs in the batch synthesis of lactulose (Figure 5), CLAGs produced by ethanol and propanol precipitation were evaluated in repeated-batch mode, since they exhibited high asp and the highest t1/2 (Tables 1 and 3). Methanol precipitated CLAGs, despite having the highest asp, was discarded because of its poor thermal stability and its difficult disintegration.

3.6. Synthesis of lactulose with CLAGs in repeated-batch operation. For the evaluation of catalyst recovery and reutilization, nine and eight consecutive batches were performed with CLAGs obtained by ethanol and propanol precipitation respectively. Figure 6 (a and b) presents the values of RC,Lu and RC,TOS produced per gram of contacted protein in CLAGs. In order to provide a fair comparison between CLAGs and the free enzyme, the values of productivity are expressed per unit mass of contacted protein in CLAGs formation. Figure 6 (c and d) presents the values of πRB,Lu and πRB,TOS attained with the free enzyme and CLAGs. Figures 6a and 6b show that higher values of RC,Lu and RC,TOS were obtained with ethanol than with propanol precipitated CLAGs. This may be due to the higher asp of the former, so that biocatalyst mass used was lower. Figure 6a shows that only two repeated batches with ethanol precipitated CLAGs were required to match the RC,Lu obtained with the free enzyme, while three were required with propanol precipitated CLAGs. In the case of RC,TOS, three repeated batches were required to match the performance of the free enzyme, 16

both in the case of using ethanol and propanol precipitated CLAGs. With conventional ammonium sulfate precipitated CLAGs, four and three repeated-batches were required to match the RC,Lu and RC,TOS of the free enzyme, respectively, both at RF/L of 4 and 1213. These results compare favorably with those obtained with A. oryzae β-galactosidase immobilized in glyoxyl-agarose and in heterofunctional amino-glyoxyl agarose, where five and three repeated-batches were required respectively to match the performance of the free enzyme32. Figure 6 With respect to πRB,Lu and πRB,TOS, values were only slightly higher with ethanol than with propanol precipitated CLAGs (Figure 6c and 6d) since YLu and YTOS were similar in both cases (Figure 5). This slight difference is due to the differences in asp, (higher for ethanol than for propanol precipitated CLAGs), which affect the amount of lactulose and TOS obtained per unit mass of protein in the CLAGs in each batch. The ratio of specific productivity obtained in each batch in repeated batch operation with respect to the one obtained with the free enzyme in batch operation was calculated and values are presented in Figure 6 (e and f). As can be seen, biocatalyst inactivation is reflected in the productivity of each batch, since progressively longer reaction times were required to attain the same final lactulose conversion batch after batch in repeated-batch operation. This occurred with both CLAGs, specific productivity being always lower than with the free enzyme, since the free enzyme is stable enough not experimenting significant activity loss in one batch. Inactivation is however relevant when the biocatalyst is used in several repeated-batches. The value of asp of both CLAGs was then determined at the beginning of each repeated-batch, so that their operational stability could be assessed. CLAGs thermal inactivation under reaction conditions was well described by a two-stage series mechanism, as in the case of inactivation under non-reactive conditions (Table 3). Values of t1/2 of ethanol and propanol precipitated CLAGS under reaction conditions were 2.34 y 2.75 d-1 respectively, which means that the latter can be reused more times in repeated-batch operation. To verify this, the long-term operation in repeated-batch was simulated, estimating the efficiency of biocatalyst use (mass of product per unit mass of biocatalyst) at different biocatalyst replacement criteria (Figure 7). 17

Figure 7 As seen in Figure 7a, at 90 and 50 % replacement criteria, more repeated-batches can be performed with propanol than with ethanol precipitated CLAGs, while the opposite occurs at replacement criteria from 5 to 40%. This may be explained in terms of the two-stage series inactivation mechanism, where the first stage of inactivation is less severe for propanol than for ethanol precipitated CLAGs, while in the second stage the situation reverses. This feature is critical in repeated-batch operation, strongly affecting the efficiency of biocatalyst use. Figures 7b and 7c show such efficiencies with respect to lactulose and TOS, respectively, where again the selection of the best biocatalyst is strongly dependent on its replacement criterion. Figure 7b shows that, despite its lower asp, the efficiency of biocatalyst use favors propanol over ethanol precipitated CLAGs for replacement criteria between 90 and 20% residual activity, because of its higher stability under reaction conditions allowing it to be used in a higher number of batches. In the case of TOS the same situation as in lactulose occurs for replacement criteria between 90 and 30% residual activity, but at values lower than 30% the efficiency of biocatalyst use is higher for ethanol precipitated CLAGs. These results highlight that having a biocatalyst obtained at a high YIA and with high asp is not the whole picture and its performance should be evaluated under the operation conditions of the reaction of interest to establish its potential for industrial application.

Acknowledgements Work financed by Chilean Fondecyt Grant 1160216 and 11180282, RED PCI-Conicyt 170057 and the postdoctoral fellowship DI 37.0/2019 of the Pontificia Universidad Católica de Valparaíso. We acknowledge the generous donation of β-galactosidase from Enzyme Development Corporation.

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23

Figure Captions

Figure 1: Effect of the precipitating agent at 85% v/v on a) immobilization yield (Y IA) and b) specific activity (asp) of CLAGs obtained at 5.5 g glutaraldehyde per gram of protein at („): 1 h, („): 3 h and ( ): 5 h of crosslinking time. Figure 2: Specific activity (asp) of CLAGs obtained at different concentrations of the precipitating agents at 5.5 g glutaraldehyde per gram of protein and („): 3 h and ( ): 5 h of crosslinking time. Figure 3: Immobilization yield (YIA) of CLAGs obtained at different concentrations of the precipitating agents at 5.5 g glutaraldehyde per gram of protein and („) 3 h and ( ) 5 h of crosslinking tim Figure 4: Thermal inactivation at 50°C under non-reactive conditions in citrate-phosphate buffer pH 4.5 of CLAGs produced by precipitation with (a) acetone, (b) methanol, (c) ethanol, (d) propanol, (e) isopropanol and tert-butanol. Dashed line represents the thermal inactivation of the free enzyme at the same temperature and pH reported by Guerrero et al.13. Figure 5: a) Lactulose yield (YLu,); b) TOS yield; c) selectivity (SLu/TOS); d) productivity (πLu) obtained in the kinetically controlled synthesis of lactulose from lactose with CLAGs produced with the selected precipitating agents (acetone, methanol, ethanol, propanol, isopropanol and tert-butanol). Reaction conditions were: 50°C, pH 4.5, 50 % w/w total sugars concentration and fructose/lactose molar ratios of 4: („), and 8: ( ). Figure 6: Cumulative mass of lactulose (RC, Lu) and transgalactosylated oligosaccharides (RC, TOS) per unit mass of contacted protein (a and b). Cumulative specific productivity of lactulose (π RB,Lu ) and TOS (πRB,TOS ) (c and d). Ratio of specific productivity of lactulose and TOS obtained with CLAGs in repeated batch operation and specific productivity of lactulose and TOS obtained with free enzyme in batch operation (π B,Lu and πB,TOS) (e and f). Reaction conditions were: 50°C, pH 4.5, 50 % w/w total sugars concentration and fructose/lactose molar ratio of 8. Batch operation with free enzyme („), repeated-batch operation with ethanol precipitated CLAGs ( ) and propanol precipitated CLAGs („). Figure 7: Number of repeated-batches at different biocatalyst replacement criteria (90 %; 75 %; 50 %; 25 %; 15 % of residual activity). (a); efficiency of biocatalyst use (kg. of lactulose per g of biocatalyst) (b); efficiency of biocatalyst use (kg. of TOS per g of biocatalyst) (c) .Simulation of lactulose synthesis with ethanol precipitated CLAGs ( „) and propanol precipitated CLAGs ( ). Reaction conditions: 50 °C, pH 4.5, 50% (w/w) total carbohydrates, 200 IUH glactose−1 and fructose/lactose molar ratio of 8.

Figure 1 30000 Acetone

4-methyl-2-pentanol

3-pentanol

2-pentanol

1-pentanol

tert-butanol

Isobutanol

2-butanol

1-butanol

Isopropanol

1-propanol

Ethanol

Methanol

Ethyl acetate

PEG-400

Acetonitrile

Hexanol

0 Heptanol

10000

Heptanol

20000 t-pentanol

b)

Hexanol

40000

t-pentanol

4-methyl-2-pentanol

3-pentanol

2-pentanol

1-pentanol

tert-butanol

Isobutanol

2-butanol

1-butanol

Isopropanol

1-propanol

Ethanol

Methanol

Ethyl acetate

PEG-400

Acetonitrile

Acetone

asp (IUH g -1) YIA (%) 75

60

45

a)

30

15

0

80000

30000

a s p (UIH g -1)

a s p (UIH g -1)

60000

20000 15000 10000

50000

b) a s p (UIH g -1)

a)

25000

60000

70000

50000 40000 30000

c)

40000 30000 20000

20000 10000

5000

10000 10 20 30 40 50 60 65 70 75 80 90

10 20 30 40 50 60 65 70 75 80 90

10 20 30 40 50 60 65 70 75 80 90

Ethanol Concetration (% v/v)

Methanol Concetration (% v/v)

Acetone Concetration (% v/v) 40000

50000

d)

50000

f)

e)

40000

40000

30000

20000

a s p (UIH g -1)

30000

a s p (UIH g -1)

a s p (UIH g -1)

0

0

0

20000

30000

20000

10000 10000

10000

0

0

0 10 20 30 40 50 60 65 70 75 80 90

Propanol Concetration (% v/v)

Figure 2

10 20 30 40 50 60 65 70 75 80 90

10 20 30 40 50 60 65 70 75 80 90

Isopropanol Concetration (% v/v)

Tert-butanol Concentration (% v/v)

100

100

a)

80

YIA (%)

YIA (%)

80

60

60

40

40

20

20

0

b)

80

YIA (%)

100

0 10 20 30 40 50 60 65 70 75 80 90

Ethanol Concetration (% v/v)

Methanol Concetration (% v/v) 100

d)

80

YIA (%)

YIA (%)

80

100

60

40

20

20

10 20 30 40 50 60 65 70 75 80 90

Propanol Concetration (% v/v)

Figure 3

80

60

40

20

0

0

f)

e) YIA (%)

100

40

40

10 20 30 40 50 60 65 70 75 80 90

Acetone Concetration (% v/v)

60

60

20

0 10 20 30 40 50 60 65 70 75 80 90

c)

0 10 20 30 40 50 60 65 70 75 80 90

Isoropanol Concetration (% v/v)

10 20 30 40 50 60 65 70 75 80 90

Tert-butanol Concentration (% v/v)

1.0

a)

0.8

e/eo

0.6

0.4

0.2

0.2

5

10

15

20

0.0 0

Time (days)

0

20

d)

e)

0.2

0.2

0.6

e/eo

0.4

0

10

Time (days)

Figure 4

20

0.4 0.2 0.0

0.0

0.0

f)

0.8

0.6

0.4

20

1.0

0.8

0.6

10

Time (days)

1.0

e/eo

e/eo

10

Time (days)

1.0 0.8

0.4 0.2

0.0 0

c)

0.6

0.6

0.4

0.0

0.8

b) e/eo

0.8

e/eo

1.0

1.0

0

10

Time (days)

20

0

10

Time (days)

20

0.4

0.10

b) 0.08

YTOS ( g g -1)

YLu ( g g -1)

a) 0.2 0

0.06

0.04

0.02 0.0

0.00

0.02

8

4

2

0

Figure 5

d) πLU (gg h-1 g -1)

mol -1)

6

S LU/TOS (mol

c)

00.01

0.00

4

4

RC,T OS (gTOSS mgPortein -1)

RC, Lu (gLuu mgPortein -1)

a) 3

2

1

0 1

2

3

4

5

6

7

8

b) 3

2

1

0

9

1

2

3

4

5

6

7

0.12

9

8

9

0.12

c) 0.08

0.04

0

d) 0.08

0.04

0 1

2

3

4

5

6

7

8

9

1

2

3

4

Batch

5

6

7

Batch 1.2

1.2

e)

f) πRB,TOS · πB,TOS -1

πRB,Luu πB,Lu -1

8

Batch πRB,TOS (gTOS· mgPortein -1· h -1 )

πRB,Lu (gLUU mgPortein -1 h -1 )

Batch

0.8

0.4

0.8

0.4

0

0 1

2

3

4

5

Batch

Figure 6

6

7

8

9

1

2

3

4

5

Batch

6

7

8

9

25

a)

200

100

20

8

b)

15

10

5

0

0 90 80 70 60 50 40 30 20 10

Replacement of biocatalyst (% residual activity)

Figure 6

5

90 80 70 60 50 40 30 20 10 5

Replacement of biocatalyst (% residual activity)

Efficiency of biocatalyst use (KgTOS· g -1)

300

Efficiency of biocatalyst use (Kg Lu· g -1 )

Number of Batches

400

c) 4

0 90 80 70 60 50 40 30 20 10 5

Replacement of biocatalyst (% residual activity)

Table 1: Effect of crosslinker (glutaraldehyde) concentration on the specific activity (a sp) and immobilization yields (YIA) obtained with CLAGs at different concentrations of the selected precipitating agents and 3h (*) and 5 h (**) of crosslinking.

Glutaraldehyde concentration (gglut ˜ gprot-1)

Acetone** (65 % v/v) asp (IU/g)

YIA

Methanol* (50 % v/v) asp

(%) ( IU /g)

Ethanol* (50 % v/v)

Propanol** (50 % v/v)

Isopropanol** (60 % v/v)

Tert-butanol** (65 % v/v)

YIA

asp

YIA

asp

YIA

asp

YIA

asp

YIA

(%)

( IU /g)

(%)

( IU /g)

(%)

( IU /g)

(%)

( IU /g)

(%)

0.5

-

-

-

-

-

-

-

-

-

-

-

-

1.5

9,776

16.8

-

-

-

-

-

-

-

-

-

-

3.5

19,946

48.5 52,253

5.5

25,220

11

26,557

100

27,147

53

42,749

67.2

23,853

51.3

-

-

64,007

100

48,374

94

44,560

90

30,852

74

42,154

80

68.3 44,615

80.5

42,811

86

41,501

71

30,542

74

28,346

56

68

Table 2: Effect of crosslinking time on the specific activity (a sp) and immobilization yields (YIA) of CLAGs obtained with the selected precipitating agents and concentrations at 5.5 g of protein per g of glutaraldehyde. Crosslinking time

Acetone (65 % v/v)

Methanol (50 % v/v)

Ethanol (50 % v/v)

Propanol (50 % v/v)

Isopropanol (60 % v/v)

Tert-butanol (65 % v/v)

Act

YIA

Act

YIA

Act

YIA

Act

YIA

Act

YIA

Act

YIA

(UI/g)

(%)

(UI/g)

(%)

(UI/g)

(%)

(UI/g)

(%)

(UI/g)

(%)

(UI/g)

(%)

1

21,330

57

80,889

100

18,280

38

28,999

50

25,150

61

21,664

40

3

22,172

59

64,007

100

48,374

94

33,697

60

27,455

65

26,222

46

5

25,220

68

35,302

67

37,695

90

44,560

90

30,852

74

42,154

80

12

21,136

54

31,610

62

31,353

67

19,243

32

27,491

56

14,166

30

24

19,475

47

29,838

56

19,546

43

25,550

37

19,757

43

24,726

51

(h)

Table 3: Parameters of thermal inactivation of CLAGs produced with different precipitating agents, at 50 °C under non-reactive conditions in citrate-phosphate buffer pH 4.5, determined according to a twostage series mechanism with no residual activity. k1 and k2 are the transition rate constants from the native to the intermediate and from the intermediate to the final enzyme species, respectively, A is the specific activity ratio of the intermediate and initial enzyme species, and t1/2 is the catalyst half-life. R2 is the adjusted correlation coefficient. Precipitating agent

k1 (days-1)

k2 (days-1)

A

R2

t 1/2 (days)

Free Enzyme*

0.076

0.0043

0.71

1.0

3.9

Acetone

6.26

0.089

0.712

0.98

4.1

Methanol

0.47

0.043

0.473

1.0

4.2

Ethanol

7.94

0.109

0.962

0.99

6.2

Propanol

0.54

0.036

0.648

0.98

9.2

Isopropanol

10.9

0.093

0.965

0.98

7.2

Terbutanol

0.43

0.024

0.510

0.98

6.2

*Data reported by Guerrero et al.(2015)

Highlights (85 caracteres) - Specific activity of CLAGs depended on type and concentration of precipitating agent - Highly active and stable CLAGs were obtained for catalyzing lactulose synthesis - Lactulose productivity with CLAGs was higher than reported with other catalysts - Repeated-batch operation allowed obtaining a high efficiency of biocatalyst use - 93 repeated-batches could be done with CLAGs for a replacement criterion of 50 %.