Molecular characterization and secreted production of basidiomycetous cell-bound β-glycosidases applicable to production of galactooligosaccharides

Abstract Cell-bound β-glycosidases of basidiomycetous yeasts show promise as biocatalysts in galactooligosaccharide (GOS) production. Using degenerated primers designed from Hamamotoa singularis (Hs) bglA gene, we newly identified three genes that encode cell-bound β-glycosidase from Sirobasidium magnum (Sm), Rhodotorula minuta (Rm), and Sterigmatomyces elviae (Se). These three genes, also named bglA, encoded family 1 glycosyl hydrolases with molecular masses of 67‒77 kDa. The BglA enzymes were approximately 44% identical to the Hs-BglA enzyme and possessed a unique domain at the N-terminus comprising 110 or 210 amino acids. The Sm-, Rm-, and Se-BglA enzymes as well as the Hs-BglA enzyme were successfully produced by recombinant Aspergillus oryzae, and all enzymes were entirely secreted to the supernatants. Furthermore, addition of some nonionic detergents (e.g. 0.4% [v/v] Triton-X) increased the production, especially of the Hs- or Se-BglA enzyme. Out of the BglA enzymes, the Se-BglA enzyme showed remarkable thermostability (∼70°C). Additionally, the Sm- and Se-BglA enzymes had better GOS yields, so there was less residual lactose than in others. Accordingly, the basidiomycetous BglA enzymes produced by recombinant A. oryzae would be applicable to GOS production, and the Se-BglA enzyme appeared to be the most promising enzyme for industrial uses.

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Post-proline endopeptidase, further characterization of the enzyme from pig kidneys

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19841846 ◽

1984 ◽

Vol 49 (8) ◽

pp. 1846-1853 ◽

Cited By ~ 4

Author(s):

Karel Hauzer ◽

Tomislav Barth ◽

Linda Servítová ◽

Karel Jošt

Keyword(s):

Amino Acids ◽

Aspartic Acid ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Relative Molecular Mass ◽

Enzyme Molecule ◽

Thiol Compounds ◽

Modified Method ◽

N Terminus

A post-proline endopeptidase (EC 3.4.21.26) was isolated from pig kidneys using a modified method described earlier. The enzyme was further purified by ion exchange chromatography on DEAE-Sephacel. The final product contained about 95% of post-proline endopeptidase. The enzyme molecule consisted of one peptide chain with a relative molecular mass of 65 600 to 70 000, containing a large proportion of acidic and alifatic amino acids (glutamic acid, aspartic acid and leucine) and the N-terminus was formed by aspartic acid or asparagine. In order to prevent losses of enzyme activity, thiol compounds has to be added.

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181 Effects of Aspergillus oryzae prebiotic on energy and nutrient digestibility of growing pigs

Journal of Animal Science ◽

10.1093/jas/skaa054.143 ◽

2020 ◽

Vol 98 (Supplement_3) ◽

pp. 80-80

Author(s):

Jinlong Zhu ◽

Gerald C Shurson ◽

Lynsey Whitacre ◽

Ignacio R Ipharraguerre ◽

Pedro E Urriola

Keyword(s):

Amino Acids ◽

Aspergillus Oryzae ◽

Nutrient Content ◽

Nutrient Digestibility ◽

Growing Pigs ◽

Adaptation Period ◽

Ileal Digestibility ◽

High Fiber ◽

Wheat Middlings ◽

Corn Distillers

Abstract The objective of this study was to determine the effects of an Aspergillus oryzae prebiotic (AOP, Amaferm®) on nutrient digestibility in growing pigs fed high fiber diets. Eighteen growing barrows (initial BW = 50.60 ± 4.90 kg) were surgically equipped with a T-cannula at the distal ileum. Three diets were formulated by including 29.65% corn-distillers dried grains with solubles (DDGS), 36.65% rice bran (RB) or 24.59% wheat middlings (WM) in corn and soybean meal-based diets to meet nutrient requirements for 50 to 75 kg growing pigs. Three additional diets were formulated by supplementing 0.05% AOP at the expense of corn in DDGS (DDGS + AOP), RB (RB + AOP), and wheat middlings (WM + AOP) diets. Pigs were allotted randomly to a triplicated 6 × 2 Youden square design with 6 diets and 2 successive periods. Feces and ileal digesta were collected for 2 d after a 21 d adaptation period, and nutrient content was analyzed to calculate apparent total tract digestibility (ATTD) and apparent ileal digestibility (AID). Standardized ileal digestibility (SID) of amino acids was calculated by correcting AID with basal endogenous amino acid losses determined from the same set of pigs. Supplementation of 0.05% AOP increased (P < 0.05) ATTD of DM, GE, CP, NDF, and ash in DDGS, RB, and WM diets. Diet DE was 35 kcal/kg greater (P < 0.05) in pigs fed AOP supplemented diets compared with those fed diets without AOP. Pigs fed DDGS+AOP diet had greater (P < 0.05) AID of ether extract compared to those fed DDGS diet. However, supplementation of AOP did not (P > 0.05) affect AID of GE, DM, CP, NDF, ash or SID of amino acids. In conclusion, supplementation of AOP in high fiber diets containing DDGS, RB, or WM increased total tract energy value and nutrient digestibility.

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Generation of Antibodies against Foot-and-Mouth-Disease Virus Capsid Protein VP4 Using Hepatitis B Core VLPs as a Scaffold

Life ◽

10.3390/life11040338 ◽

2021 ◽

Vol 11 (4) ◽

pp. 338

Author(s):

Jessica Swanson ◽

Rennos Fragkoudis ◽

Philippa C. Hawes ◽

Joseph Newman ◽

Alison Burman ◽

...

Keyword(s):

Amino Acids ◽

Hepatitis B ◽

Capsid Protein ◽

Disease Virus ◽

Foot And Mouth Disease ◽

Mouth Disease ◽

Specific Antibodies ◽

Mouth Disease Virus ◽

N Terminus ◽

Foot And Mouth

The picornavirus foot-and-mouth disease virus (FMDV) is the causative agent of the economically important disease of livestock, foot-and-mouth disease (FMD). VP4 is a highly conserved capsid protein, which is important during virus entry. Previous published work has shown that antibodies targeting the N-terminus of VP4 of the picornavirus human rhinovirus are broadly neutralising. In addition, previous studies showed that immunisation with the N-terminal 20 amino acids of enterovirus A71 VP4 displayed on the hepatitis B core (HBc) virus-like particles (VLP) can induce cross-genotype neutralisation. To investigate if a similar neutralising response against FMDV VP4 could be generated, HBc VLPs displaying the N-terminus of FMDV VP4 were designed. The N-terminal 15 amino acids of FMDV VP4 was inserted into the major immunodominant region. HBc VLPs were also decorated with peptides of the N-terminus of FMDV VP4 attached using a HBc-spike binding tag. Both types of VLPs were used to immunise mice and the resulting serum was investigated for VP4-specific antibodies. The VLP with VP4 inserted into the spike, induced VP4-specific antibodies, however the VLPs with peptides attached to the spikes did not. The VP4-specific antibodies could recognise native FMDV, but virus neutralisation was not demonstrated. This work shows that the HBc VLP presents a useful tool for the presentation of FMDV capsid epitopes.

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Optimum pH and ionic strength for the assay of cytochrome c oxidase from pea cotyledon mitochondria

Canadian Journal of Biochemistry ◽

10.1139/o77-165 ◽

1977 ◽

Vol 55 (10) ◽

pp. 1114-1117 ◽

Cited By ~ 9

Author(s):

Gerrit H. Bomhoff ◽

Mary Spencer

Keyword(s):

Ionic Strength ◽

Cytochrome C ◽

Cytochrome C Oxidase ◽

Ferrocytochrome C ◽

Optimum Ph ◽

Triton X ◽

Assay Conditions ◽

Nonionic Detergents

Cytochrome c oxidase (EC 1.9.3.1) has been solubilized by use of the nonionic detergents Triton X-114 and Triton X-100, from pea cotyledon mitochondria. Optimum assay conditions were determined for the oxidation of ferrocytochrome c in air. The results indicate that the plant cytochrome c oxidase resembles mammalian preparations in its sensitivity towards ionic strength and pH of the assay buffer.

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The efficiency of various detergents for extraction and stabilization of acetylcholinesterase from bovine erythrocytes

Biochemistry and Cell Biology ◽

10.1139/o87-002 ◽

1987 ◽

Vol 65 (1) ◽

pp. 8-18 ◽

Cited By ~ 3

Author(s):

Rex K. M. Wong ◽

Christine P. Nichol ◽

M. Chandra Sekar ◽

Basil D. Roufogalis

Keyword(s):

Chain Length ◽

Membrane Lipids ◽

Acetylcholinesterase Activity ◽

Exchange Chromatography ◽

Erythrocyte Membranes ◽

Tween 20 ◽

Bovine Erythrocyte ◽

Critical Micelle Concentrations ◽

Triton X ◽

Nonionic Detergents

The efficiency of several nonionic detergents and a homologous series of zwitterionic detergents for the extraction of acetylcholinesterase (EC 3.1.1.7) from bovine erythrocyte membranes was examined. Of the nonionic detergents examined, the polyoxyethylene-based Tweens were the least effective solubilizing agents. Within this series, increasing the length of the saturated fatty acid chain progressively decreased the efficiency of enzyme recovery, while unsaturation in the side chain reversed this trend. In the Lubrol detergents, where the chain length of the alcohol group is variable, an increase in the length of the polyoxyethylene glycol group decreased the recovery of acetylcholinesterase in the solubilized state, without affecting the efficiency of extraction of total erythrocyte protein. As with the other nonionic detergents examined, Triton X-100 and octy1 β-D-glucoside were maximally effective in solubilizing acetylcholinesterase activity at concentrations greater than their respective critical micelle concentrations. In the sulfobetaine (N-alkyldimethylaminopropane sulphonate) zwitterionic detergent series, the longer alkyl chain zwittergents Z 316 and Z 314 were more efficient than the shorter chain length members of the series (Z 310 and Z 312). In contrast to the higher chain length compounds, short chain analogs were maximally effective at or below their critical micelle concentrations. After purification by ion-exchange chromatography and affinity chromatography, the enzyme extracted with the various detergents gave sedimentation coefficients between 6.8S and 7.6S, consistent with a dimeric structure. Acetylcholinesterase could also be efficiently released by 0.2 mM EDTA or 0.5 M NaCl from bovine erythrocyte membranes previously depleted of 70–80% of the membrane lipids by butanol. Nonlinear Arrhenius plots of enzyme activity were found whether acetylcholinesterase was solubilized with Tween 20, Lubrol PX, or Triton X-100. The present work confirms that bovine erythrocyte acetylcholinesterase requires detergents to solubilize it from membranes and that its activity depends on the structure of the amphiphiles used to solubilize the enzyme.

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Role of a tryptophan anchor in human topoisomerase I structure, function and inhibition

Biochemical Journal ◽

10.1042/bj20071436 ◽

2008 ◽

Vol 411 (3) ◽

pp. 523-530 ◽

Cited By ~ 12

Author(s):

Gary S. Laco ◽

Yves Pommier

Keyword(s):

Amino Acids ◽

Topoisomerase I ◽

Electrostatic Interactions ◽

Functional Domain ◽

Site Mutation ◽

Supercoiled Dna ◽

Terminal Domain ◽

Tryptophan Residues ◽

N Terminus

Human Top1 (topoisomerase I) relaxes supercoiled DNA during cell division and transcription. Top1 is composed of 765 amino acids and contains an unstructured N-terminal domain of 200 amino acids, and a structured functional domain of 565 amino acids that binds and relaxes supercoiled DNA. In the present study we examined the region spanning the junction of the N-terminal domain and functional domain (junction region). Analysis of several published Top1 structures revealed that three tryptophan residues formed a network of aromatic stacking interactions and electrostatic interactions that anchored the N-terminus of the functional domain to sub-domains containing the nose cone and active site. Mutation of the three tryptophan residues (Trp203/Trp205/Trp206) to an alanine residue, either individually or together, in silico revealed that the individual tryptophan residue's contribution to the tryptophan ‘anchor’ was additive. When the three tryptophan residues were mutated to alanine in vitro, the resulting mutant Top1 differed from wild-type Top1 in that it lacked processivity, exhibited resistance to camptothecin and was inactivated by urea. The results indicated that the tryptophan anchor stabilized the N-terminus of the functional domain and prevented the loss of Top1 structure and function.

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The hyaluronate synthase from a eukaryotic cell line

Biochemical Journal ◽

10.1042/bj2900791 ◽

1993 ◽

Vol 290 (3) ◽

pp. 791-795 ◽

Cited By ~ 25

Author(s):

L Klewes ◽

E A Turley ◽

P Prehm

Keyword(s):

Monoclonal Antibodies ◽

Phase Separation ◽

Affinity Chromatography ◽

Cell Line ◽

Aqueous Phase ◽

Plasma Membranes ◽

Eukaryotic Cell ◽

Molecular Masses ◽

Triton X ◽

Hyaluronate Synthase

The hyaluronate synthase complex was identified in plasma membranes from B6 cells. It contained two subunits of molecular masses 52 kDa and 60 kDa which bound the precursor UDP-GlcA in digitonin solution and partitioned into the aqueous phase, together with nascent hyaluronate upon Triton X-114 phase separation. The 52 kDa protein cross-reacted with poly- and monoclonal antibodies raised against the streptococcal hyaluronate synthase and the 60 kDa protein was recognized by monoclonal antibodies raised against a hyaluronate receptor. The 52 kDa protein was purified to homogeneity by affinity chromatography with monoclonal anti-hyaluronate synthase.

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A Nonviral Peptide Can Replace the Entire N Terminus of Zucchini Yellow Mosaic Potyvirus Coat Protein and Permits Viral Systemic Infection

Journal of Virology ◽

10.1128/jvi.75.14.6329-6336.2001 ◽

2001 ◽

Vol 75 (14) ◽

pp. 6329-6336 ◽

Cited By ~ 26

Author(s):

T. Arazi ◽

Y. M. Shiboleth ◽

A. Gal-On

Keyword(s):

Amino Acids ◽

Coat Protein ◽

Foot And Mouth Disease ◽

Systemic Infection ◽

Deletion Mutants ◽

Amino Acid Residues ◽

Amino Terminal ◽

Immunogenic Epitope ◽

Mouth Disease Virus ◽

N Terminus

ABSTRACT Systematic deletion and peptide tagging of the amino-terminal domain (NT, ∼43 amino acids) of an attenuated zucchini yellow mosaic potyvirus (ZYMV-AGII) coat protein (CP) were used to elucidate its role in viral systemic infection. Deletion mutants truncated by 8, 13, and 33 amino acid residues from the CP-NT 5′ end were systemically infectious and produced symptoms similar to those of the AGII virus. Tagging these deletion mutants with either human c-Myc (Myc) or hexahistidine peptides maintained viral infectivity. Similarly, addition of these peptides to the intact AGII CP-NT did not affect viral life cycle. To determine which parts, if any, of the CP-NT are essential for viral systemic infection, a series of Myc-tagged mutants with 8 to 43 amino acids removed from the CP-NT were constructed. All Myc-tagged CP-NT deletion mutants, including those from which virtually all the viral CP-NT had been eliminated, were able to encapsidate and cause systemic infection. Furthermore, chimeric viruses with deletions of up to 33 amino acids from CP-NT produced symptoms indistinguishable from those caused by the parental AGII virus. In contrast to CP-NT Myc fusion, addition of the foot-and-mouth disease virus (FMDV) immunogenic epitope to AGII CP-NT did not permit systemic infection. However, fusion of the Myc peptide to the N terminus of the FMDV peptide restored the capability of the virus to spread systemically. We have demonstrated that all CP-NT fused peptides were exposed on the virion surface, masking natural CP immunogenic determinants. Our findings demonstrate that CP-NT is not essential for ZYMV spread and that it can be replaced by an appropriate foreign peptide while maintaining systemic infectivity.

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