The vascular endothelial growth factor (VEGF), or VEGF-A, is intimately involved in both physiological and pathological forms of angiogenesis. VEGF-A is now recognized as the founding member of a family of growth factors that has expanded to include VEGF-B, VEGF-C, VEGF-D, VEGF-E, and placental growth factor (PIGF). This family of cytokines binds differentially to at least three receptor tyrosine kinases, however, the extent to which family members other than VEGF-A contribute to physiological and pathological angiogenesis remains unclear. Issues that are of relevance include uncertainty regarding the consequences of signaling through VEGF - RI in particular, and the ability of some family members to heterodimerize, leading to the possibility ofheterodimeric receptor complexes. Structural characterization is one approach that can be used to address these issues, however, the vast majority of previous structure-function studies have only focused on VEGF-A. While these studies may provide some clues regarding the structural basis of the interaction of other family members with their receptors, studies using the ligands themselves are clearly required if highly specific interactions are to be revealed. With the recent progress toward refolding and purifying substantial' quantities of other VEGF family members, such structural studies are now possible. Here, these ~ssues are addressed with a particular emphasis on VEGF-B and its receptors.

By UV-irradiation of Eremothecium ashbyii DTl, a higWy flavinogenic mutant (UV-18-57) and a nonflavinogenic mutant (UV -85) were obtained. The physio-morphological characteristics of these three strains were studied on glucose medium in submerged fermentation. Glucose utilization and mycelial growth occurred in 0 - 2 days of fermentation. By the third day, the biomass had declined. Extracellular riboflavin excretion was distinct from the second day, reaching a maximum rate by the fourth day. The hyphae of the highly flavinogenic mutant UV-18-57 were broader than DTl, while the nonflavinogenic UV-85 hyphae were very thin. Riboflavin accumulation was high in UV-18-57 (extracellular riboflavin,$825\mu\textrm{g}/ml$ , and intracellular, $490\mu\textrm{g}/ml$) and caused the mycelia to swell into bulbous forms. Riboflavin accumulation was less in DTl ($108\mu\textrm{g}/ml$ extracellular and $24\mu\textrm{g}/ml$ intracellular) and correspondingly its hyphae were thinner than those of UV-18-57 and swollen bulbous mycelia were not prominent. UV-85 was nonflavinogenic and, accordingly, its mOlphological characteristics included long thin filaments with no intracellular riboflavin accumulation. A large number of greenish fluorescence spores were seen in UV-18-57, whereas DTI had less spores and UV-85 was nonsporulating. Sporulation is correlated with riboflavin production. UV-18-57 had better mycelial integrity and lysis started only by the seventh day, whereas DTI and UV -85 started to lyze earlier by 4 -5 days. By the late stage of fermentation (eighth day), DTl had a few long, thin filaments indicating some secondary growth, whereas UV -85 showed a compact pellet form of mycelia. Most mycelia of UV-18-57 still appeared intact.

A new alkalophilic strain of Bacillus pumilus JB¬05 producing bleach-stable and halo-tolerant alkaline protease was isolated from cement industry effluents in Karnataka, India. The effects of carbon and nitrogen sources on protease production by this alkalophilic strain were observed after a 30-h incubation. A high level of alkaline protease activity was obtained in the presence of starch as the carbon and peptone as the nitrogen sources. The partially purified enzyme showed an optimum temperature and pH activity at $58^{\circ}C$ and 10.5, respectively. The enzyme was completely inhibited by PMSF (95.0%) indicating it as a serine protease. It is bleach-stable as it retained 35% original activity in the presence of 10% (v/v) hydrogen peroxide at $30^{\circ}$C after 2 h and is halo-tolerant as it retained 70% original activity in the presence of 2.5 M sodium chloride at $30^{\circ}C$ after 2 h incubation.

This study was carried out in order to investigate the characteristics of xylitol fermentation by a xylitol dehydrogenase defective mutant PXM-4 of P stipitis CBS 5776 and to determime optimum conditions for the high yield ofxylitol production from xylose. Gluconic acid was selected as a co substrate for the xylitol fermentation, since gluconic acid neither blocked xylose transport nor repressed xylose reductase expression. An increase of gluconic acid concentration reduced the rates of xylitol production and cell growth by decreasing medium pH, and the optimal concentration of gluconic acid was determined to be 20 gll with approximately 100% xylitol conversion yield. A fed-batch cell culture resulted in a 44.8 g/l xylitol concentration with 100% yield, based on the amount of xylose consumed.

Thermophilic aerobic bacteria were applied in the degradation of industrial waste activated sludge (WAS) on a laboratory scale expreiment. The performance of digestion was estimated by measuring the reduction of total suspended solids (TSS), dissolved organic carbon (DOC), and total organic carbon (TOC). Among three strains of Bacillus stearothermophilus and three strains of Thermus species, B. stearothemophilus ATCC 31197 showed the best overall efficiency level for the degradation of industrial WAS, which was collected from a wastewater treatment plant in an oil refinery factory. Industrial WAS coul be successfully detraded in a batch digestion with ATCC 31197. The stability of the digestion process with ATCC 31197 was successfully verified by semi-continuous (fill-and-draw) digestion experiment. From the results of this study, it was shown that the thermophilic aerobic digestion process with ATCC 31197 could efficiently be applied to the degradation of industrial WAS.

An antimicrobially active bacterium which was identified as Bacillus brevis, was isolated from kimchi. The antimicrobial activity was found against various Gram-positive and Gram-negative bacteria including some pathogens food-spoilage microorganisms, and some yeast strains. The antimicrobial activity was especially strong against Bacillus anthracis and Shigella dysenteriae. The strong activity was observed during an early stationary phase in the culture when incubated at $37^{\circ}C$ with initial medium pH of 6.8. The antimicrobial activity was found to be stable at $90^{\circ}C$ for 30 min and in the pH range of 3-11, and it was insensitive to organic solvents including acetone, acetonitrile, ethanol, and methanol. Analysis of the bacterocin on tricine-sodium dodecyl sulfate-polyacrylamide gel suggested a molecular mass of approximately 4.5-6.0 kDa. The antimicrobial substance was characterized as a bacteriocin, because of its proteinaceous nature and low molecular weight. The bacteriocin could potentially be used as a food preservative, because of its thermostable property and broad antimicrobial spectrum.

To compare the p10 promoter activity of Bombyx mori nucleopolyhedrovirus (BmNPV)K1 and K4, recombinant viruses Bm101-LacZ and Bm104-LacZ with a lacZ gene under the control of each p10 promoter were constructed. The $\beta$-galactosidase activity due to Bm101-LacZ was about 5.5- and 1.1-fold higher than that due to Bm104-LacZ and BmK1-LacZ, respectively. expressing ${\beta}$-galactosidase under the control of a polyhedrin promoter. The recombinant virus BmK1-104LacZ with the same genome structure as Bm101-LacZ, except for a p10 promoter region, produced a similar ${\beta}$-galactosidase activity to that due to Bm104-LacZ and 5.5-fold lower than that due to Bm101-LacZ. The virus yield, expression level of polyhedrin, and polyhedra productivity for each recombinant virus was almost similar. These results suggested that the difference in the expression level of ${\beta}$-galactosidase resulted from a difference in the p10 promoter regions, and that an expression vector using the p10 promoter of BmNPV-K1 could be usefully exploited in the mass production of foreign proteins with silkworm larvae by means of oral ingestion.

The pyruvate dehydrogenase complex (PDC) catalyzes the oxidative decarboxylation of pyruvate with the formation of $CO_2$, acetyl-CoA, NADH, and H+. This complex contains multiple copies of three catalytic components including pyruvate dehydrogenase(E1), dihydrolipoamide acetyltransferase(E2), and dihydrolipoamide dehydrogenase (E3). Two regulatory components (E1-kinase and phospho-E1 phosphatase) and functionally less-understood protein (protein X, E3BP) are also involved in the formation of the complex. In this study, cloning and characterization of a gene for human E3BP have been carried out. A cDNA encoding the human E3BP was isolated by database search and cDNA library screening. The primary structure of E3BP has some similar characteristics with that of E2 in the lipoyl domain and the carboxyl-terminal domain, based on the nucleotide sequence and the deduced amino acid sequence. However, the conserved amino acid moiety including the histidine residue for acetyltransferase activity in E2 is not conserved in the case of human E3BP. The human E3BP was expressed and purified in E. coli. The molecular weight of the protein, excluding the mitochondrial target sequence, was about 50 kDa as determined by SDS-PAGE. Cloning of human E3BP and expression of the recombinant E3BP will facilitate the understanding of the role(s) of E3BP in mammalian PDC.

A mathematical model is proposed that can depict the kinetics of simultaneous saccharification and fermentation (SSF) using steam-exploded wood(SEW) with a glucose- and cellobiose-fermenting yeast strain. Brettanomyces custersii. An expression to describe the reduction of the relative digestibility during the hydrolysis of the SEW is introduced in the hydrolysis model. The fermentation model also takes two new factors into account, that is, the effects of the inhibitory compounds present in the SEW hydrolysates on the microorganism and the fermenting ability of Brettanomyces custersii, which can use both glucose and cellobiose as carbon sources. The model equations were used to simulate the hydrolysis of the SEW, the fermentation of the SEW hydrolysates, and a batch SSF, and the results were compared with the experimental data. The model was found to be capable of representing ethanol production over a range of substrate concentrations. Accordingly, the limiting factors in ethanol production by SSF under the high concentration of the SEW were identified as the effect of inhibitory compounds present in the SEW, the enzyme deactivation, and a limitation in the digestibility based on the physical condition of the substrate.

The biodegradation rates of diesel oil by a selected diesel-degrading bacterium, Pseudomonas stutzeri strain Y2G1, and microbial consortia composed of combinations of 5 selected diesel-degrading bacterial were determined in liquid and soil systems. The diesel degradation rate by strain Y2G1 linearly increased $(R^2=0.98)$ as the diesel concentration increased up to 12%, and a degradation rate as high as 5.64 g/l/day was obtained. The diesel degradation by strain Y2G1 was significantly affected by several environmental factors, and the optimal conditions for pH, temperature, and moisture content were at pH8, $25^{\circ}C$, and 10%, respectively. In the batch soil microcosm tests, inoculation, especially in the form of a consortium, and the addition of nutrients both significantly enhanced the diesel degradation by a factor of 1.5 and 4, respectively. Aeration of the soil columns effectively accelerated the diesel degradation, and the initial degradation rate was obviously stimulated with the addition of inorganic nutrients. Based on these results, it was concluded that the major rate-limiting factors in the tested diesel-contaminated soil were the presence of inorganic nutrients, oxygen, and diesel-degrading microorganisms. To resolve these limiting parameters, bioremediation strategies were specifically designed for the tested soil, and the successful mitigation of the limiting parameters resulted in an enhancement of the bioremediation efficiency by a factor of 11.

Low-threshold T-type $Ca^{2+}$ channels are distinctive voltage-operated gates for external $Ca^{2+}$ entry around a resting membrane potential due to their low voltage activation. These phenomena have already been extensively studied due to their relevance in diverse physiological functions. Recently, three T-type $Ca^{2+}$ channel ${\alpha}$$_1$subunits were cloned and their biophysical properties were characterized after expression in mammalian expression systems. In this study, ${\alpha_IG} and {\alpha_IH}$ low-threshold $Ca^{2+}$ channels were expressed and characterized in Xenopus oocytes after adding 5' and 3'untranslated portions of a Xenopus ${\beta}$ globin to improve their expression levels. The added portions dramatically enhanced the expression levels of the ${\alpha_IG} and {\alpha_IH}$ T-type channels. When currents were recorded in 10 mM $Ba^{2+}$ as the charge carrier, the activation thresholds were about -60 mV, peak currents appeared at -20 mV, and the reversal potentials were between +40 and +45. The activation time constants were very similar to each other, while the inactivation time constants of the ${\alpha_IG}$ currents were smaller than those of ${\alpha_IH}$. Taken together, the electrophysiological properties of the ${\alpha_IG} and {\alpha_IH}$ channels expressed in Xenopus oocytes were similar to the previously reported characteristics of low-threshold $Ca^{2+}$ channel currents.

n order to increase the virus safety of a human intravenous immunoglobulin (IVIg) that was manufactured by a successive process of cold ethanol fractionation, polyethylene glycol precipitation, and pasteurization ($60^{\circ}C$ heat treatment for 10h), a low pH incubation process (pH 3.9 at $25{\circ}C$ for 14 days) was employed as the final step. The efficacy and mechanism of the fraction III cold ethanol fractionation, pasteurization, and low pH treatment steps in the removal and/or inactivation of blood-borne viruses were closely examined. A variety of experimental model viruses for human pathogenic viruses, including the Bovine herpes virus (BHV), Bovine viral diarrhoea virus (BVDV), Murine encephalomyocarditis virus (EMCV), and Porcine parvovirus (PPV), were selected for this study. The mechanism of reduction for the enveloped viruses (BHV and BVDV) during fraction III fractionation was both inactivation and partitioning, however, it was partitioning in the case of the nonenveloped viruses (EMCV and PPV). The log reduction factors achieved during fraction III fractionation were ${\geqq}$6.7 for BHV, ${\geqq}4.7$ for BVDV, 4.5 for EMCV, and 4.4 for PPV. Pasteurization was found to be a robust and effective step in inactivating all the viruses tested. The log reduction factors achieved during the pasteurization process were ${\geqq}7.5$ for BHV, ${\geqq}4.8$ for BVDV, 3.0 for EMCV, and 3.3 for PPV. A low pH incubation was very effective in inactivating the enveloped viruses as well as EMCV. The log reduction factors achieved during low pH incubation were ${\geqq}7.4$ for BHV, ${\geqq}3.9$ for BVDV, 5.2 for EMCV, and 2.0 for PPV. These results indicate that the low pH treatment successfully improved the viral safety of the final products.

Recombinant E. coli DH5$\alpha$ harboring a dextransucrase gene (dsrB742) produced an extracellular dextransucrase in a 2% sucrose medium. The enzyme was purified by DEAE-Sepharose and Phenyl-Sepharose column chromatographies upto a 142.97-fold purification with a 11.11% recovery to near homogeneity. The enzyme had a calculated molecular mass of 168.6 kDa, which was in good agreement with the activity band of 170 kDa on a nondenaturing SDS-PAGE. An expression plasmid was constructed by inserting the dsrB742 into a pRSET expression vector. The activity after expression in E. coli BL21(DE3)pLysS increased about 6.7-fold compared to the extracellular dextransucrase from L. mesenteroides B-742CB. The expressed and purified enzyme from the clone showed similar biochemical properties (acceptor reaction, size of active dextransucrase, optimum pH, and temperature) to B-742CB dextransucrase, however, the ability to synthesize ${\alpha}$-(1$\rightarrow$3) branching decreased in comparison to that of L. mesenteroides B-742CB dextransucrase.

Bacillus sp. AIR-5, a strain from soil, produced an extracellular maltopentaose-forming amylase from amylose and soluble starch. This bacterium produced 8.9 g/l of maltopentaose from 40 g/l of soluble starch in a batch fermentation and the maltopentaose made up 90 % of the maltooligosaccharides produced (from maltose to maltoheptaose). The culture supernatant was concentrated using a 30 K molecular weight cut-off membrane and purified by DEAE-Cellulose and Sephadex G-150 column chromatographies. The purified protein showed one band on a native-PAGE and its molecular mass was estimated as 250 kDa. The 250-kDa protein was composed of tetramers of a 63-kDa protein. the isoelectric point of the purified protein was pH 6.9, and the optimum temperature for the enzyme activity was $45^{\circ}C$. The enzyme was quickly inactivated above $55^{\circ}C$, and showed a maximum activity at pH 8.5 and over 90% stability between a pH of 6 to 10. The putative N-terminal amino acid sequence of AIR-5 amylase, ATINNGTLMQYFEWYVPNDG, showed a 96% sequence similarity with that of BLA, a general liquefying amylase.

The biological control potential of entomopathogenic nematodes (EPN) can be enhanced by improved culture efficiency. Optimization of the media is a key factor for improving in vitro mass production of entomopathogenic nematodes. This study reports the effect of medium concentration. The medium is a combination of carbohydrates, lipids, proteins, sats, and growth factors, on the growth of Heterorhabditis bacteriophora and its symbiotic bacterium Photorhabdus liminescens. The overall optimal medium concentration for nematode recovery, hermaphrodite size, bacterial mass, infective juveniles (IJs) yield, and doubling time was 84 g/l. At this concentration rate, the doubling time of IJs production and the biomass of symbiotic bacteria was 1.6 days and 12.8 g/l, respectively. The maximum yield of $2.4{\times}{10^5}IJs/ml$ was attained within a one-generation cycle (eight days). The yield coefficient was $2.8{\times}{10^6}$ IJs/g medium, and the maximum productivity was $3.1{\times}{10^7}$ IJs per day. Medium concentration affected two independent factors, recovery and hermaphrodite size, which in turn influenced the final yield.

The kinetics of the thermolysin-catalyzed synthesis of N-(benzyloxycarbonyl)-L-phenylalanyl-L-leucine ethyl ester (Z-Phe-LeuOEt) from N-(benzyloxycarbonyl)-L-phyenylalanine (Z-Phe) and L-leucine ethyl ester (LeuOEt) in an ethyl acetate saturated aqueous system in a batch operation were studied. The kinetics for the synthesis of Z-Phe-LeuOEt were expressed using a rate equation for the rapid equilibrium random bireactant mechanism. The four kinetic constants involved in the rate equation were determined numerically by the quasi-Newton method so as to fit the calculated results with the experimental data. Within the pH and temperature range examined, the $K_{cat}$ value for the synthesis of Z-Phe-LeuOEt reached a maximum at pH 7.0 and $45^{\circ}C$, whereas the affinity between Z-Phe and thermolysin reached a maximum at pH 6.0 adn $40^{\circ}C$. The inhibitory effect of Z-Phe on the condensation reaction decreased as the pH and temperature decreased. In contrast, they affinity between LeuOEt and thermolysin remained unchanged within the pH and temperature range examined. Therefore, it was concluded that the protonation state of the carboxyl groups. of Z-Phe was more imprtant than that of the amono groups of LeuOEt for the synthesis of Z-Phe-LeuOEt in the present solvent system. The equilibrium yield at pH 6.0 and $30^{\circ}C$ was 8% higher than that at pH 7.0 and $40^{\circ}C$, although the rate was much slower. This result suggested that the affinity between the enzyme and the substrate rather than the overall rate was a more important factor affecting the equilibrium yield, when the peptide synthesis was carried out in a product-precipitation system.

Angiogenesis is known as a crucial process in the growth and spreading of tumor cells. Accordingly, the effective inhibition of this process would appear to be a promising way to cure angiogenesis-related diseases, including cancer. This study demonstrates that acalycixenolide E (AX-E) from the marine organism Acalycigorgia inermis exhibits a potent anti-angiogenic activity both in vitro and in vivo. AX-E inhibits the bFGF-induced proliferation of HUVECs in a dose dependent manner, along with the bFGF-induced migration, invasion, and tube formation of HUVECs. Moreover, AX-E potently inhibits the in vivo neovascularization of the chorioallantoic membranes (CAMs) of growing chick embryos. interestingly, AX-E suppresses the expression of metalloproteases 2 and 9, yet shows no effect on their activities. The novel chemical structure and potent anti-angiogenic activity of AX-E will be of great value in elucidating the molecular mechanism of angiogenesis as well as in the development of a novel anti-angiogenic drug.

A new secondary metabolite was isolated from the culture broth and mycelium of Streptomyces sp. collected from marine sediment. The structure of this compound was determined to be N-formylantimycic acid methyl ester, an acyclic derivative of antimycin, on the basis of combined chemical and spectral methods. The structure-activity relationship of antimycins is discussed.

Pseudomonas sp. strain SY5 is a PCB-degrading bacterium [24] that includes two different enzymes (BphC1 and BphC2) encoding 2,3-dihdroxybiphenyl 1,2-dioxygenase and BphD encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase. The bphC1 and bphC2 genes were found to consist of 897 based encoding 299 amino acids and 882 bases encoding 294 amino acids, respectively, whereas the bphD gene consisted of 861 bases encoding 287 amino acids. According to a homology search, a 50% and 39% similarity between the bphC1 and bphC2 genes at the nucleotide and amino acid level was shown, respectively. The bphC1 gene showed a 38% and 45% similarity at the amino acid level to Alcaligenes eutrophus A5 and Rhodococcus rhodochrous, respectively, whereas, bphC2 showed a 95% and 43% similarity, respectively. A comparison of the deduced amino acid sequence of the bphD product of Pseudomonas sp. SY5 with that of A. eutrophus A5, Pseudomons sp. KKS102, and LB400 showed a sequence identity of 92, 92, and 79%, respectively. Strain SY5 was originally isolated from municipal sewage containing recalcitrant organic compounds an found to have a high degradability of various aromatic compounds [23]. The current study found that strain SY5 had two extradiol-type dioxygenases, which did not hybridize with each other as they had a low similarity, yet a similar structure of evolutionarily conserved amino acids residues for catalytic activity between BphC1 and BphC2 was observed.

To isolate mutanse-producing bacteria, soil samples were collected from several areas in chonnam Province, South Korea. A total of 70 strains of actinomycetes were isolated from the soil samples. All isolated actinomycetes were inoculated on mutanase screening media to identify new bacterial strains producing mutanase activity. One strain in particular exhibited a strong mutanase-producing activity, and was identified as Microbispora rosea based on its morphological, cultural, and physiological characteristics, and also by 16S rDNA sequences.

A gene, Egl, from Paenibacillus sp. KCTC 8848P encoding endo-${\circ}$-1,4-glucanase was cloned and expressed in Escherichia coli. It consisted of an open reading frame of 1,191 bp for a protein that consisted of 397 amino acids with a molecular weight of 44,539 Da. The deduced amino acid sequence of the endo-${\circ}$-1,4-glucanase gene had a 94% similarity to the endo-$\beta$-1,4-glucanase of Bacillus polymyxa. The Egl gene was also expressed in Saccharomyces cerevisiae secreting Endomyces fibuliger $\beta$-glucosidase (BGL1) under the control of the alcohol dehydrogenase (ADC1) gene promoter, S. cerevisiae transformant producing both endo-${\circ}$-1,4-glucanase and ${\circ}$-glucosidase grew on carboxymethyl cellulose as the sole carbon source.

An intracellular levansucrase gene, lscR from Rahnella aquatilis ATCC 15552, was cloned and its nucleotide sequence was determined. Nucleotide sequence analysis of this gene revealed a 1,238 bp open reading frame coding for a protein of 415 amino acids. The levansucrase was expressed by using a T7 promoter in Escherichia coli BL21 (DE3) and the enzyme activity was detected in the cytoplasmic fraction. The optimum pH and temperature of this enzyme for levan formation was pH 6 and $30^{\circ}C$, respectively. The deduced amino acid sequence of the lscR gene showed a high sequence similarity (59-89%) with Gram-negative levansucrses, while the level of similarity with Gram-positive enzymes was less than 42%. Multiple alignments of levansucrase sequences reported from Gram-negative and Gram-positive bacteria revealed seven conserved regions. A comparison of the catalytic properties and deduced amino acid sequence of lscR with those of other bacterial levansucrases strongly suggest that Gram-negative and Gram-positive levansucrases have an overall different structure, but they have a similar structure at the active site.

The influence of process conditions on highly branched glucooligosaccharides production by mixed culture of Leuconostoc mesenteroides ATCC 13146 and Lipomyces starkeyi ATCC 74054 was studied. We divided the batch culture fermentations into two groups according to inoculation method. One-point inoculation was performed by coinoculation of L. mesenteroides and L. starkeyi at the ration of 10 to 1, and two-point inoculation by L. mesenteroides inoculation first and L. starkeyi inoculation after L. mesenteroides grew to the end of the log phase of growth. Two-point inoculation improved the yield of oligosaccharide by 1.5 to 20 fold more than one-point inoculation. In this process, the highest yield of oligosaccharides (48% of theoretical yield) and productivity (0.85 g/l/h) were obtained with starch as an initial substrate for L. starkeyi growth. The estimated composition of the end product consisted of 31.5% oligosaccharides, 17.6% dextran, and 46.5% mannitol.

The effect of nutrient nitrogen on the degradation of pentachlorophenol (PCP) by Phanerochaete chrysosporium in a liquid culture was investigated. PCP disappeared at almost the same rate in both nutrient nitrogen-sufficient (NS) and -limited (NL) sttionary cultures. However, more pentachloroanisole (PCA) was accumulated in the NS culture than in the NL culture. The effect of nitrogen on the degradation of PCA was also tested in both cultures. PCA disappeared faster in the NL culture than in the NS culture, indicating that the lower accumulation of PCA during the degradation of PCP in the NL culture was due to the faster degradation of PCA in the NL culture than in the NS culture. In another experiment, PCA was added to shaking cultures rather than stationary cultures to search for any other metabolite(s). While no other metabolite but PCA was found in the NS stationary culture, 2,4,5,6-tetrachloro-2,5-cyclohexadiene-1,4-dione(TCHD) was found as the only indentifiable product in the NL shaking culture. Thus, PCP would appear to be metabolized to TCHD via PCa or directly oxidized to TCHD by lignin peroxidase. Since all the above results indicate that no innocuous metabolite was formed during the degradation of PCP by the fungus, it is quite feasible to use the fungus in the biotreatment of PCP.

To elucidate the putative role of the amido group in the metal binding of the fuculose-1-phosphate aldolase from Methanococcus jannaschii, we have examined a potential targen using site-directed mutagenesis. The replacement of asparagine 25 with leucine or threonine was shown to have a negative effect, not only on catlytic efficiency, but also on substrage recognition as well. The Hill coefficient values yeilded a value of =1. All metals used with the wild-type aldolases exhibited higher activity than that of the mutants. The spectra of the mutants were quite different from the wild-type aldolase. A highly conserved amino acid of asparagine 25 in a related family of aldolase odes not appear to provide sufficient evidence for evolution.

A new combined method including the enzymatic hydrolysis of $\beta$-cyclodextrin ($\beta$-CD) and solvent extraction fo cholesterol from the hydrolyzed mixture was developed to recover cholesterol from a $\beta$-CD-cholesterol complex prepared from dairy products, such as cream, milk, and cheese. Cyclomaltodextrinase (cyclomatodextrin dextrin hydrolase, EC 3.2.1.54, DCase_ prepared form alkalophilic Bacillus sp. KJ 133 hydrolyzed the $\beta$-DC of the $\beta$-CD-cholesterol complex, and then, free cholesterol was efficiently extracted from the hydrolyzed mixture by a nonpolar solvent such as ethyl acetate. To increase the stability of free CDase, immobilized CDase was developed using sodium alginate as a carrier. The immobilized CDase showed a high recovery yield of cholesterol in a time-dependent manner compared to the free CDase. A gas chromatography analysis showed that more than 70% of cholesterol was recovered from the $\beta$-DC-cholesterol complex of cream by the immobilized CDase, whereas only 3% and 29% of cholesterol were recovered when the solvent extraction and free CDase treatment were used, respectively. The cholesterol recovered can be used as a raw material for steroid synthesis. Furthermore, this method can be an efficient way to recover cholesterol or other organic compounds that are bound in a $\beta$ -DC-cholesterol or -organic compound complex.

The mobilization and biodegradation of phenanthrene in soil was enhanced by using paraffin oil, which was stabilized by the addition of a surfactant (Brji 30). The ratio of paraffin oil/Brij 30 was determined by measuring the change in the critical micelle concentration. When only surfactant was used, the stabilized paraffin oil emulsion could dissolve more phenanthrene in the water phase. Column experiment showed increased phenanthrene mobilization from the contaminated soil. The phenanthrene mobilized in the paraffine oil/Brij 30 emulsion was biodegraded faster than that in water phase or surfactant solution. This result indicates that a paraffin oil/surfactant system can be effectively used for the removal of PAH from contaminated soil.

The effect of long-term storage on garlic antibacterial activity was investigated. A concentration of 5% or more garlic was found to be necessary to completely inhibit Eschrichia coli growth in tryptic soy broth. This value is substantially higher than the minimum inhibitory concentration of 1% for E. coli reported previously. pH-modified garlic extract was stored at different temperatures to investigate the impact of storage conditions (i.e., temperature, pH, period of storage) on the stability of the antibacterial activity of the garlic extract used against E. coli B34. The antibacterial effectiveness of the garlic extract against E. coli remained stable when both the storage temperature and the pH of the extract were kept low. When the garlic extract was stored at $40^{\circ}C and above, most or all of the garlic antibacterial activity disappeared after a 24-h storage period, regardless of the storage pH. The antibacterial activity was weakened when the pH of the garlic extract was adjusted to 8, and at low temperatures.

The growth of baceriophage T4 is inhibited by the presence of the tin gene product o bacteriophage P2. The interaction between purified Tin and gp32 proteins was observed using coimmunoprecipitation experiments. The in vivo interaction was confirmed by yeast two-hybrid experiments. A deletion analysis showed that the Asp 163 region of gp32 to DNA substrates was not affected by the presence of Tin, Thus, it would appear that the inhibition of 4 growth by Tin was due to a protein-protein interaction rather than affecting the DNA-binding ability of gp32.