In digital ASICs with predefined algorithms, the optimal word length can be defined for each internal operator, bus and register, based on the accuracy needed at the ASIC outputs. If optimal word lengths are used, rather than merely choosing 8, 16 or 32 bits, then considerable silicon area may be saved. This paper describes methods of optimizing these word lengths in a form suitable for use by a behavioral circuit-synthesis program. The estimates are based on methods used in control systems and digital filters. Methods are described for circuits which may have nonlinearities but do not contain nonlinearities within feedback loops
The partitioning methods for placement work by allocating cells to partition groups. The groups are then further partitioned until the number of cells allocated to each group is small enough to be handled efficiently by other placement methods. The authors introduce extended channel constraint (ECC) graphs, a concept extended from Lauther's channel constraint graphs. The ECC graphs lead to a methodology for compacting and reorganizing dead space generated from the partitioning methods
A bacterium capable of assimilating 3-chloro-1,2-propanediol was isolated from soil by enrichment culture. The strain was identified as Alcaligenes sp. by taxonomic studies. The crude extracts of the cells had dehalogenating activities and converted various halohydrins to the corresponding epoxides. 3-Chloro-1,2-propanediol was degraded stereospecifically by the strain, liberating chloride ion. The residual isomer was found to be the (S)-form (99.4% enantiomeric excess). (S)-3-Chloro-1,2-propanediol was obtained from the racemate by use of this strain in 38% yield, and (S)-glycidol (99.4% enantiomeric excess) was subsequently synthesized from the obtained (S)-3-chloro-1,2-propanediol by alkaline treatment.
We studied oxidative resolution with bakers' yeast for the large-scale preparation of chiral 1,2-alkanediol. First, we estimated the outline of the enantioselective oxidation, especially in relation to oxygen transfer, from the transformation of (R)-1,2-propanediol to acetol. In this yeast-mediated oxidation, we found that 1.7 mol of acetol was produced with consumption of 1 mol of oxygen (85% efficiency). The oxidation rate per gram of baker' yeast was correlated to the product of kLa, controlled by changing the concentration of bakers' yeast and/or the agitation rate, and po2. It increased with increasing kLa·po2, then became constant at a value above 100 atm/h. Treatment of racemic 1,2-propanediol,-butanediol, or -pentanediol in a 19-l bubble-column reactor afforded mixtures of the corresponding (S)-1,2-alkanediols with about 79% enantiomeric excess and the corresponding 1-hydroxy-2-alkanones. The latter were easily separated as an aqueous solution, that was directly used in the bakers' yeast-mediated bioreduction to prepare (R)-1,2-alkanediols.
A novel generation method of optically active 1,2-diols with excellent optical purity (>98% enantiomeric excess) from their racemates was developed using the resting cells of Alcaligenes sp. DS-S-7G. This method was based on microbial enantioselective oxidation of the racemic 1,2-diols. When 1% (v/v) 1,2-butanediol was used as a substrate, the degradation reaction in the presence of NAD+ could be carried out 8 times within 200 h.
An acid exo-β-1,3-d-glucanase was isolated from the commercial digestive enzyme Molsin prepared from Aspergillus saitoi. The enzyme had a molecular weight of 57,000, isoelectric point of pH 4.1, optimum pH of 3.8 and an optimum temperature of 50°C. Major amino acid components of the enzyme were Asx, Ser, Thr and Gly which comprised more than 60% of the molecule, while 11% was carbohydrates. The N-terminal amino acid was determined to be Gly. The enzyme acted on the β-1,3-d-glucan (Pachyman) or laminaritetraose in an exo-type manner and the hydrolysis product was α-d-glucose.
A β-1,3-glucanase from the culture supernatant of Oerskovia xanthineolytica TK-1 grown on minimal medium containing yeast glucan as the carbon source was purified to an electrophoretically homogeneous state by means of chromatography using DEAE-Sephacel, DEAE-Toyopearl 650 M and Bio-Gel P-2 column. The molecular mass was 40,000 Da and the pI was 6.5. The optimum pH was 7.5 and 5.5 when assayed on laminarin and yeast glucan, respectively. The enzyme had an affinity for yeast glucan and could lyse living yeast cells without the need for a second lytic component, an alkaline protease. Hydrolysis of yeast glucan was endolytic, yielding a mixture of glucose and biose. The N-terminal amino acid sequence of the enzyme showed no homology with that of a related β-1,3-glucanase from the same genus.
Endoglucanases, EGI and EgI, were produced from the same Ruminococcus albus gene in R. albus and recombinant Escherichia coli, respectively. EGI was purified from R. albus culture supernatant and EgI was extracted from the transformant E. coli (JM101/pURA1) and purified. The purified enzymes EGI and EgI revealed maximum endoglucanase activity at a same pH of 6.8 and a temperature of 37°C. Both enzymes were stable at temperatures below 30°C. In addition, about 10% of their original activities were conserved even after boiling for 10 min. Amino acid sequences of both enzymes at the N-terminal (Ala-Ala-Asp-Glu-Ser-Glu-Thr-Glu-Asn-Val-Pro-Val-Ser-Gln-Thr-His--) were consistent with each other. The antiserum against EgI reacted with both EgI and EGI, indicating that both their protein moieties were the same immunologically. However, the molecular size of EGI (43,000) was larger than that of EgI (39,000) due to the presence of sugar moiety. The specific activity (54 units/mg) of EGI was almost double that (27 units/mg) of EgI. EGI was immunologically different from the endoglucanase purified in the previous paper [Ohmiya et al.: Carbohydrate Res., 166, 145–155 (1987)].
The nucleotide sequence of a DNA fragment containing an endo-1,4-β-glucanase (EG-1) gene of Clostridium josui was determined by the dideoxy-chain termination method. The EG-1 coding sequence was an open reading frame encoding 369 amino acids, and a putative promoter sequence was located in the upstream region of the open reading frame. The N-terminal amino acid sequence of the endoglucanase (EG-1C) purified from the Escherichia coli transformant (JM103/pUCJ1) was consistent with the deduced sequence from 30Val to 44Lys. The estimated molecular weights of the precursor and the mature enzymes were 41,774 and 38,352, respectively. The region of amino acids from 61st to 335th of the enzyme revealed high homology with those of Bacillus sp. and Clostridium acetobutylicum endoglucanases.
The Michaelis constant (Km) and molecular activity (k0) of an exo-1,4-β-glucosidase (EC 22.214.171.124) from Acetobacter xylinum subsp. sucrofermentans BPR2001 for hydrolysis of cello-oligosaccharides (G2–G6) were determined by steady-state kinetic analysis. The and k0 values for G2 were much lower than those for G3–G6. The enzyme was competitively inhibited by glucono-δ-lactone and conduritol-β epoxide. Based on the theory of Hiromi et al. (Biochim. Biophys. Acta, 302: 362–375, 1973), the subsite affinities (Ai, i=1–6) and the intrinsic hydrolysis rate constant for substrate linkage in a productive complex (kint) of the enzyme were kinetically estimated: A1=2.46 kcal/mol, A2=−0.44 kcal/mol, A3=3.70 kcal/mol, A4=0.33 kcal/mol, A5=0.27 kcal/mol, A6=0.06 kcal/mol, and kint=33.4 s−1. The subsite affinities were different from those for the β-glucosidase from Aspergillus niger and for the exo-1,4-β-glucosidase from Torulopsis wickerhamii, in that the A2 value for the Acetobacter enzyme was negative. These results suggest that the enzyme possesses subsite affinities which have never previously been reported among exo-type glucosidases.
The gene encoding an endo α-1,4 polygalactosaminidase (P-GalNase) from Pseudomonas sp. 881 was cloned into Escherichia coli DH1 and MV1184 by using charomid 9–36 and a pUC vector, and then sequenced. The gene encoding the enzyme P-GalNase consisted of 882 bp encoding 294 amino acid residues including a leader sequence. The deduced amino acid sequence did not show any extensive homology with sequences of other glycanases listed in the SWISS-PROT (GenBank) data bank, but we could observe some homology between the middle segment of the P-GalNase sequence and the conserved sequences of chitinases and related glycosidases.
Cyclic α-1,4-glucan formation from synthetic amylose by hydroquinone glucosylating enzyme (HGE), which is a saccharifying α-amylase, was investigated. Upon analysis of reaction products from synthetic amylose, glucans which were not hydrolyzed by glucoamylase were detected with HPAEC in reaction mixtures. The glucans were hydrolyzed by HGE to maltooligosaccharides and hydrolyzed to glucose by the combination of HGE and glucoamylase. From these results, these glucans might be considered to be cyclic α-1,4-glucans. In order to demonstrate that these glucans were cyclic α-1,4-glucans, phenol-sulfate test, Somogyi-Nelson test, and tritium labeling test of reducing ends were conducted. From results of these tests, it was confirmed that these compounds were saccharides and did not have reducing ends. Furthermore, the molecular weight of each glucan was determined using TOF-MS spectrometry. Each molecular weight agreed with that of cyclic glucans theoretically calculated. One of these glucans was purified and was identified to be γ-cyclodextrin. These results demonstrate that HGE formed cyclic α-1,4-glucans. Cyclic α-1,4-glucans were also detected in the reaction mixtures of bacterial saccharifying α-amylase and bacterial liquefying α-amylase.
The nucleotide sequence of the ce1B gene coding for endo-1,4-β-glucanase-2 (EG-2) of Clostridium josui was determined. The structural gene consists of an open reading frame of 1,380 by encoding a 460-amino acid polypeptide. The deduced amino acid sequence of EG-2 shows high similarities with the Clostridium cellulolyticum CelCCC (92.3%) and the Clostridium thermocellum endoglucanase CelA (61.6%) in the catalytic domain, but no homology with EG-1 of C. josui. In the COOH-terminal region of EG-2, there exists a reiterated segment similar to the segments present in many clostridial cellulases and hemicellulases. Upstream and downstream of the celB gene, two incomplete open reading frames (ORF1 and ORF2) were found to encode proteins homologous to the proteins which consist of the C. cellulolyticum cellulase gene cluster. ORF1 containing a reiterated segment showed high similarities with the proteins deduced from each ORF1 from C. cellulolyticum and Caldocellum saccharolyticum. Downstream of the ce1B gene, ORF2 was found to encode a protein homologous to CeICCG of C. cellulolyticum and CelF of C. thermocellum. These results indicate that the celB gene, ORF1 and ORF2 formed a cellulase gene cluster strictly homologous to that of C. cellulolyticum, which is proposed to be part of a single operon.
A hyperthermophilic archaeon Thermococcus profundus produced two α-amylases, S and L. α-amylase L was purified to homogeneity and characterized in the present study. The enzyme hydrolyzed not only α-1,4 linkages of amylose and starch, but also α-1,6 linkages of pullulan. It exhibited maximal activity towards both starch and pullulan at pH 5.5. The temperatures for optimal amylolytic and pullulolytic activities were 80°C and 90°C, respectively, and the half-lives for these activities at 90°C in the presence of 5 mM Ca2+ were 4 h and 6.7 h, respectively. The enzyme activity was totally inhibited by 1 mM N-bromosuccinimide. The molecular mass of the enzyme was estimated to be 43,000 Da by SDS-polyacrylamide gel electrophoresis. α-Amylase L hydrolyzed soluble starch, amylose, amylopectin and glycogen to yield glucose and maltose as the predominant products. The amylase also hydrolyzed pullulan and panose to yield the same products.
The gene encoding an endo-1,4-β-glucanase from Ruminococcus albus F-40 was cloned in Escherichia coli JM109 using pBR322. The nucleotide sequence of the 1,798 bp PstI-PvuII fragment which includes a cellulase gene was determined. There was a single open reading frame (ORF) consisting of 936 bp encoding a peptide of 312 amino acid residues with a molecular weight of 35,766. The N-terminal amino acid sequence determined for the enzyme expressed in E. coli was identical to that deduced from the beginning of the ORF. A putative ribosome-binding site and a promoter were located upstream of the ORF. Activity was expressed from this fragment when it was subcloned in both orientations in pUC118 and pUC119, indicating that its own promoter functioned in E. coli. The amino acid sequence of the endoglucanase deduced from the nucleotide sequence showed 44% homology with CelA from Butyrivibrio fibrisolvens A46, suggesting that this enzyme was a member of family A2. The enzyme purified from E. coli exhibited the highest activity against carboxymethyl cellulose (CMC) at 40°C and pH around 7.0.
An endo-xylanase (1,4-β-d-xylanxylanohydrolase EC 126.96.36.199) was isolated from the culture filtrate of Paecilomyces varioti Bainier. The enzyme was purified 3.2 fold with a 60% yield by gel filtration and ion exchange chromatography. The purified enzyme had a molecular weight of 25,000 with a sedimentation coefficient of 2.2 S. The isoelectric point of the enzyme was 3.9. The enzyme was obtained in crystalline form. The optimum pH range was 5.5–7.0 and the temperature, 65°C. The Michaelis constant was 2.5 mg larchwood xylan/ml. The enzyme was found to degrade xylan by an endo mechanism producing arabinose, xylobiose, xylo- and arabinosylxylo-oligosaccharides, during the initial stages of hydrolysis. On prolonged incubation, xylotriose, arabinosylxylotriose and xylobiose were the major products with traces of xylotetraose, xylose and arabinose.
Microbial production of (2S,3S)-2,3-dihydro-3-hydroxy-2-(4-methoxyphenyl)-1,5-benzothiazepin-4(5H)-one [(2S,3S)-2], a key intermediate in the synthesis of diltiazem hydrochloride, was examined. The reduction of (RS)-2-(4-methoxyphenyl)-1,5-benzothiazepin-3,4(2H,5H)-dione [(RS)-1] by various microorganisms produced a mixture of stereoisomers with the (2S,3S) stereoisomer being the major product. Among the microorganisms tested, baker's yeast afforded the highest conversion yield and excellent stereoselectivity for the production of (2S,3S)-2. When asymmetric reduction using baker's yeast was carried out at the substrate concentration of 3.34 mM with the addition of N,N-dimethylformamide solution containing 0.0334–0.334 M (RS)-1 to the reaction mixture, the reaction gave (2S,3S)-2 with a conversion yield of 92–94% and an optical purity of >99.9% enantiomeric excess. Crystals of (2S,3S)-2 were isolated with a total yield of 85%. 3-Hydroxy-2-(4-methoxyphenyl)-1,5-benzothiazepin-4(5H)-one S-oxide [oxide-1] and (2RS,3SR)-2 were formed as by-products in the reaction mixture. The optimum conditions for (2S,3S)-2 production containing a minimum amount of by-products were pH 7.0 and 42°C. The decrease of the conversion yield of (2S,3S)-2 was attributable to the conversion to oxide-1 with increasing substrate concentration. It was concluded that the production method of diltiazem hydrochloride using microbial asymmetric reduction was an excellent method from the viewpoint of yield.
A strain (strain IsBd1) of anaerobic bacterium was isolated in pure culture from a sand sample of Lake Kasumigaura using 1,4-butanediol as a sole electron-donor source, and was characterized. The bacterium was motile with a single polar flagellum and stained gram-negative. Spores were never observed. Sulfate, sulfite, thiosulfate, and elemental sulfur were reduced with concomitant growth. Even in the presence of excess sulfate, 1,4-butanediol and 1,5-pentanediol were oxidized stoichiometrically to 4-hydroxybutyrate and 5-hydroxyvalerate, respectively; 2-propanol and 2-butanol were oxidized stoichiometrically to corresponding ketones. Interspecies hydrogen transfer occurred between the isolate and Methanospirillum hungatei with ethanol, 1,3-butanediol, 1,4-butanediol, 1-butanol, 2-butanol, 2-methoxyethanol, 1,5-pentanediol, 1,3-propanediol, 1-propanol, and 2-propanol. Chemolithoheterotrophic growth was possible with formate or hydrogen and acetate in the presence of sulfate. Acetoin was fermented to acetate, 2,3-butanediol, and ethanol. Fumarate and l-malate were fermented to acetate and succinate. S-1,2-Propanediol was fermented to 1-propanol and propionate, but a racemic mixture was not fermented. When formate or hydrogen was supplied, fumarate and l-malate were reduced stoichiometrically to succinate with growth. The DNA base ratio of the isolate was 53 mol% guanine plus cytosine. Strain IsBd1 was identified as a member of the genus Desulfovibrio.
Mutations were introduced at residues His607, Asp677, His682, and His833 in pullulanase from Klebsiella aerogenes in order to probe the role of these amino acid residues, which are located in the four conserved regions of the α-amylase family, in the action of the enzyme towards α-1,6-glucosidic linkages. For the mutations, His was replaced by Asn and Ala, and Asp by Asn and Ser. Amino acid substitutions for His607, Asp677, or His833 resulted in complete loss of enzyme activity. In contrast, the mutations at His682 still retained their activities. The binding affinity of these variants for α- or β-cyclodextrine (CD), which are competitive inhibitors for pullulanase, was measured using an α-CD Sepharose column. The mutations at His833 did not change the binding affinity for α-CD, whereas the mutations at His607 or Asp677 resulted in these two variants losing their binding ability towards pullulan. These results suggest that in Klebsiella pullulanase, His607 and Asp677 participate in substrate binding and His833 is involved in catalysis, but His682 may be not in the active site. We also found new amino acid consensus sequences specific for starch debranching enzymes in two oligo-1,6-glucosidases, several pullulanases, and an isoamylase. Two amino acid residues in the predicted consensus region of Klebsiella pullulanase, Tyr559 and Tyr564, were replaced by Ala or Phe. The Tyr559 variants resulted in complete loss of pullulanase activity without seriously affecting the binding affinities for α-CD and pullulan. The mutations at Tyr564 did not completely inactivate the enzymes, but dramatically decreased the activity. Thus, the region in Klebsiella pullulanase that includes Tyr559-Tyr564 probably participates in catalysis specific towards α-1,6-glucosidic linkages in starch debranching enzymes.
The aim of this study was to find a specific DNA probe for the detection of Salmonella in foods. A 1.8 kb HindIII DNA fragment cloned from S. typhimurium was tested for its specificity by hybridization to Salmonella of different serotypes as well as to non-Salmonella bacteria. The 35S-labeled HindIII DNA fragment hybridized with all of the common and uncommon Salmonella isolates but did not hybridize with the other bacteria tested. Hybridization efficiencies of this 1.8 kb DNA probe to Salmonella strains of different serotypes were nearly the same. When this probe was applied to the detection of Salmonella exogenously added to a variety of foods, bacterial flora naturally occurring in foods did not interfere with the hybridization assay. Thus, the hybridization specificity of this 1.8 kb DNA probe was established.
A 3-kbp DNA fragment including the l-2,3-butanediol dehydrogenase (l-BDH) gene (budC) from the chromosomal DNA of Brevibacterium saccharolyticum C-1012 was cloned in Escherichia coli JM109 after its insertion into pBluescript II SK+, and the resulting plasmid was named pLBD-SK. The budC had an open reading frame consisting of 774 bp and encoded 258 amino acids. It was not included in a 2,3-butanediol operon such as is seen in the case of the meso-BDH gene (budC) of Klebsiella pneumoniae. For the expression of the budC, the deletion plasmid pLBD2-119 was prepared from pLBD-SK. E. coli JM109/pLBD2-119 had higher l-BDH activity than that of Br. saccharolyticum C-1012. The l-BDH appeared as two bands on disc-PAGE. Isopropyl-β-d-thiogalacto-pyranoside (IPTG) influenced the quantity radio of the electrophoretic isoenzymes of l-BDH from E. coli JM109/pLBD2-119 that is, a higher relative mobility band with weak substrate specificity was abundantly produced by IPTG. The BDH was considered to belong to the short-chain dehydrogenase/reductase (SDR) family on the basis of the following distinctive features: it possessed two conservative sequences GXXXGXG and YXXXK, and it consisted of about 250 amino acids. As a result of a phylogenetic analysis of SDR family enzymes, the BDHs were considered to comprise a cluster independent from the other SDR enzymes.
A microorganism, strain M 102, capable of degrading aspergillic acid (AA), was first isolated from a soil sample in a drainage ditch and was identified as Trichoderma koningii Oudemans. This fungus degraded AA, but not hydroxyaspergillic acid (HAA) or deoxyaspergillic acid (DAA). The AA-degrading ability of M 102 was induced by incubation with AA but not with HAA or DAA. AA-degradation activity was found in a crude enzyme prepared from the mycelia induced by AA; this AA degradation reaction required NAD(P)H and oxygen.
Isolation and identification of the degradation products of aspergillic acid, a secondary metabolite of fungi, by Trichoderma koningii M 102 were undertaken. 14C-labeling experiments indicated that aspergillic acid was broken-down to a water-soluble degradation product and a chloroform-soluble one. Consequently, leucine and a new microbial metabolite, 2-hydroxyimino-3-methyl-1-pentanol, were isolated and identified as the degradation products formed by cleavage of the pyrazine ring of aspergillic acid by T. koningii M 102.
A microorganism that produces d-aspartate-oxidizing enzyme by induction was isolated from soil, and identified as Fusarium sacchari var. elongatum Y-105. The enzyme catalyzed the oxidative deamination of d-aspartate (d-Asp) and produced oxaloacetate, ammonia, and hydrogen peroxide, stoichiometrically. The enzyme is designated “d-Asp oxidase” (EC 188.8.131.52). In addition to d-Asp, the enzyme oxidized d-glutamate (d-Glu) and (NMDA). and other d- or l-amino acids, however, were inert as substrates. The optimum pH and temperature were 7.5 and 40°C, respectively. The enzyme was stable at pH 9.0 and temperature of 50°C, respectively. The enzyme activity was not inhibited by sodium benzoate which is a specific inhibitor of d-amino acid oxidase from mammals. The enzyme activity was also not affected by carboxylates such as meso- or d-tartarate, citrate, and fumarate which inhibit d-Asp oxidase from rabbits.
A novel lipase (lactonizing lipase: lipL) from Pseudomonas sp. 109 can catalyze synthesis of macrocyclic lactones in anhydrous organic solvents by transesterification. A gene situated immediately downstream from lipL was found to be essential for production of the functional lipase, and was denoted as limL (lipase modulator). Nucleotide sequence analysis of the limL region revealed a 340-amino acid open reading frame (Mr=37,658) 49 bp downstream from lipL. In the non-coding region between lipL and limL was situated a 32-base strong inverted repeat, and a putative ribosome binding site was present at 14 bases upstream from the limL initiator methionine. The deduced amino-acid sequence of LimL exhibited 29% overall homology with that of the lipase-modulator protein (LimA) of Pseudomonas cepacia. Although the homology itself was low, hydrophobicity/hydrophilicity plots of the two modulator proteins showed that the two proteins shared a similar hydropathy pattern, i.e., the amino-terminus region is highly hydrophobic and the carboxyl-terminus region is strongly hydrophilic, suggesting that the two modulator proteins may activate corresponding lipase by similar functions. When lipL and limL were cloned separately in compatible plasmids, functional lipase was produced only in E. coli hosts harboring both the plasmids, indicating that limL can act in trans toward lipL.
A gene encoding chitinase II from Aeromonas sp. no. 10S-24 was cloned into Escherichia coli DH5 a by using pUC19 and its nucleotides were sequenced. The structural gene consisted of 1626 bp encoding 542 amino acid residues with a characteristic signal peptide. A typical promoter sequence and Shine-Dalgarno (SD) region were located upstream of the initiation ATG codon. The deduced amino acid sequence of the cloned chitinase II showed sequence homology with chitinase from Saccharopolyspora erythraea (26%). The chitinase II had Pro-Thr- rich domains, consisting of repeat sequences of 47 amino acids, that are not conserved in other chitinases from bacteria. Generally, repeat sequences of amino acids rich in Pro and/or Thr appear to link discrete functional domains in many cellulases and xylanases.
Some removal characteristics of dimethyl sulfide (DMS) by a mixed culture of Hyphomicrobium sp. I55 and Pseudomonas acidovorance DMR-11 were observed. In a shaking flask experiment, the specific removal rate of DMS by the mixed culture of DMR-11 and I55 was superior to that of each single culture. As I55 oxidizes both DMS and dimethyl sulfoxide (DMSO) simultaneously, at a lower DMSO concentration, where DMR-11 oxidizes DMS to DMSO effectively, a symbiotic relationship between them was suggested. This phenomenon was also verified in peat inoculated with the two bacteria. The maximum removal rate (Vm) in a peat biofilter inoculated with strains I55 and DMR-11 was three times larger than that in I55 only, although no removal of DMS occurred on γ-ray sterilized peat inoculated with DMR-11.
Rhizopus sp. A-11 produced glucoamylase (GA, EC 184.108.40.206) to a high concentration in a basal liquid medium supplemented with zinc and calcium ions. Since zinc ions were essential for the growth of the organism, supplementation of zinc ions in the medium was essential for the growth and GA production of Rhizopus sp. A11. In addition of zinc ions, supplementation of calcium ions stimulated GA production. Supplementation of the basal liquid medium with 0.7 ppm (w/v) zinc ions and 75 ppm calcium ions was approximately resulted in GA production of 650 units per ml. Based on the activity per mg-protein, this activity was 4.6 times that recorded for extract from a conventional solid state culture of the same Rhizopus sp. A11 on wheat bran medium. Almost all of the protein secreted into the liquid culture medium was GA and this resulted in a high specific activity of GA (4,040 units/mg-protein). The results presented in this paper indicate that liquid culture of Rhizopus sp. A-11 in a basal medium supplemented with zinc and calcium ions is an efficient method for GA production with high specific activity.
A marine obligately chemolithoautotrophic aerobic hydrogen-oxidizing bacterium Hydrogenovibrio marinus strain MH-110 was cultivated in a 2-l jar fermentor with continuous supply of a mixed gas of H2/O2/CO2. Fast growth began rapidly without a lag period, even in an atmosphere of 40% oxygen. Apparent specific growth rate of the initial stage of growth was 0.60 – 0.67 h−1 irrespective of any oxygen tension tested. Under an atmosphere containing hydrogen, the growth was promoted by the addition of tetrathionate to the medium. A dense culture was achieved by increasing the tension of oxygen and adding ferrous and magnesium ions before these factors became growth limiting. Under this culture condition, exponential growth continued until the cell density reached 14 g/l dry weight. The final cell concentration reached approximately 26 g/l dry weight after 30-h cultivation.
An extracellular alkaline lipase from Pseudomonas pseudoalcaligenes F-111 was identified by screening using a rhodamine B solid medium containing Na2CO3 by detection of orange fluorescent halos around the colony. For enzyme production, the inclusion of olive oil, soymeal, Triton X-100 and sodium ion in the medium was found to be essential. The optimal culture conditions for maximum production of alkaline lipase by P. pseudoalcaligenes F-111 were investigated and shown to be as follows: a culture medium composed of (g·l−1) olive oil, 4; soymeal, 10; Bacto-peptone, 15; yeast extract, 5; Triton X-100, 2; K2HPO4, 3; MgSO4·7H2O, 0.04; Na2CO3, 1.0; with an incubation period of 24 h at 30°C under shaking conditions. The production kinetics of the enzyme were also monitored in this study. Approximately 68% of the initial crude enzyme activity was lost during storage of at 4°C for 24 h. Calcium ion added to crude enzyme broth stabilized the initial enzyme activity. Furthermore, addition of calcium ion to broth resulted in the co-precipitation of the non-protein components and facilitated the enzyme purification. The purified alkaline lipase was activated even after incubation at 30°C in 40% water-immiscible solvents for 6 h.
A cyclodextrin glucanotransferase (EC 220.127.116.11) (CGTase) from Bacillus autolyticus was purified to homogeneous, and its properties were investigated. The molecular weight was estimated to be 68,000 by gel chromatography and 70,000 by SDS-PAGE. The optimum pH of the enzyme was 5.0–6.0. The optimum reaction temperature was 60°C. HgCl2 inhibited the enzyme activity completely. The enzyme formed mainly β-cyclodextrin (β-CD) with a small amount of α-cyclodextrin (α-CD) and γ-cyclodextrin (γ-CD) from potato starch. Ethanol and Triton X-100 enhanced the specificity and yield of β-CD production. Especially, the effect of Triton X-100 was remarkable. With 4% (v/v) Triton X-100, β-CD was formed in a maximum yield of 56% from 10% (w/v) potato starch with negligible amounts of α-CD and γ-CD (both yields were less than 1%) at a later stage of the reaction.
Adenosine deaminase was induced when the cells of Klebsiella sp. LF 1202 were cultured in the medium containing adenosine as a sole source of carbon and nitrogen. The induction was partially repressed by the addition of ammonium sulfate in the medium. The amount of adenosine deaminase reached approximately 4.6% of the total intracellular soluble proteins. The enzyme was purified approximately 22-fold with a 25% activity yield. The enzyme was a monomer with a molecular weight of 26,000. The optimal activity was obtained at pH 8.0, 37°C, and the Km value for adenosine was 37 μM. Metal ions such as Zn2+, Co2+, Fe2 and Ni+ inhibited the activity of the enzyme. Sulfhydryl blocking agents such as p-chloromercuribenzoate and HgCl2 were also found to be potent inhibitors for adenosine deaminase.
A bacterial strain, Klebsiella sp. LF 1202, which was isolated from soil as an adenosine-assimilating bacterium (Ling, F. et al., Agric. Biol. Chem., 55, 573–575, 1991), was able to use polyphosphate as a source of phosphorus. The bacterium possessed a polyphosphate-phosphatase, an inducible enzyme located in a periplasmic space of the bacterium. The enzyme was purified to a homogenous state on SDS-PAGE, and found to consist of four identical subunits with a molecular weight of 24,000. The enzyme exhibited activity towards tri-, tetra-, hexa-, octa-, and decapolyphosphate at pH 8.0, but not towards pyrophosphate and metaphosphate. The enzyme did not require metal ions for activity, but its activity was strongly inhibited by Zn2+. Chelating agents such as EDTA and CDTA did not affect activity. Since the enzyme activity was not affected by diisopropylfluorophosphate, serine residues do not seem to be involved in the activity.
Continuous production of phospholipase D (PLD) in an air-lift reactor system was investigated using growing cells of Streptomyces lydicus D-121 immobilized within cross-linked chitosan beads as porous supports. In a continuous culture with the immobilized growing cells, both the activity and productivity were highest at a dilution rate of 0.330 h−1 which was higher than the maximum specific growth rate of the free cells (0.247 h−1). The highest PLD activity was obtained at pH 6.0–6.5. The average activity was 12.7 U/ml during 120–384 h in a continuous culture with immobilized cells, while the productivity of the continuous culture (4.19 × 103 U/l·h) was about 3.3–3.6-fold higher than that of the batch culture (1.16 × 103 U/l·h) and the repeated-batch culture in the reactor (1.27 × 103 U/l·h), and about 3-fold higher than that in the case of continuous culture with free cells (1.36 × 103 U/l·h).
A 5.2-kb DNA fragment containing the upstream region of the nitrite reductase gene (nirS) was cloned from Paracoccus denitrificans IFO 12442 and its DNA sequence was determined. In this fragment, four open reading frames (ORFs) were observed. Among these, two ORFs, located 3 kb upstream of the nirS gene were found to encode nitric oxide reductase (norC, norB) by homology analysis. The norC and norB encoded cytochrome c and b subunits of the enzyme, and the predicted molecular weights of the protein products were 17 kDa (150 amino acid residues) and 52.5 kDa (462 amino acid residues), respectively. The other two open reading frames, designated ORF1 (73 kDa, 681 amino acid residues) and ORF2 (32.5 kDa, 304 amino acid residues), were found between the nir and nor genes. The deduced amino acid sequence of ORF1 showed high similarity (35% identity) to that of NosR which is known to be a transcriptional regulatory protein for the N2O reductase gene cluster of Pseudomonas stutzeri. Since the ORF1 protein has a well conserved cysteine cluster and 5 hydrophobic transmembrane repeats similar to those of the NosR protein, ORF1 probably functions as a membrane bound sensor for denitrification. Northern blot analysis indicated that the ORF1–2 and norCB regions are probably transcribed as independent operons.
Gluconobacter oxydans subsp. sphaericus IFO 12467 was screened with a culture medium containing acetate for the ability to oxidize ethanol to acetate under acidic conditions. Vinegar production was investigated extensively with the selected strain under submerged conditions. This strain was found to be advantageous for the production of a vinegar containing a large amount of gluconate.
Through the screening of yeasts and bacteria, some strains of Agrobacterium radiobacter have been found to efficiently convert 7-aminocephalosporanic acid (7-ACA) to deacetyl 7-ACA. The enzyme responsible for the conversion was constitutively formed by the bacterium. The 7-ACA deacetylating enzyme was purified from a cell-free extract of A. radiobacter IFO 12607 to homogeneity. The purified enzyme was a tetramer, composed of identical subunits with a molecular mass of 26 kDa, and showed activity toward some derivatives of 7-ACA, aryl acetates, monoacetin and triacetin, but was not active towards alkyl acetates and cephalosporin C. The Km and kcat values of the enzyme for 7-ACA were 8.24 mM and 81 s−1, respectively. Serine inhibitors and sulfhydryl reagents were both found to inhibit the activity. An amino acid sequence, GDSLT, which is the active center motif of an arylesterase, was identified in the amino terminal region. These results indicate that the enzyme belongs to the family of arylesterase (EC 18.104.22.168), and is a new 7-ACA-deacetylating enzyme.
A novel arylesterase from Agrobacterium radiobacter IFO 12607 catalyzes the deacetylation of 7-aminocephalosporanic acid (7-ACA) to form deacetyl 7-ACA, but is inactive with cephalosporin C. A DNA fragment carrying the gene encoding the 7-ACA-deacetylating enzyme was cloned from the chromosomal DNA of this bacterium. The open reading frame encoding the enzyme was 642 bp long, corresponding to a protein of 214 amino acid residues (molecular mass=23,085). The deduced amino acid sequence did not contain the sequence GXSXG, typical of the many serine esterases including Bacillus cephalosporin C deacetylase, but has the pentapeptide motif sequence GDSLT (amino acid position 9–13) which is also a consensus sequence of some serine esterases. The newly cloned gene was expressed in Escherichia coli under the control of the lac promoter, and the gene product purified from E. coli exhibited the same catalytic properties as the enzyme purified from A. radiobacter. Site-directed mutagenesis of S11A or S11C within the pentapeptide motif sequence led to complete loss of the enzyme activity. Thus, the Ser-11 residue within the GDSLT motif sequence was determined to construct the catalytic center. These results together with those of our previous studies indicated that the 7-ACA-deacetylating enzyme from A. radiobacter IFO 12607 is a new member of the family of lipolytic serine esterases containing the GDSLT sequence as their catalytic center.
Methanol oxidases (MODs) were investigated in some methanol-utilizing yeasts. Pichia methanolica IAM 12901 and IAM 12481 (=Pichia cellobiosa) produced 9 forms of methanol oxidase on electrophoresis when grown on methanol but 1 form when grown on glycerol or pectin. Other strains—Candida boidinii IAM 12875, Pichia angusta IAM 12897, and Pichia pastoris IAM 12903—produced one band on electrophoresis when grown on methanol. Methanol oxidases with the smallest and largest Rm (relative mobility)—MOD A and MOD B—were purified from methanol-grown cells; another methanol oxidase (MOD P) was purified from pectin-grown cells of P. methanolica IAM 12901. All the purified enzymes were octamers composed of subunits with a molecular mass of 70,000 Da. MOD A and MOD P were mostly identical with each other in terms of their enzymatic characteristics, but MOD B differed from the other two with respect to substrate specificity, the prosthetic group, and the apparent Km for methanol. Southern analysis of the genomic DNA of P. methanolica IAM 12901 was carried out with the partial methanol oxidase gene as a probe. The genomic DNA of this yeast showed the presence of 2 methanol oxidase genes. It was concluded that the 9 forms of MOD in P. methanolica were isozymes of a chimera octamer consisting of 2 subunits encoded by different methanol oxidase genes.
The effect of exposure to heavy metal particles on the growth and survival of bacterial cells was investigated. Thiobacillus intermedius 13-1, Escherichia coli JM109, and Agrobacterium radiobacter IFO12665b1 were cultured on LB solid medium or in 5 ml of liquid medium containing 0.03 or 0.1 g respectively of the heavy metals, aluminum, cadmium, iron, lead, molybdenum, nickel, and zinc. Cadmium, nickel and zinc strongly inhibited cell growth in the three strains. In contrast, the bacterial cells were not inhibited by aluminum, iron, or lead in the solid or liquid medium. When these bacteria were exposed to heavy metals by vigorous shaking for 10 min, lead and molybdenum, but not nickel and zinc, were markedly toxic to the bacterial cells. Different reaction were thus observed under low-level, long-term and high-level, short-term exposure conditions. T. intermedius showed more resistance to zinc and nickel, and was more sensitive to molybdenum exposure, than E. coli and A. radiobacter.
Muconate cycloisomerase was purified from aniline-assimilating Rhodococcus erythropolis AN-13. The purified enzyme exhibited a novel property in that it catalyzed cycloisomerization most efficiently when the reaction mixture contained Co2+ ion as a cofactor, even though Mn2+ ion has been reported to be the best cofactor for the muconate cycloisomerase reaction.
A malic enzyme from a cell-free extract of Pseudomonas diminuta IFO-13182 was purified to electrophoretic homogeneity by DEAE-Sepharose, Sephacryl, and Blue-Sepharose chromatographies. The purified enzyme required either NAD+ or NADP+ as a coenzyme. From the results of coenzyme specificity, the enzyme should be classified as l-malate: NAD+ oxidoreductase (decarboxylating) [EC 22.214.171.124]. The purified enzyme was most active at pH 7.5 and 50°C and was stable in the pH range from 7.0 to 9.0. The isoelectric point was pH 4.3. Its molecular weight was 680,000 by COSMOSIL 5-Diol high performance liquid gel filtration on chromatography and 65,000 by SDS polyacrylamide gel electrophoresis. This indicates that the enzyme consisted of 10 subunits. The malic enzyme activity with NADP+ was about twice that measured with NAD+.
With a bioaccumulation system using Rhodococcus erythropolis CS98 for recovery of cesium-137, we found that 137Cs accumulated when a carbon source was added for energy supply. With the addition of ammonium acetate as the carbon source, almost all the 137Cs from deionized water was recovered using a cell suspension of 1 g/l with incubation for 24 h. Cell damage by radioactivity was not detected during the 24 h period. 137Cs recovery from river water samples was lower than that from deionized water, especially from river water with a very high potassium concentration (the lower reaches of the Sakura River: potassium concentration=4.3 mg/l). When 3.9 mg/l of potassium was added to a deionized water sample, 137Cs recovery decreased to 35% of that without potassium addition, suggesting that the potassium concentration is a critical factor for 137Cs recovery. We conclude that a bioaccumulation system with a semipermeable membrane tube, such as is described in this paper, is feasible for the recovery of radioactive cesium from fresh waters.
d-[1-13C]glucose is useful for studies on tracing by means of 13C-magnetic resonance imaging (MRI) the fate of a metabolite in the brain, and this technique is expected to become a sophisticated clinical tool for the diagnosis of neurological disorders. For this purpose, a more efficient method of producing inexpensive 13C-labeled d-glucose is necessary, and was investigated. When a mixture of the labeled d-fructose-6-phosphate (F-6-P) and d-glucose-6-phosphate (G-6-P) prepared from d-ribose-5-phosphate and [13C]formaldehyde is dephosphorylated by potato acid phosphatase, a combination of potato acid phosphatase, phosphoglucose isomerase, and BaCl2 was found to be effective in improving the yield of d-glucose and shortening the reaction time. The maximum yield of labeled d-glucose from the mixture of labeled F-6-P and G-6-P was 84%. Two peaks indicative of labeled d-glucose with 13C incorporated at the C-1 position were confirmed by the NMR spectroscopy.
13C-labelled l-serine was efficiently produced from [13C] formaldehyde and glycine by using partially purified l-serine hydroxymethyltransferase from Methylobacterium extorquens JCM 2804, tetrahydrofolate and a tetrahydrofolate regeneration system. The tetrahydrofolate regeneration system allowed efficient l-serine production and direct use of dihydrofolate easily prepared from folic acid. A tetrahydrofolate concentration of more than 0.020% was necessary for efficient production. Eighty seven % of the amount of [13C] formaldehyde consumed, i.e., the difference between the total amount of formaldehyde fed and the amount of formaldehyde remaining in the reaction mixture, was used for the incorporation into l-[13C] serine. Seventy two mg of added glycine was converted to l-serine when 100 mg of glycine was contained in 11.2 ml of the final reaction mixture. The l-serine was confirmed to be l-[3-13C]-serine by negative fast atom bombardment mass spectrometry (FAB-MS) and 13C-nuclear magnetic resonance spectrometry (NMR) analysis.
A gene designated lpa-14, which was cloned from Bacillus subtilis RB14 and was associated with the production of lipopeptide antibiotics, was found to be involved in the production of an iron-chelating siderophore, 2,3-dihydroxybenzoylglycine (2,3-DHBG). Although strain RΔ1, an lpa-14 defective mutant of RB14, showed no growth in iron-deficient medium in contrast to the marked growth of RB14 in the same medium, the addition of chemically synthesized 2,3-DHBG at approximately 100 ppm caused the growth of RΔ1 to be resumed.
Bacillus subtilis RB14 is a coproducer of the lipopeptide antibiotics iturin A and surfactin. Iturin A and surfactin have a similar structure consisting of a seven-amino-acid cyclic peptide, linked by either hydroxy or ester peptide linkage, respectively, to the fatty acid part. A 10-kb fragment responsible for the production of the lipopeptide antibiotics iturin A and surfactin in B. subtilis RB14 was minimized and the nucleotide sequence of the region essential for the synthesis of the lipopeptides was determined. A large open reading frame consisting of 224 amino acid residues was found. The gene, designated as lpa-14 (lipopeptide antibiotic production of RB14), showed high homology with sfp and psf-1 (regulators for surfactin production in B. subtilis and Bacillus pumilus, respectively) and an unknown open reading frame, orfX, in the upstream region of the peptide antibiotic gramicidin S biosynthesis operon of Bacillus brevis. The biosynthesis of surfactin and of iturin A was shown to be coregulated by the same gene, lpa-14. It is suggested that a common regulation system might exist among these genes for the production of peptide antibiotics in Bacillus species.
Screening for microorganisms converting stearic acid to form new compounds was conducted, Pseudomonas cepacia A-1419 isolated from soil effectively produced two compounds showing strong ultraviolet absorption when the resting cells were incubated with stearic acid. The products were isolated, and identified as (Z)-dec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (Product 1) and (Z)-dodec-3-ene-1,3,4-tricarboxylic acid 3,4-anhydride (Product 2) by infrared, mass, and nuclear magnetic resonance spectroscopies, and elemental analysis. Products 1 and 2 were produced from stearic acid at conversion rates of about 18 and 32%, respectively.
An antibacterial substance produced by Lactobacillus plantarum NRIC 149 was identified as a bacteriocin on the basis of its narrow inhibitory spectrum, proteinaceous nature and the bactericidal mode of its action. The bacteriocin, designated plantaricin-149, was produced during the log phase by the producer strain, and was most active at pH 5.0. From scanning electron microscopic observation, plantaricin-149 caused morphological changes in sensitive bacteria in the log phase. Plantaricin-149 was purified by ultrafiltration, anion-exchange chromatography and reverse-phase HPLC. The molecular mass of plantaricin-149 was estimated to be 2.2 kDa by SDS-PAGE, and the N-terminal amino acid sequence was determined to be NH2-Tyr-Ser-Leu-Gln-Met-Gly-Ala-Thr-Ala-Ile-Lys-Gln-Val-Lys-Lys-Leu-Phe-Lys-Lys-Gly-Gly.
Nitrogen deregulated manganese peroxidase (MnP)-producing mutants of Deuteromycotina IZU-154 were isolated based on their ability of decolorize synthetic melanin. One of these mutants, IZU-882, exhibited an approximately 2-fold higher level of MnP activity, 8–12 nkat/ml (nano katal/ml), under nitrogen nonlimiting conditions than that of the wild-type under nitrogen limiting conditions.