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  February 25, 2024
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Ureaka Information Enterprises of Canada

1440 Barberry Drive
Port Coquitlam
Canada
Toll free: 16049419022

Phone: 16049458408
Fax: 16049419022
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Description:

UREALYSIS OF STRAW, STOVER AND LEGUME HAULMS.

Introduction.

The identification of straw, stover and haulms as major crop residues is not new (T. A. Aredo and N.  K. R. Musimba, 2003).  Their chemical composition has determined that they are low in nitrogen (N) content and high in cell wall components with little soluble cell contents (T. A. Aredo and N. K. R. Musimba, 2003) (soluble cell contents include water-soluble carbohydrates). Chopping, wetting, ensiling with urea or animal manure, ashes and magadi soda (T. A. Aredo and N. K. R. Musimba, 2003) have been used in treatments.

With corn stover at the U. of Guelph, Ontario Canada back in the 1980s it was commented upon that approaches to feed processing/storage as more economical means for feedstock feed and energy be sought after such as those that are biological-based (P. J. Morris and D. N. Mowat, 1980), urealysis as one such biological possibility is one such approach under discussion here, that is ideal for developing tropical country settings.

We will not get into any side discussions of the chemical applications of ammoniation here but there has been considerable research of this approach in semi-developing countries including feedstock as with corn stover which is ideal for such treatments as carried out by field studies with the FAO, Rome, Italy in rural China.

Urea-ammoniation, another term for the urealytic process in toto will be used thus: autochthonous plant or soil microbes ensilo, and in future, recommended practices to be made with soybean meal addition as sources of the plant urease, and proposals for urease enzyme technology available in commercial quantities in bulk (and through urease-boosted GMOs) (this can be referred to as type-IV, indirect-type acting lignases) and with type-II lignases, yet to be further expanded upon and characterized, and to be used perhaps with added cellulases, e. g. endoglucanases, top-dressed or in the host.

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This poses the question if and when it will be possible to mechanize and even semi-automate the methods of biologically urea-ammoniation of feed in bulk by tonnage and which can be processed and contained and then sterilized (irradiation is one possibility), based on the issue of public risk and / or environmental safety of using GMOs. “Yeast Bagasse”, also in GM form, it is predicted, will be manufactured widely in industrial-scale as one livestock feed produced from sugar mills.

Enzyme Characterization in Microbial Species.

Urease catalyzes the hydrolysis of urea using a bimetallic nickel centre (S. Benini et al., 1999). It is important to increase and optimize the level of urease activity to bring about greater fibre breakdown due to delignification and to increase concentration of permeants of ammonium carbonate((NH4)2CO3), carbonate(CO3/2-) and hydroxide(OH-).

It will be important to first isolate and identify those autochthonous anoxygenic species by culturing in media with urea as the the nitrogen (N) source (selecting out various yeast, fungi and mycobacterial spp.). To process and identify the enzyme activities from a protein pellet from each candidate culture one subjects them, to further isolate them, on a 2-D electrophoretic gel and proteins assayed for urease activity with endproducts.

Klebsiella spp. is a well-studied microorganism for expression of an operon with 3 or 5 subunits to coordinate two Ni 2+ ligands.  The crystal structure of this urease enzyme has been solved and thus is determinate for manipulation by substitution including at the active site. Only native ureases in chosen autochthonous species will be cloned and boosted rather than cloned from other species proposed here.

Molecular Genetics of Cloning Urease Genes.

Plasmids or circular DNA vectors will be extracted and separated by centrifugation and analyzed for size or molecular weight (MW), mapped for origin of replication, restriction enzyme (RE) multiple cloning sites (MCS) and selectable markers inserted such as for antibiotic resistance to select out successfully transformed and expressing colonies or cells or using insertional inactivation of the MCS.

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From 2-D electrophoresis, a peptide sequence from the enzyme sequence can be derived to provide a synthesized DNA sequence to test via shotgun cloning of random fragments if the gene has been cloned in the chosen plasmid vector at the MCS and with use of Southern blot testing. However, with modern bioinformatics it is suggested that the candidate clones be sequenced with their chromosomal DNA for plasmid vectors and an appropriate fragment to be spliced in at the MCS. It is possible at this time to propose and follow an invention idea of ours, an in solution dye-detergent that could replace Coomassie blue in Mass-Spec identification and sequencing with proteomic analysis of peptides and their proteins (D. A. Flores, pers. commun. 2016)

Enzyme Assay of Urease Activity from Cultures.

The active end-products of enzymes that act in the ammoniation process are: (NH4)2CO3, CO3/2- and OH-. In culture the enzyme cloned is urease. The method of detection as to their potency are the: ammonium specific electrode, bicarbonate specific electrode and pH electrode (that can measure the potential of the H+ species concentration or conversely the OH- species), respectively. In the isolation of enzymes the proteins are pelleted and 2-D electrophoresis is applied (MW x pI) and: urease species is selected for being most potent for ammonium carbonate formation, species selected for bicarbonate ion formation and thirdly species selected for highest pH formation; the mechanisms of each of these enzymes would be: hydrolysis to ammonium ion and carbonic acid, solubilization of CO2 with OH- and H+ to form CO3/2- and reaction between ammonia and H20 to form NH4+ and OH- ions.

EFEs and Urea-Ammoniation.

Studies that measure dry matter (DM) loss with urea-ammoniation that is equal to phenolics + sugars (-microbial DM) are to be determined as to the efficiency of delignification of pretreatment on fibre.

EFEs at this time have been proven to effect no loss on phenolics in the DM mass but may improve breakage of hemicellulose-lignin bonds and can be used with steam treatment to expose fibre to microbial action in rumen digestion. Esterases are the lignases referred to here in addition to cellulases and hemicellulases as a cocktail of EFEs but perhaps in future, etherases will be engineered possibly to breakdown lignin further including lyases if the bond strengths or energies can be overcome by their enzymes. There are also other EFEs in the horizon that are in the same class (viz. type-II) that are anaerobic that contribute to breakdown of lignin in eco-habitats (e. g. waste-activated sludge) with microbial action and may be used as direct-fed probiotics, top-dressed, which will not be discussed here, any further.

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Studies of Urea-N Treatment of Straw.

It has been reported by E. A. Lufadaju and M. B.  Olayiwole (1986) that improvements in animal performance from higher digestibility and additional nitrogen is similar between urea-treated forage and supplemented untreated hay.

A study measuring the in vitro organic matter digestibility (IVOMD) increased with urea treatment.  The study included soy bean addition (as a source of plant urease) and increased measurably IVOMD (A. A. Dias-Da-Silva et al., 1988).  They demonstrated that the increase in IVOMD was correlated with dissociation of phenolics from the cell wall and increasing cell-wall digestibility.

A third study mentioned (M. J. Khan et al., 1999) involved soy bean meal:urea (1:1) (better than other soil microbial sources and a plant urease from jack bean meal) reduced the acid detergent fibre (ADF) and increased the organic matter (OM) digestibility. 

 

Ammonia-N Specific Electrode: an invention for IP incubation in the making.

Recent studies on using cellulose sourced from lignocellulose for derivitization of membranes in fuel cell technology involving proton and anions (e. g. OH-) has reached "SkyeBlue" regarding recent reestablishment of proposals to modernize the classic Kjeldahl Nitrogen Determination in proximal analyses in our case the determination of crude protein from nitrogen (N). Other samples involved can be, in addition to feeds (and digesta and faeces for animal scientists), are: wastewater, soil, fertilizers, meat, and grain.


Our interest is in developing the ion specific electrode (ISE) probe of an electronic meter in a barrel construct with its ion electrode membrane (IEM) made from cellulose.


NH3-specific electrode membrane (NEM) for an ISE (separate make) from derivitized cellulose using a hypothetical methylcellulose and limit-dextrin central X-linking node that is hydrophobic as evidenced by fact that cellulose is hydrophobic enough to block hydrophilic CH3OH (methanol) to allow passage diffusion of base applied ammonium solution causing evolution of NH3(g) and the movement across the fashioned membrane in such a way using a technique called interpenetrating and semi-interpenetrating to cause nanofibre formation against dysfunctional swelling with H2O uptake. There is also grafting and X-linking (perhaps via X-linking of limit dextrin centres) to specify pore size and rate of selective diffusion of hydrophobic NH3(g) across the hydrophobic membrane.


The interior design with cathode (+ve electrode of Cu-Selenide) in solution that is contained by the hydrophobic nature of the membrane enclosure is reduced by one e- back to ammonium (NH4+) together with a dilute solution in the chamber of the probe's metal barrel of HCl(aq) forming ammonium chloride (NH4Cl) (aq) dilute solution. The anode (-ve electrode) is possibly designed electronically we can guess passively supplies on redox rxn exchange of e-'s. It is also of selenide and completes the current conductance back into the electrode barrel's interior milieu or solution. 


Our approach to the IEM is that of methyl cellulose and limit dextrin with cellulose which we call dextrinose. It has been noted that cellulose is hydrophobic enough to block methanol (CH3OH) with methylene ether X-linking. Enzymes from microbial sources to make dextrinose from limit dextrin are available. First the dextrinose is digested to the limit dextrin. Gel exclusion chromatography by prep with Sephadex based on molecular size will allow extension of the cellulose strand (20-30 glucose units) for greater density (with controlled pore size) using 30 methylene ether molecules with 1 dextrin with 6 extensions per molecule.

 

Conclusion.

There is a need to further study interactions between the rate of urea-N application and urease specific activity level (units NH3-N/units microbial colony count) as to compare the rate of urea-N with microbial urease activity. It is hypothesized here that the acute rise in urease at a given level of urea-N application will cause improvement in delignification and increases in in vitro digestibility of fibre after which the effect on delignification will plateau.

It is suggested that a practical method in research including areas in Africa, China and Brazil where soya bean as a crop is being cultivated is to use the plant as source for urease and a common farm material such as animal manure that will lead to ammoniation upon ensilage.

The use of enhanced urealytics and ureases in future should be considered for urea-ammoniation processing including the possibility of genetically modified organisms (GMOs) and genetically engineered (GE) ureases which can be developed in industry settings.

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References.

T. A. Aredo and N. K. R. Musimba. 2003. Study on the Chemical Composition, Intake and Digestibility of Maize Stover, Tef Straw and Haricot Bean Haulms in Adami Tulu District, Ethiopia. Kasetsart (Nat. Sci.) 37: 401-407.

S. Benini, W. R. Rypniewski, K. S. Wilson, S. Miletti, S. Ciurli and S. Magani. 1999. A New Proposal for Urease Mechanism Based on the Crystal Structures of the Native and Inhibited Enzyme from Bacillus pasteurii: why urea hydrolysis costs two nickels. Structure 7: 205-216.

A. A. Dias-Da-Silva, A. M. Ferreira, C. V. M. Guedes. 1987. Effects of Moisture Level, Treatment Time and Soya Bean Addition on the Nutritive Value of Urea-treated Maize Stover. Anim. Feed Sci. & Tech. 19: 67-77.

M. J. Khan, J. R. Scaife and F. D. Hovell. 1999. The Effect of Different Sources of Urease Enzyme on the Nutritive Value of Wheat Straw Treated with Urea as a Source of Ammonia. Asian-Aus. J. Anim. Sci. 12: 1063-1069.

A. Lufadeju, M. B. Olayiwole and N. N. Umunnu. 1986. Intake and Digestibility of Urea-treated Gamba (Andropogon gayanus) Hay by Cattle. Plant Breeding and the Nutritive Value of Crop Residues – proceedings of a conference. ILRI.

J. Morris and D. N. Mowat. 1980. Nutritive Value of Ground and/or Ammoniated Corn Stover. Can. J. Anim. Sci. 60:327-336.



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