- Email: [email protected]
Fungal and bacterial lignin degraders: Purification, characterisation and structural investigation
Abstracts / New Biotechnology 33S (2016) S1–S213
Reference [1] Gerber A, et al. Microbial cell factories; 2015.
http://dx.doi.org/10.1016/j.nbt.2016.06.1419
P34-17 Production and characterization of a xylose-tolerant xylosidase (HXYLA) from Humicola grisea var. thermoidea expressed by Pichia pastoris Lorena Cintra 1,∗ , Amanda G. Fernandes 2 , Izadora C.M. de Oliveira 3 , Saulo J.L. Siqueira 4 , Fracieli Colussi 3 , Rosália S.A. Jesuíno 3 , Cirano J. Ulhoa 5 , Fabrícia P. de Faria 3 1
University of Brasília and Federal University of Goiás, Brazil University of Brasilia and Federal University of Goiás, Brazil 3 Federal University of Goiás, Brazil 4 Federal University of Goiás and State University of Goiás, Brazil 5 Federal University of Goiás and University of Brasilia, Brazil 2
One full-length -xylosidase gene was identified from the genome of Humicola grisea. The -xylosidase encoding gene, hxylA, consisted of 984 bp and the molecular mass estimated was 37 kDa. No intron and no putative signal peptide was identified. The sequence had no putative N-glycosylation site and the catalytic domain of GH43 was present. The cDNA was cloned and heterologously expressed in Pichia pastoris SMD1168 with high level of 90 mg/L in flasks without fermentation optimization. The purified enzyme, showed optimally active at pH 7.0 and 50 ◦ C. The activity was retained approximately 50% at 50 ◦ C after 12 h and 100% at pH 7.0 for about 48 h. HXYLA showed both -xylosidase and aarabinfuranosidase activities (bifunctional enzyme). The Km and Vmax values were 2.13 mM and 52.3 mmol/min/mg, respectively, against 4-nitrophenyl--xylopyranoside. HXYLA had stronger tolerance to xylose with high Ki value of 350 mM and the xylose is a non-competitive inhibitor. HXYLA was used simultaneously and sequentially with a endo-xylanase (HXYN2) from H. grisea produced by P. pastoris for synergism analysis on degradation of beechwood and oat spelt xylan. During 12-h hydrolysis, the amounts of reducing sugar released in the presence of HXYLA were about 1.10 and 1.38 fold increase for beechwood and oat spelt xylan, respectively, compared to the expected amounts for the individual enzymes acting alone. The highest degree of synergy by sequential reactions was obtained when HXYN2 was added before the HXYLA. Our results suggested that HXYLA may be useful for many biotechnology process, including the degradation of lignocellulosic biomass in bioethanol production.
S203
lignin The main objective of the study is to identify, purify and characterise several bacterial and fungal enzymes and the products obtained from their degradation of lignin. The bacterial and fungal enzymes were identified from protein databases, cloned into appropriate vectors and expressed in Escherichia coli and Pichia pastoris respectively. The enzymes were purified using a multistep purification strategy combining cation and anion exchange with size exclusion chromatography. Spectroscopic techniques with varying levels of resolution were used to investigate potentially industrially useful lignin degradation products. This includes UV–Visible, Fourier Transform Infrared (FTIR) and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS). Structural investigation using X-ray crystallography has been done on the bacterial enzymes with diffraction data obtained. All of the enzymes discovered from the study will be made available for industrial breakdown of lignin for the production of useful products. All of the completed work together with recent efforts at engineering the enzymes of interest for further investigation will be discussed in this presentation. http://dx.doi.org/10.1016/j.nbt.2016.06.1421
P34-19 Looking for rules of protein aggregation Giulia Tedeschi 1,∗ , Stefania Brocca 2 , Antonino Natalello 2 , Marina Lotti 2 1 2
University of Milano Bicocca, Italy University of Milano – Bicocca, Italy
Fatai Bello ∗ , Louise Horsfall
Protein aggregation is an issue of clinical and biotechnical relevance. It is involved in a wide variety of diseases and is a hurdle in the production of recombinant proteins in bacterial cells. Understanding determinants of aggregation might contribute to design new strategies to prevent or modulate its occurrence. Knowledge in this field might be exploited for biotechnological purposes such as the design of in/solubility tags, valuable to recover a target protein by in-vitro aggregation or, vice versa, to prevent its undesired aggregation. Our study employs Green Fluorescent Protein (GFP) and the intrinsically disordered N-terminal moiety of a viral phosphoprotein (PNT). GFP is a well-known, structured protein that can be induced to aggregate and whose fluorescence easily reports its functional status. Intrinsically disordered proteins have extended, flexible and highly-dynamic conformations, consistent with high frequency of polar/charged residues in their primary sequence. Starting from the wild-type PNT (pI ∼ 4.9) we designed variants where the contents of charged amino acids were increased to give an acidic (pI ∼ 3.4) and a basic (pI ∼ 9.9) variant of PNT. We produced as recombinant proteins the three PNT charge variants, GFP and their fusions. An array of biophysical and biochemical techniques indicates that charge mutants of PNT per se strongly react to pH and are insoluble at their own pI. Also when fused to GFP, PNTs triggers pH-dependent aggregation. Paucity of charges emerges as a leading force of aggregation overwhelming the entropic effects of conformational flexibility on solubility.
University of Edinburgh, United Kingdom
http://dx.doi.org/10.1016/j.nbt.2016.06.1422
http://dx.doi.org/10.1016/j.nbt.2016.06.1420
P34-18 Fungal and bacterial lignin degraders: Purification, characterisation and structural investigation
Lignocellulosic biomass is the most abundant renewable source of raw materials for the production of biofuels and biomaterials. Lignin, an aromatic macromolecule is one of three major component compounds of lignocellulosic biomass. Bacteria and fungi utilise a consortium of lignin degrading enzymes to break down
Reference [1] Gerber A, et al. Microbial cell factories; 2015.
http://dx.doi.org/10.1016/j.nbt.2016.06.1419
P34-17 Production and characterization of a xylose-tolerant xylosidase (HXYLA) from Humicola grisea var. thermoidea expressed by Pichia pastoris Lorena Cintra 1,∗ , Amanda G. Fernandes 2 , Izadora C.M. de Oliveira 3 , Saulo J.L. Siqueira 4 , Fracieli Colussi 3 , Rosália S.A. Jesuíno 3 , Cirano J. Ulhoa 5 , Fabrícia P. de Faria 3 1
University of Brasília and Federal University of Goiás, Brazil University of Brasilia and Federal University of Goiás, Brazil 3 Federal University of Goiás, Brazil 4 Federal University of Goiás and State University of Goiás, Brazil 5 Federal University of Goiás and University of Brasilia, Brazil 2
One full-length -xylosidase gene was identified from the genome of Humicola grisea. The -xylosidase encoding gene, hxylA, consisted of 984 bp and the molecular mass estimated was 37 kDa. No intron and no putative signal peptide was identified. The sequence had no putative N-glycosylation site and the catalytic domain of GH43 was present. The cDNA was cloned and heterologously expressed in Pichia pastoris SMD1168 with high level of 90 mg/L in flasks without fermentation optimization. The purified enzyme, showed optimally active at pH 7.0 and 50 ◦ C. The activity was retained approximately 50% at 50 ◦ C after 12 h and 100% at pH 7.0 for about 48 h. HXYLA showed both -xylosidase and aarabinfuranosidase activities (bifunctional enzyme). The Km and Vmax values were 2.13 mM and 52.3 mmol/min/mg, respectively, against 4-nitrophenyl--xylopyranoside. HXYLA had stronger tolerance to xylose with high Ki value of 350 mM and the xylose is a non-competitive inhibitor. HXYLA was used simultaneously and sequentially with a endo-xylanase (HXYN2) from H. grisea produced by P. pastoris for synergism analysis on degradation of beechwood and oat spelt xylan. During 12-h hydrolysis, the amounts of reducing sugar released in the presence of HXYLA were about 1.10 and 1.38 fold increase for beechwood and oat spelt xylan, respectively, compared to the expected amounts for the individual enzymes acting alone. The highest degree of synergy by sequential reactions was obtained when HXYN2 was added before the HXYLA. Our results suggested that HXYLA may be useful for many biotechnology process, including the degradation of lignocellulosic biomass in bioethanol production.
S203
lignin The main objective of the study is to identify, purify and characterise several bacterial and fungal enzymes and the products obtained from their degradation of lignin. The bacterial and fungal enzymes were identified from protein databases, cloned into appropriate vectors and expressed in Escherichia coli and Pichia pastoris respectively. The enzymes were purified using a multistep purification strategy combining cation and anion exchange with size exclusion chromatography. Spectroscopic techniques with varying levels of resolution were used to investigate potentially industrially useful lignin degradation products. This includes UV–Visible, Fourier Transform Infrared (FTIR) and Fourier Transform Ion Cyclotron Resonance Mass Spectrometry (FT-ICR MS). Structural investigation using X-ray crystallography has been done on the bacterial enzymes with diffraction data obtained. All of the enzymes discovered from the study will be made available for industrial breakdown of lignin for the production of useful products. All of the completed work together with recent efforts at engineering the enzymes of interest for further investigation will be discussed in this presentation. http://dx.doi.org/10.1016/j.nbt.2016.06.1421
P34-19 Looking for rules of protein aggregation Giulia Tedeschi 1,∗ , Stefania Brocca 2 , Antonino Natalello 2 , Marina Lotti 2 1 2
University of Milano Bicocca, Italy University of Milano – Bicocca, Italy
Fatai Bello ∗ , Louise Horsfall
Protein aggregation is an issue of clinical and biotechnical relevance. It is involved in a wide variety of diseases and is a hurdle in the production of recombinant proteins in bacterial cells. Understanding determinants of aggregation might contribute to design new strategies to prevent or modulate its occurrence. Knowledge in this field might be exploited for biotechnological purposes such as the design of in/solubility tags, valuable to recover a target protein by in-vitro aggregation or, vice versa, to prevent its undesired aggregation. Our study employs Green Fluorescent Protein (GFP) and the intrinsically disordered N-terminal moiety of a viral phosphoprotein (PNT). GFP is a well-known, structured protein that can be induced to aggregate and whose fluorescence easily reports its functional status. Intrinsically disordered proteins have extended, flexible and highly-dynamic conformations, consistent with high frequency of polar/charged residues in their primary sequence. Starting from the wild-type PNT (pI ∼ 4.9) we designed variants where the contents of charged amino acids were increased to give an acidic (pI ∼ 3.4) and a basic (pI ∼ 9.9) variant of PNT. We produced as recombinant proteins the three PNT charge variants, GFP and their fusions. An array of biophysical and biochemical techniques indicates that charge mutants of PNT per se strongly react to pH and are insoluble at their own pI. Also when fused to GFP, PNTs triggers pH-dependent aggregation. Paucity of charges emerges as a leading force of aggregation overwhelming the entropic effects of conformational flexibility on solubility.
University of Edinburgh, United Kingdom
http://dx.doi.org/10.1016/j.nbt.2016.06.1422
http://dx.doi.org/10.1016/j.nbt.2016.06.1420
P34-18 Fungal and bacterial lignin degraders: Purification, characterisation and structural investigation
Lignocellulosic biomass is the most abundant renewable source of raw materials for the production of biofuels and biomaterials. Lignin, an aromatic macromolecule is one of three major component compounds of lignocellulosic biomass. Bacteria and fungi utilise a consortium of lignin degrading enzymes to break down