Reduction of nucleic acid content in cell biomass of methanol-utilizing mixed bacterial culture by heat treatment

Summary A heat treatment method to reduce nucleic acid content in cell biomass of a mixed methanol-utilizing bacterial culture was studied. Maximum nucleic acid reduction in the bacterial cells was achieved by using heat shock at 65°C for 5–10 min followed by 2 h incubation at 55°C and 7.2±0.2 pH. In this treatment, 81–85% nucleic acid content was removed from the cells without affecting their true protein content and essential amino acids profile.     

Simple Process for the Reduction in the Nucleic Acid Content in Yeast

A simple one-step process for the nucleic acid reduction in Rhodotorula glutinis is described. The process consists of submitting the yeast cells to a heat treatment in an acidic (pH 2) spent medium. The optimal temperature for pH 2 medium is 90 C and the final nucleic acid content in treated yeasts was 1.2%. Heat treatment at acidic pH is preferred to that at alkaline pH because it offers a better protection for amino acids and crude protein, while being more efficient in lowering the nucleic acid level. The new process is economic and rapid and could be easily used for industrial application.     

The effect of heat shock on amino acid transport and cell volume in 3T3 cells

Summary. In 3T3 cells temperatures higher than physiological stimulated amino acid transport activity in a dose-dependent manner up to 44°C. However, the temperature increase did not induce widespread transport increase of all other nutrients tested. The activities of both amino acid transport systems A and ASC were enhanced within a few minutes following cell exposure to increased temperature. The maintenance of this effect required continuous exposure of the cells to hyperthermia. Kinetic analysis indicated that the stimulation of the activity of transport System A occurred through a mechanism affecting Vmax rather than Km. The continuous presence of cycloheximide did not prevent the transport changes induced by hyperthermia. These results suggest that the increased amino acid uptake reflects an activation or relocation of existing amino acid transport proteins. During the hyperthermic treatment, the content of ninhydrin-positive substances (NPS), mostly amino acids, increased within the cells and the accumulation of these compatible osmolytes was parallelled by an increase in cell volume. The withdrawal of amino acids from the culture medium immediately before and during the shock phase counteracted the increase and reduced the NPS content but did not prevent the increase in amino acid transport, the cell swelling and the induction of the heat shock response. Received June 30, 1999 Accepted July 27, 2000     

Changes in the radioresistance of bacteria after the inhibition of proteosynthesis in the preirradiation phase

The strain ofEscherichia coli WP₂ (try_v) was irradiated with UV light, at a dosage of 240 erg/mm. Proteosynthesis was inhibited by the elimination of the essential amino acid from the cultivation medium. Changes in radioresistance were followed during 45 minutes of starvation and during the subsequent 45 minutes of restitution after the addition of the essential amino acid. The radioresistance of the cells showed a linear increase immediately after the removal of the essential amino acids, proportional to the duration of the inhibition of proteosynthesis. The increase in radioresistance was shown to be reversible. After the addition of the essential amino acid there was an immediate decrease in radioresistance which was most marked in the first 15 minutes.     

Precipitation of nucleic acids with poly(ethyleneimine).

Removal of nucleic acids from cell extracts is a common early step in downstream processing for protein recovery. We report on the precipitation of nucleic acids from a homogenate of Saccharomyces cerevisiae by addition of the cationic polyelectrolyte poly(ethyleneimine) (PEI), focusing on the effect of PEI dosage on particle size, protein loss, and extent of nucleic acid removal in both batch and continuous mode. Better than 95% removal of nucleic acids from yeast homogenates was achieved by means of precipitation with PEI with protein losses of approximately 15% with or without previous removal of cell debris. The coprecipitated protein is predominately large molecular weight material and exhibits both low and high isoelectric points. Such treatment does not aggregate the cell debris; size distribution of the precipitated particles from a continuous precipitator is very similar to that for protein precipitation.     

Reduction of RNA and DNA in Methylococcus capsulatus by endogenous nucleases

 A method for reducing RNA and DNA in the bacterium Methylococcus capsulatus (Bath) has been developed. Endogenous RNase and DNase were activated by a 10 s heat shock at 90°C. Cells were then incubated at 60°C for 20 min to allow degradation of the nucleic acids. The optimum pH for the process was 7.0. The protein loss was less than 10% and occurred during the initial heat shock. No protein loss was found during incubation. The total dry-weight loss in connection with an 80% reduction of the nucleic acid content was 20%–25%, giving a final product with a raw protein content of approximately 75%. Reduction of both RNA and DNA was inhibited by CuSO₄ and ZnSO₄. DNA reduction was stimulated by other minerals. Optimal stimulation was found at 1 mM FeSO₄. Reduction of RNA was not increased by any of the minerals tested. Received: 29 June 1995/Received last revision: 2 October 1995/Accepted: 16 October 1995     

Studies on methanol-oxidizing yeasts

A series of yeast strains utilizing methanol as the only source of carbon and energy were isolated both from soil and waste waters. Growth of the yeasts was studied in a mineral medium with yeast extract at 30°C. Growth parameters, Q_{o 2} and composition of biomass were studied in the strain 11Bh producing in shaken flasks highest yields of biomass. The biomass was found to contain 44% of crude proteins, 2% esterified fatty acids, 3% non-esterified fatty acids, 6% RNA and 0.25% DNA. Amino acid analysis revealed a high content of essential amino acids, lysine, leucine, valine and threonine in particular.     

环链棒束孢hsp90基因的克隆及冷热胁迫下的表达特征分析

通过本地Blast筛选转录组数据库方法,首次克隆了环链棒束孢热休克蛋白90基因全长cDNA序列,命名为Ichsp90（GenBank登录号KT944289）。克隆结果表明,该序列含有2 284个碱基,包括一个含2 097个碱基的开放阅读框,编码699个氨基酸,推测蛋白的分子量为79.23kDa,等电点（pI）为4.86,且含有5个Hsp90家族特征基序和胞质特征序列MEEVD,推导的氨基酸序列与其他丝状真菌相似性在92%-96%之间。用qRT-PCR方法分析了冷热胁迫下,该基因在环链棒束孢中的相对表达情况,结果表明：在4℃冷胁迫下15min检测到Ichsp90表达量下降到最低点,为对照的-1.8倍;随后表达量开始上升,至120min表达量是对照的1.07倍。在39℃高温胁迫下,60min Ichsp90表达量达到最高峰,为对照样品的5.02倍;随后表达量开始下降,至110min为对照样品的2.46倍。因此推测,Ichsp90基因在环链棒束孢抵抗外界温度胁迫中发挥重要的作用。     

Initiation of anaerobic growth of Saccharomyces cerevisiae by amino acids or nucleic acid bases: ergosterol and unsaturated fatty acids cannot replace oxygen in minimal media

Nine out of ten industrially important strains of Saccharomyces cerevisiae did not grow in minimal media under anaerobic conditions even when ergosterol and unsaturated fatty acids were provided. Anaerobiosis was maintained either by flushing the culture flasks with prepurified nitrogen or by incubating the flasks in an anaerobic chamber. Traces of oxygen present in ‘prepurified nitrogen gas’ were sufficient to initiate yeast growth and on removal of the oxygen by catalytic means the yeasts failed to grow. The yeast grew very well anaerobically if the medium was supplemented with a mixture of amino acids or with a mixture of purines and pyrimidines. The growth initiated by including a mixture of amino acids was further enhanced when the medium was supplemented with ergosterol and an unsaturated fatty acid. Since no oxygen requirement for the synthesis of amino acids or purines and pyrimidines has been demonstrated, growth promotion by these compounds under anaerobic conditions is most likely not by eliminating the need for oxygen for their synthesis. We suggest that the amino acids and the nucleic acid bases yielded, through some hitherto unknown reactions, small amounts of a molecular or usable form of oxygen which allowed key reactions essential for ‘anaerobic’ growth to proceed. Received 16 July 1998/ Accepted in revised form 8 October 1998     

黄粉虫蛹的营养成分分析

采用现代分析技术手段对黄粉虫Tenebrio molitor（L．）蛹的营养成分进行分析,结果表明：其蛋白质、粗脂肪、碳水化合物及灰分含量分别为鲜重的16．8％、7．6％、1．2％、1．4％,其氨基酸和胆固醇含量分别为18.7mg／g和0．4mg／g;含有7种人体必需氨基酸,其必需氨基酸占总氨基酸含量的44．40％,必需氨基酸与非必需氨基酸含量的比值为79.8％,第一限制性氨基酸为含硫氨基酸,即蛋氨酸和胱氨酸;同时,其不饱和脂肪酸与必需脂肪酸分别占总脂肪的73．9％和24．1％,特别是油酸和亚油酸含量较高,分别达49．8％和23．4％：另外,该昆虫还含有K、Ca、Mg、Fe、Zn、Cu、Mn等多种矿物质和微量元素。     

Some methods for processing of single-cell protein

Methods for the production of protein concentrates, with a low content of nucleic acid, in kilogram quantities from yeast have been studied with the aid of equipment designed for operation on pilot-plant scale. The influence of drum drying and mechanical disintegration on the nutritive value of the yeast was also investigated. Drum drying and mechanical disintegration improved the nutritive value of the yeast but high extractability of protein and nucleic acid was only obtained after mechanical disintegration. Protein concentrates without and with cell walls were produced from mechanically disintegrated yeast. The different fractions which were obtained when separating cell walls and precipitating protein by heating at alkaline pH, were analyzed. After protein precipitation, about 90% of the RNA could be precipitated from the supernatant by addition of acid, giving a product containing 50% RNA of the dry weight. The protein precipitate obtained after cell wall separation had an RNA content of less than 2% and contained 70–l75% of the amino acids in the starting yeast material. Protein concentrates containing cell walls were produced by precipitating protein by heating at alkaline pH directly after mechanical disintegration. The content of RNA was about 2% and the yield of amino acids was 70–80%. It was found that the nutritive value of the protein concentrate was higher than that of the starting yeast material. To produce such a protein concentrate on a large scale, the process described can probably be employed.     

Effect of the chemical nature of the carbon source in the medium on the makeup of the biomass]

The yeast Candida tropicalis, Candida utilis, Trichosporon cutaneum were cultivated on the synthetic medium containing additions of group B vitamins (biotin, thiamin) and glucose, glycerol of lactic acid as the sole source. The protein content, amino acid composition, nucleic acids and complex B vitamins were identified in the resultant biomass. The carbon source in the medium affected these indices. The protein and nucleic acid content also depended on the yeast strain.     

鞭角华扁叶蜂幼虫的营养成分分析

采用现代分析技术手段对鞭角华扁叶蜂Chinolydaflagellicornis(F.Smith)幼虫的主要营养成分进行分析,结果表明其鲜虫浆的蛋白质、氨基酸、粗脂肪、糖类及灰分含量分别为17.1%,13.5%,6.7%,1.0%和1.5%,胆固醇含量为0.3mg/g;含有7种人体必需氨基酸,其必需氨基酸占总氨基酸含量的43.0%,必需氨基酸与非必需氨基酸含量的比值为75.3%,第一限制性氨基酸为含硫氨基酸,即蛋氨酸和胱氨酸;同时,其不饱和脂肪酸与必需脂肪酸分别占总脂肪的61.3%和24.3%,特别是油酸和亚麻酸含量较高,分别达37.0%和18.4%;此外,还含有K,Ca,Mg,Fe,Zn,Cu,Mn等多种矿物质和微量元素。最后在分析其营养价值的基础上,对鞭角华扁叶蜂幼虫的开发利用价值进行了评价。     

Reduction of the nucleic acid content of single-cell protein concentrates

Methods for reducing the content of nucleic acid in protein concentrates from disintegrated yeast and microalgae were investigated. Protein concentrates were prepared by acid precipitation of extracted protein after cell wall separation. The influence of alkaline protein extraction on the content of RNA in isoelectrically precipitated protein concentrates was studied. It was found that when a strong decrease in the RNA content was obtained, this was followed by a decrease in the yield of protein concentrate. Protein concentrates were also prepared without cell wall separation by precipitation with different agents after cell disintegration. In the precipitates from microalgae, a RNA reduction was obtained. Precipitation of yeast, protein gave no essential reduction with the precipitants used. Precipitation of yeast protein by heating at an alkaline pH gave a protein concentrate with a low content of RNA. A slightly lower RNA content was obtained when the precipitation was performed in the presence of NaCl. The yield of amino acid nitrogen was 70–80% and the RNA content was 1–2%. A process with precipitation at alkaline pH for the production of microbial protein concentrates with a low content of nucleic acid is suggested.     

The effect of acid shock on the heat resistance of Listeria monocytogenes

The effect of acid shock on the heat resistance of Listeria monocytogenes was investigated. After growth for 24 h at 30°C in tryptic soy broth containing 0.6% yeast extract, cell culture suspensions of L. monocytogenes were acidified with HCl or acetic acid over various time periods before being heated in whole milk to a temperature of 58°C. When cells were acid-shocked immediately with HCl for 1, 2 or 4 h, those acid-shocked for 1 h demonstrated the largest increase in thermotolerance as compared to control cells, when heated at 58°C in whole milk. In fact, cells acid-shocked for longer than 1 h with HCl demonstrated in some instances a decreased recovery as compared to control cells. Other types of acid-shock treatments included lowering the pH gradually either over a 4 h or a 24 h period. However, regardless of the type of acid-shock treatment, cells acid-shocked with HCl (but not acetic acid) prior to heating had significantly greater heat resistance as compared to control (non-acid-shocked) cells. It appears that acidification with HCl prior to final heating can enhance the heat resistance of L. monocytogenes.     

淮山药中氨基酸含量的测定

用氨基酸分析仪测定了淮山药中各种氨基酸的组成。淮山药样品经酸水解处理,用氨基酸分析仪进行分析,结果表明:淮山药中含有苏氨酸,缬氨酸,蛋氨酸,苯丙氨酸,异亮氨酸,亮氨酸和赖氨酸等17种氨基酸,总氨基酸质量分数为7.256％,其中人体必需氨基酸的含量占总氨基酸含量的25.32％;说明淮山药在医学和营养学上都有很高的研究价值。     

The synthesis of nucleic acids in cultivated <Emphasis Type="Italic">Polyscias filicifolia</Emphasis> cells exposed to oxidative stress

The content of nucleic acids in the cell culture of fern-leaf aralia Polyscias filicifolia (Moore ex Fournter) Bailey (Araliaceae) exposed to heat shock (3 h at 45°C) decreased significantly (by 20–30%). The decrease in DNA and RNA contents was even larger (30–40%) after longer heat shock (24 h). Cold (24 h at 7°C) caused an even more dramatic decrease in DNA (by 34.2%) and total RNA (by 48%) contents. To judge from the DNA production rate, the presence of hydrogen peroxide and phenazine methosulfate in the culture exerted a dose-dependent and differently directed action on cell proliferation.     

Nutrition of Myxococcus xanthus FBa and Some of Its Auxotrophic Mutants

A defined medium containing 15 amino acids plus salts was used to study the nutrition of Myxococcus xanthus FBa. The amino acids phenylalanine, leucine, isoleucine, valine, and methionine were essential for growth, whereas glycine, proline, asparagine, alanine, lysine, and threonine stimulated growth. An unusual pattern of requirement was found in the aromatic amino acids. Phenylalanine was essential and served as the precursor of tyrosine. Growth in the absence of tryptophan was adaptive, with cells reaching a growth rate equal to that of controls after a lag of about a week. (14)C-labeled ribose and glucose were not appreciably metabolized. Auxotrophs requiring purines and pyrimidines were isolated and were used to study the fate of externally supplied nucleic acid derivatives. Appropriate mutants could satisfy their requirements with free bases, nucleosides, and nucleotides, and could hydrolyze nucleic acids and use the products. However, studies using (14)C-ribose-labeled uridine (isolated from a Salmonella typhimurium pyrimidine auxotroph) showed that externally supplied nucleic acid derivatives were incorporated almost solely into the nucleic acids of the myxobacters, with little used either for energy-yielding oxidations or other cell anabolism.