Overexpression, purification and characterization of the Aspergillus niger endoglucanase, EglA, in Pichia pastoris (2024)

Recombinant Fungal Cellulases for the Saccharification of Sugarcane Bagasse

Biodegradation [Working Title], 2021

Cellulases are important enzymes in cellulose degradation that occurs in nature, this degradation involves a system of extracellular multienzymes and have wide application. The construction of a high-quality system for the production of these enzymes is important for its application in the process of saccharification of biomass involved in the biofuel production process. Several species of fungi are capable of synthesizing and secreting high amounts of cellulase, most studies with fungal species use linearized plasmid, since these are encompassed to chromosomal DNA, improving its stability and expression efficiency. Advances in the production of recombinant enzymes focus on the search for industrially viable microorganisms capable of producing enzymes under various conditions, expressing them in a highly efficient manner, aiming at the synthesis of several copies of genes and a strong promoter. To resay these restrictions, molecular biology combined with recombinant DNA technology i...

Cloning, expression, purification and properties of a putative multidrug resistance efflux protein from Helicobacter pylori

International journal of …, 2003

An Acidic Thermostable Recombinant Aspergillus nidulans Endoglucanase Is Active towards Distinct Agriculture Residues

Enzyme Research, 2013

Aspergillus nidulans is poorly exploited as a source of enzymes for lignocellulosic residues degradation for biotechnological purposes. This work describes the A. nidulans Endoglucanase A heterologous expression in Pichia pastoris, the purification and biochemical characterization of the recombinant enzyme. Active recombinant endoglucanase A (rEG A) was efficiently secreted as a 35 kDa protein which was purified through a two-step chromatography procedure. The highest enzyme activity was detected at 50°C/pH 4. rEG A retained 100% of activity when incubated at 45 and 55°C for 72 h. Purified rEG A kinetic parameters towards CMC were determined as Km=27.5±4.33 mg/mL, Vmax=1.185±0.11 mmol/min, and 55.8 IU (international units)/mg specific activity. Recombinant P. pastoris supernatant presented hydrolytic activity towards lignocellulosic residues such as banana stalk, sugarcane bagasse, soybean residues, and corn straw. These data indicate that rEG A is suitable for plant biomass convers...

Characterization and High Level Expression of Acidic Endoglucanase in Pichia pastoris

Applied Biochemistry and Biotechnology, 2013

Cellobiohydrolase B of Aspergillus niger over-expressed in Pichia pastoris stimulates hydrolysis of oil palm empty fruit bunches

PeerJ, 2017

Aspergillus niger, along with many other lignocellulolytic fungi, has been widely used as a commercial workhorse for cellulase production. A fungal cellulase system generally includes three major classes of enzymes i.e., β-glucosidases, endoglucanases and cellobiohydrolases. Cellobiohydrolases (CBH) are vital to the degradation of crystalline cellulose present in lignocellulosic biomass. However, A. niger naturally secretes low levels of CBH. Hence, recombinant production of A. niger CBH is desirable to increase CBH production yield and also to allow biochemical characterisation of the recombinant CBH from A. niger. In this study, the gene encoding a cellobiohydrolase B (cbhB) from A. niger ATCC 10574 was cloned and expressed in the methylotrophic yeast Pichia pastoris X-33. The recombinant CBHB was purified and characterised to study its biochemical and kinetic characteristics. To evaluate the potential of CBHB in assisting biomass conversion, CBHB was supplemented into a commercia...

Cellobiohydrolase B of<i>Aspergillus niger</i>over-expressed in<i>Pichia pastoris</i>stimulates hydrolysis of oil palm empty fruit bunches

PeerJ, 2017

Cloning, heterologous expression and characterisation of a recombinant cellobiohydrolase from Humicola insolens ATCC16454 in Pichia pastoris

Malaysian applied biology, 2015

Characterization of a novel manganese dependent endoglucanase belongs in GH family 5 from Phanerochaete chrysosporium

Journal of bioscience and bioengineering, 2015

The cDNA encoding a putative glycoside hydrolase family 5, which has been predicted to be an endoglucanase (PcEg5A), was cloned from Phanerochaete chrysosporium and expressed in Pichia pastoris. PcEg5A contains a carbohydrate-binding domain and two important amino acids, E209 and E319, playing as proton donor and nucleophile in substrate catalytic domain. SDS-PAGE analysis indicated that the recombinant endoglucanase 5A (rPcEg5A) has a molecular size of 43 kDa which corresponds with the theoretical calculation. Optimum pH and temperature were found to be 4.5-6.0, and 50°C-60°C, respectively. Moreover, rPcEg5A exhibited maximal activity in the pH range of 3.0-8.0, whereas over 50% of activity still remained at 20°C and 80°C. rPcEg5A was stable at 60°C for 12 h incubation, indicating that rPcEg5A is a thermostable enzyme. Manganese ion enhanced the enzyme activity by 77%, indicating that rPcEg5A is a metal dependent enzyme. The addition of rPcEg5A to cellobiase (cellobiohydrolase and ...

Functional characterization of GH7 endo-1,4-β-glucanase from Aspergillus fumigatus and its potential industrial application

Protein Expression and Purification, 2018

Heterologous expression and biochemicalcharacterization of a novel thermostable Sclerotiniasclerotiorum GH45 endoglucanase in Pichia pastoris

Enzymatic saccharification of lignocellulosic biomass has been widely studied. Mainly endoglucanases were found to be a prerequisite for the quick initial biomass liquefaction. In the present study, Pichia pastoris was used as a host for the heterologous expression of a Sclerotinia sclerotiorum GH45 endoglucanase, Endo2. The recombinant plasmid pPICZαA was used to transform Pichia pastoris. Pichia culture supernatants expressing the recombinant Endo2 (rEndo2) were used for the purification and biochemical characterization of this enzyme. Therefore, rEndo2 was purified 6.7 fold to homogeneity with 34% yield and gave 19 U/mg specific activity. It also showed maximum activity at pH 7.0 and 60°C (against pH 5.0 and 50°C for the native enzyme) and was thermostable at relatively high temperatures. Furthermore, rEndo2 retained its activity in a wide pH range (from 5 to 8). Besides, the recombinant endoglucanase was produced as an active 47 kDa enzyme. This molecular weight differs from the one of the native enzyme (34 kDa), which suggested a potential glycosylation of the recombinant enzyme. Moreover, rEndo2 was able to produce fermentable sugars after enzymatic assay on various cellulosic substrates with an interesting yield. Therefore, all these features offer prospects for large-scale production and industrial application of the recombinant endoglucanase.