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Microbial Enzymes for Biotechnological Applications

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

Deadline for manuscript submissions: closed (25 November 2023) | Viewed by 17561

Special Issue Editors


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Guest Editor
Department of Biotechnology, University of Verona, 37134 Verona, Italy
Interests: industrial enzymology; extremozymes; biocatalysis; biorefinery; biomass valorisation; enzymatic recycling of plastics; protein engineering; CRISPR-based applications; virus biotechnology
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Guest Editor
Associate Professor, Department of Biology, Università degli Studi di Napoli Federico II, 80138 Napoli, Italy
Interests: microbial enzymes

Special Issue Information

Dear Colleagues,

Microbial biocatalysts account for about 90% of the enzyme market shares. This mainly depends on the rapid multiplication, easy manipulation, and handling of the microbial cell factories if compared to other biocatalysts sources (i.e., animal, and vegetal tissues). Unlike chemical catalysts, enzymes show wider application potentials that are revolutionizing the agricultural, chemical, energy, and pharmaceutical industries.

This Special Issue is led by Dr. Salvatore Fusco and Prof. Patrizia Contursi, assisted by our Topical Advisory Panel Member Dr. Martina Aulitto (University of Naples Federico II). The aim is to collect original research articles, review articles, and short communications dealing with the study of microbial biocatalysts (both isolated enzymes and whole-cell biocatalysts). We welcome fundamental studies about design and optimization of microbial enzymes as well as those addressed to the exploitation of microbial enzymes for biotechnological applications (industrial, diagnostic, environmental, etc.).

Topics of interest include, but are not limited to, the following:

  • Discovery and/or characterization of new microbial enzymes (including commodity, specialty, extremophilic and polyextremophilic enzymes)
  • Functional and/or structural characterization of microbial enzymes
  • Microbial enzymes for circular economy applications
  • Design, optimization, and/or exploitation of whole-cell microbial biocatalysts
  • Bioprospecting of microbial enzymes

Dr. Salvatore Fusco
Dr. Patrizia Contursi
Guest Editors

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Published Papers (9 papers)

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Research

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28 pages, 21604 KiB  
Article
The Discovery of the Fucoidan-Active Endo-1→4-α-L-Fucanase of the GH168 Family, Which Produces Fucoidan Derivatives with Regular Sulfation and Anticoagulant Activity
by Artem S. Silchenko, Ilya V. Taran, Roza V. Usoltseva, Nikolay V. Zvyagintsev, Anastasiya O. Zueva, Nikita K. Rubtsov, Dana E. Lembikova, Olga I. Nedashkovskaya, Mikhail I. Kusaykin, Marina P. Isaeva and Svetlana P. Ermakova
Int. J. Mol. Sci. 2024, 25(1), 218; https://doi.org/10.3390/ijms25010218 - 22 Dec 2023
Viewed by 821
Abstract
Sulfated polysaccharides of brown algae, fucoidans, are known for their anticoagulant properties, similar to animal heparin. Their complex and irregular structure is the main bottleneck in standardization and in defining the relationship between their structure and bioactivity. Fucoidan-active enzymes can be effective tools [...] Read more.
Sulfated polysaccharides of brown algae, fucoidans, are known for their anticoagulant properties, similar to animal heparin. Their complex and irregular structure is the main bottleneck in standardization and in defining the relationship between their structure and bioactivity. Fucoidan-active enzymes can be effective tools to overcome these problems. In the present work, we identified the gene fwf5 encoding the fucoidan-active endo-fucanase of the GH168 family in the marine bacterium Wenyingzhuangia fucanilytica CZ1127T. The biochemical characteristics of the recombinant fucanase FWf5 were investigated. Fucanase FWf5 was shown to catalyze the endo-type cleavage of the 1→4-O-glycosidic linkages between 2-O-sulfated α-L-fucose residues in fucoidans composed of the alternating 1→3- and 1→4-linked residues of sulfated α-L-fucose. This is the first report on the endo-1→4-α-L-fucanases (EC 3.2.1.212) of the GH168 family. The endo-fucanase FWf5 was used to selectively produce high- and low-molecular-weight fucoidan derivatives containing either regular alternating 2-O- and 2,4-di-O-sulfation or regular 2-O-sulfation. The polymeric 2,4-di-O-sulfated fucoidan derivative was shown to have significantly greater in vitro anticoagulant properties than 2-O-sulfated derivatives. The results have demonstrated a new type specificity among fucanases of the GH168 family and the prospects of using such enzymes to obtain standard fucoidan preparations with regular sulfation and high anticoagulant properties. Full article
(This article belongs to the Special Issue Microbial Enzymes for Biotechnological Applications)
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21 pages, 11740 KiB  
Article
Optimization of Xylooligosaccharides Production by Native and Recombinant Xylanase Hydrolysis of Chicken Feed Substrates
by Priyashini Dhaver, Brett Pletschke, Bruce Sithole and Roshini Govinden
Int. J. Mol. Sci. 2023, 24(23), 17110; https://doi.org/10.3390/ijms242317110 - 04 Dec 2023
Viewed by 879
Abstract
Poultry production faces several challenges, with feed efficiency being the main factor that can be influenced through the use of different nutritional strategies. Xylooligosaccharides (XOS) are functional feed additives that are attracting growing commercial interest due to their excellent ability to modulate the [...] Read more.
Poultry production faces several challenges, with feed efficiency being the main factor that can be influenced through the use of different nutritional strategies. Xylooligosaccharides (XOS) are functional feed additives that are attracting growing commercial interest due to their excellent ability to modulate the composition of the gut microbiota. The aim of the study was to apply crude and purified fungal xylanases, from Trichoderma harzianum, as well as a recombinant glycoside hydrolase family 10 xylanase, derived from Geobacillus stearothermophilus T6, as additives to locally produced chicken feeds. A Box–Behnken Design (BBD) was used to optimize the reducing sugar yield. Response surface methodology (RSM) revealed that reducing sugars were higher (8.05 mg/mL, 2.81 mg/mL and 2.98 mg/mL) for the starter feed treated with each of the three enzymes compared to the treatment with grower feed (3.11 mg/mL, 2.41 mg/mL and 2.62 mg/mL). The hydrolysis products were analysed by thin-layer chromatography (TLC), and high-performance liquid chromatography (HPLC) analysis and showed that the enzymes hydrolysed the chicken feeds, producing a range of monosaccharides (arabinose, mannose, glucose, and galactose) and XOS, with xylobiose being the predominant XOS. These results show promising data for future applications as additives to poultry feeds. Full article
(This article belongs to the Special Issue Microbial Enzymes for Biotechnological Applications)
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23 pages, 5059 KiB  
Article
A Novel Lipase from Streptomyces exfoliatus DSMZ 41693 for Biotechnological Applications
by Guillermo Rodríguez-Alonso, Juan Toledo-Marcos, Lara Serrano-Aguirre, Carlos Rumayor, Beatriz Pasero, Aida Flores, Ana Saborido, Pilar Hoyos, María J. Hernáiz, Isabel de la Mata and Miguel Arroyo
Int. J. Mol. Sci. 2023, 24(23), 17071; https://doi.org/10.3390/ijms242317071 - 02 Dec 2023
Viewed by 1171
Abstract
Genome mining of Streptomyces exfoliatus DSMZ 41693 has allowed us to identify four different lipase-encoding sequences, and one of them (SeLipC) has been successfully cloned and extracellularly expressed using Rhodococcus sp. T104 as a host. SeLipC was purified by one-step [...] Read more.
Genome mining of Streptomyces exfoliatus DSMZ 41693 has allowed us to identify four different lipase-encoding sequences, and one of them (SeLipC) has been successfully cloned and extracellularly expressed using Rhodococcus sp. T104 as a host. SeLipC was purified by one-step hydrophobic interaction chromatography. The enzyme is a monomeric protein of 27.6 kDa, which belongs to subfamily I.7 of lipolytic enzymes according to its phylogenetic analysis and biochemical characterization. The purified enzyme shows the highest activity at 60 °C and an optimum pH of 8.5, whereas thermal stability is significantly improved when protein concentration is increased, as confirmed by thermal deactivation kinetics, circular dichroism, and differential scanning calorimetry. Enzyme hydrolytic activity using p-nitrophenyl palmitate (pNPP) as substrate can be modulated by different water-miscible organic cosolvents, detergents, and metal ions. Likewise, kinetic parameters for pNPP are: KM = 49.6 µM, kcat = 57 s−1, and kcat/KM = 1.15 × 106 s−1·M−1. SeLipC is also able to hydrolyze olive oil and degrade several polyester-type polymers such as poly(butylene succinate) (PBS), poly(butylene succinate)-co-(butylene adipate) (PBSA), and poly(ε-caprolactone) (PCL). Moreover, SeLipC can catalyze the synthesis of different sugar fatty acid esters by transesterification using vinyl laurate as an acyl donor, demonstrating its interest in different biotechnological applications. Full article
(This article belongs to the Special Issue Microbial Enzymes for Biotechnological Applications)
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11 pages, 3887 KiB  
Article
Enhancing the Heterologous Expression of a Thermophilic Endoglucanase and Its Cost-Effective Production in Pichia pastoris Using Multiple Strategies
by Wuling Dai, Haofan Dong, Zhaokun Zhang, Xin Wu, Tongtong Bao, Le Gao and Xiaoyi Chen
Int. J. Mol. Sci. 2023, 24(19), 15017; https://doi.org/10.3390/ijms241915017 - 09 Oct 2023
Cited by 1 | Viewed by 1130
Abstract
Although Pichia pastoris was successfully used for heterologous gene expression for more than twenty years, many factors influencing protein expression remain unclear. Here, we optimized the expression of a thermophilic endoglucanase from Thermothielavioides terrestris (TtCel45A) for cost-effective production in Pichia pastoris. To [...] Read more.
Although Pichia pastoris was successfully used for heterologous gene expression for more than twenty years, many factors influencing protein expression remain unclear. Here, we optimized the expression of a thermophilic endoglucanase from Thermothielavioides terrestris (TtCel45A) for cost-effective production in Pichia pastoris. To achieve this, we established a multifactorial regulation strategy that involved selecting a genome-editing system, utilizing neutral loci, incorporating multiple copies of the heterologous expression cassette, and optimizing high-density fermentation for the co-production of single-cell protein (SCP). Notably, even though all neutral sites were used, there was still a slight difference in the enzymatic activity of heterologously expressed TtCel45A. Interestingly, the optimal gene copy number for the chromosomal expression of TtCel45A was found to be three, indicating limitations in translational capacity, post-translational processing, and secretion, ultimately impacting protein yields in P. pastoris. We suggest that multiple parameters might influence a kinetic competition between protein elongation and mRNA degradation. During high-density fermentation, the highest protein concentration and endoglucanase activity of TtCel45A with three copies reached 15.8 g/L and 9640 IU/mL, respectively. At the same time, the remaining SCP of P. pastoris exhibited a crude protein and amino acid content of up to 59.32% and 46.98%, respectively. These findings suggested that SCP from P. pastoris holds great promise as a sustainable and cost-effective alternative for meeting the global protein demand, while also enabling the production of thermophilic TtCel45A in a single industrial process. Full article
(This article belongs to the Special Issue Microbial Enzymes for Biotechnological Applications)
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23 pages, 4728 KiB  
Article
Optimisation of β-Glucosidase Production in a Crude Aspergillus japonicus VIT-SB1 Cellulase Cocktail Using One Variable at a Time and Statistical Methods and its Application in Cellulose Hydrolysis
by Nivisti Singh, Bishop Bruce Sithole and Roshini Govinden
Int. J. Mol. Sci. 2023, 24(12), 9928; https://doi.org/10.3390/ijms24129928 - 09 Jun 2023
Cited by 1 | Viewed by 1169
Abstract
Pulp and paper mill sludge (PPMS) is currently disposed of into landfills which are reaching their maximum capacity. Valorisation of PPMS by enzymatic hydrolysis using cellulases is an alternative strategy. Existing commercial cellulases are expensive and contain low titres of β-glucosidases. In [...] Read more.
Pulp and paper mill sludge (PPMS) is currently disposed of into landfills which are reaching their maximum capacity. Valorisation of PPMS by enzymatic hydrolysis using cellulases is an alternative strategy. Existing commercial cellulases are expensive and contain low titres of β-glucosidases. In this study, β-glucosidase production was optimised by Aspergillus japonicus VIT-SB1 to obtain higher β-glucosidase titres using the One Variable at a Time (OVAT), Plackett Burman (PBD), and Box Behnken design (BBD)of experiments and the efficiency of the optimised cellulase cocktail to hydrolyse cellulose was tested. β-Glucosidase production was enhanced from 0.4 to 10.13 U/mL, representing a 25.3-fold increase in production levels after optimisation. The optimal BBD production conditions were 6 days of fermentation at 20 °C, 125 rpm, 1.75% soy peptone, and 1.25% wheat bran in (pH 6.0) buffer. The optimal pH for β-glucosidase activity in the crude cellulase cocktail was (pH 5.0) at 50 °C. Optimal cellulose hydrolysis using the crude cellulase cocktail occurred at longer incubation times, and higher substrate loads and enzyme doses. Cellulose hydrolysis with the A. japonicus VIT-SB1 cellulase cocktail and commercial cellulase cocktails resulted in glucose yields of 15.12 and 12.33 µmol/mL glucose, respectively. Supplementation of the commercial cellulase cocktail with 0.25 U/mg of β-glucosidase resulted in a 19.8% increase in glucose yield. Full article
(This article belongs to the Special Issue Microbial Enzymes for Biotechnological Applications)
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8 pages, 866 KiB  
Communication
Acuminosylation of Tyrosol by a Commercial Diglycosidase
by Peter Haluz, Peter Kis, Matej Cvečko, Mária Mastihubová and Vladimír Mastihuba
Int. J. Mol. Sci. 2023, 24(6), 5943; https://doi.org/10.3390/ijms24065943 - 21 Mar 2023
Cited by 2 | Viewed by 1231
Abstract
A commercial glycosidase mixture obtained from Penicillium multicolor (Aromase H2) was found to comprise a specific diglycosidase activity, β-acuminosidase, alongside undetectable levels of β-apiosidase. The enzyme was tested in the transglycosylation of tyrosol using 4-nitrophenyl β-acuminoside as the diglycosyl donor. The reaction was [...] Read more.
A commercial glycosidase mixture obtained from Penicillium multicolor (Aromase H2) was found to comprise a specific diglycosidase activity, β-acuminosidase, alongside undetectable levels of β-apiosidase. The enzyme was tested in the transglycosylation of tyrosol using 4-nitrophenyl β-acuminoside as the diglycosyl donor. The reaction was not chemoselective, providing a mixture of Osmanthuside H and its counterpart regioisomer 4-(2-hydroxyethyl)phenyl β-acuminoside in 58% yield. Aromase H2 is therefore the first commercial β-acuminosidase which is also able to glycosylate phenolic acceptors. Full article
(This article belongs to the Special Issue Microbial Enzymes for Biotechnological Applications)
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17 pages, 24865 KiB  
Article
An Insight into the Essential Role of Carbohydrate-Binding Modules in Enzymolysis of Xanthan
by Xin Ni, Tong Fu, Xueyan Wang, Jingjing Zhao, Zhimin Yu, Xianzhen Li and Fan Yang
Int. J. Mol. Sci. 2023, 24(6), 5480; https://doi.org/10.3390/ijms24065480 - 13 Mar 2023
Cited by 2 | Viewed by 1105
Abstract
To date, due to the low accessibility of enzymes to xanthan substrates, the enzymolysis of xanthan remains deficient, which hinders the industrial production of functional oligoxanthan. To enhance the enzymatic affinity against xanthan, the essential role of two carbohydrate binding modules—MiCBMx [...] Read more.
To date, due to the low accessibility of enzymes to xanthan substrates, the enzymolysis of xanthan remains deficient, which hinders the industrial production of functional oligoxanthan. To enhance the enzymatic affinity against xanthan, the essential role of two carbohydrate binding modules—MiCBMx and PspCBM84, respectively, derived from Microbacterium sp. XT11 and Paenibacillus sp. 62047—in catalytic properties of endotype xanthanase MiXen were investigated for the first time. Basic characterizations and kinetic parameters of different recombinants revealed that, compared with MiCBMx, PspCBM84 dramatically increased the thermostability of endotype xanthanase, and endowed the enzyme with higher substrate affinity and catalytic efficiency. Notably, the activity of endotype xanthanase was increased by 16 times after being fused with PspCBM84. In addition, the presence of both CBMs obviously enabled endotype xanthanase to produce more oligoxanthan, and xanthan digests prepared by MiXen-CBM84 showed better antioxidant activity due to the higher content of active oligosaccharides. The results of this work lay a foundation for the rational design of endotype xanthanase and the industrial production of oligoxanthan in the future. Full article
(This article belongs to the Special Issue Microbial Enzymes for Biotechnological Applications)
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Review

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12 pages, 1345 KiB  
Review
The Engineering, Expression, and Immobilization of Epimerases for D-allulose Production
by Jin Hao Tan, Anqi Chen, Jiawu Bi, Yee Hwee Lim, Fong Tian Wong and Dave Siak-Wei Ow
Int. J. Mol. Sci. 2023, 24(16), 12703; https://doi.org/10.3390/ijms241612703 - 11 Aug 2023
Cited by 1 | Viewed by 1615
Abstract
The rare sugar D-allulose is a potential replacement for sucrose with a wide range of health benefits. Conventional production involves the employment of the Izumoring strategy, which utilises D-allulose 3-epimerase (DAEase) or D-psicose 3-epimerase (DPEase) to convert D-fructose into [...] Read more.
The rare sugar D-allulose is a potential replacement for sucrose with a wide range of health benefits. Conventional production involves the employment of the Izumoring strategy, which utilises D-allulose 3-epimerase (DAEase) or D-psicose 3-epimerase (DPEase) to convert D-fructose into D-allulose. Additionally, the process can also utilise D-tagatose 3-epimerase (DTEase). However, the process is not efficient due to the poor thermotolerance of the enzymes and low conversion rates between the sugars. This review describes three newly identified DAEases that possess desirable properties for the industrial-scale manufacturing of D-allulose. Other methods used to enhance process efficiency include the engineering of DAEases for improved thermotolerance or acid resistance, the utilization of Bacillus subtilis for the biosynthesis of D-allulose, and the immobilization of DAEases to enhance its activity, half-life, and stability. All these research advancements improve the yield of D-allulose, hence closing the gap between the small-scale production and industrial-scale manufacturing of D-allulose. Full article
(This article belongs to the Special Issue Microbial Enzymes for Biotechnological Applications)
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36 pages, 2770 KiB  
Review
Microbial Enzyme Biotechnology to Reach Plastic Waste Circularity: Current Status, Problems and Perspectives
by Marco Orlando, Gianluca Molla, Pietro Castellani, Valentina Pirillo, Vincenzo Torretta and Navarro Ferronato
Int. J. Mol. Sci. 2023, 24(4), 3877; https://doi.org/10.3390/ijms24043877 - 15 Feb 2023
Cited by 12 | Viewed by 7284
Abstract
The accumulation of synthetic plastic waste in the environment has become a global concern. Microbial enzymes (purified or as whole-cell biocatalysts) represent emerging biotechnological tools for waste circularity; they can depolymerize materials into reusable building blocks, but their contribution must be considered within [...] Read more.
The accumulation of synthetic plastic waste in the environment has become a global concern. Microbial enzymes (purified or as whole-cell biocatalysts) represent emerging biotechnological tools for waste circularity; they can depolymerize materials into reusable building blocks, but their contribution must be considered within the context of present waste management practices. This review reports on the prospective of biotechnological tools for plastic bio-recycling within the framework of plastic waste management in Europe. Available biotechnology tools can support polyethylene terephthalate (PET) recycling. However, PET represents only ≈7% of unrecycled plastic waste. Polyurethanes, the principal unrecycled waste fraction, together with other thermosets and more recalcitrant thermoplastics (e.g., polyolefins) are the next plausible target for enzyme-based depolymerization, even if this process is currently effective only on ideal polyester-based polymers. To extend the contribution of biotechnology to plastic circularity, optimization of collection and sorting systems should be considered to feed chemoenzymatic technologies for the treatment of more recalcitrant and mixed polymers. In addition, new bio-based technologies with a lower environmental impact in comparison with the present approaches should be developed to depolymerize (available or new) plastic materials, that should be designed for the required durability and for being susceptible to the action of enzymes. Full article
(This article belongs to the Special Issue Microbial Enzymes for Biotechnological Applications)
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