Production of d-Aminoacylase from Alcaligenes denitrificans subsp. xylosoxydans MI-4

A bacterial strain that produces d-aminoacylase was isolated from soil and identified as Alcaligenes denitrificans subsp. xylosoxydans MI-4. l-Aminoacylase activity in this strain was only 1 to 2% of d-aminoacylase activity. d-Aminoacylase was inducibly produced. N-Acetyl-dl-leucine was the best inducer, and the d-isomer had the ability to induce the enzyme. Enzymatic resolution of N-acetyl-dl-methionine with the crude enzyme was carried out, and the d/l ratio in the resolved methionine was approximately 100/7, suggesting that resolution with crude enzymes may become possible by removing small amounts of the contaminated l-form with l-amino acid oxidase.     

Production of 5' Nucleotide by Using Halophilic Nuclease H Preferentially Adsorbed on Flocculated Cells of the Halophile Micrococcus varians subsp. halophilus

A bioreactor with a column of flocculated cells of the moderate halophile Micrococcus varians subsp. halophilus which adsorbed the halophilic nuclease H was designed to be used in the production of 5' nucleotides from RNA. A remarkable characteristic of the flocculated cells was that they preferentially adsorbed much exogenous nuclease, excluding adsorbed 5' nucleotidase. Furthermore, desalting treatment of the flocculated cells in the presence of 2% MgSO(4) . 7H(2)O gave rise to selective inactivation of 5' nucleotidase without the loss of nuclease H activity, and 5'-guanylic acid was produced with the bioreactor.     

Isolation and preliminary characterization of twenty bacteriophages infecting either brevibacterium or arthrobacter strains

Thirty-seven bacteriophages plaquing on Corynebacterium, Brevibacterium, or Arthrobacter strains were isolated from soil or vegetation samples. Restriction analysis of phage DNA indicated that 20 phages were unique; one of them produced entirely turbid plaques on Brevibacterium ketoglutamicum and was characterized as temperate. All these phages were assigned to group B of the classification of Bradley (Bacteriol. Rev. 31:230-314, 1967) and had relatively narrow host ranges.     

Frequency and characteristics of plasmids in bacteria isolated from deep-sea amphipods

Bacterial strains isolated from deep-sea amphipods were identified, classified, and screened for plasmid content. Plasmids were common, with 11 of 16 isolates carrying one or more plasmids; these ranged in size from 2.9 to 63 megadaltons. Several of the strains demonstrated distinctly different phenotypic traits yet contained plasmids of the same molecular weight. Results of agarose gel electrophoresis, DNA hybridization, and restriction analysis indicate that the plasmids detected in these deep-sea isolates are identical, suggesting that transmission may occur in the deep-sea environment and that plasmids are common in some deep-sea habitats.     

Strain Improvement of Rhodotorula graminis for Production of a Novel l-Phenylalanine Ammonia-Lyase

l-Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) from Rhodotorula rubra has been used in the commercial manufacture of l-phenylalanine from trans-cinnamic acid. In this study, R. graminis PAL was investigated. Mutant strain GX6000 was isolated after ethyl methanesulfonate mutagenesis of wild-type R. graminis GX5007 by selecting for resistance to phenylpropiolic acid, an analog of trans-cinnamic acid. Mutant strain GX6000 produced inducible PAL at levels four- to fivefold higher than had wild-type R. graminis. Furthermore, this strain had several other physiological traits that make it more commercially useful than R. rubra. For example, during fermentation, the PAL half-life was three- to fivefold longer, PAL specific activity was six to seven times higher, and PAL synthesis was significantly less inhibited by temperatures above 30 degrees C. Induction of PAL in strain GX6000 appeared to be less tightly regulated; l-leucine acted synergistically with l-phenylalanine, the physiological inducer, to increase the PAL specific activity and titer to 165 U/g (dry weight) and 3,000 U/liter, respectively, a 40% increase over the effect of l-phenylalanine alone. Strain GX6000 PAL showed significantly greater stability in bioreactors for the synthesis of l-phenylalanine, a finding that is consistent with the stability properties observed during fermentation.     

Production and Purification of d-Aminoacylase from Alcaligenes denitrificans and Taxonomic Study of the Strain

A d-aminoacylase-producing microorganism, strain DA181, isolated from soil was identified as Alcaligenes denitrificans subsp. denitrificans. This strain produced about 29,300 units (micromoles of product formed per hour) of d-aminoacylase and 2,300 units of l-aminoacylase per gram of cells (wet weight) when cultivated in a medium containing 1% N-acetyl-dl-leucine as the carbon source. The d-aminoacylase was purified 345-fold. The specific activity of the purified enzyme was 108,600 units per mg of protein when N-acetyl-d-methionine was used as a substrate. The apparent molecular weight was 58,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. N-Acetyl-d-methionine was the favored substrate, followed by N-acetyl-d-phenylalanine. This enzyme had a high stereospecificity, and its hydrolysis of N-acetyl-l-amino acids was almost negligible.     

Inhibitory Effects of Methylcellulose on Cellulose Degradation by Ruminococcus flavefaciens

Highly methylated, long-chain celluloses strongly inhibited cellulose degradation by several species of cellulolytic bacteria of ruminal origin. Specifically, the inhibitory effects of methylcellulose on the growth of Ruminococcus flavefaciens FD1 were concentration dependent, with complete inhibition at 0.1% (wt/vol). However, methylcellulose did not inhibit growth on cellobiose or cellulooligosaccharides. Mixtures of methylated cellulooligosaccharides having an average degree of polymerization of 6.7 to 9.5 inhibited cellulose degradation, but those with an average degree of polymerization of 1.0 to 4.5 did not. Similar inhibitory effects by methylcellulose and, to a lesser extent, by methyl cellulooligosaccharides were observed on cellulase activity, as measured by hydrolysis of p-nitrophenyl-beta-d-cellobioside. R. flavefaciens cultures hydrolyzed cellulooligosaccharides to cellobiose and cellotriose as final end products. Cellopentaose and cellohexaose were cleaved to these end products, but cellotetraose was also formed from cellohexaose. Methylcellulose did not inhibit hydrolysis of cellulooligosaccharides. These data are consistent with the presence of separate cellulase (beta-1,4-glucanase) and cellulodextrinase activities in R. flavefaciens.     

Biosynthesis of Territrems by Aspergillus terreus

Different radioactive precursors were added to 8-day potato-dextrose liquid cultures of Aspergillus terreus 23-1. Territrems were isolated from chloroform extracts of the cultures at day 14 and purified by thin-layer chromatography and high-pressure liquid chromatography. The territrem B obtained was treated with alkaline hydrogen peroxide, and 3, 4, 5-trimethoxy benzoic acid was isolated from an ethyl acetate extract of the reaction mixture and purified by thin-layer chromatography and high-pressure liquid chromatography. By comparison of the specific radioactivities of territrem B and its cleaved aromatic product (disintegrations per minute per micromole of compound), it was demonstrated that the radioactivity of territrem B was located mainly on its aromatic moiety when [U-C]shikimate, l-[methyl-C]methionine, and l-[methyl-H]methionine were precursors; however, the radioactivity of territrem B was located mainly on its nonaromatic moiety when [2-C]mevalonate was the precursor. Mevinolin, a specific inhibitor of beta-hydroxyl beta-methyl glutaryl coenzyme A reductase, was shown to inhibit production of territrems by A. terreus 23-1. When [U-C]acetate was used as a precursor, mevinolin inhibited the incorporation of radioactive carbon into territrem but mevinolin did not inhibit incorporation of radioactive carbon from [2-C]mevalonate into territrem.     

Detecting estrus in synchronized heifers-using tailpaint and an aerosol raddle

A synchronization treatment was initiated when each of 1227 heifers (four trials) was tailpainted. The tailpaint was sprayed with an aerosol raddle at the end of the treatment period. The heifers were in herds of 20 to 279 animals. Each herd was observed for estrus at selected post treatment intervals. A heifer was considered to be (or to have been) in estrus when the raddle was rubbed off. In three of the trials, animals which had the raddle removed were inseminated at 48h following the end of the synchronization treatment. The tailpaint of an inseminated animal was scored from 0 (less than 10% of the paint remained) to 5 (more than 90% of the paint remained) and was then reraddled with a second color. The detection-insemination sequence was always repeated at 72 and 96h, and sometimes at 120h. Animals which had been previously inseminated, but then had paint scores reduced by at least 2 units were reinseminated 24h later. Over the four trials, 94.5% of the heifers were detected in estrus through the use of the tailpaint and raddle system. The remaining 67 animals included only 10 (0.8%) which had ovulated without being detected in estrus. The reinsemination rate on consecutive days was 11.3% and was highest among animals that had a tailpaint score of 4 or 5 at 48h. The proportion of animals detected in estrus at selected posttreatment intervals varied with the different synchronization treatments used within one herd, or with the same treatment used in different herds. The combination of tailpaint, raddling, tailpaint scoring and reraddling is a simple sequence which can be effectively used to detect estrus among heifers synchronized in research or commercial herds.     

Synchronization of estrus in early diestral dairy heifers with Prostaglandin F(2)alpha and estradiol benzoate

Following observation of estrus, 134 Holstein heifers were given injections of Prostaglandin F(2)alpha (PGF(2)alpha) between Days 5 and 10 of their cycle (estrus = Day 0). They were then randomly assigned to either a group receiving 400 mug of estradiol benzoate (E(2)B) 40 h or maintained as controls. Heifers observed in estrus within 120 h of PGF(2)alpha administration were inseminated (approximately 12 h after initial observation of estrus). Blood samples for progesterone determination were drawn from the coccygeal vein on Days 15 and 21 after insemination. Pregnancy was confirmed by palpation per rectum between Days 5.0 and 60 post insemination. When control and treated heifers were compared it was found that a higher percentage of heifers treated with E(2)B exhibited estrus after PGF(2)alpha, but there had been no effect on subsequent progesterone concentrations or pregnancy rates.