Abstract:Chitinases are ubiquitous class of extracellular enzymes, which have gained attention in the past few years due to their wide biotechnological applications. The effectiveness of conventional insecticides is increasingly compromised by the occurrence of resistance; thus, chitinase offers a potential alternative to the use of chemical fungicides. The thermostable enzymes from thermophilic microorganisms have numerous industrial, medical, environmental and biotechnological applications due to their high stability… Show more
“…Within the conditions tested in this work, the enzyme expressed in P. pastoris appeared to be slightly more sensitive to temperature changes than protein previously purified from T. harzianum for which maximum activity values were reported at 40–45 °C, and maintained 50% of activity after 30 min at 50 °C [ 28 ]. However, our data are closer to what was expected because, in general, the optimal temperature for the fungal chitinase activity varies between 20 and 40 °C [ 18 ]. Fig.…”
BackgroundChitinases are ubiquitous enzymes that have gained a recent biotechnological attention due to their ability to transform biological waste from chitin into valued chito-oligomers with wide agricultural, industrial or medical applications. The biological activity of these molecules is related to their size and acetylation degree. Chitinase Chit42 from Trichoderma harzianum hydrolyses chitin oligomers with a minimal of three N-acetyl-d-glucosamine (GlcNAc) units. Gene chit42 was previously characterized, and according to its sequence, the encoded protein included in the structural Glycoside Hydrolase family GH18.ResultsChit42 was expressed in Pichia pastoris using fed-batch fermentation to about 3 g/L. Protein heterologously expressed showed similar biochemical properties to those expressed by the natural producer (42 kDa, optima pH 5.5–6.5 and 30–40 °C). In addition to hydrolyse colloidal chitin, this enzyme released reducing sugars from commercial chitosan of different sizes and acetylation degrees. Chit42 hydrolysed colloidal chitin at least 10-times more efficiently (defined by the kcat/Km ratio) than any of the assayed chitosan. Production of partially acetylated chitooligosaccharides was confirmed in reaction mixtures using HPAEC-PAD chromatography and mass spectrometry. Masses corresponding to (d-glucosamine)1–8-GlcNAc were identified from the hydrolysis of different substrates. Crystals from Chit42 were grown and the 3D structure determined at 1.8 Å resolution, showing the expected folding described for other GH18 chitinases, and a characteristic groove shaped substrate-binding site, able to accommodate at least six sugar units. Detailed structural analysis allows depicting the features of the Chit42 specificity, and explains the chemical nature of the partially acetylated molecules obtained from analysed substrates.ConclusionsChitinase Chit42 was expressed in a heterologous system to levels never before achieved. The enzyme produced small partially acetylated chitooligosaccharides, which have enormous biotechnological potential in medicine and food. Chit42 3D structure was characterized and analysed. Production and understanding of how the enzymes generating bioactive chito-oligomers work is essential for their biotechnological application, and paves the way for future work to take advantage of chitinolytic activities.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0895-x) contains supplementary material, which is available to authorized users.
“…Within the conditions tested in this work, the enzyme expressed in P. pastoris appeared to be slightly more sensitive to temperature changes than protein previously purified from T. harzianum for which maximum activity values were reported at 40–45 °C, and maintained 50% of activity after 30 min at 50 °C [ 28 ]. However, our data are closer to what was expected because, in general, the optimal temperature for the fungal chitinase activity varies between 20 and 40 °C [ 18 ]. Fig.…”
BackgroundChitinases are ubiquitous enzymes that have gained a recent biotechnological attention due to their ability to transform biological waste from chitin into valued chito-oligomers with wide agricultural, industrial or medical applications. The biological activity of these molecules is related to their size and acetylation degree. Chitinase Chit42 from Trichoderma harzianum hydrolyses chitin oligomers with a minimal of three N-acetyl-d-glucosamine (GlcNAc) units. Gene chit42 was previously characterized, and according to its sequence, the encoded protein included in the structural Glycoside Hydrolase family GH18.ResultsChit42 was expressed in Pichia pastoris using fed-batch fermentation to about 3 g/L. Protein heterologously expressed showed similar biochemical properties to those expressed by the natural producer (42 kDa, optima pH 5.5–6.5 and 30–40 °C). In addition to hydrolyse colloidal chitin, this enzyme released reducing sugars from commercial chitosan of different sizes and acetylation degrees. Chit42 hydrolysed colloidal chitin at least 10-times more efficiently (defined by the kcat/Km ratio) than any of the assayed chitosan. Production of partially acetylated chitooligosaccharides was confirmed in reaction mixtures using HPAEC-PAD chromatography and mass spectrometry. Masses corresponding to (d-glucosamine)1–8-GlcNAc were identified from the hydrolysis of different substrates. Crystals from Chit42 were grown and the 3D structure determined at 1.8 Å resolution, showing the expected folding described for other GH18 chitinases, and a characteristic groove shaped substrate-binding site, able to accommodate at least six sugar units. Detailed structural analysis allows depicting the features of the Chit42 specificity, and explains the chemical nature of the partially acetylated molecules obtained from analysed substrates.ConclusionsChitinase Chit42 was expressed in a heterologous system to levels never before achieved. The enzyme produced small partially acetylated chitooligosaccharides, which have enormous biotechnological potential in medicine and food. Chit42 3D structure was characterized and analysed. Production and understanding of how the enzymes generating bioactive chito-oligomers work is essential for their biotechnological application, and paves the way for future work to take advantage of chitinolytic activities.Electronic supplementary materialThe online version of this article (10.1186/s12934-018-0895-x) contains supplementary material, which is available to authorized users.
“…Several factors define this property such as the number of hydrogen bonds, salt bridges, stabilization of secondary structures, occurrence of disulfide bonds, higher number of proline residues, higher polar surface area, shortening of loops, and stabilization of the lid domain (Pack and Yoo, 2004; Santarossa et al, 2005; Zhou et al, 2008; Khan et al, 2015a,b). …”
Lipases are important industrial enzymes. Most of the lipases operate at lipid–water interfaces enabled by a mobile lid domain located over the active site. Lid protects the active site and hence responsible for catalytic activity. In pure aqueous media, the lid is predominantly closed, whereas in the presence of a hydrophobic layer, it is partially opened. Hence, the lid controls the enzyme activity. In the present review, we have classified lipases into different groups based on the structure of lid domains. It has been observed that thermostable lipases contain larger lid domains with two or more helices, whereas mesophilic lipases tend to have smaller lids in the form of a loop or a helix. Recent developments in lipase engineering addressing the lid regions are critically reviewed here. After on, the dramatic changes in substrate selectivity, activity, and thermostability have been reported. Furthermore, improved computational models can now rationalize these observations by relating it to the mobility of the lid domain. In this contribution, we summarized and critically evaluated the most recent developments in experimental and computational research on lipase lids.
“…The homology modeling is helpful in predicting the 3D protein models, and to establish structure–function relationships . The Modeler software was used for 3D modeling of PLA 1 wild‐type using the crystal structures of GZEL (PDB ID: 3NGM), TLL (PDB ID: 1EIN), and RML (PDB ID: 4TGL) as templates.…”
Enzymatic activity is important characteristic of enzymes for industrial application, which can be improved by protein engineering. PLA1 is a biocatalyst applied in phospholipid modification for its phospholipase activity and had obtained board attention for its application in oil degumming. Bioinformatics analysis suggested that three charged residues, R81, R84, and E87 located in the lid of the protein, may affect the conformational change of lid thus influence the activity of the enzyme. In the current study, mutagenesis of these residues was conducted with protein engineering. Five mutants such as R81A, R84A, R84M, R84K, and E87Q with higher phospholipase activity were screened out. Biochemical properties analysis showed that all of them had identical optimal pH value with wild‐type, while the optimal temperature was decreased to be 50°C and the kcat/KM was improved. Degumming soybean oil, three of five mutants, R81A, R84M, and E87Q, decreased phosphorous content lower than 8.3 mg/kg within 3 h, which was highly improved compared with wild‐type. R84M decrease phosphorus content less than 5 mg/kg within 5 h. These findings not only permit optimization of enzyme performance in degumming but also shed light on the application of bioinformatics techniques and protein engineering techniques on industry application.
Practical applications: The results support that design proteins according to bioinformatics and protein engineering is desirable and the phospholipase with higher activity is more suitable for oil degumming.
The phospholipase activity of PLA1 can be improved by protein engineering based on bioinformatics analysis. With its properties of hydrolyzing sn‐1 position ester bond of phospholipids, nonhydratable phospholipids were converted into their hydratable forms, PLA1 can be applied in oil degumming. PLA1 with higher phospholipase activity is more likely to suitable for oil degumming since it could decrease the phosphorous content of oil lower than 10 mg/kg within shorter reaction, which showed great potencial for degumming application.
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