Enzyme engineering and its industrial applications

2021 ◽  
Author(s):  
Isabela Victorino da Silva Amatto ◽  
Nathalia Gonsales da Rosa‐Garzon ◽  
Flávio Antônio de Oliveira Simões ◽  
Fernanda Santiago ◽  
Nathália Pereira da Silva Leite ◽  
...  
Keyword(s):  
Industrial Applications ◽  
Enzyme Engineering

scholarly journals Structure-based enzyme engineering improves donor-substrate recognition of Arabidopsis thaliana glycosyltransferases

2020 ◽  
Vol 477 (15) ◽  
pp. 2791-2805
Author(s):  
Aishat Akere ◽  
Serena H. Chen ◽  
Xiaohan Liu ◽  
Yanger Chen ◽  
Sarath Chandra Dantu ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Biochemical Characterization ◽  
Structural Information ◽  
Substrate Recognition ◽  
Industrial Applications ◽  
Enzyme Engineering ◽  
Donor Substrate ◽  
Substrate Preferences ◽  
Dynamics Simulations ◽  
Experimental Crystal

Glycosylation of secondary metabolites involves plant UDP-dependent glycosyltransferases (UGTs). UGTs have shown promise as catalysts in the synthesis of glycosides for medical treatment. However, limited understanding at the molecular level due to insufficient biochemical and structural information has hindered potential applications of most of these UGTs. In the absence of experimental crystal structures, we employed advanced molecular modeling and simulations in conjunction with biochemical characterization to design a workflow to study five Group H Arabidopsis thaliana (76E1, 76E2, 76E4, 76E5, 76D1) UGTs. Based on our rational structural manipulation and analysis, we identified key amino acids (P129 in 76D1; D374 in 76E2; K275 in 76E4), which when mutated improved donor substrate recognition than wildtype UGTs. Molecular dynamics simulations and deep learning analysis identified structural differences, which drive substrate preferences. The design of these UGTs with broader substrate specificity may play important role in biotechnological and industrial applications. These findings can also serve as basis to study other plant UGTs and thereby advancing UGT enzyme engineering.

scholarly journals Protein Engineering with A Glycosylation Circuit Enables Improved Enzyme Characteristics

2021 ◽  
Author(s):  
Eray Bozkurt ◽  
Irem Cagil ◽  
Ebru Kehribar ◽  
Musa Isilak ◽  
Urartu Ozgur Safak Seker
Keyword(s):  
Recombinant Proteins ◽  
Industrial Applications ◽  
Enzyme Engineering ◽  
Protein Glycosylation ◽  
Post Translational Modifications ◽  
E Coli ◽  
Proteolytic Stability ◽  
New Perspective ◽  
Harsh Conditions ◽  
Bacterial Glycosylation

Protein glycosylation is one of the most crucial and common post-translational modifications. It plays a fate-determining role and can alter many properties of proteins, making it an interesting for many biotechnology applications. The discovery of bacterial glycosylation mechanisms, opened a new perspective and transfer of C.jejuni N-linked glycosylation into laboratory work-horse E. coli increased research pace in the field exponentially. It has been previously showed that utilizing N-Linked Glycosylation, certain recombinant proteins have been furnished with improved features, such as stability and solubility. In this study, we utilized N-linked Glycosylation to glycosylate alkaline phosphatase (ALP) enzyme in E. coli and investigate the effects of glycosylation on an enzyme. Considering the glycosylation mechanism is highly dependent on the acceptor protein, ALP constructs carrying glycosylation tag at different locations of the gene has been created and glycosylation rates have been calculated. The most glycosylated construct has been selected for comparison with the native enzyme. We investigated the performance of glycosylated ALP in terms of its thermostability, proteolytic stability, tolerance to suboptimal pH and under denaturing conditions. Studies showed that glycosylated ALP performed remarkably better at optimal and harsh conditions Therefore, N-linked Glycosylation mechanism can be employed for enzyme engineering purposes and is a useful tool for industrial applications that require enzymatic activity.

scholarly journals Enzyme Functional Screening, Discovery and Engineering; Automation, Metagenomics and High-throughput Approaches

2020 ◽  
pp. 1-12
Author(s):  
Sikander Ali ◽  
Syed Shahid Hussain
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Industrial Applications ◽  
Enzyme Engineering ◽  
Functional Screening ◽  
Functional Metagenomics ◽  
Immense Potential

Concerned with the construction and design of novel biocatalysts, the enzyme engineering served to overcome the limitations of native enzymes, in order to create biocatalysts with tailored functions, to facilitate industrial applications. The enzymes, being recognized by screening and discovery workflows and further tailored by engineering platforms, are of immense potential as improved biocatalysts. Functional metagenomics is a powerful tool to identify novel enzymes followed by the construction of metagenome-based enzyme libraries. And the subsequent screening of these enzyme libraries is in turn facilitated by ultra-high-throughput-based, for example FACS or microfluidics, enzyme engineering technologies. Relies on the compartmentalization of reaction components, in order to detect and measure assay signal within the reaction compartments, the enzyme engineering platforms are designed which include cell-as-compartment platforms, droplet-based platforms and micro-chamber-based platforms. The metagenomics approach and high-throughput screening by these three prime enzyme engineer platforms are the focus of this review.

scholarly journals Improvement of the Stability and Activity of an LPMO Through Rational Disulfide Bonds Design

2022 ◽  
Vol 9 ◽  
Author(s):  
Xiaoli Zhou ◽  
Zhiqiang Xu ◽  
Yueqiu Li ◽  
Jia He ◽  
Honghui Zhu
Keyword(s):  
Disulfide Bonds ◽  
Specific Activity ◽  
Fold Increase ◽  
Catalytic Performance ◽  
Industrial Applications ◽  
Enzyme Engineering ◽  
Wild Type ◽  
Polysaccharide Monooxygenases ◽  
Saccharification Efficiency ◽  
The Stability

Lytic polysaccharide monooxygenases (LPMOs) oxidatively break down the glycosidic bonds of crystalline polysaccharides, significantly improving the saccharification efficiency of recalcitrant biomass, and have broad application prospects in industry. To meet the needs of industrial applications, enzyme engineering is needed to improve the catalytic performance of LPMOs such as enzyme activity and stability. In this study, we engineered the chitin-active CjLPMO10A from Cellvibrio japonicus through a rational disulfide bonds design. Compared with the wild-type, the variant M1 (N78C/H116C) exhibited a 3-fold increase in half-life at 60°C, a 3.5°C higher T5015, and a 7°C rise in the apparent Tm. Furthermore, the resistance of M1 to chemical denaturation was significantly improved. Most importantly, the introduction of the disulfide bond improved the thermal and chemical stability of the enzyme without causing damage to catalytic activity, and M1 showed 1.5 times the specific activity of the wild-type. Our study shows that the stability and activity of LPMOs could be improved simultaneously by selecting suitable engineering sites reasonably, thereby improving the industrial adaptability of the enzymes, which is of great significance for applications.

scholarly journals Microbial Production of Biodegradable Lactate-Based Polymers and Oligomeric Building Blocks From Renewable and Waste Resources

2021 ◽  
Vol 8 ◽  
Author(s):  
John Masani Nduko ◽  
Seiichi Taguchi
Keyword(s):  
Plant Biomass ◽  
Building Blocks ◽  
Industrial Applications ◽  
Enzyme Engineering ◽  
Energy Reserves ◽  
Renewable Biomass ◽  
The Past ◽  
Naturally Occurring ◽  
Secretory Production ◽  
The Cost

Polyhydroxyalkanoates (PHAs) are naturally occurring biopolymers produced by microorganisms. PHAs have become attractive research biomaterials in the past few decades owing to their extensive potential industrial applications, especially as sustainable alternatives to the fossil fuel feedstock-derived products such as plastics. Among the biopolymers are the bioplastics and oligomers produced from the fermentation of renewable plant biomass. Bioplastics are intracellularly accumulated by microorganisms as carbon and energy reserves. The bioplastics, however, can also be produced through a biochemistry process that combines fermentative secretory production of monomers and/or oligomers and chemical synthesis to generate a repertoire of biopolymers. PHAs are particularly biodegradable and biocompatible, making them a part of today’s commercial polymer industry. Their physicochemical properties that are similar to those of petrochemical-based plastics render them potential renewable plastic replacements. The design of efficient tractable processes using renewable biomass holds key to enhance their usage and adoption. In 2008, a lactate-polymerizing enzyme was developed to create new category of polyester, lactic acid (LA)–based polymer and related polymers. This review aims to introduce different strategies including metabolic and enzyme engineering to produce LA-based biopolymers and related oligomers that can act as precursors for catalytic synthesis of polylactic acid. As the cost of PHA production is prohibitive, the review emphasizes attempts to use the inexpensive plant biomass as substrates for LA-based polymer and oligomer production. Future prospects and challenges in LA-based polymer and oligomer production are also highlighted.

Next Generation Feedstocks From New Frontiers in Oilseed Engineering

2005 ◽  
Author(s):  
J. Grushcow ◽  
M. A. Smith
Keyword(s):  
Fatty Acids ◽  
Industrial Applications ◽  
Enzyme Engineering ◽  
Hydroxy Fatty Acids ◽  
Alternative Source ◽  
High Oleic ◽  
Genomics And Proteomics ◽  
New Research ◽  
Oil Modification ◽  
Breeding Techniques

Recent advances in molecular breeding techniques along with developing tools for Genomics and Proteomics are delivering new oil seed profiles for industrial applications. Ultra high Oleic, Erucic and blends including Hydroxy fatty acids are now, or will be shortly, available in a variety of oilseed crops including Soybean and Canola as well as Flax. As a result, vegetable oils need to be re-examined by industry for specific applications. Feedstocks and base oils derived from oil seeds are renewable as well as biodegradable. A brief summary of recent progress is presented together with a description of new research into the development of an alternative source of Hydroxy fatty acids to replace castor oil and an overview of an enzyme engineering approach to create new enzymes for seed oil modification.

scholarly journals Extremophilic SHMTs: From Structure to Biotechnology

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Sebastiana Angelaccio
Keyword(s):  
Structural Changes ◽  
Extreme Environments ◽  
Industrial Applications ◽  
Enzyme Engineering ◽  
Suitable Model ◽  
Functional Studies ◽  
Metabolic Role ◽  
Enzyme Adaptation ◽  
High And Low Temperatures ◽  
Data Banks

Recent advances in molecular and structural biology have improved the availability of virtually any biocatalyst in large quantity and have also provided an insight into the detailed structure-function relationships of many of them. These results allowed the rational exploitation of biocatalysts for use in organic synthesis. In this context, extremophilic enzymes are extensively studied for their potential interest for many biotechnological and industrial applications, as they offer increased rates of reactions, higher substrate solubility, and/or longer enzyme half-lives at the conditions of industrial processes. Serine hydroxymethyltransferase (SHMT), for its ubiquitous nature, represents a suitable model for analyzing enzyme adaptation to extreme environments. In fact, many SHMT sequences from Eukarya, Eubacteria and Archaea are available in data banks as well as several crystal structures. In addition, SHMT is structurally conserved because of its critical metabolic role; consequently, very few structural changes have occurred during evolution. Our research group analyzed the molecular basis of SHMT adaptation to high and low temperatures, using experimental and comparativein silicoapproaches. These structural and functional studies of SHMTs purified from extremophilic organisms can help to understand the peculiarities of the enzyme activity at extreme temperatures, indicating possible strategies for rational enzyme engineering.

scholarly journals Rational Engineering of a Cold-Adapted α-Amylase from the Antarctic Ciliate Euplotes focardii for Simultaneous Improvement of Thermostability and Catalytic Activity

2017 ◽  
Vol 83 (13) ◽  
Author(s):  
Guang Yang ◽  
Hua Yao ◽  
Matteo Mozzicafreddo ◽  
Patrizia Ballarini ◽  
Sandra Pucciarelli ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Enzyme Engineering ◽  
Content Type ◽  
Cold Adapted ◽  
Wide Range ◽  
Cold Active ◽  
Surface Loops ◽  
The Antarctic

ABSTRACT The α-amylases are endo-acting enzymes that hydrolyze starch by randomly cleaving the 1,4-α-d-glucosidic linkages between the adjacent glucose units in a linear amylose chain. They have significant advantages in a wide range of applications, particularly in the food industry. The eukaryotic α-amylase isolated from the Antarctic ciliated protozoon Euplotes focardii (EfAmy) is an alkaline enzyme, different from most of the α-amylases characterized so far. Furthermore, EfAmy has the characteristics of a psychrophilic α-amylase, such as the highest hydrolytic activity at a low temperature and high thermolability, which is the major drawback of cold-active enzymes in industrial applications. In this work, we applied site-directed mutagenesis combined with rational design to generate a cold-active EfAmy with improved thermostability and catalytic efficiency at low temperatures. We engineered two EfAmy mutants. In one mutant, we introduced Pro residues on the A and B domains in surface loops. In the second mutant, we changed Val residues to Thr close to the catalytic site. The aim of these substitutions was to rigidify the molecular structure of the enzyme. Furthermore, we also analyzed mutants containing these combined substitutions. Biochemical enzymatic assays of engineered versions of EfAmy revealed that the combination of mutations at the surface loops increased the thermostability and catalytic efficiency of the enzyme. The possible mechanisms responsible for the changes in the biochemical properties are discussed by analyzing the three-dimensional structural model. IMPORTANCE Cold-adapted enzymes have high specific activity at low and moderate temperatures, a property that can be extremely useful in various applications as it implies a reduction in energy consumption during the catalyzed reaction. However, the concurrent high thermolability of cold-adapted enzymes often limits their applications in industrial processes. The α-amylase from the psychrophilic Antarctic ciliate Euplotes focardii (named EfAmy) is a cold-adapted enzyme with optimal catalytic activity in an alkaline environment. These unique features distinguish it from most α-amylases characterized so far. In this work, we engineered a novel EfAmy with improved thermostability, substrate binding affinity, and catalytic efficiency to various extents, without impacting its pH preference. These characteristics can be considered important properties for use in the food, detergent, and textile industries and in other industrial applications. The enzyme engineering strategy developed in this study may also provide useful knowledge for future optimization of molecules to be used in particular industrial applications.

The Application of Ultra-Thin Sectioning Techniques to Non-Biological Samples

1968 ◽  
Vol 26 ◽  
pp. 332-333
Author(s):  
C. F. Oster
Keyword(s):  
Biological Sciences ◽  
Epoxy Resin ◽  
Cross Sections ◽  
Biological Samples ◽  
Industrial Applications ◽  
Photographic Film ◽  
Silver Particles ◽  
Thin Sectioning ◽  
Embedding Medium

Although ultra-thin sectioning techniques are widely used in the biological sciences, their applications are somewhat less popular but very useful in industrial applications. This presentation will review several specific applications where ultra-thin sectioning techniques have proven invaluable.The preparation of samples for sectioning usually involves embedding in an epoxy resin. Araldite 6005 Resin and Hardener are mixed so that the hardness of the embedding medium matches that of the sample to reduce any distortion of the sample during the sectioning process. No dehydration series are needed to prepare our usual samples for embedding, but some types require hardening and staining steps. The embedded samples are sectioned with either a prototype of a Porter-Blum Microtome or an LKB Ultrotome III. Both instruments are equipped with diamond knives.In the study of photographic film, the distribution of the developed silver particles through the layer is important to the image tone and/or scattering power. Also, the morphology of the developed silver is an important factor, and cross sections will show this structure.

The mensuration of interfaces

1992 ◽  
Vol 50 (1) ◽  
pp. 216-217
Author(s):  
W.M. Stobbs
Keyword(s):  
Electron Microscopy ◽  
50Th Anniversary ◽  
Numerical Data ◽  
Industrial Applications ◽  
Dynamical Theory ◽  
Large Strains ◽  
Reasonable Approach ◽  
Lattice Resolution ◽  
Analogue To Digital ◽  
The Way

I do not have access to the abstracts of the first meeting of EMSA but at this, the 50th Anniversary meeting of the Electron Microscopy Society of America, I have an excuse to consider the historical origins of the approaches we take to the use of electron microscopy for the characterisation of materials. I have myself been actively involved in the use of TEM for the characterisation of heterogeneities for little more than half of that period. My own view is that it was between the 3rd International Meeting at London, and the 1956 Stockholm meeting, the first of the European series , that the foundations of the approaches we now take to the characterisation of a material using the TEM were laid down. (This was 10 years before I took dynamical theory to be etched in stone.) It was at the 1956 meeting that Menter showed lattice resolution images of sodium faujasite and Hirsch, Home and Whelan showed images of dislocations in the XlVth session on “metallography and other industrial applications”. I have always incidentally been delighted by the way the latter authors misinterpreted astonishingly clear thickness fringes in a beaten (”) foil of Al as being contrast due to “large strains”, an error which they corrected with admirable rapidity as the theory developed. At the London meeting the research described covered a broad range of approaches, including many that are only now being rediscovered as worth further effort: however such is the power of “the image” to persuade that the above two papers set trends which influence, perhaps too strongly, the approaches we take now. Menter was clear that the way the planes in his image tended to be curved was associated with the imaging conditions rather than with lattice strains, and yet it now seems to be common practice to assume that the dots in an “atomic resolution image” can faithfully represent the variations in atomic spacing at a localised defect. Even when the more reasonable approach is taken of matching the image details with a computed simulation for an assumed model, the non-uniqueness of the interpreted fit seems to be rather rarely appreciated. Hirsch et al., on the other hand, made a point of using their images to get numerical data on characteristics of the specimen they examined, such as its dislocation density, which would not be expected to be influenced by uncertainties in the contrast. Nonetheless the trends were set with microscope manufacturers producing higher and higher resolution microscopes, while the blind faith of the users in the image produced as being a near directly interpretable representation of reality seems to have increased rather than been generally questioned. But if we want to test structural models we need numbers and it is the analogue to digital conversion of the information in the image which is required.

Sign in / Sign up

Export Citation Format

Share Document