International Journal of Molecular Sciences

Research

Jump to: Review

22 pages, 3352 KiB

Open AccessArticle

Influence of Lytic Polysaccharide Monooxygenase Active Site Segments on Activity and Affinity

by Christophe V.F.P. Laurent, Peicheng Sun, Stefan Scheiblbrandner, Florian Csarman, Pietro Cannazza, Matthias Frommhagen, Willem J.H. van Berkel, Chris Oostenbrink, Mirjam A. Kabel and Roland Ludwig

Int. J. Mol. Sci. 2019, 20(24), 6219; https://doi.org/10.3390/ijms20246219 - 10 Dec 2019

Cited by 49 | Viewed by 5231

Abstract

In past years, new lytic polysaccharide monooxygenases (LPMOs) have been discovered as distinct in their substrate specificity. Their unconventional, surface-exposed catalytic sites determine their enzymatic activities, while binding sites govern substrate recognition and regioselectivity. An additional factor influencing activity is the presence or [...] Read more.

In past years, new lytic polysaccharide monooxygenases (LPMOs) have been discovered as distinct in their substrate specificity. Their unconventional, surface-exposed catalytic sites determine their enzymatic activities, while binding sites govern substrate recognition and regioselectivity. An additional factor influencing activity is the presence or absence of a family 1 carbohydrate binding module (CBM1) connected via a linker to the C-terminus of the LPMO. This study investigates the changes in activity induced by shortening the second active site segment (Seg2) or removing the CBM1 from Neurospora crassa LPMO9C. NcLPMO9C and generated variants have been tested on regenerated amorphous cellulose (RAC), carboxymethyl cellulose (CMC) and xyloglucan (XG) using activity assays, conversion experiments and surface plasmon resonance spectroscopy. The absence of CBM1 reduced the binding affinity and activity of NcLPMO9C, but did not affect its regioselectivity. The linker was found important for the thermal stability of NcLPMO9C and the CBM1 is necessary for efficient binding to RAC. Wild-type NcLPMO9C exhibited the highest activity and strongest substrate binding. Shortening of Seg2 greatly reduced the activity on RAC and CMC and completely abolished the activity on XG. This demonstrates that Seg2 is indispensable for substrate recognition and the formation of productive enzyme-substrate complexes. Full article

(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)

► Show Figures

Figure 1

16 pages, 2047 KiB

Open AccessArticle

Disulfide Engineered Lipase to Enhance the Catalytic Activity: A Structure-Based Approach on BTL2

by César A. Godoy, Javier Klett, Bruno Di Geronimo, Juan A. Hermoso, José M. Guisán and César Carrasco-López

Int. J. Mol. Sci. 2019, 20(21), 5245; https://doi.org/10.3390/ijms20215245 - 23 Oct 2019

Cited by 14 | Viewed by 3730

Abstract

Enhancement, control, and tuning of hydrolytic activity and specificity of lipases are major goals for the industry. Thermoalkaliphilic lipases from the I.5 family, with their native advantages such as high thermostability and tolerance to alkaline pHs, are a target for biotechnological applications. Although [...] Read more.

Enhancement, control, and tuning of hydrolytic activity and specificity of lipases are major goals for the industry. Thermoalkaliphilic lipases from the I.5 family, with their native advantages such as high thermostability and tolerance to alkaline pHs, are a target for biotechnological applications. Although several strategies have been applied to increase lipases activity, the enhancement through protein engineering without compromising other capabilities is still elusive. Lipases from the I.5 family suffer a unique and delicate double lid restructuration to transition from a closed and inactive state to their open and enzymatically active conformation. In order to increase the activity of the wild type Geobacillus thermocatenulatus lipase 2 (BTL2) we rationally designed, based on its tridimensional structure, a mutant (ccBTL2) capable of forming a disulfide bond to lock the open state. ccBTL2 was generated replacing A191 and F206 to cysteine residues while both wild type C64 and C295 were mutated to serine. A covalently immobilized ccBTL2 showed a 3.5-fold increment in esterase activity with 0.1% Triton X-100 (2336 IU mg⁻¹) and up to 6.0-fold higher with 0.01% CTAB (778 IU mg⁻¹), both in the presence of oxidizing sulfhydryl agents, when compared to BTL2. The remarkable and industrially desired features of BTL2 such as optimal alkaliphilic pH and high thermal stability were not affected. The designed disulfide bond also conferred reversibility to the enhancement, as the increment on activity observed for ccBTL2 was controlled by redox pretreatments. MD simulations suggested that the most stable conformation for ccBTL2 (with the disulfide bond formed) was, as we predicted, similar to the open and active conformation of this lipase. Full article

(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)

► Show Figures

Graphical abstract

26 pages, 11537 KiB

Open AccessArticle

Comparative Study of Extracellular Proteolytic, Cellulolytic, and Hemicellulolytic Enzyme Activities and Biotransformation of Palm Kernel Cake Biomass by Lactic Acid Bacteria Isolated from Malaysian Foods

by Fu Haw Lee, Suet Ying Wan, Hooi Ling Foo, Teck Chwen Loh, Rosfarizan Mohamad, Raha Abdul Rahim and Zulkifli Idrus

Int. J. Mol. Sci. 2019, 20(20), 4979; https://doi.org/10.3390/ijms20204979 - 9 Oct 2019

Cited by 28 | Viewed by 5283

Abstract

Biotransformation via solid state fermentation (SSF) mediated by microorganisms is a promising approach to produce useful products from agricultural biomass. Lactic acid bacteria (LAB) that are commonly found in fermented foods have been shown to exhibit extracellular proteolytic, β-glucosidase, β-mannosidase, and β-mannanase activities. [...] Read more.

Biotransformation via solid state fermentation (SSF) mediated by microorganisms is a promising approach to produce useful products from agricultural biomass. Lactic acid bacteria (LAB) that are commonly found in fermented foods have been shown to exhibit extracellular proteolytic, β-glucosidase, β-mannosidase, and β-mannanase activities. Therefore, extracellular proteolytic, cellulolytic, and hemicellulolytic enzyme activities of seven Lactobacillus plantarum strains (a prominent species of LAB) isolated from Malaysian foods were compared in this study. The biotransformation of palm kernel cake (PKC) biomass mediated by selected L. plantarum strains was subsequently conducted. The results obtained in this study exhibited the studied L. plantarum strains produced versatile multi extracellular hydrolytic enzyme activities that were active from acidic to alkaline pH conditions. The highest total score of extracellular hydrolytic enzyme activities were recorded by L. plantarum RI11, L. plantarum RG11, and L. plantarum RG14. Therefore, they were selected for the subsequent biotransformation of PKC biomass via SSF. The hydrolytic enzyme activities of treated PKC extract were compared for each sampling interval. The scanning electron microscopy analyses revealed the formation of extracellular matrices around L. plantarum strains attached to the surface of PKC biomass during SSF, inferring that the investigated L. plantarum strains have the capability to grow on PKC biomass and perform synergistic secretions of various extracellular proteolytic, cellulolytic, and hemicellulolytic enzymes that were essential for the effective biodegradation of PKC. The substantial growth of selected L. plamtraum strains on PKC during SSF revealed the promising application of selected L. plantarum strains as a biotransformation agent for cellulosic biomass. Full article

(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)

► Show Figures

Figure 1

13 pages, 1430 KiB

Open AccessArticle

Multipoint TvDAAO Mutants for Cephalosporin C Bioconversion

by Denis L. Atroshenko, Mikhail D. Shelomov, Sophia A. Zarubina, Nikita Y. Negru, Igor V. Golubev, Svyatoslav S. Savin and Vladimir I. Tishkov

Int. J. Mol. Sci. 2019, 20(18), 4412; https://doi.org/10.3390/ijms20184412 - 7 Sep 2019

Cited by 4 | Viewed by 3266

Abstract

d-amino acid oxidase (DAAO, EC 1.4.3.3) is used in many biotechnological processes. The main industrial application of DAAO is biocatalytic production of 7-aminocephalosporanic acid from cephalosporin C with a two enzymes system. DAAO from the yeast Trigonopsis variabilis (TvDAAO) shows the best [...] Read more.

d-amino acid oxidase (DAAO, EC 1.4.3.3) is used in many biotechnological processes. The main industrial application of DAAO is biocatalytic production of 7-aminocephalosporanic acid from cephalosporin C with a two enzymes system. DAAO from the yeast Trigonopsis variabilis (TvDAAO) shows the best catalytic parameters with cephalosporin C among all known DAAOs. We prepared and characterized multipoint TvDAAO mutants to improve their activity towards cephalosporin C and increase stability. All TvDAAO mutants showed better properties in comparison with the wild-type enzyme. The best mutant was TvDAAO with amino acid changes E32R/F33D/F54S/C108F/M156L/C298N. Compared to wild-type TvDAAO, the mutant enzyme exhibits a 4 times higher catalytic constant for cephalosporin C oxidation and 8- and 20-fold better stability against hydrogen peroxide inactivation and thermal denaturation, respectively. This makes this mutant promising for use in biotechnology. The paper also presents the comparison of TvDAAO catalytic properties with cephalosporin C reported by others. Full article

(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)

► Show Figures

Graphical abstract

13 pages, 1857 KiB

Open AccessArticle

Characterization of the First Bacterial and Thermostable GDP-Mannose 3,5-Epimerase

by Ophelia Gevaert, Stevie Van Overtveldt, Koen Beerens and Tom Desmet

Int. J. Mol. Sci. 2019, 20(14), 3530; https://doi.org/10.3390/ijms20143530 - 19 Jul 2019

Cited by 14 | Viewed by 4514

Abstract

GDP-mannose 3,5-epimerase (GM35E) catalyzes the conversion of GDP-mannose towards GDP-l-galactose and GDP-l-gulose. Although this reaction represents one of the few enzymatic routes towards the production of l-sugars and derivatives, it has not yet been exploited for that purpose. [...] Read more.

GDP-mannose 3,5-epimerase (GM35E) catalyzes the conversion of GDP-mannose towards GDP-l-galactose and GDP-l-gulose. Although this reaction represents one of the few enzymatic routes towards the production of l-sugars and derivatives, it has not yet been exploited for that purpose. One of the reasons is that so far only GM35Es from plants have been characterized, yielding biocatalysts that are relatively unstable and difficult to express heterologously. Through the mining of sequence databases, we succeeded in identifying a promising bacterial homologue. The gene from the thermophilic organism Methylacidiphilum fumariolicum was codon optimized for expression in Escherichia coli, resulting in the production of 40 mg/L of recombinant protein. The enzyme was found to act as a self-sufficient GM35E, performing three chemical reactions in the same active site. Furthermore, the biocatalyst was highly stable at temperatures up to 55 °C, making it well suited for the synthesis of new carbohydrate products with application in the pharma industry. Full article

(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)

► Show Figures

Figure 1

15 pages, 1794 KiB

Open AccessArticle

A Thermostable Aspergillus fumigatus GH7 Endoglucanase Over-Expressed in Pichia pastoris Stimulates Lignocellulosic Biomass Hydrolysis

by Aline Vianna Bernardi, Deborah Kimie Yonamine, Sergio Akira Uyemura and Taisa Magnani Dinamarco

Int. J. Mol. Sci. 2019, 20(9), 2261; https://doi.org/10.3390/ijms20092261 - 7 May 2019

Cited by 28 | Viewed by 5729

Abstract

In the context of avoiding the use of non-renewable energy sources, employing lignocellulosic biomass for ethanol production remains a challenge. Cellulases play an important role in this scenario: they are some of the most important industrial enzymes that can hydrolyze lignocellulose. This study [...] Read more.

In the context of avoiding the use of non-renewable energy sources, employing lignocellulosic biomass for ethanol production remains a challenge. Cellulases play an important role in this scenario: they are some of the most important industrial enzymes that can hydrolyze lignocellulose. This study aims to improve on the characterization of a thermostable Aspergillus fumigatus endo-1,4-β-glucanase GH7 (Af-EGL7). To this end, Af-EGL7 was successfully expressed in Pichia pastoris X-33. The kinetic parameters K_m and V_max were estimated and suggested a robust enzyme. The recombinant protein was highly stable within an extreme pH range (3.0–8.0) and was highly thermostable at 55 °C for 72 h. Low Cu²⁺ concentrations (0.1–1.0 mM) stimulated Af-EGL7 activity up to 117%. Af-EGL7 was tolerant to inhibition by products, such as glucose and cellobiose. Glucose at 50 mM did not inhibit Af-EGL7 activity, whereas 50 mM cellobiose inhibited Af-EGL7 activity by just 35%. Additionally, the Celluclast^® 1.5L cocktail supplemented with Af-EGL7 provided improved hydrolysis of sugarcane bagasse “in natura”, sugarcane exploded bagasse (SEB), corncob, rice straw, and bean straw. In conclusion, the novel characterization of Af-EGL7 conducted in this study highlights the extraordinary properties that make Af-EGL7 a promising candidate for industrial applications. Full article

(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)

► Show Figures

Graphical abstract

9 pages, 1866 KiB

Open AccessCommunication

Laccases with Variable Properties from Different Strains of Steccherinum ochraceum: Does Glycosylation Matter?

by Olga A. Glazunova, Konstantin V. Moiseenko, Inna A. Kamenihina, Tatyana U. Isaykina, Alexander I. Yaropolov and Tatyana V. Fedorova

Int. J. Mol. Sci. 2019, 20(8), 2008; https://doi.org/10.3390/ijms20082008 - 24 Apr 2019

Cited by 15 | Viewed by 2734

Abstract

Laccases are blue multi-copper oxidases with an extensive number of actual and potential industrial applications. It is known that laccases from different fungal strains may vary in properties; however, the reason of this remains unclear. In the current study we have isolated and [...] Read more.

Laccases are blue multi-copper oxidases with an extensive number of actual and potential industrial applications. It is known that laccases from different fungal strains may vary in properties; however, the reason of this remains unclear. In the current study we have isolated and characterized seven laccases from different strains of Steccherinum ochraceum obtained from regions of central Russia. Although all seven laccases had the same primary sequences, there was a little variation in their molecular weights and thermostabilities. Moreover, statistically significant differences in laccases’ catalytic parameters of oxidation of phenolic substrates and ABTS were observed. After the deglycosylation of four selected laccases by Endo H and PNGase F, their affinities to pyrocatechol and ABTS became the same, suggesting a substantial role of N-linked glycosylation in moderation of enzymatic properties of laccases. Full article

(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)

► Show Figures

Graphical abstract

20 pages, 2579 KiB

Open AccessArticle

Characterization and Dye Decolorization Potential of Two Laccases from the Marine-Derived Fungus Pestalotiopsis sp.

by Saowanee Wikee, Juliette Hatton, Annick Turbé-Doan, Yann Mathieu, Marianne Daou, Anne Lomascolo, Abhishek Kumar, Saisamorn Lumyong, Giuliano Sciara, Craig B. Faulds and Eric Record

Int. J. Mol. Sci. 2019, 20(8), 1864; https://doi.org/10.3390/ijms20081864 - 15 Apr 2019

Cited by 29 | Viewed by 4422

Abstract

Two laccase-encoding genes from the marine-derived fungus Pestalotiopsis sp. have been cloned in Aspergillus niger for heterologous production, and the recombinant enzymes have been characterized to study their physicochemical properties, their ability to decolorize textile dyes for potential biotechnological applications, and their activity [...] Read more.

Two laccase-encoding genes from the marine-derived fungus Pestalotiopsis sp. have been cloned in Aspergillus niger for heterologous production, and the recombinant enzymes have been characterized to study their physicochemical properties, their ability to decolorize textile dyes for potential biotechnological applications, and their activity in the presence of sea salt. The optimal pH and temperature of PsLac1 and PsLac2 differed in relation to the substrates tested, and both enzymes were shown to be extremely stable at temperatures up to 50 °C, retaining 100% activity after 3 h at 50 °C. Both enzymes were stable between pH 4–6. Different substrate specificities were exhibited, and the lowest K_m and highest catalytic efficiency values were obtained against syringaldazine and 2,6-dimethoxyphenol (DMP) for PsLac1 and PsLac2, respectively. The industrially important dyes—Acid Yellow, Bromo Cresol Purple, Nitrosulfonazo III, and Reactive Black 5—were more efficiently decolorized by PsLac1 in the presence of the redox mediator 1-hydroxybenzotriazole (HBT). Activities were compared in saline conditions, and PsLac2 seemed more adapted to the presence of sea salt than PsLac1. The overall surface charges of the predicted PsLac three-dimensional models showed large negatively charged surfaces for PsLac2, as found in proteins for marine organisms, and more balanced solvent exposed charges for PsLac1, as seen in proteins from terrestrial organisms. Full article

(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)

► Show Figures

Figure 1

22 pages, 1441 KiB

Open AccessArticle

Extracellular Proteolytic Activity and Amino Acid Production by Lactic Acid Bacteria Isolated from Malaysian Foods

by Cui Jin Toe, Hooi Ling Foo, Teck Chwen Loh, Rosfarizan Mohamad, Raha Abdul Rahim and Zulkifli Idrus

Int. J. Mol. Sci. 2019, 20(7), 1777; https://doi.org/10.3390/ijms20071777 - 10 Apr 2019

Cited by 60 | Viewed by 5416

Abstract

Amino acids (AAs) are vital elements for growth, reproduction, and maintenance of organisms. Current technology uses genetically engineered microorganisms for AAs production, which has urged the search for a safer food-grade AA producer strain. The extracellular proteolytic activities of lactic acid bacteria (LAB) [...] Read more.

Amino acids (AAs) are vital elements for growth, reproduction, and maintenance of organisms. Current technology uses genetically engineered microorganisms for AAs production, which has urged the search for a safer food-grade AA producer strain. The extracellular proteolytic activities of lactic acid bacteria (LAB) can be a vital tool to hydrolyze extracellular protein molecules into free AAs, thereby exhibiting great potential for functional AA production. In this study, eight LAB isolated from Malaysian foods were determined for their extracellular proteolytic activities and their capability of producing AAs. All studied LAB exhibited versatile extracellular proteolytic activities from acidic to alkaline pH conditions. In comparison, Pediococcus pentosaceus UP-2 exhibited the highest ability to produce 15 AAs extracellularly, including aspartate, lysine, methionine, threonine, isoleucine, glutamate, proline, alanine, valine, leucine, tryptophan, tyrosine, serine, glycine, and cystine, followed by Pediococcus pentosaceus UL-2, Pediococcus acidilactici UB-6, and Pediococcus acidilactici UP-1 with 11 to 12 different AAs production detected extracellularly. Pediococcus pentosaceus UL-6 demonstrated the highest increment of proline production at 24 h of incubation. However, Pediococcus acidilactici UL-3 and Lactobacillus plantarum I-UL4 exhibited the greatest requirement for AA. The results of this study showed that different LAB possess different extracellular proteolytic activities and potentials as extracellular AA producers. Full article

(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)

► Show Figures

Figure 1

12 pages, 1751 KiB

Open AccessArticle

Structure-Guided Immobilization of an Evolved Unspecific Peroxygenase

by Patricia Molina-Espeja, Paloma Santos-Moriano, Eva García-Ruiz, Antonio Ballesteros, Francisco J. Plou and Miguel Alcalde

Int. J. Mol. Sci. 2019, 20(7), 1627; https://doi.org/10.3390/ijms20071627 - 2 Apr 2019

Cited by 16 | Viewed by 4352

Abstract

Unspecific peroxygenases (UPOs) are highly promiscuous biocatalyst with self-sufficient mono(per)oxygenase activity. A laboratory-evolved UPO secreted by yeast was covalently immobilized in activated carriers through one-point attachment. In order to maintain the desired orientation without compromising the enzyme’s activity, the S221C mutation was introduced [...] Read more.

Unspecific peroxygenases (UPOs) are highly promiscuous biocatalyst with self-sufficient mono(per)oxygenase activity. A laboratory-evolved UPO secreted by yeast was covalently immobilized in activated carriers through one-point attachment. In order to maintain the desired orientation without compromising the enzyme’s activity, the S221C mutation was introduced at the surface of the enzyme, enabling a single disulfide bridge to be established between the support and the protein. Fluorescence confocal microscopy demonstrated the homogeneous distribution of the enzyme, regardless of the chemical nature of the carrier. This immobilized biocatalyst was characterized biochemically opening an exciting avenue for research into applied synthetic chemistry. Full article

(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)

► Show Figures

Figure 1

14 pages, 2938 KiB

Open AccessArticle

Disulfide Bond Engineering of an Endoglucanase from Penicillium verruculosum to Improve Its Thermostability

by Anna Bashirova, Subrata Pramanik, Pavel Volkov, Aleksandra Rozhkova, Vitaly Nemashkalov, Ivan Zorov, Alexander Gusakov, Arkady Sinitsyn, Ulrich Schwaneberg and Mehdi D. Davari

Int. J. Mol. Sci. 2019, 20(7), 1602; https://doi.org/10.3390/ijms20071602 - 30 Mar 2019

Cited by 43 | Viewed by 6079

Abstract

Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial [...] Read more.

Endoglucanases (EGLs) are important components of multienzyme cocktails used in the production of a wide variety of fine and bulk chemicals from lignocellulosic feedstocks. However, a low thermostability and the loss of catalytic performance of EGLs at industrially required temperatures limit their commercial applications. A structure-based disulfide bond (DSB) engineering was carried out in order to improve the thermostability of EGLII from Penicillium verruculosum. Based on in silico prediction, two improved enzyme variants, S127C-A165C (DSB2) and Y171C-L201C (DSB3), were obtained. Both engineered enzymes displayed a 15–21% increase in specific activity against carboxymethylcellulose and β-glucan compared to the wild-type EGLII (EGLII-wt). After incubation at 70 °C for 2 h, they retained 52–58% of their activity, while EGLII-wt retained only 38% of its activity. At 80 °C, the enzyme-engineered forms retained 15–22% of their activity after 2 h, whereas EGLII-wt was completely inactivated after the same incubation time. Molecular dynamics simulations revealed that the introduced DSB rigidified a global structure of DSB2 and DSB3 variants, thus enhancing their thermostability. In conclusion, this work provides an insight into DSB protein engineering as a potential rational design strategy that might be applicable for improving the stability of other enzymes for industrial applications. Full article

(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)

► Show Figures

Figure 1

21 pages, 2849 KiB

Open AccessArticle

New Recombinant Cold-Adapted and Organic Solvent Tolerant Lipase from Psychrophilic Pseudomonas sp. LSK25, Isolated from Signy Island Antarctica

by Leelatulasi Salwoom, Raja Noor Zaliha Raja Abd. Rahman, Abu Bakar Salleh, Fairolniza Mohd. Shariff, Peter Convey and Mohd Shukuri Mohamad Ali

Int. J. Mol. Sci. 2019, 20(6), 1264; https://doi.org/10.3390/ijms20061264 - 13 Mar 2019

Cited by 29 | Viewed by 4784

Abstract

In recent years, studies on psychrophilic lipases have become an emerging area of research in the field of enzymology. The study described here focuses on the cold-adapted organic solvent tolerant lipase strain Pseudomonas sp. LSK25 isolated from Signy Station, South Orkney Islands, maritime [...] Read more.

In recent years, studies on psychrophilic lipases have become an emerging area of research in the field of enzymology. The study described here focuses on the cold-adapted organic solvent tolerant lipase strain Pseudomonas sp. LSK25 isolated from Signy Station, South Orkney Islands, maritime Antarctic. Strain LSK25 lipase was successfully cloned, sequenced, and over-expressed in an Escherichia coli system. Sequence analysis revealed that the lipase gene of Pseudomonas sp. LSK25 consists of 1432 bp, lacks an N-terminal signal peptide and encodes a mature protein consisting of 476 amino acids. The recombinant LSK25 lipase was purified by single-step purification using Ni-Sepharose affinity chromatography and had a molecular mass of approximately 65 kDa. The final recovery and purification fold were 44% and 1.3, respectively. The LSK25 lipase was optimally active at 30 °C and at pH 6. Stable lipolytic activity was reported between temperatures of 5–30 °C and at pH 6–8. A significant enhancement of lipolytic activity was observed in the presence of Ca²⁺ ions, the organic lipids of rice bran oil and coconut oil, a synthetic C12 ester and a wide range of water immiscible organic solvents. Overall, lipase strain LSK25 is a potentially desirable candidate for biotechnological application, due to its stability at low temperatures, across a range of pH and in organic solvents. Full article

(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)

► Show Figures

Figure 1

14 pages, 3312 KiB

Open AccessArticle

Characterization of Stackebrandtia nassauensis GH 20 Beta-Hexosaminidase, a Versatile Biocatalyst for Chitobiose Degradation

by Meng Wang, Feng Zheng, Ting Wang, Yong-Mei Lyu, Matthew G. Alteen, Zhi-Peng Cai, Zhong-Li Cui, Li Liu and Josef Voglmeir

Int. J. Mol. Sci. 2019, 20(5), 1243; https://doi.org/10.3390/ijms20051243 - 12 Mar 2019

Cited by 16 | Viewed by 4777

Abstract

An unstudied β-N-acetylhexosaminidase (SnHex) from the soil bacterium Stackebrandtia nassauensis was successfully cloned and subsequently expressed as a soluble protein in Escherichia coli. Activity tests and the biochemical characterization of the purified protein revealed an optimum pH of 6.0 and [...] Read more.

An unstudied β-N-acetylhexosaminidase (SnHex) from the soil bacterium Stackebrandtia nassauensis was successfully cloned and subsequently expressed as a soluble protein in Escherichia coli. Activity tests and the biochemical characterization of the purified protein revealed an optimum pH of 6.0 and a robust thermal stability at 50 °C within 24 h. The addition of urea (1 M) or sodium dodecyl sulfate (1% w/v) reduced the activity of the enzyme by 44% and 58%, respectively, whereas the addition of divalent metal ions had no effect on the enzymatic activity. PUGNAc (O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate) strongly inhibited the enzyme in sub-micromolar concentrations. The β-N-acetylhexosaminidase was able to hydrolyze β1,2-linked, β1,3-linked, β1,4-linked, and β1,6-linked GlcNAc residues from the non-reducing end of various tested glycan standards, including bisecting GlcNAc from one of the tested hybrid-type N-glycan substrates. A mutational study revealed that the amino acids D306 and E307 bear the catalytically relevant side acid/base side chains. When coupled with a chitinase, the β-N-acetylhexosaminidase was able to generate GlcNAc directly from colloidal chitin, which showed the potential of this enzyme for biotechnological applications. Full article

(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)

► Show Figures

Graphical abstract

18 pages, 16150 KiB

Open AccessArticle

β-Galactosidase from Lactobacillus helveticus DSM 20075: Biochemical Characterization and Recombinant Expression for Applications in Dairy Industry

by Suwapat Kittibunchakul, Mai-Lan Pham, Anh-Minh Tran and Thu-Ha Nguyen

Int. J. Mol. Sci. 2019, 20(4), 947; https://doi.org/10.3390/ijms20040947 - 22 Feb 2019

Cited by 24 | Viewed by 6161

Abstract

β-Galactosidase encoding genes lacLM from Lactobacillus helveticus DSM 20075 were cloned and successfully overexpressed in Escherichia coli and Lactobacillus plantarum using different expression systems. The highest recombinant β-galactosidase activity of ∼26 kU per L of medium was obtained when using an expression system [...] Read more.

β-Galactosidase encoding genes lacLM from Lactobacillus helveticus DSM 20075 were cloned and successfully overexpressed in Escherichia coli and Lactobacillus plantarum using different expression systems. The highest recombinant β-galactosidase activity of ∼26 kU per L of medium was obtained when using an expression system based on the T7 RNA polymerase promoter in E. coli, which is more than 1000-fold or 28-fold higher than the production of native β-galactosidase from L. helveticus DSM 20075 when grown on glucose or lactose, respectively. The overexpression in L. plantarum using lactobacillal food-grade gene expression system resulted in ∼2.3 kU per L of medium, which is approximately 10-fold lower compared to the expression in E. coli. The recombinant β-galactosidase from L. helveticus overexpressed in E. coli was purified to apparent homogeneity and subsequently characterized. The K_m and v_max values for lactose and o-nitrophenyl-β-d-galactopyranoside (oNPG) were 15.7 ± 1.3 mM, 11.1 ± 0.2 µmol D-glucose released per min per mg protein, and 1.4 ± 0.3 mM, 476 ± 66 µmol o-nitrophenol released per min per mg protein, respectively. The enzyme was inhibited by high concentrations of oNPG with K_i,s = 3.6 ± 0.8 mM. The optimum pH for hydrolysis of both substrates, lactose and oNPG, is pH 6.5 and optimum temperatures for these reactions are 60 and 55 °C, respectively. The formation of galacto-oligosaccharides (GOS) in discontinuous mode using both crude recombinant enzyme from L. plantarum and purified recombinant enzyme from E. coli revealed high transgalactosylation activity of β-galactosidases from L. helveticus; hence, this enzyme is an interesting candidate for applications in lactose conversion and GOS formation processes. Full article

(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)

► Show Figures

Figure 1

18 pages, 2650 KiB

Open AccessArticle

A Computational Method to Propose Mutations in Enzymes Based on Structural Signature Variation (SSV)

by Diego César Batista Mariano, Lucianna Helene Santos, Karina dos Santos Machado, Adriano Velasque Werhli, Leonardo Henrique França de Lima and Raquel Cardoso de Melo-Minardi

Int. J. Mol. Sci. 2019, 20(2), 333; https://doi.org/10.3390/ijms20020333 - 15 Jan 2019

Cited by 11 | Viewed by 4506

Abstract

With the use of genetic engineering, modified and sometimes more efficient enzymes can be created for different purposes, including industrial applications. However, building modified enzymes depends on several in vitro experiments, which may result in the process being expensive and time-consuming. Therefore, computational [...] Read more.

With the use of genetic engineering, modified and sometimes more efficient enzymes can be created for different purposes, including industrial applications. However, building modified enzymes depends on several in vitro experiments, which may result in the process being expensive and time-consuming. Therefore, computational approaches could reduce costs and accelerate the discovery of new technological products. In this study, we present a method, called structural signature variation (SSV), to propose mutations for improving enzymes’ activity. SSV uses the structural signature variation between target enzymes and template enzymes (obtained from the literature) to determine if randomly suggested mutations may provide some benefit for an enzyme, such as improvement of catalytic activity, half-life, and thermostability, or resistance to inhibition. To evaluate SSV, we carried out a case study that suggested mutations in β-glucosidases: Essential enzymes used in biofuel production that suffer inhibition by their product. We collected 27 mutations described in the literature, and manually classified them as beneficial or not. SSV was able to classify the mutations with values of 0.89 and 0.92 for precision and specificity, respectively. Then, we used SSV to propose mutations for Bgl1B, a low-performance β-glucosidase. We detected 15 mutations that could be beneficial. Three of these mutations (H228C, H228T, and H228V) have been related in the literature to the mechanism of glucose tolerance and stimulation in GH1 β-glucosidase. Hence, SSV was capable of detecting promising mutations, already validated by in vitro experiments, that improved the inhibition resistance of a β-glucosidase and, consequently, its catalytic activity. SSV might be useful for the engineering of enzymes used in biofuel production or other industrial applications. Full article

(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)

► Show Figures

Figure 1

17 pages, 1328 KiB

Open AccessArticle

Kinetics and Predicted Structure of a Novel Xylose Reductase from Chaetomium thermophilum

by Julian Quehenberger, Tom Reichenbach, Niklas Baumann, Lukas Rettenbacher, Christina Divne and Oliver Spadiut

Int. J. Mol. Sci. 2019, 20(1), 185; https://doi.org/10.3390/ijms20010185 - 6 Jan 2019

Cited by 13 | Viewed by 5285

Abstract

While in search of an enzyme for the conversion of xylose to xylitol at elevated temperatures, a xylose reductase (XR) gene was identified in the genome of the thermophilic fungus Chaetomium thermophilum. The gene was heterologously expressed in Escherichia coli as a [...] Read more.

While in search of an enzyme for the conversion of xylose to xylitol at elevated temperatures, a xylose reductase (XR) gene was identified in the genome of the thermophilic fungus Chaetomium thermophilum. The gene was heterologously expressed in Escherichia coli as a His6-tagged fusion protein and characterized for function and structure. The enzyme exhibits dual cofactor specificity for NADPH and NADH and prefers D-xylose over other pentoses and investigated hexoses. A homology model based on a XR from Candida tenuis was generated and the architecture of the cofactor binding site was investigated in detail. Despite the outstanding thermophilicity of its host the enzyme is, however, not thermostable. Full article

(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)

► Show Figures

Figure 1

Review

Jump to: Research

20 pages, 2898 KiB

Open AccessReview

Production of Hexaric Acids from Biomass

by Riku Sakuta and Nobuhumi Nakamura

Int. J. Mol. Sci. 2019, 20(15), 3660; https://doi.org/10.3390/ijms20153660 - 26 Jul 2019

Cited by 22 | Viewed by 7658

Abstract

Sugar acids obtained by aldohexose oxidation of both the terminal aldehyde group and the hydroxy group at the other end to carboxyl groups are called hexaric acids (i.e., six-carbon aldaric acids). Because hexaric acids have four secondary hydroxy groups that are stereochemically diverse [...] Read more.

Sugar acids obtained by aldohexose oxidation of both the terminal aldehyde group and the hydroxy group at the other end to carboxyl groups are called hexaric acids (i.e., six-carbon aldaric acids). Because hexaric acids have four secondary hydroxy groups that are stereochemically diverse and two carboxyl groups, various applications of these acids have been studied. Conventionally, hexaric acids have been produced mainly by nitric acid oxidation of aldohexose, but full-scale commercialization has not been realized; there are many problems regarding yield, safety, environmental burden, etc. In recent years, therefore, improvements in hexaric acid production by nitric acid oxidation have been made, while new production methods, including biocatalytic methods, are actively being studied. In this paper, we summarize these production methods in addition to research on the application of hexaric acids. Full article

(This article belongs to the Special Issue Industrial Enzymes: Structure, Function and Applications)

► Show Figures

Graphical abstract

Journal Menu

Journal Browser

Industrial Enzymes: Structure, Function and Applications

Share This Special Issue

Special Issue Editors

Special Issue Information

Keywords

Benefits of Publishing in a Special Issue

Published Papers (17 papers)

Research

Review

Further Information

Guidelines

MDPI Initiatives

Follow MDPI