scholarly journals Molecular Identification of Keratinase DgokerA from Deinococcus gobiensis for Feather Degradation

2022 ◽  
Vol 12 (1) ◽  
pp. 464
Author(s):  
Yong Meng ◽  
Yin Tang ◽  
Xiuhong Zhang ◽  
Jin Wang ◽  
Zhengfu Zhou
Keyword(s):  
Enzyme Activity ◽  
Disulfide Bonds ◽  
Agricultural Waste ◽  
Extreme Environments ◽  
Industrial Applications ◽  
Site Directed Mutagenesis ◽  
Gobi Desert ◽  
Structural Protein ◽  
Feather Degradation

Keratin is a tough fibrous structural protein that is difficult to digest with pepsin and trypsin because of the presence of a large number of disulfide bonds. Keratin is widely found in agricultural waste. In recent years, especially, the development of the poultry industry has resulted in a large accumulation of feather keratin resources, which seriously pollute the environment. Keratinase can specifically attack disulfide bridges in keratin, converting them from complex to simplified forms. The keratinase thermal stability has drawn attention to various biotechnological industries. It is significant to identify keratinases and improve their thermostability from microorganism in extreme environments. In this study, the keratinases DgoKerA was identified in Deinococcus gobiensis I-0 from the Gobi desert. The amino acid sequence analysis revealed that DgoKerA was 58.68% identical to the keratinase MtaKerA from M. thermophila WR-220 and 40.94% identical to the classical BliKerA sequence from B. licheniformis PWD-1. In vitro enzyme activity analysis showed that DgoKerA exhibited an optimum temperature of 60 °C, an optimum pH of 7 and a specific enzyme activity of 51147 U/mg. DgoKerA can degrade intact feathers at 60 °C and has good potential for industrial applications. The molecular modification of DgoKerA was also carried out using site-directed mutagenesis, in which the mutant A350S enzyme activity was increased by nearly 30%, and the results provide a theoretical basis for the development and optimization of keratinase applications.

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 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 Quantitative Disassembly and Reassembly of Human Papillomavirus Type 11 Viruslike Particles In Vitro

1998 ◽  
Vol 72 (1) ◽  
pp. 32-41 ◽  
Author(s):  
Michael P. McCarthy ◽  
Wendy I. White ◽  
Frances Palmer-Hill ◽  
Scott Koenig ◽  
Joann A. Suzich
Keyword(s):  
Human Papillomavirus ◽  
Ionic Strength ◽  
Self Assembly ◽  
Disulfide Bonds ◽  
Structural Protein ◽  
Homogeneous Population ◽  
Carbonate Buffer ◽  
Viruslike Particles

ABSTRACT The human papillomavirus (HPV) capsid is primarily composed of a structural protein denoted L1, which forms both pentameric capsomeres and capsids composed of 72 capsomeres. The L1 protein alone is capable of self-assembly in vivo into capsidlike structures referred to as viruslike particles (VLPs). We have determined conditions for the quantitative disassembly of purified HPV-11 L1 VLPs to the level of capsomeres, demonstrating that disulfide bonds alone are essential to maintaining long-term HPV-11 L1 VLP structure at physiological ionic strength. The ionic strength of the disassembly reaction was also important, as increased NaCl concentrations inhibited disassembly. Conversely, chelation of cations had no effect on disassembly. Quantitative reassembly to a homogeneous population of 55-nm, 150S VLPs was reliably achieved by the re-formation of disulfide linkages following removal of reducing agent at near-neutral pH and moderate NaCl concentration. HPV-11 L1 VLPs could also be dissociated by treatment with carbonate buffer at pH 9.6, but VLPs could not be regenerated following carbonate treatment. When probed with conformationally sensitive and/or neutralizing monoclonal antibodies, both capsomeres generated by disulfide reduction of purified VLPs and reassembled VLPs formed from capsomeres upon removal of reducing agents exhibited epitopes found on the surface of authentic HPV-11 virions. Antisera raised against either purified VLP starting material or reassembled VLPs similarly neutralized infectious HPV-11 virions. The ability to disassemble and reassemble VLPs in vitro and in bulk allows basic features of capsid assembly to be studied and also opens the possibility of packaging selected exogenous compounds within the reassembled VLPs.

scholarly journals Cleavage of the Feline Calicivirus Capsid Precursor Is Mediated by a Virus-Encoded Proteinase

1998 ◽  
Vol 72 (4) ◽  
pp. 3051-3059 ◽  
Author(s):  
Stanislav V. Sosnovtsev ◽  
Svetlana A. Sosnovtseva ◽  
Kim Y. Green
Keyword(s):  
Cleavage Site ◽  
Site Directed Mutagenesis ◽  
In Vitro Translation ◽  
Feline Calicivirus ◽  
Cdna Clones ◽  
Bacterial Cells ◽  
Structural Protein ◽  
Critical Determinant ◽  
Capsid Precursor

ABSTRACT Feline calicivirus (FCV), a member of theCaliciviridae, produces its major structural protein as a precursor polyprotein from a subgenomic-sized mRNA. In this study, we show that the proteinase responsible for processing this precursor into the mature capsid protein is encoded by the viral genome at the 3′-terminal portion of open reading frame 1 (ORF1). Protein expression studies of either the entire or partial ORF1 indicate that the proteinase is active when expressed either in in vitro translation or in bacterial cells. Site-directed mutagenesis was used to characterize the proteinase Glu-Ala cleavage site in the capsid precursor, utilizing an in vitro cleavage assay in which mutant precursor proteins translated from cDNA clones were used as substrates fortrans cleavage by the proteinase. In general, amino acid substitutions in the P1 position (Glu) of the cleavage site were less well tolerated by the proteinase than those in the P1′ position (Ala). The precursor cleavage site mutations were introduced into an infectious cDNA clone of the FCV genome, and transfection of RNA derived from these clones into feline kidney cells showed that efficient cleavage of the capsid precursor by the virus-encoded proteinase is a critical determinant in the growth of the virus.

scholarly journals Biochemical insight into redox regulation of plastidial 3-phosphoglycerate dehydrogenase from Arabidopsis thaliana

2020 ◽  
Vol 295 (44) ◽  
pp. 14906-14915
Author(s):  
Keisuke Yoshida ◽  
Kinuka Ohtaka ◽  
Masami Yokota Hirai ◽  
Toru Hisabori
Keyword(s):  
Arabidopsis Thaliana ◽  
Enzyme Activity ◽  
Disulfide Bond ◽  
Protein Modification ◽  
Redox Regulation ◽  
Site Directed Mutagenesis ◽  
Reductive Activation ◽  
Wide Range ◽  
Phosphoglycerate Dehydrogenase

Thiol-based redox regulation is a post-translational protein modification for controlling enzyme activity by switching oxidation/reduction states of Cys residues. In plant cells, numerous proteins involved in a wide range of biological systems have been suggested as the target of redox regulation; however, our knowledge on this issue is still incomplete. Here we report that 3-phosphoglycerate dehydrogenase (PGDH) is a novel redox-regulated protein. PGDH catalyzes the first committed step of Ser biosynthetic pathway in plastids. Using an affinity chromatography-based method, we found that PGDH physically interacts with thioredoxin (Trx), a key factor of redox regulation. The in vitro studies using recombinant proteins from Arabidopsis thaliana showed that a specific PGDH isoform, PGDH1, forms the intramolecular disulfide bond under nonreducing conditions, which lowers PGDH enzyme activity. MS and site-directed mutagenesis analyses allowed us to identify the redox-active Cys pair that is mainly involved in disulfide bond formation in PGDH1; this Cys pair is uniquely found in land plant PGDH. Furthermore, we revealed that some plastidial Trx subtypes support the reductive activation of PGDH1. The present data show previously uncharacterized regulatory mechanisms of PGDH and expand our understanding of the Trx-mediated redox-regulatory network in plants.

scholarly journals Coupled Site-Directed Mutagenesis/Transgenesis Identifies Important Functional Domains of the Mouse Agouti Protein

Genetics ◽  
1996 ◽  
Vol 144 (1) ◽  
pp. 255-264 ◽  
Author(s):  
William L Perry ◽  
Takuro Nakamura ◽  
Deborah A Swing ◽  
Lisa Secrest ◽  
Bryn Eagleson ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Coat Color ◽  
Signal Sequence ◽  
Biological Activities ◽  
Normal Function ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Functional Domains ◽  
Basic Domain

Abstract The agouti locus encodes a novel paracrine signaling molecule containing a signal sequence, an N-linked glycosylation site, a central lysine-rich basic domain, and a C-terminal tail containing 10 cysteine (Cys) residues capable of forming five disulfide bonds. When overexpressed, agouti causes a number of pleiotropic effects including yellow coat and adult-onset obesity. Numerous studies suggest that agouti causes yellow coat color by antagonizing the binding of a-melanocyte-stimulating hormone (α-MSH) to the a-MSH-(melanocortin-1) receptor. With the goal of identifying functional domains of agouti important for its diverse biological activities, we have generated 14 agouti mutations by in vitro sitedirected mutagenesis and analyzed these mutations in transgenic mice for their effects on coat color and obesity. These studies demonstrate that the signal sequence, the N-linked glycosylation site, and the C-terminal Cys residues are important for full biological activity, while at least a portion of the lysine-rich basic domain is dispensable for normal function. They also show that the same functional domains of agouti important in coat color determination are important for inducing obesity, consistent with the hypothesis that agouti induces obesity by antagonizing melanocortin binding to other melanocortin receptors.

scholarly journals The C-terminal part of diacylglycerol kinase α lacking zinc fingers serves as a catalytic domain

1996 ◽  
Vol 318 (2) ◽  
pp. 583-590 ◽  
Author(s):  
Fumio SAKANE ◽  
Masahiro KAI ◽  
Ikuo WADA ◽  
Shin-ichi IMAI ◽  
Hideo KANOH
Keyword(s):  
Enzyme Activity ◽  
Binding Sites ◽  
Enzyme Activities ◽  
Catalytic Domain ◽  
Zinc Fingers ◽  
Diacylglycerol Kinase ◽  
Site Directed Mutagenesis ◽  
Atp Binding ◽  
Wild Type

All mammalian diacylglycerol kinase (DGK) isoenzymes so far cloned consist of four conserved regions, namely C1, C2 (tandem EF-hand structures), C3 (tandem cysteine-rich zinc finger sequences) and the C-terminal C4 domains. To determine the catalytic domain we expressed in COS-7 cells various truncation mutants of pig DGKα and assessed their enzyme activities. We found that the C4 domain lacking the whole N-terminal region including the zinc fingers possessed DGK activity that was dependent on the concentrations of diacylglycerol and ATP very similarly, as did the wild-type DGKα. Furthermore the DGK activity of the wild-type DGK and that expressed by the C4 domain were similarly activated by anionic amphiphiles such as phosphatidylserine, phosphatidylinositol and deoxycholate. It was also shown that a DGK mutant consisting of the zinc fingers and the C4 domain has enzymological properties very similar to those expressed by the C4 domain alone. We also confirmed that the intact DGKs α, β and γ expressed in COS-7 cells displayed no detectable phorbol ester binding. These results show that the C4 domain of DGK is the catalytic region that is responsible for the enzyme activities sensitive to different activators. We cannot exclude the possibility that the N-terminal portion including the zinc fingers can still interact with diacylglycerol and activators without affecting the enzyme activity measured in vitro. However, it is quite likely that the DGK zinc fingers do not serve as diacylglycerol-binding sites, in contrast with those present in other proteins such as protein kinases C and n-chimaerin. Site-directed mutagenesis of all six putative ATP binding sites (Lys248, Lys383, Lys395, Lys483, Lys492, and Lys554) did not significantly affect the enzyme activity. We therefore suggest that DGK does not contain a typical P-loop of ATP binding sites.

Crystal structures of plant inorganic pyrophosphatase, an enzyme with a moonlighting autoproteolytic activity

2019 ◽  
Vol 476 (16) ◽  
pp. 2297-2319 ◽  
Author(s):  
Marta Grzechowiak ◽  
Milosz Ruszkowski ◽  
Joanna Sliwiak ◽  
Kamil Szpotkowski ◽  
Michal Sikorski ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Size Exclusion ◽  
Inorganic Pyrophosphatase ◽  
Prolonged Storage ◽  
Mitochondrial Targeting ◽  
Cleavage Rate ◽  
Inorganic Pyrophosphate ◽  
Putative Signal Peptide ◽  
Targeting Peptide

Abstract Inorganic pyrophosphatases (PPases, EC 3.6.1.1), which hydrolyze inorganic pyrophosphate to phosphate in the presence of divalent metal cations, play a key role in maintaining phosphorus homeostasis in cells. DNA coding inorganic pyrophosphatases from Arabidopsis thaliana (AtPPA1) and Medicago truncatula (MtPPA1) were cloned into a bacterial expression vector and the proteins were produced in Escherichia coli cells and crystallized. In terms of their subunit fold, AtPPA1 and MtPPA1 are reminiscent of other members of Family I soluble pyrophosphatases from bacteria and yeast. Like their bacterial orthologs, both plant PPases form hexamers, as confirmed in solution by multi-angle light scattering and size-exclusion chromatography. This is in contrast with the fungal counterparts, which are dimeric. Unexpectedly, the crystallized AtPPA1 and MtPPA1 proteins lack ∼30 amino acid residues at their N-termini, as independently confirmed by chemical sequencing. In vitro, self-cleavage of the recombinant proteins is observed after prolonged storage or during crystallization. The cleaved fragment corresponds to a putative signal peptide of mitochondrial targeting, with a predicted cleavage site at Val31–Ala32. Site-directed mutagenesis shows that mutations of the key active site Asp residues dramatically reduce the cleavage rate, which suggests a moonlighting proteolytic activity. Moreover, the discovery of autoproteolytic cleavage of a mitochondrial targeting peptide would change our perception of this signaling process.

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