Microbial keratinases: An overview of biochemical characterization and its eco-friendly approach for industrial applications

2020 ◽  
Vol 252 ◽  
pp. 119847 ◽  
Author(s):  
Binti Srivastava ◽  
Madhu Khatri ◽  
Gursharan Singh ◽  
Shailendra Kumar Arya
Keyword(s):  
Biochemical Characterization ◽  
Industrial Applications

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 Biochemical Characterization of Cellulase From Bacillus subtilis Strain and its Effect on Digestibility and Structural Modifications of Lignocellulose Rich Biomass

2021 ◽  
Vol 9 ◽  
Author(s):  
Waseem Ayoub Malik ◽  
Saleem Javed
Keyword(s):  
Bacillus Subtilis ◽  
Biochemical Characterization ◽  
Biomass Conversion ◽  
Industrial Applications ◽  
Partial Purification ◽  
Subtilis Strain ◽  
Complex Nature ◽  
Structural Modifications ◽  
Optimum Activity

Microbial cellulases have become the mainstream biocatalysts due to their complex nature and widespread industrial applications. The present study reports the partial purification and characterization of cellulase from Bacillus subtilis CD001 and its application in biomass saccharification. Out of four different substrates, carboxymethyl cellulose, when amended as fermentation substrate, induced the highest cellulase production from B. subtilis CD001. The optimum activity of CMCase, FPase, and amylase was 2.4 U/ml, 1.5 U/ml, and 1.45 U/ml, respectively. The enzyme was partially purified by (NH4)2SO4 precipitation and sequenced through LC-MS/MS. The cellulase was found to be approximately 55 kDa by SDS-PAGE and capable of hydrolyzing cellulose, as confirmed by zymogram analysis. The enzyme was assigned an accession number AOR98335.1 and displayed 46% sequence homology with 14 peptide-spectrum matches having 12 unique peptide sequences. Characterization of the enzyme revealed it to be an acidothermophilic cellulase, having an optimum activity at pH 5 and a temperature of 60°C. Kinetic analysis of partially purified enzyme showed the Km and Vmax values of 0.996 mM and 1.647 U/ml, respectively. The enzyme activity was accelerated by ZnSO4, MnSO4, and MgSO4, whereas inhibited significantly by EDTA and moderately by β-mercaptoethanol and urea. Further, characterization of the enzyme saccharified sugarcane bagasse, wheat straw, and filter paper by SEM, ATR-FTIR, and XRD revealed efficient hydrolysis and structural modifications of cellulosic materials, indicating the potential industrial application of the B. subtilis CD001 cellulase. The findings demonstrated the potential suitability of cellulase from B. subtilis CD001 for use in current mainstream biomass conversion into fuels and other industrial processes.

scholarly journals Biochemical Characterization of the Amylase Activity from the New Haloarchaeal Strain Haloarcula sp. HS Isolated in the Odiel Marshlands

Biology ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 337
Author(s):  
Patricia Gómez-Villegas ◽  
Javier Vigara ◽  
Luis Romero ◽  
Cecilia Gotor ◽  
Sara Raposo ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Amylase Activity ◽  
Extreme Environments ◽  
Three Dimensional ◽  
Large Family ◽  
Industrial Applications ◽  
Dimensional Structure ◽  
Glycosyl Hydrolases ◽  
Calcium Dependent ◽  
Alpha Amylases

Alpha-amylases are a large family of α,1-4-endo-glycosyl hydrolases distributed in all kingdoms of life. The need for poly-extremotolerant amylases encouraged their search in extreme environments, where archaea become ideal candidates to provide new enzymes that are able to work in the harsh conditions demanded in many industrial applications. In this study, a collection of haloarchaea isolated from Odiel saltern ponds in the southwest of Spain was screened for their amylase activity. The strain that exhibited the highest activity was selected and identified as Haloarcula sp. HS. We demonstrated the existence in both, cellular and extracellular extracts of the new strain, of functional α-amylase activities, which showed to be moderately thermotolerant (optimum around 60 °C), extremely halotolerant (optimum over 25% NaCl), and calcium-dependent. The tryptic digestion followed by HPLC-MS/MS analysis of the partially purified cellular and extracellular extracts allowed to identify the sequence of three alpha-amylases, which despite sharing a low sequence identity, exhibited high three-dimensional structure homology, conserving the typical domains and most of the key consensus residues of α-amylases. Moreover, we proved the potential of the extracellular α-amylase from Haloarcula sp. HS to treat bakery wastes under high salinity conditions.

scholarly journals Degradation Patterns of GH11 xylanases for Efficiently Hydrolyzing xylan in Aspergillus Niger An76

2021 ◽  
Author(s):  
Shu Zhang ◽  
Sha Zhao ◽  
Weihao Shang ◽  
Xiuyun Wu ◽  
Yingjie Li ◽  
...  
Keyword(s):  
Aspergillus Niger ◽  
Filamentous Fungi ◽  
Biochemical Characterization ◽  
Catalytic Performance ◽  
Industrial Applications ◽  
Transcriptional Analysis ◽  
Degrading Enzymes ◽  
Beechwood Xylan ◽  
Catalytic Functions ◽  
Encoding Genes

Abstract Background: Xylan is the most abundant hemicellulose polysaccharide in nature. Endo-xylanases from GH10 and GH11 families are the most critical xylan degrading enzymes. Filamentous fungi are highly effective xylan degraders and possess numerous xylan degrading isoenzyme-encoding genes, especially Aspergillus niger. Most noteworthy, the amplification of the GH11 xylanase-encoding genes occurs frequently in an organism, but the knowledge of each GH11 xylanases is little known. Results: A. niger An76 encoded a comprehensive set of xylan-degrading enzymes, including five endo-xylanases (one GH10 and four GH11). Quantitative transcriptional analysis showed that three xylanases were up-regulated by xylose substrates, and the order and amount of enzyme secretion differed. Specifically, XynA and XynB were initially secreted successively, followed by XynC. Structural bioinformatics analysis indicated that the different modes of action of the three GH11 xylanases may be due to intricate hydrogen bonding between substrates and functional residues in the active site architectures. Heterologous expression and biochemical characterization of three GH11 xylanases (XynA, XynB and XynD) revealed differences in catalytic performance and product profiles. Furthermore, XynA and XynB displayed obvious synergistic action against beechwood xylan. Conclusions: We investigated subtle differences in the functions of different isoenzymes in the same family using a combination of physiological and biochemical experiments. The transcriptional regulation and catalytic functions of enzymes could be the result of long-term evolutionary adaptation. The finding further expanded our understanding of GH-encoding genes amplification in filamentous fungi, which could guide the design of the optimal enzyme cocktails in industrial applications.

scholarly journals Peroxidase from Coleus Forskohlii: Purification and Biochemical Characterization

2020 ◽  
Vol 5 (1) ◽  
pp. 9-20
Author(s):  
Yaaser Q. Almulaiky ◽  
Yaaser Q. Almulaiky
Keyword(s):  
Ion Exchange ◽  
Peroxidase Activity ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Gel Filtration ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Coleus Forskohlii ◽  
Sds Page ◽  
Optimum Ph

In this study, a peroxidase from new source was purified using ion exchange and gel filtration techniques. The recovery for peroxidase activity was 19% with 11-fold purification and specific activity of 749 unit/mg protein. Purified peroxidase demonstrated a molecular mass of 39 kDa using gel filtration and was confirmed as a single band on SDS-PAGE. The purified peroxidase revealed a broad optimum pH activity at 6.0-6.5 and 50°C temperature. The kinetic parameters for purified peroxidase toward H2O2 and guaiacol as substrates were found to be Km = 3.355, 5.395 mM, Kcat = 99.52, 79.56 s-1 and Vmax =1.531, 1.242 µmole ml-1 min-1, respectively. The catalytic efficiency (kcat/Km) of the purified peroxidase was 14.75 and 29.66 s−1 mM−1 for guaiacol and H2O2, respectively. Peroxidase activity was observed to be enhanced by Cu2+, Co2+, Ni2+ and inhibited in the presence of Sn2+, Al3+, Hg2+, NaN3, EDTA and urea. Characterization showed that peroxidase purified from C. forskohlii has the ability to be used for food industrial applications.

scholarly journals A Novel Method of Affinity Tag Cleavage in the Purification of a Recombinant Thermostable Lipase from Aneurinibacillus thermoaerophilus Strain HZ

Catalysts ◽  
2018 ◽  
Vol 8 (10) ◽  
pp. 479 ◽  
Author(s):  
Malihe Masomian ◽  
Raja Rahman ◽  
Abu Salleh
Keyword(s):  
Organic Solvents ◽  
High Efficiency ◽  
Biochemical Characterization ◽  
Acyl Chain ◽  
Industrial Applications ◽  
Start Codon ◽  
High Yield ◽  
Medium Size ◽  
Affinity Tag ◽  
Purification Method

The development of an efficient and economical purification method is required to obtain a pure and mature recombinant protein in a simple process with high efficiency. Hence, a new technique was invented to cleave the tags from the N-terminal region of recombinant fusion HZ lipase in the absence of protease treatment. The recombinant pET32b/rHZ lipase was overexpressed into E. coli BL21 (DE3). Affinity chromatography was performed as the first step of purification. The stability of the protein was then tested in different temperatures. The fused Trx-His-S-tags to the rHZ lipase was cleaved by treatment of the fusion protein at 20 °C in 100 mM Tris-HCl buffer, pH 8.0. The precipitated tag was removed, and the mature recombinant enzyme was further characterized to specify its properties. A purification yield of 78.9% with 1.3-fold and 21.8 mg total purified mature protein was obtained from 50 mL starting a bacterial culture. N-terminal sequencing of purified recombinant HZ lipase confirmed the elimination of the 17.4 kDa tag from one amino acid before the native start codon (Methionine) of the protein. The mature rHZ lipase was highly active at 65 °C and a pH of 7.0, with a half-life of 2 h 15 min at 55 °C and 45 min at 60 °C. The rHZ lipase showed a preference for the hydrolysis of natural oil with a long carbon chain (C18) and medium-size acyl chain p-nitrophenyl esters (C10). The enzyme remained stable in the presence of nonpolar organic solvents, and its activity was increased by polar organic solvents. This study thus demonstrates a simple and convenient purification method which resulted in the high yield of mature enzyme along with unique and detailed biochemical characterization of rHZ lipase, making the enzyme favorable in various industrial applications.

scholarly journals Partial purification and biochemical characterization of an alkaline esterase from Sorghum bicolor

2020 ◽  
Vol 42 ◽  
pp. e52115
Author(s):  
Fabiana Guillen Moreira Gasparin ◽  
Marcio de Barros ◽  
Gabriela Alves Macedo
Keyword(s):  
Biochemical Characterization ◽  
Low Cost ◽  
Reaction Medium ◽  
Short Chain Fatty Acids ◽  
Relative Activity ◽  
Industrial Applications ◽  
Biochemical Properties ◽  
Water Soluble ◽  
Partial Purification ◽  
Alternative Source

Esterases are enzymes that present good potential for industrial applications since they catalyze the formation or cleavage of ester bonds in water-soluble substrates, and sorghum seeds can represent an alternative source of this enzyme. The extraction of esterase from sorghum seeds is an economical alternative to obtain an enzyme of great interest. Esterases may improve the quality or accelerate the maturation of cheeses, cured bacon and fermented sausages and may also resolve racemic mixtures. Recently, seed esterases have been the focus of much attention as biocatalysts. In some cases, these enzymes present advantages over animal and microbial lipases due to some quite interesting features such as specificity and low cost, being a great alternative for their commercial exploitation as industrial enzymes The esterase studied here was extracted from sorghum seeds and some of its biochemical properties determined using synthetic substrates (p-nitrophenyl butyrate, caprylate, laurate and palmitate). The enzyme presented optimum activity at pH 8.0 and was stable in all the pH ranges studied. The optimum temperature for its activity was 40ºC but it showed low stability at this temperature (40% relative activity). The values derived for Km and Vmax were 0.67mM and 125 U.mg-1, respectively, obtained using p-nitrophenyl butyrate as the substrate. The enzyme showed an increase in activity when K2HPO4 was added to the reaction medium, but the ions Mn2+, CO+, Hg+ and Fe2+ strongly inhibited the enzyme activity. This enzyme showed a preference for the hydrolysis of short chain fatty acids. The characteristics of sorghum esterase are very similar to those of the microbial esterases used in detergent processing.

scholarly journals Comparative Analysis and Biochemical Characterization of Two Endo-β-1,3-Glucanases from the Thermophilic Bacterium Fervidobacterium sp.

Catalysts ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 830 ◽  
Author(s):  
Burkhardt ◽  
Schäfers ◽  
Claren ◽  
Schirrmacher ◽  
Antranikian
Keyword(s):  
Elevated Temperatures ◽  
Biochemical Characterization ◽  
Hot Spring ◽  
Biomass Conversion ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Thermophilic Bacterium ◽  
Metagenomic Sample ◽  
Wide Range ◽  
Active Site Cleft

Laminarinases exhibit potential in a wide range of industrial applications including the production of biofuels and pharmaceuticals. In this study, we present the genetic and biochemical characteristics of FLamA and FLamB, two laminarinases derived from a metagenomic sample from a hot spring in the Azores. Sequence comparison revealed that both genes had high similarities to genes from Fervidobacterium nodosum Rt17-B1. The two proteins showed sequence similarities of 62% to each other and belong to the glycoside hydrolase (GH) family 16. For biochemical characterization, both laminarinases were heterologously produced in Escherichia coli and purified to homogeneity. FLamA and FLamB exhibited similar properties and both showed highest activity towards laminarin at 90 °C and pH 6.5. The two enzymes were thermostable but differed in their half-life at 80 °C with 5 h and 1 h for FLamA and FLamB, respectively. In contrast to other laminarinases, both enzymes prefer β-1,3-glucans and mixed-linked glucans as substrates. However, FLamA and FLamB differ in their catalytic efficiency towards laminarin. Structure predictions were made and showed minor differences particularly in a kink adjacent to the active site cleft. The high specific activities and resistance to elevated temperatures and various additives make both enzymes suitable candidates for application in biomass conversion.

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