A mannose-binding protein from the cell surface of flocculent Saccharomyces cerevisiae (NCIM 3528): its role in flocculation.

A cell surface lectin was isolated and purified to homogeneity from the cell walls of a highly flocculent strain of Saccharomyces cerevisiae (NCIM 3528) by chromatography on DEAE-cellulose, phenyl Sepharose and Sephacryl S-300. It showed a molecular mass of 40 kDa on SDS-PAGE. It is an acidic protein with a pI of 4.0 and contains 44% hydrophobic amino acids. The N-terminal sequence up to 10 amino acid residues showed at least 70% homology with the predicted N-terminal sequence of the putative FLO1 as well as FLO5 gene products. The mannose-binding nature of the lectin was indicated by its high affinity and specificity towards the branched trisaccharide of mannose, a ligand which also inhibits the flocculation of yeast cells. Immunofluorescence studies confirmed the presence of lectin on the yeast cell surface and lectin-specific IgGs prevented flocculation of the cells. This cell surface mannose-specific lectin probably plays an important role in flocculation, with the branched trimannoside on the cell wall being the apparent carbohydrate receptor. The N-terminal sequence data gives a primary indication that the lectin could be a product of one of the FLO genes.

Purification and characterization of a cell-surface lectin (Lectin II) from Agrobacterium radiobacter NCIM 2443.

A lectin was isolated from Agrobacterium radiobacter cell surface and purified. It is a monomer of 40 kDa as shown by SDS-PAGE. The lectin has a pI of 9.15 and amino acid composition of the lectin shows that 44% of the amino acids are hydrophobic. The lectin agglutinates rabbit erythrocytes but does not agglutinate human erythrocytes. It does not show specificity for monosaccharides except for D-glucosamine. Fetuin and its N-linked glycopeptide also inhibit the activity of the lectin but greater inhibition is shown by locust bean gum and Nicotiana tobaccum (tobacco) tissue extracts.

Purification and characterization of an extracellular lectin (Lectin I) from Agrobacterium radiobacter NCIM 2443.

A lectin from culture filtrate of Agrobacterium radiobacter NCIM 2443 is purified to homogeneity by ion exchange chromatography on a DEAE cellulose column followed by hydrophobic chromatography on phenyl sepharose and hydroxyapatite column chromatography. The protein (Lectin I) is a monomer of relative molecular mass 37,000, as determined by denaturing gel electrophoresis as well as size exclusion chromatography. Lectin I is stable at pH 5.0 and its isoelectric point is pH 4.0. Amino acid analysis reveals that acidic amino acids and glycine are predominant amino acids and cysteine is absent in the lectin. Chemical modification of tryptophan residues causes more than 80% loss of haemagglutination activity of the lectin and 60% loss of activity is caused by modification of carboxyl groups. Lectin I agglutinates rabbit erythrocytes but does not agglutinate human A, B and O types of erythrocytes. It is specific for N-acetyl D-glucosamine, chitobiose, pNP-beta-mannoside as well as high mannose type glycopeptides. The relative inhibition by disaccharides, oligosaccharides and glycoproteins indicates that Lectin I recognizes Man3-GlcNAc-GlcNAc core carbohydrate structure of asparagine linked glycopeptides. Tobacco tissue extracts also inhibit the haemagglutination activity of Lectin I.

Bacillus sphaericus penicillin V acylase: purification, substrate specificity, and active-site characterization.

Penicillin V acylase from Bacillus sphaericus was purified to homogeneity with an overall yield of 15%. The enzyme exhibited comparatively high specificity for penicillin V, penicillin G, and other related compounds being hydrolyzed at less than 10% of the rate of penicillin V. Moreover, the high rate of hydrolysis was observed when the side chain of the substrate molecule was unsubstituted. Lysine-modifying reagents inactivated the enzyme rapidly. Kinetics and titration studies indicated the involvement of lysine in the catalytic activity of the enzyme.

Industrial yeast strain improvement: construction of a highly flocculent yeast with a killer character by protoplast fusion.

Conditions were optimized for rapid release and improved regeneration of protoplasts of Saccharomyces cerevisiae NCIM 3458. Rapid protoplast release was also obtained with representatives of several other yeast genera under the modified conditions of treatment. The application of the procedure in construction of a highly flocculent Saccharomyces cerevisiae with a killer character is described. Fusion was effected between UV-killed protoplasts of S. cerevisiae NCIM 3578 with a killer character and live protoplasts of the highly flocculent S. cerevisiae NCIM 3528 in the presence of polyethylene glycol (PEG) 6000. Fusants were selected using benomyl resistance as marker, the killer toxin producer rather than the highly flocculent yeast being resistant to the fungicide at a concentration of 100 micrograms ml-1. Fusants were also characterized by their DNA contents, capacity for ethanolic fermentation of molasses sugar and levels of invertase, alcohol dehydrogenase and pyruvate decarboxylase activities.

Studies of operational variables in batch mode for genetically engineered Escherichia coli cells containing penicillin acylase.

A recombinant Escherichia coli was constructed by cloning the penicillin acylase gene from E. coli ATCC 11105. The cloning was carried out using a recombinant plasmid pUSAD2 harboring the pac gene. The recombinant E. coli DH 5 cells were used as a biocatalyst and were studied in a batch reactor for determination of optimum value for some of the process parameters, such as effect of pH, temperature, substrate concentration, kLa and effect of carbon and nitrogen source on penicillin acylase production. These values were then compared with the values obtained with the standard parent strain. Whereas the cloned pac gene was found to produce higher levels of penicillin acylase constitutively, the process parameters remained about the same for both the parent and the recombinant.

Immobilization of permeabilized Escherichia coli cells with penicillin acylase activity.

Escherichia coli cells with penicillin acylase activity were sequentially treated at pH 7.8 with aqueous solutions of N-cetyl-N,N,N-trimethylammonium bromide and glutaraldehyde and then immobilized within porous polyacrylamide beads. The immobilized whole cells showed enhanced hydrolysis rates in the conversion of benzylpenicillin to 6-aminopenicillanic acid (6-APA) compared to untreated cells immobilized and used under identical conditions. The immobilized system showed no apparent loss in enzyme activity when used repeatedly over 90 cycles for 6-APA production from 4% benzylpenicillin.

Immobilization of penicillin acylase in porous beads of polyacrylamide gel.

A procedure is described for the immobilization of benzylpenicillin acylase from Escherichia coli within uniformly spherical, porous polyacrylamide gel beads. Aqueous solutions of the enzyme and sodium alginate and of acrylamide monomer, N,N'-methylene-bis-acrylamide, N,N,N,N'-tetramethylethylenediamine (TEMED) and sodium alginate are cooled separately, mixed, and dropped immediately into ice-cold, buffered calcium formate solution, pH 8.5, to give calcium alginate-coated beads. The beads are left for 30-60 min in the cold calcium formate solution for polyacrylamide gel formation. The beads are then treated with a solution of glutaraldehyde and the calcium alginate subsequently leached out with a solution of potassium phosphate. Modification of the native enzyme with glutaraldehyde results in a slight enhancement in the rate of hydrolysis of benzylpenicillin at pH 7.8 and 0.05M substrate concentration. The enzyme entrapped in porous polyacrylamide gel beads shows no measurable diffusional limitation in stirred reactors, catalyzing the hydrolysis of the substrate at a rate comparable to that of the glutaraldehyde-modified native enzyme. The immobilized enzyme preparation has been used in batch mode over 90 cycles without any apparent loss in hydrolytic activity.

Evidence for involvement of arginyl residue at the catalytic site of penicillin acylase from Escherichia coli.

Incubation of penicillin acylase from Escherichia coli with phenylglyoxal or 2,3-butanedione results in enzyme inactivation. Both benzylpenicillin and phenylacetate protect the enzyme against the inactivation, indicating the presence of arginine at or near the catalytic site. The reactions follow pseudofirst order kinetics and the inactivation kinetics indicate the presence of a single essential arginine moiety.