Preliminary investigations into the synthesis of lipase cross-linked enzyme aggregates (CLEA) for hydrolysis of lipid-rich wastewater

doi:10.21203/rs.3.rs-2576515/v1

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Preliminary investigations into the synthesis of lipase cross-linked enzyme aggregates (CLEA) for hydrolysis of lipid-rich wastewater

https://doi.org/10.21203/rs.3.rs-2576515/v1

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Cross-linked enzyme aggregates (CLEA) from Rhizopus oryzae lipase (RoL) was prepared using ammonium sulphate and glutaraldehyde as the precipitant and cross-linker respectively. The lipase CLEA was characterized via scanning electron microscopy. Thereafter, the CLEA was utilized in the hydrolysis of lipid-rich wastewater. The purified CLEA could be reused up to five cycles and its catalysis increased free fatty acid levels in wastewater by 84.8% and 142.1% at 0.5% and 1% (w/v) CLEA respectively. RoL CLEA is a unique biocatalyst in treatment of oil-contaminated wastewater. Hence, the possible large-scale remediation of oil-contaminated water bodies using this immobilized enzyme could be exploited.

Applied & Industrial Microbiology

Lipase

Rhizopus oryzae

cross-linked enzyme aggregates

wastewater treatment

The hydrolysis of triglycerides to free fatty acids and glycerol is catalyzed by lipases (triacylglycerol acylhydrolases; EC 3.1.1.3) (Ayinla et al., 2017). Besideshydrolysis, lipases catalyze various synthetic reactions, such as transesterification, in low water activity media (Ferreira-Dias et al., 2013).

It is widely known that severe environmental pollution can result from oil-contaminated wastewater. The diffusion of oxygen from air into water is prevented by the formation of oil film on water surfaces, causing the death of numerous aquatic lives (Mendes et al., 2010). Also, particles in wastewater, especially aggregates formed by oil droplets, can also cause blockage in drainage lines (Demirel et al., 2005). Asides the aforementioned environmental problems, there are also operational problems, which may arise from the non-treatment of wastewater with high fat contents, such as granular biomass flotation (Mendes et al., 2006), fat scum layers forming at the surface of the reactor as well as fats solidifying at lower temperatures leading to clogging and unpleasant odours (Masse et al., 2001).

Employment of specific enzymes such as lipases has gained wide attention due to the clean and friendly nature of enzyme applications, as well as the firm environmental regulations (Mendes et al., 2010). Previous studies have shown the feasibility of enzymatic treatment of lipid-rich wastewater which aids in increasing its anaerobic biodegradability (Mendes et al., 2006).

Free enzymes can only be utilized once in solutions because they are generally soluble and unstable. They are also susceptible to and frequently inactivated by various environmental conditions including inhibitors, ionic strength and pH (Jeganathan et al., 2007). Most importantly, they are too expensive to be used in wastewater treatment. These challenges can be overcome by immobilization which has its unique benefits.

Immobilization confers certain advantages on the enzymes such as increased stability at different temperature, pH and ionic strength, as well as recyclability from the reaction mixture (El-Sayed et al., 2016). Different methods have been employed for lipase immobilization over the years, including cross-linking, adsorption, multipoint covalent attachment and physical entrapment (Villeneuve et al., 2000), with each having its advantages, disadvantages and peculiarities. The cross-linking of enzyme molecules' physical aggregates results in the formation of cross-linked enzyme aggregates (CLEAs) (Ademakinwa, 2021). Some advantages over carrier-bound immobilized enzyme is the important savings on the cost of the manufactured superstructure due to the lack of support, the simplicity and broad applicability of the technique (Cao, 2005) and the reality that no previous purification of the enzyme is required (Guauque Torres et al., 2013; Ademakinwa, 2021). The study aimed to immobilize Rhizopus oryzae lipase by cross-linking and evaluate the application of the immobilized enzyme in wastewater hydrolysis.

Materials

p-nitrophenyl butyrate (pNPB), ammonium sulfate, p-nitrophenol, glutaraldehyde, tritonX-100, oleic acid, isopropanol, toluene, sodium dihydrogen phosphate, disodium hydrogen phosphate and methyl heptadecanoate were purchasedfrom Sigma-Aldrich (St. Louis, USA). Pierce^TMBCA (bicinchoninic acid) protein assay kit was obtained fromThermoFisherScientific™(Massachusetts, USA). All the chemicals and reagents usedwere of analytical grade.

Preparation of Cross-linked Enzyme Aggregates (CLEAs) of RoL-ZAC3

Crude (Ayinla et al., 2017) and purified RoL-ZAC3 (Ayinla et al., 2021) were prepared as previously reported and immobilized by cross linking with glutaraldehyde to form CLEAs, which is a simple and robust immobilization method to provide highly stable enzymes with superior retention of activity. Ammonium sulfate was progressively added to the enzyme solution to reach 50% saturation, and the mixture was stirred on a magnetic stirrer at 4 ^oC for 30 min before glutaraldehyde was added in a dropwise manner to reach a final concentration of 0.125% (v/v) (Ademakinwa et al., 2018; Ademakinwa, 2021). The mixture was again subjected to magnetic stirring at 4 ^oC for 24 h, centrifuged at 4000 rpm and the recovered pellets were washed with phosphate buffer (50 mM, pH 8), dried and stored at 4 ^oC (Lai et al., 2012).The activity of the CLEA was determined by the modified copper soap colorimetry assay method (Wang et al., 2017) which estimates the amount of fatty acids released from the hydrolysis of triglycerides. The substrate consisted of 1 ml of emulsion of olive oil–isopropanol (1:10 v/v) and 1 ml of phosphate buffer (50 mM, pH 8) which was pre-incubated for 5 min at 37 ^oC. Then, 0.1 mg of CLEA was added to initiate the reaction. The reaction was stopped with 1 ml of toluene after 15 min and the upper organic phase containing the free fatty acids was separated. To this phase, 1 ml of 100 mM copper acetate was added and the absorbance was read at 710nm and oleic acid standard curve was used to estimate the fatty acid concentration. After each cycle of catalysis, the CLEA was washed and reused to catalyze another round of olive oil hydrolysis. The CLEA was also characterized by obtaining a scanning electron micrograph of the CLEA and its size was determined.

$$\text{C}\text{L}\text{E}\text{A} \text{A}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y} (\text{U}/\text{g})=\frac{\text{A}\text{b}\text{s} \times \text{V}}{\text{m}\times \text{M}\times \text{t}}$$

where Abs = Absorbance at 710nm

V = Volume of fatty acid solution

m = slope of oleic acid standard curve

M = mass of CLEA

t = reaction time

$$\text{A}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y} \text{R}\text{e}\text{c}\text{o}\text{v}\text{e}\text{r}\text{y} \left(\text{%}\right)=\frac{\text{t}\text{o}\text{t}\text{a}\text{l} \text{a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y} \text{o}\text{f} \text{C}\text{L}\text{E}\text{A} \left(\text{U}\right)}{\text{t}\text{o}\text{t}\text{a}\text{l} \text{f}\text{r}\text{e}\text{e} \text{e}\text{n}\text{z}\text{y}\text{m}\text{e} \text{a}\text{c}\text{t}\text{i}\text{v}\text{i}\text{t}\text{y} \text{u}\text{s}\text{e}\text{d} \text{f}\text{o}\text{r} \text{C}\text{L}\text{E}\text{A} \text{p}\text{r}\text{o}\text{d}\text{u}\text{c}\text{t}\text{i}\text{o}\text{n} \left(\text{U}\right)}\times 100$$

Waste Water Characterization

Lipid Content Determination

The determination of the lipid content of the treated and pre-treated wastewater was done according to the method of Prasad and Manjunath (2011). Diluted HCl (1:1) was used to acidify 250 ml of wastewater sample to pH 2.0. Thirty milliliters of 1,1,2-trichlorotrifluoroethane was repeatedly used to extract the lipids in the wastewater. This was done until the solvent phase was clear and there was no oil layer shown in the aqueous phase. The solvent extracts were combined and dried by evaporating at 70^oC.The amount of lipid present in the sample was indicated by the dry weight obtained following evaporation.

Determination of Chemical Oxygen Demand (COD)

The wastewater sample (100 ml) was digested overnight using 10ml of 0.1N potassium dichromate and 20ml of concentrated sulphuric acid. After 24 hours of digestion, 100 ml of distilled water and 5 ml of orthophosphoricwere added to the mixture. The blank was obtained by using distilled water in place of the sample. The sample mixture was then titrated against 0.1N ferrous ammonium sulphate with a few drops of 5% barium diphenylamine sulphonate as the indicator (Goltermanet al., 1978; Ademakinwa et al., 2020).

Enzymatic Wastewater Hydrolysis

The hydrolysis reactions were carried out in 250 ml flasks containing 50 ml of raw wastewater (from vegetable oil factory). The wastewater pH was adjusted to 8, followed by the addition of the required lipase amount (0.5 or 1% w/v of the immobilized CLEA) and sodium ions (0.1% w/v) as additive for the lipase activity. Reactions were carried out at 40 ^oC at a constant speed of 150 rpm in a shaker for 4 h. An aliquot of sample (1 ml) was withdrawn from the reaction and transferred to a new tube. Toluene (1 ml) was added to the sample to extract the fatty acid and to inactivate the enzyme. This was followed by the addition of 1 ml of 100 mM copper acetate in order to determine the concentration of free fatty acids released. Absorbance was read at 710nm and fatty acid concentration was estimated from the oleic acid standard curve. The free fatty acids produced during the hydrolysis reaction were used to calculate the percentage hydrolysis.

Cross-linking the lipase with glutaraldehyde was a form of immobilization to create stable cross-links (Schiff bases) between the aldehyde group of the glutaraldehyde and the lipase amino group (Lai et al., 2012; Rodrigues et al., 2016a, Ademakinwa, 2021). The scanning electron micrograph of the cross-linked lipase (RoL-ZAC3-CLEA) showed that it is an amorphous structure with many cavities and a high surface area, an indication that the cross-linked enzyme molecules are easily accessible to the substrates in order for reactions to proceed (Fig. 1).

Both crude and purified lipases were cross-linked and aggregated and both were active in catalyzing the hydrolysis of olive oil to oleic acid. Though the purified CLEA gave higher activity per cycle compared to the crude, it lost activity completely after the fifth cycle. The crude did not show as much activity as the purified, however it was still active up to the eighth cycle (Fig. 2A and 2B). The fact that the crude enzyme could be used directly makes the procedure an economically viable one. The crude also showed full activity recovery consistent all through the eight cycles (Fig. 2C).

Table 1

Wastewater Characterization
Parameter	Value
pH	5.9
Initial COD (mg/ml)	23.7
Lipids (mg/ml)	2.0
Free fatty acids (µmol)	55.4

The loss of activity observed as the number of cycles progressed may be due to leaching of the enzyme molecules during the washing steps (Rodrigues et al, 2016a). Reports in literature have it that CLEAs present higher stability than free un-immobilized enzymes because of the rigidity conferred on the enzyme structure by the cross-linking. The immobilization also confers on the enzyme conformation protection from distortion that may arise from extremes of pH and/or temperature, thus the enzyme is less prone to activity loss even in tough environments (Lai et al., 2012). A summary of the characterization of the wastewater is shown in Table 1.

In terms of the parameters assessed following enzymatic hydrolysis of lipid-rich wastewater with RoL-ZAC3-CLEA, an increase in free fatty acids and a decrease in lipid content and chemical oxygen demand (COD) were observed after incubating for 4 h. Increasing the CLEA amount from 0.5–1% w/v increased the free fatty acid levels from 102.4 to 134.1 µmol, which corresponds to an increment of 84.8% and 142.1% respectively compared to the control (Table 2).

Table 2

Wastewater degradation with RoL-ZAC3 at two different concentrations
RoL-ZAC3 (% w/v)	Wastewater	Free fatty acid (µmol)	Lipids (mg/ml)	COD (mg/ml)
0	Control, C	55.4 ± 3.2	2.0 ± 0.1	23.7 ± 1.5
0.5	Wastewater, W1	102.4 ± 9.5	1.9 ± 0.1	21.9 ± 0.8
1	Wastewater, W2	134.1 ± 3.3	1.7 ± 0.2	16.7 ± 3.0

Untreated lipid-rich wastewaters normally have high concentrations of lipid and chemical oxygen demand levels (Nakhla et al., 2003). Decreased COD is an indication of decreased organic content.This decrease in COD, tells the quantity of oxidizable/organic pollutants in wastewater expressed as the mass of consumed oxygen over the solution volume (Geerdink et al., 2017). There was an increase in COD removal by 7.6% and 29.5% from the wastewater when 0.5% (w/v) and 1% (w/v) RoL-ZAC3-CLEA were used respectively (Table 3).

Table 3

Summary of wastewater degradation parameters
ROZAC3-CLEA (%w/v)	% Free Fatty acids	% Lipid Hydrolysis	% COD Removal
0.5	84.8	4.5	7.6
1	142.1	18.0	29.5

The efficiency of the enzymatic degradation was verified by comparing with the control (which was wastewater not pretreated with enzyme). Oily wastewater treatment helps to improve its biodegradability and reduce its toxicity (Song et al., 2011). Prasad and Manjunath (2011) obtained a 22% increase in the COD removal from swine and bovine meat industry wastewater following treatment with bacterial lipase. The successful utilization of RoL-ZAC3-CLEA in the degradation of lipid-rich wastewater makes the immobilized enzyme a promising alternative for this application.

Cross-linked RoL-ZAC3 at 0.5% and 1% w/v hydrolyzed lipid-rich wastewater, decreasing the total lipid by 4.5% and 18% respectively and improving other wastewater properties such as free fatty acids and COD. The immobilized enzyme showed potential in wastewater degradation that might be exploited on a large scale remediation of oil-contaminated wastewater.

Declaration of interests

The authors declare no potential conflict of interests

Ademakinwa A.N. (2021). A heat resistant intracellular laccase immobilized via cross-linked enzyme aggregate preparation: Characterization, Bisphenol A removal and phytotoxicity evaluation. Journal of Hazardous Materials 419: 126480.
Ademakinwa, A. N., Agunbiade, M. O., & Fagbohun, O. (2021). Biodegradation of cyanide in cassava wastewater using a novel thermodynamically-stable immobilized rhodanese. Preparative Biochemistry & Biotechnology, 51(6), 607-617
Ademakinwa, A. N., & Agboola, F. K. (2015). Bioremediation of textile dye solutions, textile dye mixtures and textile effluents by laccase from Aureobasidium pullulans (de Bary) G. Arnaud (1918)(Fungi: Ascomycota).
Ademakinwa N.A., Ayinla, Z.A., Omitogun, O.G. and Agboola, F.K. (2018). Preparation, Characterization and Optimization of Cross-Linked Fructosyltransferase Aggregates for the Production of Prebiotic Fructooligosaccharides. BioTechnologia 99(4): 418-435.
Ayinla, Z.A., Ademakinwa, A. N. and Agboola, F. K. (2017). Studies on the Optimization of Lipase Production by Rhizopus sp. ZAC3 Isolated from the Contaminated Soil of a Palm Oil Processing Shed. J. Appl. Biol. Biotechnol., 5(2): 030-037.
Ayinla, Z. A., Ademakinwa, A. N., Gross, R. A. and Agboola, F. K. (2021). Biochemical and biophysical characterisation of a small purified lipase from Rhizopus oryzae ZAC3. Biocatalysis and Biotransformation, DOI: 10.1080/10242422.2021.1883006
Ayinla, Z. A., Ademakinwa, A. N., & Agboola, F. K. (2022). Comparative modelling, molecular docking and immobilization studies on Rhizopus oryzae lipase: evaluation of potentials for fatty acid methyl esters synthesis. Journal of Biomolecular Structure and Dynamics, 1-13.
Basso, A., Hesseler, M. and Serban, S. (2016). Hydrophobic microenvironment optimization forefficient immobilization of lipases on octadecylfunctionalised resins.Tetrahedron, 72(46): 7323–7328.
Bornscheuer, U., Huisman, G., Kazlauskas, R., Lutz, S., Moore, J. and Robins, K. (2012). Engineering the third wave of biocatalysis.Nature, 485(7397): 185–194.
Cao, L. (2005). Immobilised enzymes: science or art? Current Opinion in Chemical Biology, 3: 217–226.
Ćorović, M.,Mihailović, M., Banjanac, K., Carević, M., Milivojević, A., Milosavić, N. andBezbradica, D. (2017). Immobilization of Candida antarctica lipase B onto Purolite® MN102and its application in solvent-free and organic media esterification.Bioprocess Biosyst. Eng. 40 (1): 23–34.
Demirel, B., Yenigun, O. and Onay, T. T. (2005), Anaerobic treatment of dairy wastewaters: A review. Process Biochem., 40: 2583–2595.
El-Sayed, A. M., Eweda, W. E., El-Tayeb, T. S. and Abdel Azeiz, A. Z. (2016). Production, Characterization and Immobilization of a FusariumsolaniLipase by Chitosan Magnetic Nanoparticles. Egypt J. Microbiol. 51: 63–75.
Fan, X., Niehus, X. and Sandoval, G.(2012). In: Sandoval, G. (Ed.). Lipases andPhospholipases: Methods and Applications. Methods in Molecular Biology(Walker J. M Ed.). Springer-Humana Press, NY USA. pp. 471-483.
Ferreira-Dias, S., Sandoval, G., Plou, F. G. and Valero, F.(2013). The potentialuse of lipases in the production of fatty acid derivatives for the food andnutraceutical industries - Review, Electron. J. Biotechn., 16: 38.
Geerdink, R. B., Sebastiaa V. D. H. and Epema, O. J. (2017). Chemical oxygen demand: Historical perspectives and future challenges. Anal. Chim. Acta, 961: 1 – 11.
Golterman, H. L., Clymo, R. S. and Ohnstad, M. (1978). Methods for Physical and Chemical Analysis of Fresh Waters. Blackwell, London, pg. 213.
Guauque Torres, M. P., Foresti, M. L. and Ferreira, M. L. (2013). Cross-linked enzyme aggregates (CLEAs) of selected lipases: a procedure for the proper calculation of their recovered activity. AMB Express, 3: 25 – 37.
Jeganathan, J. Nakhla, G. and Bassi, A. (2007). Hydrolytic pretreatment of oily wastewater by immobilized lipase. J. Hazard. Mater., 145: 127–135.
Kurtovic, I., Marshall, S. N. and Zhao, X. (2011). Hydrophobic immobilization of a bile salt activatedlipase from Chinook salmon (Oncorhynchustshawytscha), J. Mol. Catal. B: Enzym. 72 (3): 168–174.
Liu, T., Liu, Y., Wang, X., Li, Q., Wang, J. and Yan, Y. (2011). Improving catalytic performance ofBurkholderiacepacia lipase immobilized on macroporous resin NKA, J. Mol. Catal. B: Enzym., 71 (1): 45–50.
Manoel, E. A., Ribeiro, M. F. P., Dos Santos, J. C. S., Coelho, M. A. Z., Simas, A. B. C., Fernandez-Lafuente, R. and Freire, D. M. G. (2015). Accurel MP 1000 as a support for the immobilizationof lipase from Burkholderiacepacia: application to the kinetic resolutionof myo-inositol derivatives, Process Biochem. 50 (10): 1557–1564.
Masse, L., Kennedy, K. J. and Chou, S. (2001). Testing of alkaline and enzymatic pretreatment for fat particles in slaughterhouses wastewater. Bioresour. Technol., 77: 145–155.
Mie, Y., Miller, L., Gao, W. and Gross, R. A. (2003). Imaging the distribution and secondary structure of immobilized enzymes using infrared microspectroscopy. Biomacromolecules, 4(1): 70 – 74.
Mendes, A. A.; Pereira, E. B. and Castro, H. F. (2006). Effect of the enzymatic hydrolysis pretreatment of lipids-rich wastewater on the anaerobic biodigestion. Biochem. Eng. J., 32: 185–190.
Mendes, A. A., Pereira, E. B., Furigo Jr, A. and Castro, A. F. (2010). Anaerobic biodegradability of dairy wastewater pretreated with porcine pancreas lipase. Braz. Arch. Biol., 53(6): 1279 – 1284.
Nakhla, G., Al-Sabawi, M., Bassi, A. and Liu, V. (2003). Anaerobic treatability of highoil and grease rendering wastewater. J. Hazard.Mater., 102: 243–255.
Panzavolta, F., Soro, S., D’Amato, R., Palocci, C., Cernia, E. and Russo, M. V. (2005). Acetylenicpolymers as new immobilization matrices for lipolytic enzymes.J. Mol. Catal. B: Enzym. 32(3): 67–76.
Prasad, M. P. and Manjunath, K. (2011). Comparative study on biodegradation of lipid-rich wastewater using lipase producing bacterial species. Indian J. Biotechnol., 10: 121-124.
Rodrigues, R. C., Ortiz, C., Berenguer-Murcia, Á.,Torres, R. and Fernández-Lafuente, R. (2013). Modifying enzyme activity and selectivity by immobilization.ChemSoc Rev. 42: 6290–6307.
Sivozhelezov, V., Bruzzese, D., Pastorino, L., Pechkova, E. and Nicolini, C. (2009). Increase of catalytic activity of lipase towards olive oil by Langmuir-film immobilization of lipase. Enzyme Microb. Technol., 44(2): 72 – 76.
Smith, P. K., Krohn, R. I., Hermanson, G. T., Mallia, A. K., Gartner, F. H., Provenzano, M. D., Fujimoto, E. K., Goeke, N. M., Olson, B. J. and Klenk, D. C. (1985). Measurement of protein using bicinchoninic acid. Anal. Biochem., 150(1): 76 – 85.
Song, H., Zhou, L., Zhang, L., Gao, B., Wei, D., Shen, Y., Wang, R., Madzak, C. and Jiang, Z. (2011). Construction of a whole-cell catalyst displaying a fungal lipase for effective treatment of oily wastewaters. J. Mol. Catal. BEnzym., 71(3-4): 166 – 170.
Su, A., Shirke, A., Baik, J. Zou, Y. and Gross, R. (2018). Immobilized cutinases: preparation, solvent tolerance and thermal stability. Enzyme Microb. Tech., 116: 33–40.
Tecelão, C., Guillén, M., Valero, F. and Ferreira-Dias, S. (2012). Immobilized heterologousRhizopus oryzae lipase: a feasible biocatalyst for the production of human milk fatsubstitutes, Biochem. Eng. J., 67: 104–110.
Vikbjerg, A. F., Mu, H. and Xu, X. (2007). Synthesis of structured phospholipids by immobilized phospholipase A2 catalyzed acidolysis, J. Biotechnol., 128(3): 545–554.
Villalba, M., Verdasco-Martín, C. M., Dos Santos, J. C. S., Fernandez-Lafuente, R. and Otero, C. (2016). Operational stabilities of different chemical derivatives of Novozym 435in an alcoholysis reaction, Enzyme Microb. Technol., 90: 35–44.
Villeneuve, P., Muderhwa, J. M., Graille, J., Haasc, M. J. (2000). Customizing lipases for biocatalysis: a survey of chemical, physical and molecular biological approaches.J. Mol. Catal B: Enzymatic. 9: 113–148.
Wang, S., Zheng, D., Yin, L. and Wang, F. (2017). Preparation, activity and structure of cross-linked enzyme aggregates (CLEAs) with nanoparticle. Enzyme Microb. Technol., 107 (22-31).

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Preliminary investigations into the synthesis of lipase cross-linked enzyme aggregates (CLEA) for hydrolysis of lipid-rich wastewater

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