Directed Evolution of Clostridium phytofermentans Glycoside Hydrolase Family 9 Endoglucanase for Enhanced Specific Activity on Solid Cellulosic Substrate

Ahmad, Sajjad; Ma, Hui; Akhtar, Muhammad; Zhang, Y.‐H. Percival; Zhang, Xiaozhou

doi:10.1007/s12155-013-9382-8

Cited by 9 publications

(6 citation statements)

References 30 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Multimeric cloning approach, which involves the use of POE-PCR, was opted for the construction of expression of plasmid pNWPH-GCSF (containing ~0.5 kb GCSF gene downstream of the P HbaII promoter). This technique, originally described by You et al [ 23 ], is relatively new but is simple and cost-effective and has certain advantages over the conventional cloning strategies, in particular the direct transformation of host without additional steps of restriction digestion and DNA ligation [ 22 ].…”

Section: Discussionmentioning

confidence: 99%

“…Plasmid pNWPH-mini-scaf [ 22 ] containing chloramphenicol resistance gene, a strong P HpaII promoter and SPymwC signal sequence, was used for the construction of pNWPH-GCSF ( Figure 1 ). The primers used for the multimer cloning were comprised of 50 nucleotides (nt) each, having 25 nt overlapping region of the insert and 25 nt of the vector ( Table 1 ).…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Enhanced and Secretory Expression of Human Granulocyte Colony Stimulating Factor byBacillus subtilis SCK6

Bashir

Sadaf

Ahmad

et al. 2015

BioMed Research International

Self Cite

View full text Add to dashboard Cite

This study describes a simplified approach for enhanced expression and secretion of a pharmaceutically important human cytokine, that is, granulocyte colony stimulating factor (GCSF), in the culture supernatant of Bacillus subtilis SCK6 cells. Codon optimized GCSF and pNWPH vector containing SpymwC signal sequence were amplified by prolonged overlap extension PCR to generate multimeric plasmid DNA, which was used directly to transform B. subtilis SCK6 supercompetent cells. Expression of GCSF was monitored in the culture supernatant for 120 hours. The highest expression, which corresponded to 17% of the total secretory protein, was observed at 72 hours of growth. Following ammonium sulphate precipitation, GCSF was purified to near homogeneity by fast protein liquid chromatography on a QFF anion exchange column. Circular dichroism spectroscopic analysis showed that the secondary structure contents of the purified GCSF are similar to the commercially available GCSF. Biological activity, as revealed by the regeneration of neutrophils in mice treated with ifosfamine, was also similar to the commercial preparation of GCSF. This, to our knowledge, is the first study that reports secretory expression of human GCSF in B. subtilis SCK6 with final recovery of up to 96 mg/L of the culture supernatant, without involvement of any chemical inducer.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Enhanced and Secretory Expression of Human Granulocyte Colony Stimulating Factor byBacillus subtilis SCK6

Bashir

Sadaf

Ahmad

et al. 2015

BioMed Research International

Self Cite

View full text Add to dashboard Cite

show abstract

“…Similarly, the fusion of CBM and dockerin domains using different linker lengths to Thermobifida fusca cellulases helps to demonstrate that CBMs play a major role in the degradation of crystalline cellulosic substrates 5 . The mutations in CBM3 of Cel9 showed a synergistic effect to enhance the activity almost three times that of the native cellulase of Clostridium phytofermentans 6 . The linker length also alters the enzymatic activity; generally, a flexible linker region is favorable for the proper synergism between CBM and the catalytic domain.…”

Section: Introductionmentioning

confidence: 95%

Engineering processive cellulase of Clostridium thermocellum to divulge the role of the carbohydrate‐binding module

Ahmad

Sajjad

Altayb

et al. 2022

Biotech and App Biochem

Self Cite

View full text Add to dashboard Cite

The processive cellulase (CelO) is an important modular enzyme of Clostridium thermocellum. To study the effect of the carbohydrate-binding module (CBM3b) on the catalytic domain of CelO (GH5), four engineered derivatives of CelO were designed by truncation and terminal fusion of CBM3b. These are CBM at the N-terminus, native form (CelO-BC, 62 kDa); catalytic domain only (CelO-C, 42 kDa); CBM at the C-terminus (CelO-CB, 54 kDa) and CBM attached at both termini (CelO-BCB, 73 kDa). All constructs were cloned into pET22b (+) and expressed in Escherichia coli BL21 (DE3) star. The expression levels of CelO-C, CelO-CB, CelO-BC, and CelO-BCB were 35%, 35%, 30%, and 20%, respectively.The enzyme activities of CelO-C, CelO-CB, CelO-BC, and CelO-BCB against 1% regenerated amorphous cellulose (RAC) were 860, 758, 985, and 1208 units per μmole of the enzyme, respectively. The enzymes were partially purified from the lysate of E. coli cells by heat treatment followed by anion exchange FPLC purification. Against RAC, CelO-C, CelO-CB, CelO-BC, and CelO-BCB showed K M values of 32, 33, 45, and 43 mg⋅mL -1 and V max values of 3571, 3846, 3571, and 4545 U⋅min -1 , respectively. CBM3b at the N-terminus of GH5 linked through a P/Trich linker was found to enhance the catalytic activity and thermostability of the enzyme.

show abstract

“…The highest wild-type bacterial free cellulase activity was reported at about 2.5 U/mg protein on Avicel for a GH9 processive endoglucanase originated from Clostridium phytofermentans [78] and a GH5 bacterial endoglucanase screened from the Buffalo rumen metagenomic library [55]. Directed evolution of this Clostridium phytofermentans GH9 processive endoglucanase further enhanced its activity to about 6 U/mg protein on Avicel [79]. The highest fungal free cellulase activities were reported at 9.7 and 29.8 U/mg protein for two exocellulases on Avicel and these free cellulases were respectively originated from ruminal Neocallimastix patriciarum [80] and the White Rot fungus Irpex lacteus [75].…”

Section: Diversity Of Microbial Cellulasesmentioning

confidence: 99%

“…Directed evolution is a very powerful protein-engineering tool for improving enzyme properties such as specific activity and thermostability without in-depth immediate understanding of enzyme protein structure and enzyme-substrate interactions [78]. Given the fact that the GH9 Clostridium Phytofermentans processive endocellulase had a large molecular weight (over 100 kDa) and was multi-modular, Ahmad et al [79] demonstrated this by using B. subtilis as a host and applying directed evolution for improving the processive GH9 endocellulase activity by about two-fold from the Clostridium Phytofermentans on the solid cellulosic substrate. Efforts ought to be made to further enhance both GH5-tCel5A1 and GH5-p4818Cel5_2A endocellulase activity by directed evolution and using B. subtilis as a host.…”

Section: Structure and Functionality Of The Newly Characterized Processive Endocellulasesmentioning

confidence: 99%

Metagenomic Discovery and Characterization of Multi-Functional and Monomodular Processive Endoglucanases as Biocatalysts

et al. 2021

View full text Add to dashboard Cite

Biomass includes cellulose, hemicelluloses, pectin and lignin; constitutes the components of dietary fibre of plant and alge origins in animals and humans; and can potentially provide inexhaustible basic monomer compounds for developing sustainable biofuels and biomaterials for the world. Development of efficacious cellulases is the key to unlock the biomass polymer and unleash its potential applications in society. Upon reviewing the current literature of cellulase research, two characterized and/or engineered glycosyl hydrolase family-5 (GH5) cellulases have displayed unique properties of processive endoglucanases, including GH5-tCel5A1 that was engineered and was originally identified via targeted genome sequencing of the extremely thermophilic Thermotoga maritima and GH5-p4818Cel5_2A that was screened out of the porcine hindgut microbial metagenomic expression library. Both GH5-tCel5A1 and GH5-p4818_2A have been characterized as having small molecular weights with an estimated spherical diameter at or < 4.6 nm; being monomodular without a required carbohydrate-binding domain; and acting as processive β-1,4-endoglucanases. These two unique GH5-tCel5A1 and GH5-p4818_2A processive endocellulases are active in hydrolyzing natural crystalline and pre-treated cellulosic substrates and have multi-functionality towards several hemicelluloses including β-glucans, xylan, xylogulcans, mannans, galactomannans and glucomannans. Therefore, these two multifunctional and monomodular GH5-tCel5A1 and GH5-p4818_2A endocellulases already have promising structural and functional properties for further optimization and industrial applications.

show abstract

Directed Evolution of Clostridium phytofermentans Glycoside Hydrolase Family 9 Endoglucanase for Enhanced Specific Activity on Solid Cellulosic Substrate

Cited by 9 publications

References 30 publications

Enhanced and Secretory Expression of Human Granulocyte Colony Stimulating Factor byBacillus subtilis SCK6

Enhanced and Secretory Expression of Human Granulocyte Colony Stimulating Factor byBacillus subtilis SCK6

Engineering processive cellulase of Clostridium thermocellum to divulge the role of the carbohydrate‐binding module

Metagenomic Discovery and Characterization of Multi-Functional and Monomodular Processive Endoglucanases as Biocatalysts

Contact Info

Product

Resources

About