Cocoa pod husk, a new source of hydrolase enzymes for preparation of cross‑linked enzyme aggregate

Cocoa pod husk (CPH) is a by-product of cocoa production obtained after removing the beans from the fruit. The analysis of CPH has shown that it contains high amounts of protein. This study is aimed to utilize this protein source in hydrolase enzyme production. In this study, seven hydrolase enzymes (amylase, fructosyltransferase, mannanase,glucosidase, glucanase, lipase and protease) were screened from CPH for the first time for feasible industrial production. Among these hydrolases, lipase was chosen for the next steps of experiments as it has a lot of applications in different industries.The extraction of high active lipase from CPH has been done under optimum conditions.The condition that was optimum for the three major factors was achieved using Face centered central composite design (FCCCD) with response surface methodology (RSM) to obtain the highest enzyme activity of crude lipase from CPH. The optimum condition of extraction is used for preparation of cross-linked enzyme aggregate (CLEA). For the production of immobilized biocatalyst, the technique of CLEA is considered as an effective technique for its industrially attractive advantages. Referring to the results of OFAT, CLEA-lipase was prepared in the best condition at the presence of 30 mM ammonium sulphate, 70 mM glutaraldehyde with 0.23 mM Bovine serum albumin as an additive. Immobilization effectively improved the stability of lipase against various organic solvent

Development of an immobilized biocatalyst with lipase and protease activities as a multipurpose cross-linked enzyme aggregate (multi-CLEA)

This study focused on the production of a novel multi-CLEA comprising the enzyme activities of lipase and protease from fish viscera. A multi-CLEA is a single biocatalyst that can catalyze separate unrelated reactions, but these reactions can be conducted in one application. Tests pertaining to the effect of various additives on the multi-CLEA’s activity were performed. Response Surface Methodology’s Face Centered Central Composite Design (FCCCD) was employed to optimize the preparation parameters of the multi-CLEA in an aqueous medium. It was found that 55% (w/v) of ammonium sulfate, 65 mM of glutaraldehyde, and 0.113 mM of bovine serum albumin were the optimum levels of additives to prepare the multi-CLEA with the protease and lipase recovery activity of 43.82% and 99.91%, respectively. Multi-CLEAs were found to retain an average of more than 34% of the initial activity after five consecutive batches for both enzymes. Finally, the multi-CLEA was utilized to catalyze two reactions: improved washing process and biodiesel production. The stain removal percentage of a commercial detergent was improved by 67.78% after adding multi-CLEA. In addition, the multi-CLEA catalyzed biodiesel production from vegetable oil with a percentage conversion of 51.7%. Such results demonstrated that the multi-CLEA is a promising catalyst for biotechnological applications

Optimizing the preparation conditions of cross-linked enzyme aggregates (CLEA)-protease

Background: Cross-linked enzyme aggregate (CLEA) is considered as an effective technique in the production of immobilized biocatalysts for its industrially attractive advantages. Simplicity, stability, low cost, time saving and reusability are proved to be some of CLEA’s main advantages. Results: In this study, an active, stable and recyclable CLEA-protease from the viscera of channel catfish Ictalurus punctatus has been prepared. Optimization of the preparation parameters is carried out with the help of Response Surface Methodology. This methodology helped in studying the interaction between the most contributing factors such as cross-linker, precipitant and the additive concentrations. The optimum specific activity for CLEA-protease of 4.512 U/mg protein has shown a high stability against the denaturation forces such as temperature and pH as compared to free protease. It is further found from the study that the highest activity was achieved at the pH of 6.8 and at the temperature of 45 °C. After six cycles, CLEA-protease maintained 28 % of its original activity. Additionally, Michaelis–Menten models were used to determine the kinetic parameters i.e. Km and Vmax that helped in showing a significant difference after immobilization as compared to free protease. Conclusion: This work found that this novel CLEA-protease can be used as a very active biocatalyst in industrial applications

Cocoa pod husk: a new source of CLEA-lipase for preparation of low-cost biodiesel: an optimized process

Cocoa pod husk (CPH) is a by-product of cocoa production obtained after removing the beans from the fruit. The analysis of CPH has shown that it contains high amounts of protein. This study is aimed to utilize this protein source in hydrolase enzyme production. In this study, seven hydrolase enzymes (amylase, fructosyltransferase, mannanase,glucosidase, glucanase, lipase and protease) were screened from CPH for the first time for feasible industrial production. Among these hydrolases, lipase was chosen for the next steps of experiments as it has a lot of applications in different industries.The extraction of high active lipase from CPH has been done under optimum conditions.The condition that was optimum for the three major factors was achieved using Face centered central composite design (FCCCD) with response surface methodology (RSM) to obtain the highest enzyme activity of crude lipase from CPH. The optimum condition of extraction is used for preparation of cross-linked enzyme aggregate (CLEA). For the production of immobilized biocatalyst, the technique of CLEA is considered as an effective technique for its industrially attractive advantages. Referring to the results of OFAT, CLEA-lipase was prepared in the best condition at the presence of 30 mM ammonium sulphate, 70 mM glutaraldehyde with 0.23 mM Bovine serum albumin as an additive. Immobilization effectively improved the stability of lipase against various organic solvent

Optimized preparation and characterization of CLEA-lipase from cocoa pod husk

Cross-linked enzyme aggregate (CLEA), a new method of carrier free enzyme immobilization has many advantages and considered as an economical method in the context of industrial biocatalysis. In this research, a highly active and stable CLEA-lipase has been successfully prepared from cocoa pod husk (CPH), a by-product of the cocoa industry. Based on the Face Centered Central Composite Design (FCCCD) under Response Surface Methodology (RSM) using three important parameters, the optimal preparation condition of CLEA-lipase shows that the highest activity achieved is 9.407Uor 83% of the activity of the free lipase. It was prepared using 20% saturated (NH4)2SO4 as the precipitant, 60mMglutaraldehyde as the cross-linker and 0.169mM BSA as the feeder. The optimal reaction temperature and pH for both CLEA-lipase and free lipase differed, where they were 60 ◦C and 8.2 and 45 ◦C and 8 respectively. A systematic study of temperature and pH stability showed that CLEA-lipase is more stable than free lipase. Results also show that the prepared CLEA-lipase retained more than 50% of the initial activity after five repeated runs. The observed high stability and recyclability of CLEA-lipase prepared from CPH demonstrated that it has potential to be used in different industrial applications

Optimized preparation and characterization of CLEA-lipase from cocoa pod husk

Cross-linked enzyme aggregate (CLEA) is easily prepared from crude enzyme and has many advantagesto the environment and it is considered as an economic method in the context of industrial biocatalysiscompared to free enzyme. In this work, a highly active and stable CLEA-lipase from cocoa pod husk (CPH)which is a by-product after removal of cocoa beans, were assayed for their hydrolytic activity and char-acterized under the optimum condition successfully. Face centered central composite design (FCCCD)under response surface methodology (RSM) was used to get the optimal conditions of the three signif-icant factors (concentration of ammonium sulfate, concentration of glutaraldehyde and concentrationof additive) to achieve higher enzyme activity of CLEA. From 20 runs, the highest activity recorded wasaround 9.407 U (83% recovered activity) under the condition of using 20% saturated ammonium sulfate,60 mM glutaraldehyde as cross-linker and 0.17 mM bovine serum albumin as feeder. Moreover, the opti-mal reaction temperature and pH value in enzymatic reaction for both crude enzyme and immobilizedwere found to be 45◦C at pH 8 and 60◦C at pH 8.2, respectively. A systematic study of the stability of CLEAand crude enzyme was taken with regards to temperature (25–60◦C) and pH (5–10) value and in bothfactors, CLEA-lipase showed more stability than free lipase. The Kmvalue of CLEA was higher comparedto free enzyme (0.55 mM vs. 0.08 mM). The CLEA retained more than 60% of the initial activity after sixcycles of reuse compared to free enzyme. The high stability and recyclability of CLEA-lipase from CPHmake it efficient for different industrial applications

Simultaneous enhanced antibacterial and osteoblast cytocompatibility performance of Ti6Al7Nb implant by nano-silver/graphene oxide decorated mixed oxide nanotube composite

The self-ordered architecture allows for the exact design and control of geometrical features, to achieve materials with unique properties. For this reason, mixed oxide nanotube arrays have been highly regarded by the scientific community in recent years. In the present study, a hybrid approach of an optimized physical vapor deposition magnetron sputtering (PVDMS), electrochemical anodization as well as spin coating is proposed to improve the mechanical properties, corrosion resistance, antibacterial and osteoblast cytocompatibility performance of Ti6Al7Nb implant (Ti67IMP). Accordingly, controlled decorations of mixed oxide nanotube with silver nanoparticles/graphene oxide (AgNPs/GO) were designed to assess the biofunctionality of the modified Ti6Al7Nb implant. The results show that the surface modification has dramatically reduced the viability of Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) cells. Besides, the AgNPs/GO loaded mixed oxide nanotube has significantly promoted cell adhesion and spreading, compared to the bare substrate. The proposed hybrid approach can also be extended to fabricate highly complex nanoarchitectures with controlled shape and biofunctionality for various orthopedic applications. © 2019 Elsevier B.V

Carrier-free enzyme immobilization by cross-linked enzyme aggregates (CLEA) technology

Biocatalyst in the form of enzymes is widely used in diverse applications. Unfortunately, free enzymes are quite unstable and may undergo denaturation even under mild conditions, thus hampering their usefulness, and this may lead to higher cost in enzyme based applications. A credible solution is to immobilize the enzymes prior to usages. This procedure was proven to improve the performances in term of stability, activity and selectivity of the enzymes. In addition, separation of product from the used enzyme was made easier and enzyme recyclability was possible. However, carrier-supported enzyme immobilization suffers from many disadvantages, such as large amounts of non-catalytic mass and expensive carrier beads. Thus, to overcome this problem, cross-linked enzyme aggregates (CLEA) has been since widely researched. It involves simple procedure and has many benefits; for example, this procedure does not need purified enzyme. The technique involves an initial precipitation of enzymes using, either organic solvents, salts, non-ionic polymers or acids to obtain aggregates. It is then followed by cross-linking the aggregates by polyfunctional reagents, such as glutaryldehyde, whereby the enzyme molecules react among themselves, leading to the formation of ‘solid biocatalyst’. This chapter aims at deliberating the CLEA technique for enzyme immobilization. Lipase extracted from cocoa pod husk (CPH), an agricultural waste product, has been chosen as the model enzyme, and upon immobilization, the biocatalyst is termed as CLEA-lipase. The production of CLEA-lipase was carried out under an optimum condition and this was followed by experimental comparison with the free-form, on the temperature and pH optima and stabilities. Additionally, recyclability of CLEA-lipase was also studied. Finally, the morphology of the solid biocatalyst, which has bearings towards its activity, was examined by Field Emission Scanning Electron Microscopy (FESEM)