Expression and characterization of a family 45 glycosyl hydrolase from Fomitopsis pinicola and comparison to Phanerochaete chrysosporium Cel45A

Amengual, Neus Gacias; Csarman, Florian; Wohlschlager, Lena; Ludwig, Roland

doi:10.1016/j.enzmictec.2022.110000

Cited by 6 publications

(5 citation statements)

References 35 publications

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“…Figure 1a shows the optimum temperatures of MtEG5A, MtEG7A, MtEG12A, and MtEG45A were 75 °C, 70 °C, 65 °C, and 65 °C, respectively. The optimum temperatures of MtEG5A and the MtEG7A showed slight variations compared to the previously reported optimum temperatures of EG 5 and EG 7 (70 °C and 60 °C, respectively) [26,27,29], which may be due to the difference in the expression host. Figure 1b shows that the optimum pH for MtEG5A, MtEG7A, and MtEG12A was pH 5.0, and MtEG45A showed the highest activity levels at pH 4.0.…”

Section: Enzymatic Properties Of Different Egs From M Thermophilacontrasting

confidence: 64%

“…On the other hand, it was also reported the EGs from M. thermophila were heat resistant; for example, MtEG5A, MtEG7A, and MtEG45A showed optimum temperatures of 70 °C, 60 °C, and 65 °C, respectively. After pre-incubation in 100 mM phosphate–citrate buffer (pH 5.0) for 8 h, MtEG5A at 50 °C and MtEG7A at 40 °C remained fairly stable, whereas MtEG45A was stable up to 24 h at temperatures up to 70 °C [ 26 , 27 , 28 , 29 ]; therefore, these enzymes are expected to accelerate the preparation of CNCs by enzymatic hydrolysis at a relatively high temperature due to their temperature resistance and stability characteristics.…”

Section: Introductionmentioning

confidence: 99%

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Production and Characterization of Cellulose Nanocrystals from Eucalyptus Dissolving Pulp Using Endoglucanases from Myceliophthora thermophila

Waghmare

et al. 2023

IJMS

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Endoglucanase (EG) is a key enzyme during enzymatic preparation of cellulose nanocrystals (CNCs). Myceliophthora thermophila is a thermophilic fungus that has thermal properties and a high secretion of endoglucanases (EGs), and could serve as potential sources of EGs for the preparation of CNCs. In this work, four different GH families (GH5, GH7, GH12, and GH45) of EGs from M. thermophila were expressed and purified, and their enzymatic characteristics and feasibility of application in CNC preparation were investigated. It was shown that the MtEG5A from M. thermophila has good potential in the enzymatic preparation of CNCs using eucalyptus dissolving pulp as feedstock. It was also observed that there was a synergistic effect between the MtEG5A and other MtEGs in the preparation of CNCs, which improved the yield and properties of CNCs obtained by enzymatic hydrolysis. This study provides a reference for understanding the enzymatic characteristics of different families of EGs from M. thermophile and their potential application in nanocellulose production.

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Section: Enzymatic Properties Of Different Egs From M Thermophilacontrasting

confidence: 64%

Section: Introductionmentioning

confidence: 99%

Production and Characterization of Cellulose Nanocrystals from Eucalyptus Dissolving Pulp Using Endoglucanases from Myceliophthora thermophila

Waghmare

et al. 2023

IJMS

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“…Our studies showed comparatively slower hydrolysis rates and lower reducing end yields than on betaglucan or glucomannan. Interestingly, this pattern is not observed across studies, particularly in reference to subfamily C. Multiple studies show higher product yields on CMC compared to betaglucan in subfamily C (Amengual et al, 2022;. Subfamily C members have been studied under multiple incubation settings with aeration ranging from 100 to 1200 rpm, different incubation times with discoveries of optimal pH values of 4.0 -6.5.…”

Section: Konjac Glucomannanmentioning

confidence: 99%

Structure and function studies of GH45 glycoside hydrolases

Okmane¹

2023

Acta Universitatis Agriculturae Sueciae

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The enzymatic hydrolysis of lignocellulosic material in nature is carried out by a plethora of cellulases. Glycoside hydrolase family 45 (GH45) enzymes are small cellulases most commonly found in fungi that catalyse the hydrolysis of β(1→4) linked glucans. Additionally, GH45 enzymes display a structural resemblance to non-hydrolytic protein groups such as expansins and loosenins. In this thesis, the distinctness of GH45 enzymes from different subfamilies was explored. The enzymatic activity of GH45 enzymes from Humicola insolens (HiCel45A), Mytilus edulis (MeCel45A), Trichoderma reesei (TrCel45A), Phanerochaete chrysosporium (PcCel45A), Gloeophyllum trabeum (GtCel45A) was demonstrated. Among the tested substrates were barley betaglucan, konjac glucomannan, carboxymethyl cellulose, and cellohexaose. Initial hydrolysis rates and hydrolysis yields were determined by reducing sugar assays, product formation was analysed using NMR spectroscopy and HPLC. The subfamily B and C enzymes exhibited mannanase activity, and the subfamily B enzyme MeCel45A appeared cold adapted in comparison to TrCel45A. GH45 enzymes are known to act using an inverting action mechanism. This action mechanism had not been experimentally demonstrated in subfamilies B and C, however. Here experimental evidence is provided for the inverting nature of GH45 enzymes from all subfamilies. Comparisons of GH45 enzyme structures were carried out and the first crystal structure of a GH45 subfamily C enzyme from the brown-rot fungus G. trabeum was reported at 1.3 Å resolution. Furthermore, structure and function investigations were done on an isotopically labelled Cel45A from the white-rot fungus P. chrysosporium using NMR spectroscopy. PcCel45A was expressed in Pichia pastoris with 13C and 15N labelling. A nearly complete assignment of 1 H, 13C and 15N backbone resonances was obtained and the interaction of 15Nlabelled PcCel45A with cellobiose was studied.

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“…The heterologous production of recombinant endoglucanases, cellobiohydrolases, β-glucosidases, and laccases by the yeast K. phaffii is discussed in detail in another review [ 174 ]. Among recent research articles, we can cite examples of the successful expression (in the K. phaffii system) of genes of endoglucanases from Colletotrichum graminicola [ 305 , 306 ], Fomitopsis pinicola [ 307 ], and Penicillium funiculosum [ 308 ]; the synthesis of recombinant cellobiohydrolases from A. niger [ 309 ], Lentinula edodes [ 310 ], and Phanerochaete chrysosporium [ 308 ]; the production of β-glucosidases from Coptotermes formosanus [ 311 ] and Chaetomella raphigera [ 62 ]; and the synthesis of laccases from Pleurotus ostreatus [ 312 ], C. cinerea [ 313 ], Laccaria bicolor [ 314 ], Streptomyces coelicolor [ 315 ], Trametes versicolor [ 316 ], and other microbes.…”

Section: K Phaffii As a Producer Of Enzymes For Increasing N...mentioning

confidence: 99%

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry

Khlebodarova,

Bogacheva,

Zadorozhny

et al. 2024

Microorganisms

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In the 1980s, Escherichia coli was the preferred host for heterologous protein expression owing to its capacity for rapid growth in complex media; well-studied genetics; rapid and direct transformation with foreign DNA; and easily scalable fermentation. Despite the relative ease of use of E. coli for achieving the high expression of many recombinant proteins, for some proteins, e.g., membrane proteins or proteins of eukaryotic origin, this approach can be rather ineffective. Another microorganism long-used and popular as an expression system is baker’s yeast, Saccharomyces cerevisiae. In spite of a number of obvious advantages of these yeasts as host cells, there are some limitations on their use as expression systems, for example, inefficient secretion, misfolding, hyperglycosylation, and aberrant proteolytic processing of proteins. Over the past decade, nontraditional yeast species have been adapted to the role of alternative hosts for the production of recombinant proteins, e.g., Komagataella phaffii, Yarrowia lipolytica, and Schizosaccharomyces pombe. These yeast species’ several physiological characteristics (that are different from those of S. cerevisiae), such as faster growth on cheap carbon sources and higher secretion capacity, make them practical alternative hosts for biotechnological purposes. Currently, the K. phaffii-based expression system is one of the most popular for the production of heterologous proteins. Along with the low secretion of endogenous proteins, K. phaffii efficiently produces and secretes heterologous proteins in high yields, thereby reducing the cost of purifying the latter. This review will discuss practical approaches and technological solutions for the efficient expression of recombinant proteins in K. phaffii, mainly based on the example of enzymes used for the feed industry.

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Expression and characterization of a family 45 glycosyl hydrolase from Fomitopsis pinicola and comparison to Phanerochaete chrysosporium Cel45A

Cited by 6 publications

References 35 publications

Production and Characterization of Cellulose Nanocrystals from Eucalyptus Dissolving Pulp Using Endoglucanases from Myceliophthora thermophila

Production and Characterization of Cellulose Nanocrystals from Eucalyptus Dissolving Pulp Using Endoglucanases from Myceliophthora thermophila

Structure and function studies of GH45 glycoside hydrolases

Komagataella phaffii as a Platform for Heterologous Expression of Enzymes Used for Industry

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