scholarly journals New Approach of Highly Efficient Fermentation Process for Bioethanol using Xylose as Agriculture Residues

2017 ◽  
Vol 26 (1) ◽  
pp. 1-12
Author(s):  
Abu Saleh Ahmed ◽  
Seiya Watanabe ◽  
Sinin Hamdan ◽  
Tsutomu Kodaki ◽  
Keisuke Makino
Keyword(s):  
Enzyme Activity ◽  
Agricultural Waste ◽  
Xylose Reductase ◽  
Pichia Stipitis ◽  
Ethanol Conversion ◽  
Waste Biomass ◽  
Wild Type ◽  
Coenzyme Specificity ◽  
Recombinant Yeasts

Agricultural waste biomass has already been transferred to bioethanol and used as energy related products, although many issues such as efficiency and productivity still exist to be overcome. In this study, the protein engineering was applied to generate enzymes with completely reversed coenzyme specificity and developed recombinant yeasts containing those engineered enzymes for construction of an efficient biomass-ethanol conversion system. Recombinant yeasts were constructed with the genes encoding a wild type xylose reductase (XR) and the protein engineered xylitol dehydrogenase (XDH) (with NADP) of Pichia stipitis.  These recombinant yeasts were characterized based on the enzyme activity and fermentation ability of xylose to ethanol. The protein engineered enzymes were expressed significantly in Saccharomyces cerevisiae as judged by the enzyme activity in vitro. Ethanol fermentation was measured in batch culture under anaerobic conditions. The significant enhancement was found in Y-ARS strain, in which NADP+-dependent XDH was expressed; 85% decrease of unfavorable xylitol excretion with 26% increased ethanol production, when compared with the reference strain expressing the wild–type XDH.      

scholarly journals New Approach of Highly Efficient Fermentation Process for Bio ethanol using Xylose as Agricultur e Residues

2017 ◽  
Vol 26 (1) ◽  
Author(s):  
Abu Saleh Ahmed ◽  
Seiya Watanabe ◽  
Sinin Hamdan ◽  
Tsutomu Kodaki ◽  
Keisuke Makino
Keyword(s):  
Enzyme Activity ◽  
Agricultural Waste ◽  
Xylose Reductase ◽  
Ethanol Conversion ◽  
Wild Type ◽  
Coenzyme Specificity ◽  
Genes Encoding ◽  
Efficient Fermentation ◽  
Recombinant Yeasts

Agricultural waste biomasshas already been transferred to bioethanol and used as energy related products, although many issues such as efficiency and productivity still exist to be overcome. In this study, the protein engineering was applied to generate enzymes with completely reversed coenzyme specificity and developed recombinant yeasts containing those engineered enzymes for construction of an efficient biomass-ethanol conversion system. Recombinant yeasts were constructed with the genes encoding a wild type xylose reductase (XR)and the protein engineered xylitol dehydrogenase (XDH)(with NADP) of Pichiastipitis. These recombinant yeasts were characterized based on the enzyme activity and fermentation ability of xylose to ethanol. The protein engineered enzymes were expressed significantly in Saccharomycescerevisiaeas judged by the enzyme activity in vitro. Ethanol fermentation was measured in batch culture under anaerobic conditions. The significant enhancement was found in Y-ARSstrain, in which NADP+-dependentXDH was expressed; 85% decrease of unfavorable xylitol excretion with 26% increased ethanol production, when compared with the reference strain expressing the wild–type XDH. 

scholarly journals Conversion of Waste Agriculture Biomass to Bioethanol by Recombinant Saccharomyces cerevisiae

2010 ◽  
Vol 2 (2) ◽  
pp. 351-361
Author(s):  
A. A. Saleh ◽  
S. Hamdan ◽  
N. Annaluru ◽  
S. Watanabe ◽  
M. R. Rahman ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Enzyme Activity ◽  
Protein Engineering ◽  
Agricultural Waste ◽  
Xylitol Dehydrogenase ◽  
Ethanol Conversion ◽  
Waste Biomass ◽  
Wild Type ◽  
Coenzyme Specificity ◽  
Recombinant Yeasts

Agricultural waste biomass has already been transferred to bioethanol and used as energy related products, although many issues such as efficiency and productivity still to be overcome. In this study, the protein engineering was applied to generate enzymes with completely reversed coenzyme specificity and developed recombinant yeasts containing those engineered enzymes for construction of an efficient biomass-ethanol conversion system. Recombinant yeasts were constructed with the genes encoding a wild type xylose reductase (XR) and the protein engineered xylitol dehydrogenase (XDH) (with NADP) of Pichia stipitis.  These recombinant yeasts were characterized based on the enzyme activity and fermentation ability of xylose to ethanol. The protein engineered enzymes were expressed significantly in Saccharomyces cerevisiae as judged by the enzyme activity in vitro. Ethanol fermentation was measured in batch culture under anaerobic conditions. The significant enhancement was found in Y-ARS strain, in which NADP+-dependent XDH was expressed; 85% decrease of unfavorable xylitol excretion with 26% increased ethanol production, when compared with the reference strain expressing the wild-type XDH.  Keywords: Agricultural waste biomass; Protein engineering; Xylitol dehydrogenase; Xylose-fermentation; Eethanol production. © 2010 JSR Publications. ISSN: 2070-0237 (Print); 2070-0245 (Online). All rights reserved. DOI: 10.3329/jsr.v2i2.2882               J. Sci. Res. 2 (2), 351-361 (2010) 

scholarly journals The effects of the site-directed removal of N-glycosylation sites from β-1,4-N-acetylgalactosaminyltransferase on its function

1995 ◽  
Vol 312 (1) ◽  
pp. 273-280 ◽  
Author(s):  
M Haraguchi ◽  
S Yamashiro ◽  
K Furukawa ◽  
K Takamiya ◽  
H Shiku ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Intracellular Localization ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Kinetics Analysis ◽  
Glycosylation Sites ◽  
In The Wild ◽  
Polymerase Chain ◽  
The Stability

The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T; EC 2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction (PCR)-mediated site-directed mutagenesis. Using transcription/translation in vitro, we confirmed that all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal beta 1-->4Glc-Cer (GM2) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of Km among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.

scholarly journals Genetically Engineered SaccharomycesYeast Capable of Effective Cofermentation of Glucose and Xylose

1998 ◽  
Vol 64 (5) ◽  
pp. 1852-1859 ◽  
Author(s):  
Nancy W. Y. Ho ◽  
Zhengdao Chen ◽  
Adam P. Brainard
Keyword(s):  
Xylose Reductase ◽  
Pichia Stipitis ◽  
Genetically Engineered ◽  
Cellulosic Biomass ◽  
Aerobic Growth ◽  
Metabolizing Enzymes ◽  
E Coli ◽  
Dehydrogenase Gene ◽  
Reductase Gene ◽  
Recombinant Yeasts

ABSTRACT Xylose is one of the major fermentable sugars present in cellulosic biomass, second only to glucose. However, Saccharomycesspp., the best sugar-fermenting microorganisms, are not able to metabolize xylose. We developed recombinant plasmids that can transformSaccharomyces spp. into xylose-fermenting yeasts. These plasmids, designated pLNH31, -32, -33, and -34, are 2μm-based high-copy-number yeast-E. coli shuttle plasmids. In addition to the geneticin resistance and ampicillin resistance genes that serve as dominant selectable markers, these plasmids also contain three xylose-metabolizing genes, a xylose reductase gene, a xylitol dehydrogenase gene (both from Pichia stipitis), and a xylulokinase gene (from Saccharomyces cerevisiae). These xylose-metabolizing genes were also fused to signals controlling gene expression from S. cerevisiae glycolytic genes. Transformation of Saccharomyces sp. strain 1400 with each of these plasmids resulted in the conversion of strain 1400 from a non-xylose-metabolizing yeast to a xylose-metabolizing yeast that can effectively ferment xylose to ethanol and also effectively utilizes xylose for aerobic growth. Furthermore, the resulting recombinant yeasts also have additional extraordinary properties. For example, the synthesis of the xylose-metabolizing enzymes directed by the cloned genes in these recombinant yeasts does not require the presence of xylose for induction, nor is the synthesis repressed by the presence of glucose in the medium. These properties make the recombinant yeasts able to efficiently ferment xylose to ethanol and also able to efficiently coferment glucose and xylose present in the same medium to ethanol simultaneously.

scholarly journals Replacement of the catalytic nucleophile cysteine-296 by serine in class II polyhydroxyalkanoate synthase from Pseudomonas aeruginosa-mediated synthesis of a new polyester: identification of catalytic residues

2003 ◽  
Vol 374 (2) ◽  
pp. 413-421 ◽  
Author(s):  
Amro A. AMARA ◽  
Bernd H. A. REHM
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Enzyme Activity ◽  
Chain Length ◽  
Class Ii ◽  
Base Catalyst ◽  
Wild Type ◽  
General Base ◽  
Medium Chain Length

The class II PHA (polyhydroxyalkanoate) synthases [PHAMCL synthases (medium-chain-length PHA synthases)] are mainly found in pseudomonads and catalyse synthesis of PHAMCLs using CoA thioesters of medium-chain-length 3-hydroxyfatty acids (C6–C14) as a substrate. Only recently PHAMCL synthases from Pseudomonas oleovorans and Pseudomonas aeruginosa were purified and in vitro activity was achieved. A threading model of the P. aeruginosa PHAMCL synthase PhaC1 was developed based on the homology to the epoxide hydrolase (1ek1) from mouse which belongs to the α/β-hydrolase superfamily. The putative catalytic residues Cys-296, Asp-452, His-453 and His-480 were replaced by site-specific mutagenesis. In contrast to class I and III PHA synthases, the replacement of His-480, which aligns with the conserved base catalyst of the α/β-hydrolases, with Gln did not affect in vivo enzyme activity and only slightly in vitro enzyme activity. The second conserved histidine His-453 was then replaced by Gln, and the modified enzyme showed only 24% of wild-type in vivo activity, which indicated that His-453 might functionally replace His-480 in class II PHA synthases. Replacement of the postulated catalytic nucleophile Cys-296 by Ser only reduced in vivo enzyme activity to 30% of wild-type enzyme activity and drastically changed substrate specificity. Moreover, the C296S mutation turned the enzyme sensitive towards PMSF inhibition. The replacement of Asp-452 by Asn, which is supposed to be required as general base catalyst for elongation reaction, did abolish enzyme activity as was found for the respective amino acid residue of class I and III enzymes. In the threading model residues Cys-296, Asp-452, His-453 and His-480 reside in the core structure with the putative catalytic nucleophile Cys-296 localized at the highly conserved γ-turns of the α/β-hydrolases. Inhibitor studies indicated that catalytic histidines reside in the active site. The conserved residue Trp-398 was replaced by Phe and Ala, respectively, which caused inactivation of the enzyme indicating an essential role of this residue. In the threading model this residue was found to be surface-exposed. No evidence for post-translational modification by 4-phosphopantetheine was obtained. Overall, these data suggested that in class II PHA synthases the conserved histidine which was found as general base catalyst in the catalytic triad of enzymes related to the α/β-hydrolase superfamily, was functionally replaced by His-453 which is conserved among all PHA synthases.

scholarly journals Knockout of Arabidopsis Serotonin N-Acetyltransferase-2 Reduces Melatonin Levels and Delays Flowering

Biomolecules ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 712 ◽  
Author(s):  
Hyoung Yool Lee ◽  
Kyungjin Lee ◽  
Kyoungwhan Back
Keyword(s):  
Arabidopsis Thaliana ◽  
Enzyme Activity ◽  
Amino Acid ◽  
Plant Growth ◽  
Wild Type ◽  
Knockout Mutant ◽  
Flowering Locus T ◽  
Amino Acid Homology ◽  
Delayed Flowering

Melatonin plays roles in both plant growth and defense. Serotonin N-acetyltransferase (SNAT) catalyzes formation of N-acetylserotonin (NAS) from serotonin. Plants contain two SNAT isogenes, which exhibit low-level amino acid homology. We studied the Arabidopsis thaliana SNAT2 (AtSNAT2) gene; we prepared recombinant SNAT2 protein and characterized a snat2 knockout mutant. The SNAT2 protein exhibited 27% amino acid homology with SNAT1; the Km was 232 μM and the Vmax was 2160 pmol/min/mg protein. Melatonin inhibited SNAT enzyme activity in vitro. SNAT2 mRNA was abundantly expressed in flowers; the melatonin content of flowers of the snat2 mutant was significantly less than that of wild-type flowers. The mutant exhibited delayed flowering and reductions in leaf area and biomass compared to the wild type. Delayed flowering was attributable to reductions in the expression levels of the gibberellin biosynthetic genes ent-kaurene synthase (KS) and FLOWERING LOCUS T (FT).

scholarly journals The coenzyme specificity of Candida tenuis xylose reductase (AKR2B5) explored by site-directed mutagenesis and X-ray crystallography

2004 ◽  
Vol 385 (1) ◽  
pp. 75-83 ◽  
Author(s):  
Barbara PETSCHACHER ◽  
Stefan LEITGEB ◽  
Kathryn L. KAVANAGH ◽  
David K. WILSON ◽  
Bernd NIDETZKY
Keyword(s):  
Double Mutant ◽  
Profile Analysis ◽  
Xylose Reductase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Binary Complex ◽  
Wild Type ◽  
Coenzyme Specificity ◽  
X Ray ◽  
X Ray Crystallography

CtXR (xylose reductase from the yeast Candida tenuis; AKR2B5) can utilize NADPH or NADH as co-substrate for the reduction of D-xylose into xylitol, NADPH being preferred approx. 33-fold. X-ray structures of CtXR bound to NADP+ and NAD+ have revealed two different protein conformations capable of accommodating the presence or absence of the coenzyme 2′-phosphate group. Here we have used site-directed mutagenesis to replace interactions specific to the enzyme–NADP+ complex with the aim of engineering the co-substrate-dependent conformational switch towards improved NADH selectivity. Purified single-site mutants K274R (Lys274→Arg), K274M, K274G, S275A, N276D, R280H and the double mutant K274R–N276D were characterized by steady-state kinetic analysis of enzymic D-xylose reductions with NADH and NADPH at 25 °C (pH 7.0). The results reveal between 2- and 193-fold increases in NADH versus NADPH selectivity in the mutants, compared with the wild-type, with only modest alterations of the original NADH-linked xylose specificity and catalytic-centre activity. Catalytic reaction profile analysis demonstrated that all mutations produced parallel effects of similar magnitude on ground-state binding of coenzyme and transition state stabilization. The crystal structure of the double mutant showing the best improvement of coenzyme selectivity versus wild-type and exhibiting a 5-fold preference for NADH over NADPH was determined in a binary complex with NAD+ at 2.2 Å resolution.

scholarly journals Biological and Biochemical Functions of RNA in the Tetrahymena Telomerase Holoenzyme

2005 ◽  
Vol 25 (11) ◽  
pp. 4442-4454 ◽  
Author(s):  
Doreen D. Cunningham ◽  
Kathleen Collins
Keyword(s):  
Enzyme Activity ◽  
Telomere Length ◽  
Affinity Purification ◽  
In Vitro Activity ◽  
Wild Type ◽  
Cell Extracts ◽  
Simple Sequence ◽  
Telomere Length Homeostasis

ABSTRACT Telomerase extends chromosome ends by the synthesis of tandem simple-sequence repeats. Studies of minimal recombinant telomerase ribonucleoprotein (RNP) reconstituted in vitro have revealed sequences within the telomerase RNA subunit (TER) that are required to establish its internal template and other unique features of enzyme activity. Here we test the significance of these motifs following TER assembly into telomerase holoenzyme in vivo. We established a method for stable expression of epitope-tagged TER and TER variants in place of wild-type Tetrahymena TER. We found that sequence substitutions in nontemplate regions of TER altered telomere length maintenance in vivo, with an increase or decrease in the set point for telomere length homeostasis. We also characterized the in vitro activity of the telomerase holoenzymes reconstituted with TER variants, following RNA-based RNP affinity purification from cell extracts. We found that nontemplate sequence substitutions imposed specific defects in the fidelity and processivity of template use. These findings demonstrate nontemplate functions of TER that are critical for the telomerase holoenzyme catalytic cycle and for proper telomere length maintenance in vivo.

Sign in / Sign up

Export Citation Format

Share Document