Microbial proteases constitute one of the most important groups of industrially relevant enzymes. Proline iminopeptidases (PIPs) that specifically release amino-terminal proline from peptides are of major interest for applications in food biotechnology. Proline iminopeptidase has been extensively characterised in bacteria and filamentous fungi. However, no similar reports exist for yeasts. In this study, a protease gene from Glaciozyma antarctica designated as GaPIP was cloned and overexpressed in Escherichia coli. Sequence analyses of the gene revealed a 960 bp open reading frame encoding a 319 amino acid protein (35,406 Da). The purified recombinant GaPIP showed a specific activity of 3561 Umg⁻¹ towards L-proline-p-nitroanilide, confirming its identity as a proline iminopeptidase. GaPIP is a cold-active enzyme with an optimum activity of 30 °C at pH 7.0. The enzyme is stable between pH 7.0 and 8.0 and able to retain its activity at 10–30 °C. Although GaPIP is a serine protease, only 25% inhibition by the serine protease inhibitor, phenylmethanesulfonylfluoride (PMSF) was recorded. This enzyme is strongly inhibited by the presence of EDTA, suggesting that it is a metalloenzyme. The dimeric structure of GaPIP was determined at a resolution of 2.4 Å. To date, GaPIP is the first characterised PIP from yeasts and the structure of GaPIP is the first structure for PIP from eukaryotes.

The formation of cross-linked enzyme aggregates (CLEAs) using macromolecular cross-linkers improves substrate accessibility and enhances enzyme retention. However, there have been few studies exploring the use of macromolecular cross-linkers due to the challenges related to cross-linker screening. In compliance with our previous computational and experimental screening, dextran is the optimal macromolecular cross-linker to develop CLEAs of endolevanase from Bacillus lehensis G1 (rlevblg1-dex-CLEA) for levan-type-fructooligosaccharides (L-FOS) production. In this study, rlevblg1-dex-CLEAs was optimized, and the activity recovery continued to increase and reached 90.5%. Subsequently, the rlevblg1-dex-CLEAs were characterized and they displayed higher thermal stability after 1 h of incubation in comparison to the free enzyme. Moreover, the rlevblg1-dex-CLEAs were reusable for five cycles and exhibited greater storage stability over 180 days at 4 °C (60.9%) than that of free rlevblg1. In addition, the rlevblg1-dex-CLEAs demonstrated similar catalytic efficiency as the free enzyme and generated a substantial amount of L-FOS with a longer degree of polymerization, which is more beneficial for industrial use.

Surface chemistry of magnetic nanoparticles (MNP) is crucial to provide a strong protein-support interaction for the immobilization process. The stability and biocompatibility of the MNP can be structurally enhanced by integrating with organic materials. In this study, MNP from KI/FeCl3 has successfully synthesized that showed a stronger magnetic strength (72.5 emu/g) compared to common standard precursors, FeCl2/FeSO4 (< 60 emu/g). The synthesized MNP was then incorporated via in-situ with functionalized starch; dialdehyde (DAS-MNP), thiol (TS-MNP), and carboxymethyl (CMS-MNP) for Laccase (Lac) immobilization. From docking analysis, CMS-MNP portrayed the highest binding affinity and interacted with highest number of Lac amino acids residues compared to DAS- and TS-MNP. Aligned with this result, immobilized Lac using CMS-MNP achieved the highest recovery activity (80.3%), highly stable at 75 °C for 4 h, and retained more than 50% of its initial activity after 10 cycles. The CMS-MNP-Lac also showed about the same catalytic efficiency with free Lac (1.19 and 1.58 mM-1s-1, respectively). It is demonstrated that the functional group of the starch-MNP plays a crucial role in attaining a stable immobilized Lac. Therefore, yield a promising biocatalyst to be applied in various fields.

Cross-linked enzymes aggregate (CLEA) is a versatile carrier free-immobilization technique that has gained much attention in the development of biocatalyst technology. However, there is no precise and accurate method of using this technique that will lead to an expected outcome that meets the requirements of the industrial standard. Therefore, the objective of this study is to investigate the effect of a few methods of executing cross-linked enzyme aggregates approach using maltogenic amylase by measuring the activity recovery of the developed CLEA. Some factors that are considered in developing the methodologies are the interaction between cross-linkers and enzymes, size of the cross-linked enzyme aggregates and substrate diffusion. The addition of precipitant and the cross-linking agent steps has been manipulated and four different methodologies were developed. Based on the results, Method 2 showed the highest activity recovery (57.9%) whilst Method 4 gave the lowest activity recovery (15.7%). Method 2 is an improvised method that removed supernatant after centrifugation before proceeding to the cross-linking step. The characterization of cross-linked enzyme aggregates such as morphological characterization and Fourier Transform-Infrared Spectroscopy was also determined. In conclusion, the best and most productive preparation method was determined based on the highest activity recovery.