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2 pages, 205 KiB

Open AccessEditorial

Novel Microbial Enzymes with Industrial Applications

by María-Isabel González-Siso and Manuel Becerra

Microorganisms 2023, 11(4), 986; https://doi.org/10.3390/microorganisms11040986 - 10 Apr 2023

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Abstract

Eberhardt et al [...] Full article

(This article belongs to the Special Issue Novel Microbial Enzymes with Industrial Applications)

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19 pages, 2669 KiB

Open AccessArticle

Reactivity of a Recombinant Esterase from Thermus thermophilus HB27 in Aqueous and Organic Media

by Roberto González-González, Pablo Fuciños, Elisa Beneventi, Olalla López-López, Begoña Pampín, Ramón Rodríguez, María Isabel González-Siso, Jacobo Cruces and María Luisa Rúa

Microorganisms 2022, 10(5), 915; https://doi.org/10.3390/microorganisms10050915 - 27 Apr 2022

Cited by 1 | Viewed by 2104

Abstract

The thermoalkalophilic membrane-associated esterase E34Tt from Thermus thermophilus HB27 was cloned and expressed in Kluyveromyces lactis (KLEST-3S esterase). The recombinant enzyme was tested as a biocatalyst in aqueous and organic media. It displayed a high thermal stability and was active in the presence [...] Read more.

The thermoalkalophilic membrane-associated esterase E34Tt from Thermus thermophilus HB27 was cloned and expressed in Kluyveromyces lactis (KLEST-3S esterase). The recombinant enzyme was tested as a biocatalyst in aqueous and organic media. It displayed a high thermal stability and was active in the presence of 10% (v/v) organic solvents and 1% (w/v) detergents. KLEST-3S hydrolysed triglycerides of various acyl chains, which is a rare characteristic among carboxylic ester hydrolases from extreme thermophiles, with maximum activity on tributyrin. It also displayed interfacial activation towards triacetin. KLEST-3S was also tested as a biocatalyst in organic media. The esterase provided high yields for the acetylation of alcohols. In addition, KLEST-3S catalyzed the stereoselective hydrolysis of (R,S)-ibuprofen methyl ester (87% ee). Our results indicate that KLEST-3S may be a robust and efficient biocatalyst for application in industrial bioconversions. Full article

(This article belongs to the Special Issue Novel Microbial Enzymes with Industrial Applications)

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14 pages, 2257 KiB

Open AccessArticle

Synergy of Cellulase Systems between Acetivibrio thermocellus and Thermoclostridium stercorarium in Consolidated-Bioprocessing for Cellulosic Ethanol

by Na Wang, Zhihua Yan, Na Liu, Xiaorong Zhang and Chenggang Xu

Microorganisms 2022, 10(3), 502; https://doi.org/10.3390/microorganisms10030502 - 24 Feb 2022

Cited by 10 | Viewed by 1896

Abstract

Anaerobes harbor some of the most efficient biological machinery for cellulose degradation, especially thermophilic bacteria, such as Acetivibrio thermocellus and Thermoclostridium stercorarium, which play a fundamental role in transferring lignocellulose into ethanol through consolidated bioprocessing (CBP). In this study, we compared activities [...] Read more.

Anaerobes harbor some of the most efficient biological machinery for cellulose degradation, especially thermophilic bacteria, such as Acetivibrio thermocellus and Thermoclostridium stercorarium, which play a fundamental role in transferring lignocellulose into ethanol through consolidated bioprocessing (CBP). In this study, we compared activities of two cellulase systems under varying kinds of hemicellulose and cellulose. A. thermocellus was identified to contribute specifically to cellulose hydrolysis, whereas T. stercorarium contributes to hemicellulose hydrolysis. The two systems were assayed in various combinations to assess their synergistic effects using cellulose and corn stover as the substrates. Their maximum synergy degrees on cellulose and corn stover were, respectively, 1.26 and 1.87 at the ratio of 3:2. Furthermore, co-culture of these anaerobes on the mixture of cellulose and xylan increased ethanol concentration from 21.0 to 40.4 mM with a high cellulose/xylan-to-ethanol conversion rate of up to 20.7%, while the conversion rates of T. stercorarium and A. thermocellus monocultures were 19.3% and 15.2%. The reason is that A. thermocellus had the ability to rapidly degrade cellulose while T. stercorarium co-utilized both pentose and hexose, the metabolites of cellulose degradation, to produce ethanol. The synergistic effect of cellulase systems and metabolic pathways in A. thermocellus and T. stercorarium provides a novel strategy for the design, selection, and optimization of ethanol production from cellulosic biomass through CBP. Full article

(This article belongs to the Special Issue Novel Microbial Enzymes with Industrial Applications)

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27 pages, 16792 KiB

Open AccessArticle

Novel Feather Degrading Keratinases from Bacillus cereus Group: Biochemical, Genetic and Bioinformatics Analysis

by Arwa Ali Almahasheer, Amal Mahmoud, Hesham El-Komy, Amany I. Alqosaibi, Sultan Aktar, Sayed AbdulAzeez and J. Francis Borgio

Microorganisms 2022, 10(1), 93; https://doi.org/10.3390/microorganisms10010093 - 01 Jan 2022

Cited by 18 | Viewed by 3504

Abstract

In this study, five keratinolytic bacteria were isolated from poultry farm waste of Eastern Province, Saudi Arabia. The highest keratinase activity was obtained at 40–45 °C, pH 8–9, feather concentration 0.5–1%, and using white chicken feather as keratin substrate for 72 h. Enhancement [...] Read more.

In this study, five keratinolytic bacteria were isolated from poultry farm waste of Eastern Province, Saudi Arabia. The highest keratinase activity was obtained at 40–45 °C, pH 8–9, feather concentration 0.5–1%, and using white chicken feather as keratin substrate for 72 h. Enhancement of keratinase activity through physical mutagen UV radiation and/or chemical mutagen ethyl methanesulfonate (EMS) resulted in five mutants with 1.51–3.73-fold increased activity over the wild type. When compared with the wild type, scanning electron microscopy validated the mutants’ effectiveness in feather degradation. Bacterial isolates are classified as members of the S8 family peptidase Bacillus cereus group based on sequence analysis of the 16S rRNA and keratinase genes. Interestingly, keratinase KerS gene shared 95.5–100% identity to keratinase, thermitase alkaline serine protease, and thermophilic serine protease of the B. cereus group. D₁₃₇N substitution was observed in the keratinase KerS gene of the mutant strain S13 (KerS13uv+ems), and also seven substitution variations in KerS26 and KerS26uv of strain S26 and its mutant S26uv. Functional analysis revealed that the subtilisin-like serine protease domain containing the Asp/His/Ser catalytic triad of KerS gene was not affected by the predicted substitutions. Prediction of physicochemical properties of KerS gene showed instability index between 17.5–19.3 and aliphatic index between 74.7–75.7, which imply keratinase stability and significant thermostability. The docking studies revealed the impact of substitutions on the superimposed structure and an increase in binding of mutant D₁₃₇N of KerS13uv+ems (affinity: −7.17; S score: −6.54 kcal/mol) and seven mutants of KerS26uv (affinity: −7.43; S score: −7.17 kcal/mol) compared to the wild predicted structure (affinity: −6.57; S score: −6.68 kcal/mol). Together, the keratinolytic activity, similarity to thermostable keratinases, and binding affinity suggest that keratinases KerS13uv+ems and KerS26uv could be used for feather processing in the industry. Full article

(This article belongs to the Special Issue Novel Microbial Enzymes with Industrial Applications)

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14 pages, 2128 KiB

Open AccessArticle

Novel Thermotolerant Amylase from Bacillus licheniformis Strain LB04: Purification, Characterization and Agar-Agarose

by Anaid Silva-Salinas, Melissa Rodríguez-Delgado, Jesús Gómez-Treviño, Ulrico López-Chuken, Clarita Olvera-Carranza and Edgar Allan Blanco-Gámez

Microorganisms 2021, 9(9), 1857; https://doi.org/10.3390/microorganisms9091857 - 01 Sep 2021

Cited by 10 | Viewed by 3434

Abstract

This study analyzed the thermostability and effect of calcium ions on the enzymatic activity of α-amylase produced by Bacillus licheniformis strain LB04 isolated from Espinazo Hot springs in Nuevo Leon, Mexico. The enzyme was immobilized by entrapment on agar-agarose beads, with an entrapment [...] Read more.

This study analyzed the thermostability and effect of calcium ions on the enzymatic activity of α-amylase produced by Bacillus licheniformis strain LB04 isolated from Espinazo Hot springs in Nuevo Leon, Mexico. The enzyme was immobilized by entrapment on agar-agarose beads, with an entrapment yield of 19.9%. The identification of the bacteria was carried out using 16s rDNA sequencing. The enzyme was purified through ion exchange chromatography (IEX) in a DEAE-Sephadex column, revealing a protein with a molecular weight of ≈130 kDa. The enzyme was stable at pH 3.0 and heat stable up to 80 °C. However, the optimum conditions were reached at 65 °C and pH 3.0, with a specific activity of 1851.7 U mg⁻¹ ± 1.3. The agar-agarose immobilized α-amylase had a hydrolytic activity nearly 25% higher when compared to the free enzyme. This study provides critical information for the understanding of the enzymatic profile of B. licheniformis strain LB04 and the potential application of the microorganisms at an industrial level, specifically in the food industry. Full article

(This article belongs to the Special Issue Novel Microbial Enzymes with Industrial Applications)

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16 pages, 5167 KiB

Open AccessArticle

Characterization of Two Unique Cold-Active Lipases Derived from a Novel Deep-Sea Cold Seep Bacterium

by Chenchen Guo, Rikuan Zheng, Ruining Cai, Chaomin Sun and Shimei Wu

Microorganisms 2021, 9(4), 802; https://doi.org/10.3390/microorganisms9040802 - 10 Apr 2021

Cited by 8 | Viewed by 2395

Abstract

The deep ocean microbiota has unexplored potential to provide enzymes with unique characteristics. In order to obtain cold-active lipases, bacterial strains isolated from the sediment of the deep-sea cold seep were screened, and a novel strain gcc21 exhibited a high lipase catalytic activity, [...] Read more.

The deep ocean microbiota has unexplored potential to provide enzymes with unique characteristics. In order to obtain cold-active lipases, bacterial strains isolated from the sediment of the deep-sea cold seep were screened, and a novel strain gcc21 exhibited a high lipase catalytic activity, even at the low temperature of 4 °C. The strain gcc21 was identified and proposed to represent a new species of Pseudomonas according to its physiological, biochemical, and genomic characteristics; it was named Pseudomonas marinensis. Two novel encoding genes for cold-active lipases (Lipase 1 and Lipase 2) were identified in the genome of strain gcc21. Genes encoding Lipase 1 and Lipase 2 were respectively cloned and overexpressed in E. coli cells, and corresponding lipases were further purified and characterized. Both Lipase 1 and Lipase 2 showed an optimal catalytic temperature at 4 °C, which is much lower than those of most reported cold-active lipases, but the activity and stability of Lipase 2 were much higher than those of Lipase 1 under different tested pHs and temperatures. In addition, Lipase 2 was more stable than Lipase 1 when treated with different metal ions, detergents, potential inhibitors, and organic solvents. In a combination of mutation and activity assays, catalytic triads of Ser, Asp, and His in Lipase 1 and Lipase 2 were demonstrated to be essential for maintaining enzyme activity. Phylogenetic analysis showed that both Lipase 1 and Lipase 2 belonged to lipase family III. Overall, our results indicate that deep-sea cold seep is a rich source for novel bacterial species that produce potentially unique cold-active enzymes. Full article

(This article belongs to the Special Issue Novel Microbial Enzymes with Industrial Applications)

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24 pages, 2388 KiB

Open AccessArticle

Genome-Driven Discovery of Enzymes with Industrial Implications from the Genus Aneurinibacillus

by Majid Rasool Kamli, Nada A. Y. Alzahrani, Nahid H. Hajrah, Jamal S. M. Sabir and Adeel Malik

Microorganisms 2021, 9(3), 499; https://doi.org/10.3390/microorganisms9030499 - 26 Feb 2021

Cited by 10 | Viewed by 2500

Abstract

Bacteria belonging to the genus Aneurinibacillus within the family Paenibacillaceae are Gram-positive, endospore-forming, and rod-shaped bacteria inhabiting diverse environments. Currently, there are eight validly described species of Aneurinibacillus; however, several unclassified species have also been reported. Aneurinibacillus spp. have shown the potential [...] Read more.

Bacteria belonging to the genus Aneurinibacillus within the family Paenibacillaceae are Gram-positive, endospore-forming, and rod-shaped bacteria inhabiting diverse environments. Currently, there are eight validly described species of Aneurinibacillus; however, several unclassified species have also been reported. Aneurinibacillus spp. have shown the potential for producing secondary metabolites (SMs) and demonstrated diverse types of enzyme activities. These features make them promising candidates with industrial implications. At present, genomes of 9 unique species from the genus Aneurinibacillus are available, which can be utilized to decipher invaluable information on their biosynthetic potential as well as enzyme activities. In this work, we performed the comparative genome analyses of nine Aneurinibacillus species representing the first such comprehensive study of this genus at the genome level. We focused on discovering the biosynthetic, biodegradation, and heavy metal resistance potential of this under-investigated genus. The results indicate that the genomes of Aneurinibacillus contain SM-producing regions with diverse bioactivities, including antimicrobial and antiviral activities. Several carbohydrate-active enzymes (CAZymes) and genes involved in heavy metal resistance were also identified. Additionally, a broad range of enzyme classes were also identified in the Aneurinibacillus pan-genomes, making this group of bacteria potential candidates for future investigations with industrial applications. Full article

(This article belongs to the Special Issue Novel Microbial Enzymes with Industrial Applications)

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14 pages, 1424 KiB

Open AccessArticle

β-Galactosidases from a Sequence-Based Metagenome: Cloning, Expression, Purification and Characterization

by María Florencia Eberhardt, José Matías Irazoqui and Ariel Fernando Amadio

Microorganisms 2021, 9(1), 55; https://doi.org/10.3390/microorganisms9010055 - 28 Dec 2020

Cited by 10 | Viewed by 2770

Abstract

Stabilization ponds are a common treatment technology for wastewater generated by dairy industries. Large proportions of cheese whey are thrown into these ponds, creating an environmental problem because of the large volume produced and the high biological and chemical oxygen demands. Due to [...] Read more.

Stabilization ponds are a common treatment technology for wastewater generated by dairy industries. Large proportions of cheese whey are thrown into these ponds, creating an environmental problem because of the large volume produced and the high biological and chemical oxygen demands. Due to its composition, mainly lactose and proteins, it can be considered as a raw material for value-added products, through physicochemical or enzymatic treatments. β-Galactosidases (EC 3.2.1.23) are lactose modifying enzymes that can transform lactose in free monomers, glucose and galactose, or galactooligosacharides. Here, the identification of novel genes encoding β-galactosidases, identified via whole-genome shotgun sequencing of the metagenome of dairy industries stabilization ponds is reported. The genes were selected based on the conservation of catalytic domains, comparing against the CAZy database, and focusing on families with β-galactosidases activity (GH1, GH2 and GH42). A total of 394 candidate genes were found, all belonging to bacterial species. From these candidates, 12 were selected to be cloned and expressed. A total of six enzymes were expressed, and five cleaved efficiently ortho-nitrophenyl-β-galactoside and lactose. The activity levels of one of these novel β-galactosidase was higher than other enzymes reported from functional metagenomics screening and higher than the only enzyme reported from sequence-based metagenomics. A group of novel mesophilic β-galactosidases from diary stabilization ponds’ metagenomes was successfully identified, cloned and expressed. These novel enzymes provide alternatives for the production of value-added products from dairy industries’ by-products. Full article

(This article belongs to the Special Issue Novel Microbial Enzymes with Industrial Applications)

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