Production of Extracellular l-Asparaginases by Microorganisms

A variety of microorganisms were tested for their extracellular l-asparaginase productivity and it was found that many bacteria, fungi and yeasts are positive for it. Especially some strains in the genera Pseudomonas, Candida and Rhodotorula were able to produce a large amounts of the enzyme. Escherichia coli, however, that contained intracellular enzyme was unable to produce extracellular one. In enzymological properties some differences were noted among these extracellular enzymes. Pseudomonas asparaginase showed glutaminase activity too, but the asparaginases of Candida and Rhodotorula were unable to hydrolyze glutamine. Candida l-asparaginase was most stable to heat-treatment.     

Dramatic alteration of sphingolipid bases of Hansenula ciferrii by exogenous fatty acid

$\text{Hansenula}\text{ciferrii}$, a yeast which normally excretes large amounts of C₁₈-phytosphingosine in the form of its tetra-acetyl derivative, was grown in basal medium containing added pentadecanoic acid (0.1%) and Brij 58 (1%). Analyses of the sphingolipid bases recovered from the culture medium indicated the presence of significant quantities of C₁₇-phytosphingosine (39%) and C₁₉-phytosphingosine (15%) in addition to the normal C₁₈-phytosphingosine (46%). The sphingolipid bases were characterized by gas-liquid chromatography combined gasliquid chromatography-mass spectrometry, and chemical degradation.     

Pilot-Plant Production of Protease by Alternaria tenuissima

Agar slants of a selected Alternaria tenuissima strain were illuminated to give conidia suitable for further propagation. For production of protease with an optimal caseolytic activity in the region of pH 8 to 10, the fungus was cultivated in steel fermentors with 6- and 60-liter working capacity. Maximal activity, 1.5 enzyme units per liter, was attained in a medium based on liver after about 60 hr of cultivation. The protease was secreted parallel with, or slightly after, the main growth phase. The process could be run favorably with a relatively low aeration rate. The pH of the culture decreased during the process from 7.0 to about 6.3. This was the optimal region also when pH was kept constant by automatic pH control. Optimal temperature was about 28 C, which resulted in a maximal productivity of 0.057 enzyme units per liter per hr during the protease secretion phase. Replacement of the liver in the medium with skim milk, meat scrap, or rapeseed oil meal resulted in a decrease of the protease yield.     

Survey of Microorganisms for the Production of Extracellular Phytase

A culture enrichment technique was used to isolate phytase-producing microorganisms. Also, microorganisms from various culture collections were tested for their phytase-producing ability. A number of the Aspergillus niger group produced extracellular phytase which dephosphorylated calcium phytate in acidic solution. A soil isolate, A. ficuum NRRL 3135, produced the most active phytase in a cornstarch-based medium. Production of phytase was strongly repressed by inorganic phosphates and required a high carbon to phosphorus ratio in the medium.     

Extracellular Accumulation of Organic Acids by Isopropanol-utilizing Microorganisms

Isopropanol-utilizing microorganisms were newly isolated from soils and several of them accumulated two acids in the culture broth, α-ketoglutaric acid being a major one and succinic acid a minor one. Two strains (N–79 and S–1), classified as the genus Mycobacterium, were examined for the cultural conditions with respect to the accumulation of the acids. The accumulation of α-ketoglutaric acid depended greatly on the pH value in the broth, which is required to be kept at around 4 for the maximum accumulation. By means of the pH-controlled culture (at 3.5) with a jar fermentor, strain N–79 accumulated α-ketoglutaric acid at a rate of 0.015 g/liter/hr. The data obtained in this work indicate that the metabolism of isopropanol by strain N–79 probably proceeds via the acetone pathway without the inter-conversion between isopropanol and n-propanol.     

木糖醇微生物转化的研究

一、引言 木糖醇是一种被广泛用于国防、医药、食品和化学等工业的五碳糖醇。木糖醇也是一种具有营养价值的甜味剂,由于它的代谢不需胰岛素,可作为糖尿病人的食糖代用品。另外木糖醇还可以作为防龋食品。目前国内外生产木糖醇的方法是化学法,即由农业纤维废料如玉米芯和甘蔗渣等经酸水解和提     

Production of catalase-free alcohol oxidase by Hansenula polymorpha

Summary Many of the potential technical applications of alcohol oxidase (MOX; EC 1.1.3.13) are limited by the presence of high activities of catalase in the enzyme preparations. In order to circumvent laborious and costly purification or inactivation procedures, the induction of MOX in a catalase-negative mutant of Hansenula polymorpha has been studied. Emphasis was laid on the induction of activities of MOX and the dissimilatory enzymes in continuous cultures grown on various mixtures of formate/glucose and formaldehyde/glucose. In continuous cultures of the catalase-negative mutant grown on these mixtures, MOX can be induced efficiently. To obtain a stable and productive process, the ratio of the substrates is of critical importance. The optimal ratios of the mixtures for the catalase-negative strain for formate/glucose and formaldehyde/glucose were 3:1 and 1–2:1, respectively. Under identical cultivation conditions the wild-type strain showed similar induction patterns for MOX and the dissimilatory enzymes formaldehyde dehydrogenase (FaDH) and formate dehydrogenase (FoDH). The MOX levels in the catalase-negative strain were approx. 50% of those in the wild-type strain.     

Formation of Glycerol Dehydrogenase by Microorganisms

The distribution of glycerol dehydrogenase activity was studied with cell-free extracts of bacteria, yeasts, molds and actinomycetes. High activity was found in 4 strains of bacteria and in 3 strains of molds. The enzymes of bacteria were dependent on NAD⁺ and those of molds were dependent on NADP⁺. An isolated gram-positive bacterium, which showed the high activity, was identified as Cellulomonas sp. NT3060. The total and specific activities were associated with growth of this strain and reached the maximum at the early stationary phase. Significant high level activity was detected in cell-free extracts from glycerol and glucose media.     

Production of mead by immobilized cells of Hansenula anomala

Summary Mead was produced by immobilized cells of Hansenula anomala in calcium alginate gels. The immobilized cell beads of 3 mm diameter packed in column reactors of dimensions 2.2x60, 4x40 and 8x80 cm, produced mead containing maximum concentrations of ethanol and ethyl acetate of 70 g/l and 730 mg/l, respectively at a dilution rate of 0.1 h^–1. The maximum alcohol productivity achieved was 23.1 g/l·h at a dilution rate of 0.33 h^–1. With intermittent regenerations of the cells the reactor operated continuously for 110 days. This process enables the quick production of matured mead by a single culture and the elimination of the traditionally used long aging periods.     

l-Glutamate Formation from Hydrocarbons by Microorganisms

Large quantities of l-glutamic acid from liquid paraffins by microorganisms were produced with an addition of penicillin to the growing culture, and the action of penicillin to the glutamate production was studied. One of main effects of penicillin seems to exist in the cellular permeability of l-glutamic acid.     

Extracellular Formation of Alanine by Anaerobes

The possibility of extracellular formation of amino acids by anaerobes was investigated. In general, anaerobes were able to produce 20 to 50 mg of alanine per dl of medium extracellularly. Clostridium saccharoperbutylacetonicum, the anaerobic bacterium for acetone-butanol fermentation, accumulated 100 mg of alanine per dl of medium containing 5 g of glucose, 1 g of ammonium acetate, 0.1 g of potassium dihydrogen phosphate, 0.04 g of magnesium sulfate, 0.001 g of ferrous sulfate, 1 μg of biotin, 0.1 g of yeast extract and 1 g of calcium carbonate in tap water.

Relationships between alanine formation and solvent yields or sporulation were investigated. Spore formation was not active in the medium for alanine formation and yield of acetone increased in this medium.     

Extracellular Formation of O-Butylhomoserine by Anaerobes

Clostridium BB–264, Cl. acetobutyricum 314–48, Cl. kaneboi, Cl. saccharoperbutylacetonicum and other two strains of Cl isolated recently produced an unidentified ninhydrin-positive compound in medium containing 5 % glucose, 1 % ammonium acetate, 0.1 % potassium dihydrogen phosphate, 0.04 % magnesium sulfate, 0.001 % ferrous sulfate, 0.1 % yeast extract, 10 μg/liter of biotin and 1 % calcium carbonate.

This ninhydrin-positive compound was eluted with solvent composed of butanol: acetic acid: water (4: 1: 2) by chromatography on cellulose powder column. It was crystallized from ethanol and then identified as an amino acid, O-butylhomoserine (Abbrev. as O-BHSer). Yield of this amino acid increased by adding homoserine or butanol to the medium. The increase was also recognized with addition of glycine, lysine, serine, threonine or valine. The formation of this amino acid was repressed by adding methionine to the medium.

Gas pressure to the culture is one of the important factors that make the amino acid formation by anaerobes possible.     

Extracellular Deoxyribonuclease Production by Yeasts

A total of 20 genera of yeasts and yeastlike organisms were tested for their ability to produce an extracellular deoxyribonuclease. Results indicate that ability to produce the enzyme appears to be a specific characteristic of the three genera Rhodotorula, Cryptococcus, and Tremella. A single strain of Endomycopsis fibuligera was also shown to be positive for the enzyme. In comparing the ability of the organisms to excrete extracellular deoxyribonuclease with their ability to produce urease, a surprisingly close correlation was found. With the exception of Lipomyces starkeyi, all the organisms which were deoxyribonuclease-negative were also urease-negative. Of those organisms which were deoxyribonuclease-positive, only E. fibuligera was urease-negative. The ability of cryptococci to produce extracellular deoxyribonuclease is discussed in relation to the implication which this finding may have for the taxonomy and phylogeny of the genus.