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16 pages, 2948 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Production of Human Milk Fat Substitutes by Lipase-Catalyzed Acidolysis: Immobilization, Synthesis, Molecular Docking and Optimization Studies

by Cleide M. F. Soares, Milson S. Barbosa, Samuel B. Santos, Silvana Mattedi, Álvaro S. Lima, Matheus M. Pereira, Carla Tecelão and Suzana Ferreira-Dias

Catalysts 2023, 13(5), 825; https://doi.org/10.3390/catal13050825 - 29 Apr 2023

Cited by 3 | Viewed by 1938

Abstract

Human milk fat (HMF) triacylglycerols (TAGs) mainly contain palmitic acid esterified at the sn-2 position while oleic and other unsaturated fatty acids are located at positions sn-1,3. This study aimed at the production of HMF substitutes (HMFS) by lipase-catalyzed acidolysis of [...] Read more.

Human milk fat (HMF) triacylglycerols (TAGs) mainly contain palmitic acid esterified at the sn-2 position while oleic and other unsaturated fatty acids are located at positions sn-1,3. This study aimed at the production of HMF substitutes (HMFS) by lipase-catalyzed acidolysis of tripalmitin with oleic acid, in a solvent-free medium. Burkholderia cepacia lipase (BCL) was immobilized in silica (prepared with protic or aprotic ionic liquids) by covalent binding or encapsulation and used as biocatalyst. The supports and immobilized biocatalysts were characterized by FTIR, TGA, and SEM. Molecular docking analysis showed that BCL preferentially attacks oleic acid rather than tripalmitin, due to the lower free energy of hydrophobic binding with this acid (−6.5 kcal·mol⁻¹) than with tripalmitin (5.4 kcal·mol⁻¹). Therefore, the tripalmitin attack by BCL and subsequent HMFS production only occurs after the binding to most of the oleic acid molecules. The highest acidolysis activity was obtained with BCL immobilized by covalent binding in prepared silica with aprotic ionic liquid. A central composite rotatable design, as a function of temperature (58–72 °C) and oleic acid/tripalmitin molar ratio (MR = 2:1–6.8:1), was performed for acidolysis optimization. Under optimized conditions (58 °C and MR = 4:1 or 60 °C and MR = 2:1), the oleic acid incorporation of 28 mol.% was achieved after 48 h. Full article

(This article belongs to the Special Issue Enzyme Engineering for Improving the Enzymatic Catalysis, Stability and Substrate Specificity and Application)

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16 pages, 4838 KiB

Open AccessArticle

Immobilized KDN Lipase on Macroporous Resin for Isopropyl Myristate Synthesis

by Ming Song, Yuhan Xin, Sulan Cai, Weizhuo Xu and Wei Xu

Catalysts 2023, 13(4), 772; https://doi.org/10.3390/catal13040772 - 19 Apr 2023

Cited by 2 | Viewed by 1713

Abstract

Free enzymes often face economic problems because of their non-repeatability and variability, which limit their application in industrial production. In this study, KDN lipase was immobilized with the macroporous resin LXTE-1000 and glutaraldehyde. The optimal conditions of enzyme immobilization were defined by a [...] Read more.

Free enzymes often face economic problems because of their non-repeatability and variability, which limit their application in industrial production. In this study, KDN lipase was immobilized with the macroporous resin LXTE-1000 and glutaraldehyde. The optimal conditions of enzyme immobilization were defined by a single factor experiment and response surface methodology (RSM). The concentration of the cross-linking agent glutaraldehyde was 0.46% (v/v), the cross-linking temperature was 25.0 °C, and the cross-linking time was 157 min. The enzyme activity of the immobilized KDN lipase after adsorption/cross-linking was 291.36 U/g, and the recovery of the enzyme activity was 9.90%. The optimal conditions for the synthesis of isopropyl myristate were catalyzed by the immobilized KDN lipase in a solvent-free system: immobilized enzyme 53 mg, reaction temperature 36.1 °C, myristic acid 228.4 mg, isopropanol 114 µL, and reaction time 18 h. The yield of isopropyl myristate was 66.62%. After ten cycles, the activity of the immobilized KDN lipase preserved more than 46.87% of its initial enzyme activity, and it demonstrated high tolerance to solvents compared to free KDN lipase. Full article

(This article belongs to the Special Issue Enzyme Engineering for Improving the Enzymatic Catalysis, Stability and Substrate Specificity and Application)

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16 pages, 3644 KiB

Open AccessArticle

Biochemical Characterization of an Endoglucanase GH7 from Thermophile Thermothielavioides terrestris Expressed on Aspergillus nidulans

by Robson C. Alnoch, Jose C. S. Salgado, Gabriela S. Alves, Diandra de Andrades, Luana P. Meleiro, Fernando Segato, Gabriela Leila Berto, Richard J. Ward, Marcos S. Buckeridge and Maria de Lourdes T. M. Polizeli

Catalysts 2023, 13(3), 582; https://doi.org/10.3390/catal13030582 - 14 Mar 2023

Cited by 3 | Viewed by 1964

Abstract

Endoglucanases (EC 3.2.1.4) are important enzymes involved in the hydrolysis of cellulose, acting randomly in the β-1,4-glycosidic bonds present in the amorphous regions of the polysaccharide chain. These biocatalysts have been classified into 14 glycosyl hydrolase (GH) families. The GH7 family is of [...] Read more.

Endoglucanases (EC 3.2.1.4) are important enzymes involved in the hydrolysis of cellulose, acting randomly in the β-1,4-glycosidic bonds present in the amorphous regions of the polysaccharide chain. These biocatalysts have been classified into 14 glycosyl hydrolase (GH) families. The GH7 family is of particular interest since it may act on a broad range of substrates, including cellulose, β-glucan, and xylan, an attractive feature for biotechnological applications, especially in the renewable energy field. In the current work, a gene from the thermophilic fungus Thermothielavioides terrestris, encoding an endoglucanase GH7 (TtCel7B), was cloned in the secretion vector pEXPYR and transformed into the high-protein-producing strain Aspergillus nidulans A773. Purified TtCel7B has a molecular weight of approximately 66 kDa, evidenced by SDS-PAGE. Circular dichroism confirmed the high β-strand content consistent with the canonical GH7 family β-jellyroll fold, also observed in the 3D homology model of TtCel7B. Biochemical characterization assays showed that TtCel7B was active over a wide range of pH values (3.5–7.0) and temperatures (45–70 °C), with the highest activity at pH 4.0 and 65 °C. TtCel7B also was stable over a wide range of pH values (3.5–9.0), maintaining more than 80% of its activity after 24 h. The K_M and Vmax values in low-viscosity carboxymethylcellulose were 9.3 mg mL⁻¹ and 2.5 × 10⁴ U mg⁻¹, respectively. The results obtained in this work provide a basis for the development of applications of recombinant TtCel7B in the renewable energy field. Full article

(This article belongs to the Special Issue Enzyme Engineering for Improving the Enzymatic Catalysis, Stability and Substrate Specificity and Application)

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17 pages, 4865 KiB

Open AccessArticle

Improved Stability and Hydrolysates of Hyperthermophilic GH57 Type II Pullulanase from the Deep-Sea Archaeon Thermococcus siculi HJ21 by Truncation

by Xudong Wu, Baojie Dou, Boyan Wang, Mingwang Liu, Ruxue Shao, Jing Lu, Mingsheng Lyu and Shujun Wang

Catalysts 2023, 13(3), 453; https://doi.org/10.3390/catal13030453 - 21 Feb 2023

Cited by 6 | Viewed by 2283

Abstract

Pullulanase (EC 3.2.1.41) belongs to the amylase family and is often used alone or in combination with other amylases in the industrial production of starch-based products. This enzyme is often required in industrial production because of its better stability. We here truncated the [...] Read more.

Pullulanase (EC 3.2.1.41) belongs to the amylase family and is often used alone or in combination with other amylases in the industrial production of starch-based products. This enzyme is often required in industrial production because of its better stability. We here truncated the pullulanase gene from the deep-sea hydrothermal anaerobic archaeon Thermococcus siculi HJ21 and obtained Pul-HJΔ782, which is a member of the α-amylase family GH57. The results revealed that the optimum temperature for Pul-HJΔ782 was 100 °C, and its thermostability at 100 °C improved after truncation. Less than 15% of its enzyme activity was lost after 1 h of incubation at 100 °C, and 57% activity remained after 5 h of treatment. Truncation significantly improved the overall pH tolerance range of Pul-HJΔ782, and its stability in the pH range 4–8 was over 80% relative activity from an average of 60%. The sequence and structural model of Pul-HJΔ782 was analyzed, and its instability index was reduced significantly. Furthermore, the hydrolysates of the truncated and wild-type pullulanase were analyzed, and the enzymatic digestion efficiency of the truncated Pul-HJΔ782 was higher. Full article

(This article belongs to the Special Issue Enzyme Engineering for Improving the Enzymatic Catalysis, Stability and Substrate Specificity and Application)

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12 pages, 15400 KiB

Open AccessArticle

Production of Biomodified Bleached Kraft Pulp by Catalytic Conversion Using Penicillium verruculosum Enzymes: Composition, Properties, Structure, and Application

by Aleksandr R. Shevchenko, Irina V. Tyshkunova, Dmitry G. Chukhchin, Alexey V. Malkov, Evgeniy A. Toptunov, Vadim D. Telitsin, Aleksandra M. Rozhkova, Olga A. Sinitsyna, Iosif V. Gofman and Andrey S. Aksenov

Catalysts 2023, 13(1), 103; https://doi.org/10.3390/catal13010103 - 3 Jan 2023

Cited by 4 | Viewed by 2353

Abstract

The global development of the bioeconomy is impossible without technologies for comprehensive processing of plant renewable resources. The use of proven pretreatment technologies raises the possibility of the industrial implementation of the enzymatic conversion of polysaccharides from lignocellulose considering the process’s complexity. For [...] Read more.

The global development of the bioeconomy is impossible without technologies for comprehensive processing of plant renewable resources. The use of proven pretreatment technologies raises the possibility of the industrial implementation of the enzymatic conversion of polysaccharides from lignocellulose considering the process’s complexity. For instance, a well-tuned kraft pulping produces a substrate easily degraded by cellulases and hemicelulases. Enzymatic hydrolysis of bleached hardwood kraft pulp was carried out using an enzyme complex of endoglucanases, cellobiohydrolases, β-glucosidases, and xylanases produced by recombinant strains of Penicillium verruculosum at a 10 FPU/g mixture rate and a 10% substrate concentration. As a result of biocatalysis, the following products were obtained: sugar solution, mainly glucose, xylobiose, xylose, as well as other minor reducing sugars; a modified complex based on cellulose and xylan. The composition of the biomodified kraft pulp was determined by HPLC. The method for determining the crystallinity on an X-ray diffractometer was used to characterize the properties. The article shows the possibility of producing biomodified cellulose cryogels by amorphization with concentrated 85% H₃PO₄ followed by precipitation with water and supercritical drying. The analysis of the enzymatic hydrolysate composition revealed the predominance of glucose (55–67%) among the reducing sugars with a maximum content in the solution up to 6% after 72 h. The properties and structure of the modified kraft pulp were shown to change during biocatalysis; in particular, the crystallinity increased by 5% after 3 h of enzymatic hydrolysis. We obtained cryogels based on the initial and biomodified kraft pulp with conversion rates of 35, 50, and 70%. The properties of these cryogels are not inferior to those of cryogels based on industrial microcrystalline cellulose, as confirmed by the specific surface area, degree of swelling, porosity, and SEM images. Thus, kraft pulp enzymatic hydrolysis offers prospects not only for producing sugar-rich hydrolysates for microbiological synthesis, but also cellulose powders and cryogels with specified properties. Full article

(This article belongs to the Special Issue Enzyme Engineering for Improving the Enzymatic Catalysis, Stability and Substrate Specificity and Application)

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13 pages, 1003 KiB

Open AccessFeature PaperArticle

Mineralization of Lipase from Thermomyces lanuginosus Immobilized on Methacrylate Beads Bearing Octadecyl Groups to Improve Enzyme Features

by José R. Guimarães, Diego Carballares, Javier Rocha-Martin, Paulo W. Tardioli and Roberto Fernandez-Lafuente

Catalysts 2022, 12(12), 1552; https://doi.org/10.3390/catal12121552 - 1 Dec 2022

Cited by 5 | Viewed by 1743

Abstract

Lipase from Thermomyces lanuginosus (TLL) has been immobilized on Purolite Lifetech^® ECR8806F (viz. methacrylate macroporous resin containing octadecyl groups, designated as Purolite C18-TLL), and the enzyme performance has been compared to that of the enzyme immobilized on octyl-agarose, designated as agarose C8-TLL. [...] Read more.

Lipase from Thermomyces lanuginosus (TLL) has been immobilized on Purolite Lifetech^® ECR8806F (viz. methacrylate macroporous resin containing octadecyl groups, designated as Purolite C18-TLL), and the enzyme performance has been compared to that of the enzyme immobilized on octyl-agarose, designated as agarose C8-TLL. The hydrolytic activity versus p-nitrophenol butyrate decreased significantly, and to a lower extent versus S-methyl mandelate (more than twofold), while versus triacetin and R-methyl mandelate, the enzyme activity was higher for the biocatalyst prepared using Purolite C18 (up to almost five-fold). Regarding the enzyme stability, Purolite C18-TLL was significantly more stable than the agarose C8-TLL. Next, the biocatalysts were mineralized using zinc, copper or cobalt phosphates. Mineralization increased the hydrolytic activity of Purolite C18-TLL versus triacetin and R-methyl mandelate, while this activity decreased very significantly versus the S-isomer, while the effects using agarose C8-TLL were more diverse (hydrolytic activity increase or decrease was dependent on the metal and substrate). The zinc salt treatment increased the stability of both biocatalysts, but with a lower impact for Purolite C18-TLL than for agarose-C8-TLL. On the contrary, the copper and cobalt salt treatments decreased enzyme stability, but more intensively using Purolite C18-TLL. The results show that even using enzymes immobilized following the same strategy, the differences in the enzyme conformation cause mineralization to have diverse effects on enzyme stability, hydrolytic activity, and specificity. Full article

(This article belongs to the Special Issue Enzyme Engineering for Improving the Enzymatic Catalysis, Stability and Substrate Specificity and Application)

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19 pages, 4126 KiB

Open AccessArticle

Rational Engineering of 3α-Hydroxysteroid Dehydrogenase/Carbonyl Reductase for a Biomimetic Nicotinamide Mononucleotide Cofactor

by Yan-Liang Chen, Yun-Hao Chou, Chia-Lin Hsieh, Shean-Jaw Chiou, Tzu-Pin Wang and Chi-Ching Hwang

Catalysts 2022, 12(10), 1094; https://doi.org/10.3390/catal12101094 - 21 Sep 2022

Cited by 2 | Viewed by 2152

Abstract

Enzymes are powerful biological catalysts for natural substrates but they have low catalytic efficiency for non-natural substrates. Protein engineering can be used to optimize enzymes for catalysis and stability. 3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidoreduction reaction of NAD⁺ with androsterone. Based [...] Read more.

Enzymes are powerful biological catalysts for natural substrates but they have low catalytic efficiency for non-natural substrates. Protein engineering can be used to optimize enzymes for catalysis and stability. 3α-Hydroxysteroid dehydrogenase/carbonyl reductase (3α-HSD/CR) catalyzes the oxidoreduction reaction of NAD⁺ with androsterone. Based on the structure and catalytic mechanism, we mutated the residues of T11, I13, D41, A70, and I112 and they interacted with different portions of NAD⁺ to switch cofactor specificity to biomimetic cofactor nicotinamide mononucleotide (NMN⁺). Compared to wild-type 3α-HSD/CR, the catalytic efficiency of these mutants for NAD⁺ decreased significantly except for the T11 mutants but changed slightly for NMN⁺ except for the A70K mutant. The A70K mutant increased the catalytic efficiency for NMN⁺ by 8.7-fold, concomitant with a significant decrease in NAD⁺ by 1.4 × 10⁴-fold, resulting in 9.6 × 10⁴-fold cofactor specificity switch toward NMN⁺ over NAD⁺. Meanwhile, the I112K variant increased the thermal stability and changed to a three-state transition from a two-state transition of thermal unfolding of wild-type 3α-HSD/CR by differential scanning fluorimetry. Molecular docking analysis indicated that mutations on these residues affect the position and conformation of the docked NAD⁺ and NMN⁺, thereby affecting their activity. A70K variant sterically blocks the binding with NAD⁺, restores the H-bonding interactions of catalytic residues of Y155 and K159 with NMN⁺, and enhances the catalytic efficiency for NMN⁺. Full article

(This article belongs to the Special Issue Enzyme Engineering for Improving the Enzymatic Catalysis, Stability and Substrate Specificity and Application)

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Review

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21 pages, 3206 KiB

Open AccessReview

Role and Application of Biocatalysts in Cancer Drug Discovery

by Sounok Sengupta, Prathama Das, Samridhi Sharma, Monu Kumar Shukla, Rajesh Kumar, Rajiv Kumar Tonk, Sadanand Pandey and Deepak Kumar

Catalysts 2023, 13(2), 250; https://doi.org/10.3390/catal13020250 - 21 Jan 2023

Cited by 8 | Viewed by 3938

Abstract

A biocatalyst is an enzyme that speeds up or slows down the rate at which a chemical reaction occurs and speeds up certain processes by 10⁸ times. It is used as an anticancer agent because it targets drug activation inside the tumor [...] Read more.

A biocatalyst is an enzyme that speeds up or slows down the rate at which a chemical reaction occurs and speeds up certain processes by 10⁸ times. It is used as an anticancer agent because it targets drug activation inside the tumor microenvironment while limiting damage to healthy cells. Biocatalysts have been used for the synthesis of different heterocyclic compounds and is also used in the nano drug delivery systems. The use of nano-biocatalysts for tumor-targeted delivery not only aids in tumor invasion, angiogenesis, and mutagenesis, but also provides information on the expression and activity of many markers related to the microenvironment. Iosmapinol, moclobemide, cinepazide, lysine dioxygenase, epothilone, 1-homophenylalanine, and many more are only some of the anticancer medicines that have been synthesised using biocatalysts. In this review, we have highlighted the application of biocatalysts in cancer therapies as well as the use of biocatalysts in the synthesis of drugs and drug-delivery systems in the tumor microenvironment. Full article

(This article belongs to the Special Issue Enzyme Engineering for Improving the Enzymatic Catalysis, Stability and Substrate Specificity and Application)

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