Robust cross-linked cyclodextrin glucanotransferase from Bacillus lehensis G1 aggregates using an improved cross-linker and a new co-aggregant for the production of cyclodextrins.

One of the potentials of carrier-free cross-linked enzyme aggregates (CLEA) immobilization is the ability to be separated and reuse. Yet, it might be impeded by the poor mechanical stability resulting low recyclability. CLEA of CGTase from Bacillus lehensis G1 (CGTase G1-CLEA) using chitosan (CS) as a cross-linker demonstrated high activity recovery however, displayed poor reusability. Therefore, the relationship between mechanical strength and reusability is studied by enhancing the CS mechanical properties and applying a new co-aggregation approach. Herein, CS was chemically cross-linked with glutaraldehyde (GA) and GA was introduced as a co-aggregant (coGA). CGTase G1-CLEA developed using an improved synthesized chitosan-glutaraldehyde (CSGA) cross-linker and a new coGA technique showed to increase its mechanical stability which retained 63.4% and 52.2%, respectively compared to using CS that remained 33.1% of their initial activity after stirred at 500 rpm. The addition of GA impacted the morphology and interaction consequently stabilizing the CLEAs durability in production of cyclodextrins. As a result, the reusability of CGTase G1-CLEA with CSGA and coGA increased by 56.6% and 42.8%, respectively compared to previous CLEA after 5 cycles for 2 h of reaction. This verifies that the mechanical strength of immobilized enzyme influences the improvement of its operational stability.

Protein surface engineering and interaction studies of maltogenic amylase towards improved enzyme immobilisation.

A combined strategy of computational, protein engineering and cross-linked enzyme aggregates (CLEAs) approaches was performed on Bacillus lehensis G1 maltogenic amylase (Mag1) to investigate the preferred amino acids and orientation of the cross-linker in constructing stable and efficient biocatalyst. From the computational analysis, Mag1 exhibited the highest binding affinity towards chitosan (-7.5 kcal/mol) and favours having interactions with aspartic acid whereas glutaraldehyde was the least favoured (-3.4 kcal/mol) and has preferences for lysine. A total of eight Mag1 variants were constructed with either Asp or Lys substitutions on different secondary structures surface. Mutant Mag1-mDh exhibited the highest recovery activity (82.3%) in comparison to other Mag1 variants. Mutants-CLEAs exhibited higher thermal stability (20-30% activity) at 80 °C whilst Mag1-CLEAs could only retain 9% of activity at the same temperature. Reusability analysis revealed that mutants-CLEAs can be recovered up to 8 cycles whereas Mag1-CLEAs activity could only be retained for up to 6 cycles. Thus, it is evident that amino acids on the enzyme's surface play a crucial role in the construction of highly stable, efficient and recyclable CLEAs. This demonstrates the necessity to determine the preferential amino acid by the cross-linkers in advance to facilitate CLEAs immobilisation for designing efficient biocatalysts.

Cross-linked cyclodextrin glucanotransferase aggregates from Bacillus lehensis G1 for cyclodextrin production: Molecular modeling, developmental, physicochemical, kinetic and thermodynamic properties.

Type of cross-linking agents influence the stability and active cross-linked enzyme aggregates (CLEA) immobilization. The information of molecular interaction between enzyme-cross linker is not well explored thus screening wide numbers of cross-linker is crucial in CLEA development. This study combined the molecular modeling and experimental optimization to investigate the influences of different cross-linking agents in developing CLEA of cyclodextrin glucanotranferase G1 (CGTase G1) for cyclodextrins (CDs) synthesis. Seven types of cross-linkers were tested and CGTase G1 cross-linked with chitosan (CS-CGTG1-CLEA) displayed the highest activity recovery (84.6 ± 0.26%), aligning with its highest binding affinity, radius of gyration and flexibility through in-silico analysis towards CGTase G1. CS-CGTG1-CLEA was characterized and showed a longer half-life (30.06 ± 1.51 min) and retained a greater thermal stability (52.73 ± 0.93%) after 30 min incubation at optimal conditions compared to free enzyme (10.30 ± 1.34 min and 5.51 ± 2.10% respectively). CS-CGTG1-CLEA improved CDs production by 33% and yielded cumulative of 52.62 g/L CDs after five cycles for 2 h of reaction. This study reveals that abundant of hydroxyl group on chitosan interacted with CGTase G1 surface amino acid residues to form strong and stable CLEA thus can be a promising biocatalyst in CDs production.

From the Front or Back Door? Quantitative analysis of direct and indirect extractions of α-mangostin from mangosteen (Garcinia mangostana).

The pulp and pericarp of mangosteen (Garcinia mangostana) fruit are popular food, beverage and health products whereby 60% of the fruit consist of the pericarp. The major metabolite in the previously neglected or less economically significant part of the fruit, the pericarp, is the prenylated xanthone α-mangostin. This highly bioactive secondary metabolite is typically isolated using solvent extraction methods that involve large volumes of halogenated solvents either via direct or indirect extraction. In this study, we compared the quantities of α-mangostin extracted using three different extraction methods based on the environmentally friendly solvents methanol and ethyl acetate. The three solvent extractions methods used were direct extractions from methanol (DM) and ethyl acetate (DEA) as well as indirect extraction of ethyl acetate obtained via solvent partitioning from an initial methanol extract (IEA). Our results showed that direct extraction afforded similar and higher quantities of α-mangostin than indirect extraction (DM: 318 mg; DEA: 305 mg; IEA: 209 mg per 5 g total dried pericarp). Therefore, we suggest that the commonly used method of indirect solvent extraction using halogenated solvents for the isolation of α-mangostin is replaced by single solvent direct extraction using the environmentally friendly solvents methanol or ethyl acetate.