Won Gi Bang

Functional characterization of a bacterial expansin from Bacillus subtilis for enhanced enzymatic hydrolysis of cellulose

Expansin is a plant protein family that induces plant cell wall‐loosening and cellulose disruption without exerting cellulose‐hydrolytic activity. Expansin‐like proteins have also been found in other eukaryotes such as nematodes and fungi. While searching for an expansin produced by bacteria, we found that the BsEXLX1 protein from Bacillus subtilis had a structure that was similar to that of a β‐expansin produced by maize. Therefore, we cloned the BsEXLX1 gene and expressed it in Escherichia coli to evaluate its function. When incubated with filter paper as a cellulose substrate, the recombinant protein exhibited both cellulose‐binding and cellulose‐weakening activities, which are known functions of plant expansins. In addition, evaluation of the enzymatic hydrolysis of filter paper revealed that the recombinant protein also displayed a significant synergism when mixed with cellulase. By comparing the activity of a mixture of cellulase and the bacterial expansin to the additive activity of the individual proteins, the synergistic activity was found to be as high as 240% when filter paper was incubated with cellulase and BsEXLX1, which was 5.7‐fold greater than the activity of cellulase alone. However, this synergistic effect was observed when only a low dosage of cellulase was used. This is the first study to characterize the function of an expansin produced by a non‐eukaryotic source. Biotechnol. Bioeng. 2009;102: 1342–1353.

Enhanced production of recombinant B-domain deleted factor VIII from Chinese hamster ovary cells by propionic and butyric acids

Sodium propionate, as well as sodium butyrate, enhanced the production of recombinant B-domain-deleted, factor VIII (rFVIIIdB) by Chinese hamster ovary cells growing in a spinner-flask with a protein-free medium by more than six-fold. The two acids, however, had different cytotoxicities.

Molecular characterization of a novel bacterial aryl acylamidase belonging to the amidase signature enzyme family

In seeking aryl acylamidase (EC 3.5.1.13) acting on an amide bond in p-acetaminophenol (Tylenol™), we identified a novel gene encoding 496 residues of a protein. The gene revealed a conserved amidase signature region with a canonical catalytic triad. The gene was expressed in E. coli and characterized for its biochemical properties. The optimum pH and temperature for the activity on p-acetaminophenol were 10 and 37°C, respectively. The half-life of enzyme activity at 37°C was 192 h and 90% of its activity remained after 3 h incubation at 40°C. Divalent metals was found to inhibit the activity of enzyme. The Km values for various aryl acylamides such as 4-nitroacetanilide, p-acetaminophenol, phenacetin, 4-chloroacetanilide and acetanilide were 0.10, 0.32, 0.83, 1.9 and 19 mM, respectively. The reverse reaction activity (amide synthesis) was also examined using various chain lengths (C1∼C4 and C10) of carboxylic donors and aniline as substrates. These kinetic parameters and substrate specificity in forward and reverse reaction indicated that the aryl acylamidase in this study has a preference for aryl substrate having polar functional groups and hydrophobic carboxylic donors.

Random sequence analysis of genomic DNA of a hyperthermophile: Aquifex pyrophilus

AbstractAquifex pyrophilus is one of the hyperthermophilic bacteria that can grow at temperatures up to 95°C. To obtain information about its genomic structure, random sequencing was performed on plasmid libraries containing 0.5–2 kb genomic DNA fragments of A. pyrophilus. Comparison of the obtained sequence tags with known proteins revealed that 123 tags showed strong similarity to previously identifed proteins in the PIR or Genebank databases. These included three proteases, two amino acid racemases, and three enzymes utilizing oxygen as substrate. Although the GC ratio of the genome is about 40%, the codon usage of A. pyrophilus showed biased occurrence of G and C at the third position of codons, especially those for amino acids such as asparagine, aspartic acid, cysteine, glutamine, glutamic acid, histidine, lysine, and tyrosine. A higher ratio of positively charged amino acids in A. pyrophilus proteins as compared with proteins from mesophiles suggested that Aquifex proteins might contain increased ion-pair interaction that could help to maintain heat stability.

Crystal structure of fatty acid-CoA racemase from Mycobacterium tuberculosis H37Rv.

Introduction. The classic catabolic pathway by which fatty acids are degraded is -oxidation, and a mitochondrial as well as a peroxisomal -oxidation pathway is known. Very long-chain fatty acids, 2-methyl-branched fatty acids, the side-chains of bile acid intermediates, and eicosanoids are mainly handled by the peroxisomal pathway, whereas shortand medium-chain fatty acids are oxidized mainly in mitochondria. Phytanic acid and other 3-methyl-branched fatty acids cannot undergo -oxidation, because the 3-methyl group prevents the formation of a 3-keto substituent in the dehydrogenation step. Therefore, 3-methyl-branched fatty acids first undergo -oxidation. In the case of phytanic acid, this results in the generation of 2-methyl-branched pristanic acid (2,6,10,14tetramethyl pentadecanoic acid), which is then shortened to 4,8-dimethyl nonanoic acid via peroxisomal -oxidation. The dimethyl fatty acid is then degraded further via mitochondrial -oxidation. The -oxidation of fatty acids is not a stereoselective process, so that after -oxidation of branched-chain fatty acids, both (R,R,R)and (S,R,R)isomers are formed. However, the -oxidation system is stereoselective because only the (2S)-isomer is accepted as substrate by branched-chain acyl–coenzyme A (CoA) oxidase, the first enzyme of the -oxidation system. Thus, the (R,R,R)-isomers of the metabolites of -oxidation need to be converted to their (S,R,R)-isomers by enzymatic racemization prior to further degradation. The fatty acid-CoA racemase (FAR) encoded by the far gene of Mycobacterium tuberculosis H37Rv converts several (2R)-branched-chain fatty acid-CoA’s to their (2S)stereoisomers in the metabolism of fatty acids. FAR is thought to be involved in this racemization prior to the stereospecific -oxidation, although this has not yet been demonstrated experimentally. Bhaumik et al. found three genes—mcr, far, and Rv3727—in the M. tuberculosis genome that encode proteins that are homologous to mammalian -methylacyl-CoA racemase (AMACR), which is a mitochondrial and peroxisomal enzyme that is essential in the -oxidation of bile acid intermediates and branchedchain fatty acids. Among them, only one crystal structure of -methylacyl-CoA racemase (MCR) from M. tuberculosis relative to mammalian AMACR has been reported to date. Thus, the FAR from M. tuberculosis was taken as a model protein to provide the structural information of another protein corresponding to mammalian AMACR. Moreover, regarding the strong sequence similarity of AMACR with CoA transferases of family III, the question arises whether this enzyme also catalyzes CoA trans-

Use of plant protein hydrolysates for varicella virus production in serum-free medium

AbstractSerum-free media containing no animal-derived components were assessed for their efficacy to produce live attenuated varicella virus. Serum-free medium containing an ultrafiltrate of soy protein acid hydrolysate and lipid resulted in a viral production yield comparable to media containing fetal bovine serum, indicating that varicella virus can be produced without the risk of contamination associated with the use of bovine serum.

Retinoic acid modulates the level of nerve growth factor gene expression and proliferation of cultured human keratinocytes

Retinoic acid (RA) inhibited proliferation of cultured human keratinocytes. Southern blot analysis at 24 h showed that RA inhibited nerve growth factor (NGF) mRNA synthesis in a dose-dependent manner. However, RA stimulated the production of NGF protein up to 187% of control after 96 h and the treatment of cells with RA did not enhance apoptosis, either in the presence of low or high concentration of Ca2+, when compared to the control with DMSO.

Bleeding Strategy for the Long-Term Perfusion Culture of Hybridoma

We cultivated the hybridoma producing vWF antibody in a 10L bioreactor in order to investigate the characteristics oftwo different cultures, intermittent and continuous bleeding. To compare with bleeding culture non-bleeding culture was also performed. Non-bleeding culture achieved a cell density of 2.1 x 107 cells/ml at 16th day with 110 mg/l ofvwF mAb titer. However, the culture was stopped after the 18 days, due to membrane clogging. Intermittent and continuous bleeding cultures were maintained the cell density around 1 x10 cell/ml. The vWF mAb titer was 45mg/l and 53mg/l for intermittent and continuous bleeding culture. As for volumetric productivity, the continuous bleeding culture was higher than intermittent bleeding culture by 4 1%. For glucose consumption rate (GCR), lactate production rate (LPR), glutamine consumption rate (GnCR), and ammoniaproduction rate (APR), continuous bleeding culture showed 28%, 70%, 31% and 40% higher than intermittent bleeding culture. These results suggest that physiological activity ofthe continuous bleeding culture was higher than that of intermittent bleeding culture.