Comparative characterization of a bifunctional endo-1,4- ß-mannanase/1,3-1,4- ß-glucanase and its individual domains.

A fusion gene isolated from a microbial metagenome encodes a N-terminal endo-1,4- ß-mannanase and a C-terminal 1,3-1,4- ß -glucanase,. The full-length gene and the individual N- and C-domains were separately cloned and expressed in E coli. The purified whole enzyme hydrolyzed glucomannan, galactomannan, and ß-glucan with Km and kcat values 2.2, 2.6, 3.6 mg/ml, and 302, 130, 337 min -1 , respectively. The hydrolysis of ß-glucan by the C domain enzyme decreased significantly with added glucomannan to the reaction, suggesting inhibition effect. Analogous result was not observed with the N domain enzyme when ß-glucan was added to the reaction. The whole enzyme did not show improvement of efficiency compared to the individual or additive total hydrolysis of the two domain enzymes using single or mixed substrates.

Functional Cloning and Expression of the Schizophyllum commune Glucuronoyl Esterase Gene and Characterization of the Recombinant Enzyme.

The gene encoding Schizophyllum commune glucuronoyl esterase was identified in the scaffold 17 of the genome, containing two introns of 50 bp and 48 bp, with a transcript sequence of 1179 bp. The gene was synthesized and cloned into Pichia pastoris expression vector pGAPZα to achieve constitutive expression and secretion of the recombinant enzyme in soluble active form. The purified protein was 53 kD with glycosylation and had an acidic pI of 3.7. Activity analysis on several uronic acids and their derivatives suggests that the enzyme recognized only esters of 4-O-methyl-D-glucuronic acid derivatives, even with a 4-nitrophenyl aglycon but did not hydrolyze the ester of D-galacturonic acid. The kinetic values were K(m) 0.25 mM, V(max) 16.3 µM·min(-1), and k(cat) 9.27 s(-1) with 4-nitrophenyl 2-O-(methyl 4-O-methyl-α-D-glucopyranosyluronate)-ß-D-xylopyranoside as the substrate.

Transgenic oncogenes induce oncogene-independent cancers with individual karyotypes and phenotypes.

Cancers are clones of autonomous cells defined by individual karyotypes, much like species. Despite such karyotypic evidence for causality, three to six synergistic mutations, termed oncogenes, are generally thought to cause cancer. To test single oncogenes, they are artificially activated with heterologous promoters and spliced into the germ line of mice to initiate cancers with collaborating spontaneous oncogenes. Because such cancers are studied as models for the treatment of natural cancers with related oncogenes, the following must be answered: 1) which oncogenes collaborate with the transgenes in cancers; 2) how do single transgenic oncogenes induce diverse cancers and hyperplasias; 3) what maintains cancers that lose initiating transgenes; 4) why are cancers aneuploid, over- and underexpressing thousands of normal genes? Here we try to answer these questions with the theory that carcinogenesis is a form of speciation. We postulate that transgenic oncogenes initiate carcinogenesis by inducing aneuploidy. Aneuploidy destabilizes the karyotype by unbalancing teams of mitosis genes. This instability thus catalyzes the evolution of new cancer species with individual karyotypes. Depending on their degree of aneuploidy, these cancers then evolve new subspecies. To test this theory, we have analyzed the karyotypes and phenotypes of mammary carcinomas of mice with transgenic SV40 tumor virus- and hepatitis B virus-derived oncogenes. We found that (1) a given transgene induced diverse carcinomas with individual karyotypes and phenotypes; (2) these karyotypes coevolved with newly acquired phenotypes such as drug resistance; (3) 8 of 12 carcinomas were transgene negative. Having found one-to-one correlations between individual karyotypes and phenotypes and consistent coevolutions of karyotypes and phenotypes, we conclude that carcinogenesis is a form of speciation and that individual karyotypes maintain cancers as they maintain species. Because activated oncogenes destabilize karyotypes and are dispensable in cancers, we conclude that they function indirectly, like carcinogens. Such oncogenes would thus not be valid models for the treatment of cancers.

Cloning and characterization of an exo-xylogucanase from rumenal microbial metagenome.

A novel exo-glucanase gene (xeg5B) was isolated from a rumenal microbial metagenome, cloned, and expressed in E. coli. The 1548 bp gene coded for a protein of 516 amino acids, which assumed an (a/b)(8) fold typical of glycoside hydrolase (GH) family 5. The protein molecule consisted of a loop segment blocking one end of the active site, which potentially provided the enzyme with exo-acting property. The recombinant enzyme showed exclusive specificity towards only xyloglucan and oligoxyloglucan substrates with no detectable activity on unsubstituted linear glucans, CMC, laminarin, and lichenan. The major end products of exhaustive hydrolysis were XX (tetrasaccharide) and XG (trisaccharide). The hydrolysis of tamarind xyloglucan followed the Michaelis-Menten kinetics, yielding K(m) and V(max) of 2.12+/-0.13 mg/ml and 0.17+/-0.01 mg/ml/min (37 degrees C, pH 6.0), respectively.

A novel xyloglucan-specific endo-beta-1,4-glucanase: biochemical properties and inhibition studies.

A novel xyloglucan-specific endo-beta-1,4-glucanase gene (xeg5A) was isolated, cloned, and expressed in Esherichia coli. The enzyme XEG5A consisted of a C-terminal catalytic domain and N-terminal sequence of approximately 90 amino acid residues with unknown function. The catalytic domain assumed an (alpha/beta)(8)-fold typical of glycoside hydrolase (GH) family 5, with the two catalytic residues Glu240 and Glu362 located on opposite sides of the surface groove of the molecule. The recombinant enzyme showed high specificity towards tamarind xyloglucan and decreasing activity towards xyloglucan oligosaccharide (HDP-XGO), carboxymethyl cellulose, and lichenan. Tamarind xyloglucan was hydrolyzed to three major fragments, XXXG, XXLG/XLXG, and XLLG. The hydrolysis followed the Michaelis-Menten kinetics, yielding K (m) and V (max) of 3.61 +/- 0.23 mg/ml and 0.30 +/- 0.01 mg/ml/min, respectively. However, the hydrolysis of HDP-XGO showed a decrease in the rate at high concentrations suggesting appearance of excess substrate inhibition. The addition of XXXG resulted in linear noncompetitive inhibition on the hydrolysis of tamarind xyloglucan giving a K (i) of 1.46 +/- 0.13 mM. The enzyme was devoid of transglycosylase activities.

Functional cloning and expression of a novel Endo-alpha-1,5-L-arabinanase from a metagenomic library.

A novel endo-alpha-L-arabinanase gene (arn2) was isolated, and expressed in E. coli in active form. The recombinant enzyme (ARN2) had optimum activity at pH 6.0 and 45-50( degrees )C with stability between pH 5.0-8.0 and at temperatures up to 40( degrees )C. The recombinant ARN2 catalyzed internal cleavage of alpha-1,5 glycosidic bonds of CM-arabinan, debranched arabinan, linear arabinan, and sugar beet (native) arabinan at rates of decreasing order, and was inactive on wheat arabinoxylan and p-nitrophenyl-alpha-L-arabinofuranoside. Kinetic analysis showed that branching in the arabinan did not significantly affect the apparent K(m) values, and the difference in the reaction rates was likely due to the chemical step after substrate binding. The enzyme hydrolyzed arabino-oligosaccharides of DP> or =6 to smaller oligomers and mostly arabinotriose. Natural and modified arabinans were cleaved to oligomers of various chain lengths, which were progressively hydrolyzed to yield arabinotriose. The pattern of degradation revealed an endo-acting mechanism with arabinotriose as the end product.

Cancer-causing karyotypes: chromosomal equilibria between destabilizing aneuploidy and stabilizing selection for oncogenic function.

The chromosomes of cancer cells are unstable, because of aneuploidy. Despite chromosomal instability, however, cancer karyotypes are individual and quasi-stable, as is evident especially from clonal chromosome copy numbers and marker chromosomes. This paradox would be resolved if the karyotypes in cancers represent chromosomal equilibria between destabilizing aneuploidy and stabilizing selection for oncogenic function. To test this hypothesis, we analyzed the initial and long-term karyotypes of seven clones of newly transformed human epithelial, mammary, and muscle cells. Approximately 1 in 100,000 such cells generates transformed clones at 2-3 months after introduction of retrovirus-activated cellular genes or the tumor virus SV40. These frequencies are too low for direct transformation, so we postulated that virus-activated genes initiate transformation indirectly, via specific karyotypes. Using multicolor fluorescence in situ hybridization with chromosome-specific DNA probes, we found individual clonal karyotypes that were stable for at least 34 cell generations-within limits, as follows. Depending on the karyotype, average clonal chromosome numbers were stable within +/- 3%, and chromosome-specific copy numbers were stable in 70-100% cells. At any one time, however, relative to clonal means, per-cell chromosome numbers varied +/-18% and chromosome-specific copy numbers varied +/-1 in 0-30% of cells; unstable nonclonal markers were found within karyotype-specific quotas of <1% to 20% of the total chromosome number. For two clones, karyotypic ploidies also varied. With these rates of variation, the karyotypes of transformed clones would randomize in a few generations unless selection occurs. We conclude that individual aneuploid karyotypes initiate and maintain cancers, much like new species. These cancer-causing karyotypes are in flexible equilibrium between destabilizing aneuploidy and stabilizing selection for transforming function. Karyotypes as a whole, rather than specific mutations, explain the individuality, fluidity, and phenotypic complexity of cancers.