Abstract: Novel purified agarase enzymes from Flavobacterium sp. strain NR19 and cloned genes encoding the agarase enzymes are disclosed. Transformed host cells which express the novel agarase enzymes in isolatable quantities are also described. Also disclosed are antibodies specifically reactive with the novel agarases.

Abstract: Formation of an intramolecular cross-link in enzyme donor polypeptide fragments of .beta.-galactosidase, thereby forming a cyclic enzyme donor which is hindered from complementation with an enzyme acceptor fragment to form active of .beta.-galactosidase. The cyclic enzyme donor can be linearized by cleaving to restore complementation ability. Assays in which such cyclic enzyme donors are linearized by specific analytes are disclosed, as well as novel homobifunctional bis-maleimido cross-linking agents of the formula ##STR1## wherein R is hydroxy or acetate.

Abstract: An isolated SDS-resistant hyperthermostable .beta.-galactosidase gene derived from Pyrococcus furiosus. Examples thereof include genes respectively shown in SEQ ID NO: 1 and SEQ ID NO: 2 and genes each hybridizable with the above gene shown in SEQ ID NO: 1. A method of cloning the hyperthermostable .beta.-galactosidase gene in which each of the above genes or pacts thereof is used as a probe or primer. A process for producing a hyperthermostable .beta.-galactosidase by culturing a transformant into which a plasmid containing each of the above gene has been introduced.

Abstract: A diagnostic assay for detecting the presence of an infectious herpesvirus in a specimen and a genetically engineered cell line for use in such assay are disclosed. The cell line used in the assay expresses a reporter gene only if infectious herpesvirus is present in the specimen. The assay involves inoculating a DNA-transfected cell line with a specimen suspected of containing a herpesvirus, allowing a sufficient period of time for the herpesvirus infectious cycle to proceed, and detecting and quantifying the number of herpesvirus-infected cells to determine the number of infectious herpesvirus virions in the specimen. The cell line is a DNA-transfected cell line susceptible to infection by a herpesvirus which is stably transformed with a chimeric gene comprising a herpesvirus inducible promoter and a gene coding for an enzyme, the expression of the enzyme being dependent upon and quantitatively proportional to the presence of herpesvirus. A kit for such assay is also provided.

Abstract: The present invention relates to methods of producing a polypeptide, comprising: (a) introducing into a respiratory-defective mutant of a cell (i) one or more first nucleic acid sequences which complement the respiratory defect and (ii) a second nucleic acid sequence which encodes the polypeptide; (b) cultivating the cell containing the first and second nucleic acid sequences in a culture medium under aerobic conditions suitable for expression of the first and second nucleic acid sequences; and (c) isolating the polypeptide from the cultivation medium of the cell. The present invention also relates to methods for disrupting a gene in a respiratory-deficient mutant cell. The present invention further relates to respiratory-deficient mutant cells and methods for obtaining such mutant cells.

Abstract: .beta.-Galactosides are synthesized using a transglycosylation reaction catalyzed by .beta.-galactosidase. The reaction employs a carbohydrate donor having a glycosidic leaving group attached to its anomeric carbon and an oxo group attached to the C-6 carbon. Strong leaving groups are preferred over weak leaving groups. The method can be carried out in aqueous solution without organic solvents to give the transglycosylation product in high yields and high regioselectivity. The synthesis of lactosamine using this methodology with galactose oxidase (GO) and .beta.-galactosidase has been accomplished. (FIG. 3). The methodology affords simple reaction conditions and minimal purification steps. In addition, the intermediate substrates maintain high stability, the process affords high yields and the enzymes and reagents employed are commercially available with high stability and low costs.

Abstract: Novel purified agarase enzymes from Flavobacterium sp. strain NR19 and cloned genes encoding the agarase enzymes are disclosed. Transformed host cells which express the novel agarase enzymes in isolatable quantities are also described. Also disclosed are antibodies specifically reactive with the novel agarases.

Abstract: The invention relates to a plasmid carrying a selenium-specifying DNA seqce of the E. coli fdhF gene upstream of the E. coli lac'Z gene which permits the incorporation of selenocystein into .beta.-galactosidase. Particular plasmids according to the invention are pRM2, deposited at the ATCC under No. 75594 and pRM4, deposited at the ATCC under No. 75595. The invention also relates to microorganisms transformed with a plasmid according to the invention having selenium dependent .beta.-galactosidase activity. The invention provides a method for the quantitative determine of selenium in selenium derivatives in a biological sample comprising incubating microorganisms according to the invention in a suitable medium also containing said sample and measuring the level of .beta.-galactosidase activity. The biological sample may be, e.g. a blood sample or a food sample.

Abstract: This invention discloses novel methods of making fermented food products such as yogurt. It also discloses novel Lactobacillus bulgaricus organisms for making fermented food products which are conditionally sensitive, that is, operate to metabolize a desired compound normally under the processing conditions for fermented food products but slow or decrease in activity beyond what is normal under the routine storage temperatures for the fermented food products. Such fermented food products exhibits improved shelf life and long-term taste.

Abstract: The present invention provides a biomarker system for the in vivo and in vitro assessment of cell senescence. In the method of the present invention, .beta.-galactosidase activity is utilized as a means by which cell senescence may be assessed either in vitro cell cultures or in vivo.

Abstract: A method of producing and purifying an enzyme which comprises selecting a spore forming host organism, preparing a genetic construct consisting of a DNA sequence encoding the desired enzyme and a DNA sequence directing synthesis of the desired enzyme during sporulation, inserting the genetic construct into the host organism, culturing the transformed host organism under sporulating conditions to obtain host organism spores with the enzyme integrally associated to the spores, and then treating the host organism and enzyme combination to remove any impurities, if necessary. The free enzyme can be obtained by cleaving the connection between the host organism and the enzyme. The combination of the enzyme and host organism is both a stabilized and an immobilized enzyme preparation.

Abstract: An isolated hyperthermostable .beta.-galactosidase gene derived from Pyrococcus furiosus is described. Examples include DNA sequences shown in SEQ ID NO. 2 and SEQ ID NO. 4 and DNAs hybridizable with these DNAs. A method of cloning the hyperthermostable .beta.-galactosidase gene in which each of the above sequences or portions thereof is used as a probe or primer is described. A process for producing a hyperthermostable .beta.-galactosidase by culturing a transformant into which a plasmid containing each of the above genes has been introduced is described. A particularly preferred gene encodes an SDS-resistant hyperthermostable .beta.-galactosidase.

Abstract: Novel methods are disclosed for the enhanced expression and secretion of lactase from filamentous fungi. Specifically the novel processes cause enhanced production of lactase from an Aspergillus and preferably enhanced production of A. oryzae lactase from A. oryzae host strains transformed with necessary DNA. Also described are the DNA sequence encoding the lactase gene from A. oryzae and the deduced amino acid sequence of the lactase therefrom.

Abstract: A diagnostic assay for detecting the presence of an infectious herpesvirus in a specimen and a genetically engineered cell line for use in such assay are disclosed. The cell line used in the assay expresses a reporter gene only if infectious herpesvirus is present in the specimen. The assay involves inoculating a DNA-transfected cell line with a specimen suspected of containing a herpesvirus, allowing a sufficient period of time for the herpesvirus infectious cycle to proceed, and detecting and quantifying the number of herpesvirus-infected cells to determine the number of infectious herpesvirus virions in the specimen. The cell line is a DNA-transfected cell line susceptible to infection by a herpesvirus which is stably transformed with a chimeric gene comprising a herpesvirus inducible promoter and a gene coding for an enzyme, the expression of the enzyme being dependent upon and quantitatively proportional to the presence of herpesvirus. A kit for such assay is also provided.

Abstract: A chemically commercially sterilized enzyme composition for addition to a pasteurized dairy product to reduce gradually the lactose content of the dairy product and to introduce functioning enzymes into the digestive tract of an individual when the dairy product is ingested.

Abstract: Expression of a desired gene and production of a product from the desired gene can be obtained effectively by cultivating a microorganism containing a hybrid plasmid comprising the desired gene, a vector and a promoter in its cell, and adding an inducer and a nutriment simultaneously to the culture medium at the time when the nutriment originally present in the culture medium is almost consumed.

Abstract: A non-radioactive method of detecting the enzymes beta galactosidase or beta glucosidase directly or for the detection of a ligand and antiligand complex is provided wherein beta galactosidase or the complex labelled with beta galactosidase or a tracer having beta galactosidase conjugated thereto is reacted with 5-bromo-4-chloro-3-indolyl-B-D-galactoside and a tetrazolium salt to produce a colored formazan or a color change indicative of the presence of beta galactosidase, or wherein beta glucosidase or the complex labelled with the beta glucosidase or a tracer having beta glucosidase conjugated thereto is reacted with 5-bromo-4-chloro-3-indolyl-B-D-glucoside and a tetrazolium salt to produce a colored formazan or a color change indicative of the presence of beta glucosidase. Optionally, the galactosidase-galactoside determination or the glucosidase-glucoside determination may further include catalyst phenazine methosulfate (PMS) as a reactant.

Abstract: This invention discloses novel methods of making fermented food products such as yogurt. It also discloses novel Lactobacillus bulgaricus organisms for making fermented food products which are conditionally sensitive, that is, operate to metabolize a desired compound normally under the processing conditions for fermented food products but slow or decrease in activity beyond what is normal under the routine storage temperatures for the fermented food products. Such fermented food products exhibits improved shelf life and long-term taste.

Abstract: The invention is directed to purified and isolated concanavalin A-binding proteins from chondrocytes that are not present in dedifferentiated cells from chondrocytes. The invention is also directed to a purified and isolated chondrocyte membrane protein, (CMP), which is a concanavalin A-binding protein, with a molecular weight of about 76 kD in SDS-PAGE. After treatment with endoglycosidase, CMP has an apparent molecular weight of about 67 kD. The N-terminal amino acid sequence and several internal amino acid sequences are given for CMP. These proteins can be used in assays, methods, or treatments involving differentiation of chondrocytes and the control of cartilaginous osteogenesis.

Abstract: This invention relates to improved methods and novel compositions for enzyme complementation assays for qualitative and quantitative determination of a suspected analyte in a sample. The use of enzyme-acceptor and enzyme-donor polypeptides prepared by recombinant DNA techniques, DNA synthesis or chemical polypeptide synthesis techniques which are capable of interacting to form an active enzyme complex having catalytic activity characteristic of .beta.-galactosidase is described. Both homogeneous and heterogeneous assays utilizing these polypeptides are described.

Abstract: The invention concerns a complex between at least one negatively charged nucleic acid and at least one positively charged polymeric conjugate, the link between the nucleic acid and the polymeric conjugate being electrostatic in nature, the polymeric conjugate containing a polymer formed from monomer components having free NH.sub.3.sup.+ functions of the aforementioned components and being as follows:--the free NH.sub.3.sup.+ functions from the aforementioned components are substituted in a ratio of at least 10%, advantageously from 45% to 70%, particularly 60%, by noncharged residues leading to a reduction of positive charges in comparison to the same nonsubstituted polymeric conjugate, facilitating the release of nucleic acid by the dissociation of the complex,--the aforementioned residues possess in addition the following properties:.fwdarw.they contain at least one hydroxyl group,.fwdarw.they do not correspond to a recognition signal recognized by a cellular membrane receptor,--the free NH.sub.3.sup.

Abstract: A transient expression system is disclosed that utilizes bacteriophage RNA polymerase in the presence of a DNA-based cytoplasmic virus to facilitate expression of a foreign gene in the cytoplasm of a eukaryotic cell.A method of expressing a foreign gene in the cytoplasm of a eukaryotic cell is also disclosed which comprises incorporating into the cytoplasm a DNA-based cytoplasmic virus, a suitable carrier comprising a gene for an RNA polymerase which gene is foreign to the carrier and to the cells, and a suitable carrier comprising a functional, cistron including a foreign gene flanked by a promotor sequence which is recognized by the RNA polymerase.

Abstract: Recombinant mycobacterial vaccine vehicles capable of expressing DNA of interest which encodes at least one protein antigen for at least one pathogen against which an immune response is desired and which can be incorporated into the mycobacteria or stably integrated into the mycobacterial genome. The vaccine vehicles are useful for administration to mammalian hosts for purposes of immunization. A recombinant vector which replicates in E. coli but not in mycobacteria is also disclosed. The recombinant vector includes 1) a mycobacterial gene or portions thereof, necessary for recombination with homologous sequences in the genome of mycobacteria transformed with the recombinant plasmid; 2) all or a portion of a gene which encodes a polypeptide or protein whose expression is desired in mycobacteria transformed with the recombinant plasmid; 3) DNA sequences necessary for replication and selection in E. coli; and 4) DNA sequences necessary for selection in mycobacteria (e.g., drug resistance).

Abstract: Muteins of enzyme acceptor polypeptide fragments of .beta.-galactosidase are provided which exhibit substantially increased kinetic complementation activity with no significant loss in stability. A preferred enzyme acceptor fragment has an amino acid other than cysteine located at position 500 of the natural sequence. An especially preferred substitution is serine or valine. Other preferred muteins have an amino acid other than methionine located at position 443, with leucine being especially preferred, or an amino acid other than cysteine at position 76, with leucine being an especially preferred substitution. Also provided are methods for producing the novel muteins, reagent compositions comprising the novel muteins, and immunoassay methods for determining an analyte in which the novel mutein recombines with an enzyme donor polypeptide fragment to form enzymatically active .beta.-galactosidase.

Abstract: The present invention provides a biomarker system for the in vivo and in vitro assessment of cell senescence. In the method of the present invention, .beta.-galactosidase activity is utilized as a means by which cell senescence may be assessed either in in vitro cell cultures or in vivo.

Abstract: The present invention relates to .beta.-1,4-galactanase derived from A. aculeatus which have (a) a pH-optimum between 3.0 and 5.0, (b) an isoelectric point of 2.5-3.5, (c) a molecular weight of between 30,000 and 50,000, and (d) a temperature optimum between 10.degree. and 50.degree. C.

Abstract: A mutein of an enzyme acceptor polypeptide fragment of .beta.-galactosidase which is resistant to oxidation is provided. The enzyme acceptor fragment has an amino acid other than cysteine located at position 602 of the natural sequence. An especially preferred substitution is serine. Also provided are a method for producing the novel mutein, a reagent composition comprising the novel mutein, and an immunoassay method for determining an analyte in which the novel mutein recombines with an enzyme donor polypeptide fragment to form enzymatically active .beta.-galactosidase.

Abstract: A formulation consisting essentially of -1,4-xylanase and -1,3-xylosidase, but essentially free of -1,4-glucanase and -1,4-cellobiohydrolase and on or more lactic acid-producing bacteria is disclosed. The formulation can be used in a silage composition to stabilize silage from cereal and other crops and to enhance its nutritive value and digestibility in a ruminant or other animal.

Abstract: Biologically pure strains of Thermus sp. FERM BP-1678, FERM BP-1679 and FERM BP-1680 have been isolated, and are capable of producing a beta-galactosidase. The beta-galactosidase produced by these strains has specific characteristics wherein the enzyme is optimum at a temperature of 75 to 85 degrees celsius and pH of 4.5 to 6.5. Further, the enzymatic activity is reduced by at most 10% in the presence of 50 millimoles (mM) of galactose and glucose, respectively.

Abstract: A diagnostic assay for detecting the presence of an infectious herpes virus in a specimen and a genetically engineered cell line for use in such assay are disclosed. The cell line used in the assay expresses a reporter gene only if infectious herpes virus is present in the specimen. The assay involves inoculating a DNA-transfected cell line with a specimen suspected of containing a herpes virus, allowing a sufficient period of time for the herpes virus infectious cycle to proceed, and detecting and quantifying the number of herpes virus-infected cells to determine the number of infectious herpes virus virions in the specimen. The cell line is a DNA-transfected cell line susceptible to infection by a herpes virus which is stably transformed with a chimeric gene comprising a herpes virus inducible promoter and a gene coding for an enzyme, the expression of the enzyme being dependent upon and quantitatively proportional to the presence of herpes virus. A kit for such assay is also provided.

Abstract: An enzymatic process is disclosed for the preparation of galactosyl.beta.1,3glycal disaccharides such as Gal.beta.1,3Glucal, an intermediate useful in Le.sup.a preparation and an inhibitor of .beta.-galactosidase. The process utilizes .beta.-galactosidase, an enzyme usually used for bond breaking, to form a bond between a galactoside and a glucal such as glycal, a 6-O--C.sub.1 -C.sub.6 acylglucal or 6-O--C.sub.1 -C.sub.6 acetylgalactal.

Abstract: A method for the hydrolysis of only the lacto-N-biosidic (Gal.beta.1-3GlcNAc.beta.1-) linkages at the non-reducing termini of sugar compounds, characterized by the use of glycosidase that is specific for only the lacto-N-biosidic linkage in said sugar compounds. And disclosed is a reagent for use in hydrolysis of only the lacto-N-biosidic linkage at the non-reducing termini of sugar compounds.

Abstract: Antigens and vaccines containing purified oligomers (1-50 units) of the repeating pentasaccharide unit of type III Group B Stretococcus (III GBS) polysaccharide capsule. Methods of making the antigen by recovering polysaccharide from cultured III GBS or medium and digesting the polysaccharide with a specific endo-.beta.-galactosidase. Enzymatic cleavage of bacterial polysaccharide to make purified oligomers. A purified trypsin-resistant C surface protein, m.w. about 14,000 and vaccine. Passive immunization using the above vaccines. Immunoassays for GBS immunodeterminants or anti-GBS antibodies.

Abstract: A method of producing a growth promoting factor for Bifidobacterium species from lactose which comprises contacting lactose with resting cells of a lactose-utilizing yeast strain having activity to rearrange lactose to galacto-oligosaccharides.

Abstract: A rapid process for detecting pathogenic microorganisms in products for human consumption comprises contacting the microorganisms with a methylumbelliferone substrate. The substrate is hydrolyzed into methylumbelliferone by an enzyme given off by the microorganisms. Hydrolysis is accelerated by sodium lauryl sulfate, which renders the microorganisms more permeable to the substrate, the enzyme, or both. The methylumbelliferone is detected by its fluorescence, either in solution or on an agar medium supporting microcolonies formed from individual microorganisms.

Abstract: A .beta.-galactosidase having a high thermostability is produced by Thermus sp. FERM BP-1678, FERM BP-1679, and FERM BP-1680. The .beta.-galactosidase has an optimum temperature of 75 to 85 degrees celsius, and an optimum pH of 4.5 to 6.5 and an enzymatic activity which is not lowered substantially by 50 mM each of galactose and glucose. The .beta.-galactosidase is used to hydrolyze lactose in foods, such as milk, at a high temperature at which the putrefaction by saprophytes is difficult.

Abstract: It has been possible to show for a first time with the process according to the invention that the synthesis of glyco conjugates is possible by direct linkage of sugars with serine, serine derivatives and serine peptides with the aid of glucosidases.

Abstract: Described herein are a novel heat-resistant .beta.-galactosyltransferase, a production process of the enzyme and a utilization method of the enzyme. The enzyme is produced preferably by produced by a microorganism belonging to the family of Actinomycetaceae, which may be selected from fungi belonging to the genus Saccharopolyspora, the genus Thermomonospora or the genus Thermoactinomyces.

Abstract: The invention relates to a protein stabilizing sequence particularly useful for stabilization of proteolytically sensitive proteins. The sequence includes a relatively small number of amino acids that may be expressed fused with a proteolytically sensitive protein. The most effective stabilization sequences assume .alpha.-helix structures with a hydrophobic face and a positively charged polar face which appear to require proper orientation with respect to each other. Other aspects of the invention include cloning vectors incorporating a gene sequence encoding the stabilization polypeptide and production of stabilized antigenic proteins.

Abstract: Enzyme-donor fragments and other unstructured peptides are stabilized against proteolytic degradation by including a mixture of soluble, random-sequence peptides in the assay medium or in the medium in which the enzyme-donor fragment or other peptide is stored.

Abstract: In order to increase the enzymatic reactivity of .beta.-galactosidase, an azide, thiocyanate, cyanate and/or thiosulphate is added to the reaction mixture.

Abstract: A polypeptide having amino acid sequence 172-192 of a Mycobacterium bovis BCG 64 kD polypeptide, said sequence having the formula ##STR1## as well as polypeptides derived therefrom, in the amino acid sequence of which sequence 172-179 and/or sequence 189-192 is (are) entirely or partially absent, were found to be useful as immunogens inducing resistance to auto-immune arthritis and similar auto-immune diseases.The invention relates to these polypeptides, to polypeptides showing sequential homology with these polypeptides, and to derivatives and multimers thereof. Also, microorganisms expressing the polypeptides either as such or as part of a fusion protein or as a multimer form part of the invention.Finally, the invention relates to pharmaceutical compositions, diagnostic compositions and test kits comprising a compound according to the invention.

Abstract: Described herein are a novel heat-resistant .beta.-galactosyltransferase, a production process of the enzyme and a utilization method of the enzyme. The enzyme is produced preferably by produced by a microorganism belonging to the family of Actinomycetaceae, which may be selected from fungi belonging to the genus Saccharopolyspora, the genus Thermomonospora or the genus Thermoactinomyces.

Abstract: Cultivation of microorganisms or animal cells or plant cells is carried out to produce high density cultivation, high cell yield and high production of desired metabolite products by monitoring acetate concentration in culture broth and regulating assimilation of acetate in the culture broth by the microorganisms or animal cells or plant cells to control the acetate concentration to a set value or less. Preferably, an acetic acid-producing bacterium that is inhibited by acetic acid and is capable of assimilating acetic acid is cultured to produce biologically active substances such as enzymes. The bacterium may be a recombinant Escherichia coli and an inducer which acts on a promoter in an expression vector is added to produce the desired metabolite product. The inducer is 3-.beta.- indolylacrylic acid when the promoter is trp-promoter or isopropyl-.beta.-D-thiogalactoside when the promoter is lac-promoter or tacpromoter.

Abstract: A method for producing a polyprotein having at least 10 units, and often as many as 50 to 100 and more units held together by sulfur to sulfur or sulfur to carbon bonds is disclosed. Each unit comprises a protein and one or more heterobifunctional reagents. One functional group of the reagent is capable of forming a covalent bond with an amino group, permitting the reagent to bind to a protein. The other functional group of the reagent is capable of forming a covalent bond with a thiol group so as to form the covalent sulfur-carbon or sulfur-sulfur bond with another heterobifunctional reagent bonded to another protein.

Abstract: A polypeptide-labeled analyte analog for use in an immunoassay is prepared which is capable of binding with a polypeptide partner to provide enzymatic activity such as a ribonuclease or .beta.-galactosidase activity. The polypeptide analog provides a highly sensitive, immunoassay method for determining the amount of an analyte in a sample containing a known analyte in an unknown concentration. To carry out an immunoassay, there is brought together in a medium a sample, the polypeptide-labeled analog of the analyte, an antibody specific for said analyte, a polypeptide partner capable of non-covalently binding with the polypeptide-labeled analyte analog to form a complex having catalytic activity, and a substrate capable of being converted to a reporter molecule by the catalytic activity of said complex.

Abstract: The invention concerns antigens for the production of antibodies to Platelet Activating Factor (PAF). The antigens are PAF analogues of formula (I) ##STR1## wherein X comprises a high molecular weight group, R.sup.1 is a linking group and R.sup.2 to R.sup.5 are selected from C.sub.1 to C.sub.6 alkyl.Other aspects of the invention include PAF-antibodies produced using said antigens, labelled PAF analogues, intermediates for the preparation of PAF analogues and methods and a kit for the immunoassay of PAF.

Abstract: The present invention provides a hapten-protein conjugate, wherein a hapten is bound to the reducing end of a sugar which consists of up to 10 monosaccharide units and on a free CH.sub.2 OH group on the other end of the sugar, which is in the .alpha.-position to a hydroxyl group, is bound a protein.

Abstract: Novel methods for covalent attachment of antibodies, antigens, or other molecules to solid phases using extended length heterobifunctional crosslinking reagents are disclosed. The resulting derivatized solid phases can be used in diagnostic assays.

Abstract: A reagent comprising a fraction obtained from plasma of an insect such as silkworm larvae and capable of reacting specifically with .beta.-1,3-glucan or peptidoglycan can be used for determining .beta.-1,3-glucan or peptidoglycan.