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The Characterization and Application of Enzymes in Bioprocesses
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The Characterization and Application of Enzymes in Bioprocesses

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: 20 August 2025 | Viewed by 4377

Special Issue Editor

Dr. Loredana Marcolongo

E-Mail Website
Guest Editor
Research Institute on Terrestrial Ecosystems-IRET-CNR, Via Pietro Castellino 111, 80131 Naples, Italy
Interests: protein biochemistry; enzyme and protein purification; enzyme characterization; extremozymes; extremophilic microorganisms; biotechnology for sustainability; biocatalysts; biorefinery; biomass and waste valorization; bioresources; bioactive molecules for well-being

Special Issue Information

Dear Colleagues,

Enzymes are protein catalysts that are found in all forms of organisms and play a crucial role in several bioprocesses; this is a vital area of biotechnology that utilizes living organisms and their enzymes to produce various types of products. The aim of biocatalysts is to accelerate the speed and efficiency of chemical reactions with high substrate specificity and without being consumed. In recent years, enzymes and biological molecules have garnered increased research interest due to the importance and versatility of their utilization. They are employed as catalysts in a variety of fields, and for their application in the technological, medical, environmental and industrial domains, they require an enhanced capacity in order to fulfil the operating conditions of such processes. To determine the feasibility of applying enzymes in various fields, they must be characterized, and their function and application studied via multidisciplinary approaches. The objective of this Special Issue is to collect original research articles, reviews, communications, and so on addressing the study, characterization and application of biocatalysts of different origins. Studies on the bioprocessing, biotechnological design, optimization and application (diagnostic/medical, industrial, agro-industrial, environmental, etc.) of biocatalysts are welcome.

Dr. Loredana Marcolongo
Guest Editor

Manuscript Submission Information

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Keywords

  • biocatalysts
  • enzymatic characterization
  • enzymatic action
  • protein engineering
  • biocatalysts application
  • bioprocessing

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Published Papers (4 papers)

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Research

19 pages, 9280 KiB  
Article
Site-Directed Mutagenesis Increased the Catalytic Activity and Stability of Oenococcus oeni β-Glucosidase: Characterization of Enzymatic Properties and Exploration of Mechanisms
by Junpeng Zuo, Jie Zhang, Hongyu Ma, Yanqi Zhang, Pengyan Li, Ying Wu, Pingping Tian, Qiuxia Fan, Li Cao, Jianrui Sun and Shaobin Gu
Int. J. Mol. Sci. 2025, 26(9), 3983; https://doi.org/10.3390/ijms26093983 - 23 Apr 2025
Viewed by 147
Abstract
Oenococcus oeni β-glucosidase can significantly improve food flavor, but its catalytic activity and stability performance need to be enhanced. In this study, the two dominant mutants III and IV were obtained by site-directed mutation of key amino acid residues in the catalytic pocket [...] Read more.
Oenococcus oeni β-glucosidase can significantly improve food flavor, but its catalytic activity and stability performance need to be enhanced. In this study, the two dominant mutants III and IV were obtained by site-directed mutation of key amino acid residues in the catalytic pocket of Oenococcus oeni β-glucosidase. Compared with the wild-type enzyme, the activities of mutants III and IV were increased by 2.81 and 3.18 times, respectively. Their thermal stability was also significantly improved. Mutants III and IV showed a significant increase in affinity for the standard substrate p-NPG, with the Km value decreasing by 18.2% and 33.3%, respectively. Molecular docking analysis indicated that hydrogen bonding and π–π aromatic interactions were the primary factors influencing the changes in enzyme properties, with F133 and N181 identified as key amino acids affecting catalytic activity and stability. This research is of great significance for enhancing food flavor and expands the potential industrial applications of Oenococcus oeni β-glucosidase. Full article
(This article belongs to the Special Issue The Characterization and Application of Enzymes in Bioprocesses)
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20 pages, 1030 KiB  
Article
Optimization and Bioreactor Scale-Up of Cellulase Production in Trichoderma sp. KMF006 for Higher Yield and Performance
by Seongwoo Myeong, Yun-Yeong Lee and Jeonghee Yun
Int. J. Mol. Sci. 2025, 26(8), 3731; https://doi.org/10.3390/ijms26083731 - 15 Apr 2025
Viewed by 303
Abstract
This study optimized operating parameters to enhance cellulase production and evaluated scale-up feasibility in submerged fermentation (SmF) using Trichoderma sp. KMF006. Flask-scale experiments assessed the effects of Avicel:cellulose ratios (4:0–0:4), agitation speeds (150–210 rpm), and turbulence (baffled vs. non-baffled flasks), with optimized conditions [...] Read more.
This study optimized operating parameters to enhance cellulase production and evaluated scale-up feasibility in submerged fermentation (SmF) using Trichoderma sp. KMF006. Flask-scale experiments assessed the effects of Avicel:cellulose ratios (4:0–0:4), agitation speeds (150–210 rpm), and turbulence (baffled vs. non-baffled flasks), with optimized conditions applied to a 10 L bioreactor. A 3:1 Avicel:cellulose ratio (A3C1) significantly accelerated cellulase production, reaching peak activity 6 days earlier than Avicel alone. An agitation speed of 180 rpm was optimal, balancing enzyme activity and energy efficiency. Turbulence enhanced cellulase yields, with baffled flasks increasing EG, BGL, and CBH activities 19.9-, 6.2-, and 8.9-fold, respectively, compared to the control. Biochar further improved cellulase production but only under turbulent conditions, demonstrating a synergistic effect. At the bioreactor scale, the A3-180_Imp (A3C1, 180 rpm, impeller-induced turbulence) achieved the highest enzymatic activity (33.60 U/mL EG, 3.46 U/mL BGL, and 0.63 U/mL CBH). The filter paper unit (FPU) was 84 FPU/mL, a two-fold increase compared to the control. However, excessive turbulence at 210 rpm reduced enzyme stability, emphasizing the importance of balancing shear stress. These findings provide a systematic framework for optimizing SmF conditions, highlighting the significance of balancing hydrodynamic conditions for efficient cellulase production at an industrial scale. Full article
(This article belongs to the Special Issue The Characterization and Application of Enzymes in Bioprocesses)
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16 pages, 4866 KiB  
Article
Polyphosphate Kinase from Burkholderia cenocepacia, One Enzyme Catalyzing a Two-Step Cascade Reaction to Synthesize ATP from AMP
by Dianelis T. Monterrey, Leire Azcona, Julia Revuelta, Israel Sánchez-Moreno and Eduardo García-Junceda
Int. J. Mol. Sci. 2024, 25(23), 12995; https://doi.org/10.3390/ijms252312995 - 3 Dec 2024
Viewed by 1026
Abstract
This study characterizes a novel polyphosphate kinase from Burkholderia cenocepacia (BcPPK2-III), an enzyme with potential applications in ATP regeneration processes. Bioinformatic and structural analyses confirmed the presence of conserved motifs characteristic of PPK2 enzymes, including Walker A and B motifs, and [...] Read more.
This study characterizes a novel polyphosphate kinase from Burkholderia cenocepacia (BcPPK2-III), an enzyme with potential applications in ATP regeneration processes. Bioinformatic and structural analyses confirmed the presence of conserved motifs characteristic of PPK2 enzymes, including Walker A and B motifs, and the subclass-specific residue E137. Molecular docking simulations showed AMP had the highest binding affinity (−7.0 kcal/mol), followed by ADP (−6.5 kcal/mol), with ATP having the lowest affinity (−6.3 kcal/mol). It was overexpressed in Escherichia coli, after purification enzymatic activity assays revealed that BcPPK2-III needed divalent cations (Mg2⁺, Mn2⁺, Co2⁺) as cofactors to be active. Functional assays revealed its ability to synthesize ATP from AMP through a stepwise phosphorylation mechanism, forming ADP as an intermediate, achieving 70% ATP conversion (TTN 4354.7) after 24 h. Kinetic studies indicated cooperative behavior and substrate preference, with AMP phosphorylation to ADP being the most efficient step. The enzyme demonstrated high thermostability (T50 = 62 °C) and a broad pH stability range (pH 6.0–9.0), making it suitable for diverse biocatalytic applications. The study highlights BcPPK2-III as a robust and versatile candidate for cost-effective ATP regeneration, offering advantages in industrial processes requiring stoichiometric amounts of ATP. Full article
(This article belongs to the Special Issue The Characterization and Application of Enzymes in Bioprocesses)
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20 pages, 3533 KiB  
Article
Exploring the Kinetics and Thermodynamics of a Novel Histidine Ammonia-Lyase from Geobacillus kaustophilus
by Francisco Manuel Salas-Garrucho, Alba Carrillo-Moreno, Lellys M. Contreras, Felipe Rodríguez-Vico, Josefa María Clemente-Jiménez and Francisco Javier Las Heras-Vázquez
Int. J. Mol. Sci. 2024, 25(18), 10163; https://doi.org/10.3390/ijms251810163 - 21 Sep 2024
Viewed by 2037
Abstract
Histidine ammonia-lyase (HAL) plays a pivotal role in the non-oxidative deamination of L-histidine to produce trans-urocanic, a crucial process in amino acid metabolism. This study examines the cloning, purification, and biochemical characterization of a novel HAL from Geobacillus kaustophilus (GkHAL) [...] Read more.
Histidine ammonia-lyase (HAL) plays a pivotal role in the non-oxidative deamination of L-histidine to produce trans-urocanic, a crucial process in amino acid metabolism. This study examines the cloning, purification, and biochemical characterization of a novel HAL from Geobacillus kaustophilus (GkHAL) and eight active site mutants to assess their effects on substrate binding, catalysis, thermostability, and secondary structure. The GkHAL enzyme was successfully overexpressed and purified to homogeneity. Its primary sequence displayed 40.7% to 43.7% similarity with other known HALs and shared the same oligomeric structure in solution. Kinetic assays showed that GkHAL has optimal activity at 85 °C and pH 8.5, with high thermal stability even after preincubation at high temperatures. Mutations at Y52, H82, N194, and E411 resulted in a complete loss of catalytic activity, underscoring their essential role in enzyme function, while mutations at residues Q274, R280, and F325 did not abolish activity but did reduce catalytic efficiency. Notably, mutants R280K and F325Y displayed novel activity with L-histidinamide, expanding the substrate specificity of HAL enzymes. Circular dichroism (CD) analysis showed minor secondary structure changes in the mutants but no significant effect on global GkHAL folding. These findings suggest that GkHAL could be a promising candidate for potential biotechnological applications. Full article
(This article belongs to the Special Issue The Characterization and Application of Enzymes in Bioprocesses)
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