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.     

Properties of intracellular ribonuclease utilized for RNA reduction in disintegrated cells of Saccharomyces cerevisiae.

The properties of intracellular RNase in disintegrated cell suspensions of Saccharomyces cerevisiae have been studied. The influence of salt addition and/or incubation of the suspension on the activity of RNase and on the degradation of endogenous RNA was determined. No significant change in the RNase activity in the disintegrated suspensions was obtained by addition of 3% NaCl or by incubation at 50 degrees C with 3% NaCl. During the incubation with NaCl the active RNase was able to degrade endogenous RNA. By incubation without salt the RNase was inactivated. Inactivation also occurred after extraction at alkaline pH. The RNase had an optima at pH 5-6 and temperatures between 50-60 degrees C. The main part of the RNase in the unincubated suspension was soluble also at pH 4.0. No serious protein degradation occurred during the short time incubation needed for RNA reduction. 70% of the protein in the suspensions was recovered in the precipitate at pH 4.0 after 20 min of incubation. The corresponding protein recovery from unincubated suspensions was 77%.     

The influence of alkali and heat treatment on yeast protein

The soluble components in disintegrated cells of Saccharomyces cereivisiae have been characterized by means of extraction, centrifugation, dialysis, and gel filtration. The influence of alkali and heat treatment on the protein and RNA in the soluble fraction from disintegrated yeast cells and on functional properties of protein concentrates have been studied. After water extraction and centrifugation at 100000 g 42% of the nitrogen containing components of the disintegrated cells were recovered in the supernatant. By extraction at pH 11.5 an additional 31% of the nitrogen was solubilized. Half of the water-soluble nitrogen-containing components has a molecular weight lower than 5000. In the water- and alkali-soluble fractions about 80% of each amino acid was recovered The water-soluble protein was separated into 3 fractions by gel filtration on Sephadex G 200. The major portion of the protein had a molecular weight about 100,000. The amount of protein in this fraction was decreased after treatment at increasing pH and temperature. No degradation of protein to low molecular peptides occurred. The amount of RNA in the soluble fraction was only slightly influenced by alkali treatment and by heat treatment at pH 7.5 in the presence of 5% NaCl. RNA was not degraded to low molecular components of the treatments. The solubility of protein concentrates decreased after treatment at alkaline pH and after heat precipitation.     

Regulatory Influences on the Production of Gamma-Aminobutyric Acid by a Marine Pseudomonad

A pseudomonad capable of producing γ-aminobutyric acid (GABA) was isolated from seawater via an enrichment in which glutamate was the sole carbon and nitrogen source. The organism grew optimally at pH 7.3 and at 25°C. Putrescine, alanine, and glucose-nitrate also served as effective growth substrates. The isolate grew poorly on GABA. Cell suspensions of the organism in 0.02 M phosphate buffer (pH 7.6) containing NaCl (19.4 g liter^-1) and MgCl₂. 6H₂O(3 g liter^-1) produced GABA from succinic semialdehyde in combination with glutamate or alanine but not from any substrate alone. Little or no GABA was produced with putrescine or glucose-nitrate as substrates. GABA production in the amino acid cosubstrate systems was transitory with optimum levels occurring in the suspension fluid after 3 h of incubation (0.3 and 0.03 mM for glutamate and alanine cosubstrates, respectively). However, yields of GABA in the cell suspension fluid were low, and quantities near that predicted from stoichiometry could be obtained only by extracting cell suspensions with methanol. GABA release in the suspension fluid was increased with higher pH or by decreasing NaCl. Substitution of the salt by the equivalent Tris-HCl or KCl likewise resulted in increased GABA release. When nigericin (10 μg ml^-1) was added to cell suspensions in which NaCl was not decreased, GABA release increased in a way similar to that observed in suspensions with decreased NaCl. The ionophore also decreased GABA uptake by cell suspensions of GABA-grown cells, and the effect was duplicated by lowering NaCl in cell suspensions. The results indicate a role for an Na⁺-dependent transport system in GABA release.     

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.     

Production,characterization, and immobilization of partially purified surfactant–detergent and alkali-thermostable protease from newly isolated Aeromonas caviae

Proteolytic Aeromonas caviae P-1-1 growing at wide-ranging pH (7.0–11.0) and moderate salinity (0–5% NaCl) was isolated from cattle shed of Thanjavur, India. It produced lipase, gelatinase, and polyhydroxybutyrate. Different culture conditions, incubation time, carbon and nitrogen sources, vitamins, amino acids, surfactants, and metal ions for optimal growth and protease production of P-1-1 were examined. Maximum protease (0.128?U/mL) production was achieved with 1% fructose, 1% yeast extract, 0.1% ammonium sulfate, 3% NaCl, 0.1% CaCl₂?·?2H₂O, 1% glycine, 0.1% vitamin E, and 0.1% Tween-40 at pH 8.0 after 42?hr of incubation at 37°C. It was active over broad range of pH (7.0–12.0), temperature (15–100°C), and salinity (0–9% NaCl) with optima at pH 10.0, 55°C, and 3% NaCl. It retained 65 and 48% activities at pH 12.0 and 100°C, respectively. Partially purified protease was highly stable (100%) within pH range 7.0–12.0 and salinities of 0–5% NaCl for 48?hr. Cu²⁺, Mn²⁺, Co²⁺, and Ca²⁺ did not inhibit its activity. Its stability at extreme pHs, temperatures, and in the presence of surfactants and commercial detergents suggests its possible application in laundry detergents. Partially purified protease was immobilized and reused. This is the first report of alkali-thermotolerant, surfactant–detergent-stable partially purified extracellular protease from A. caviae.     

Changes in Sugars, Sucrose Synthase Activity and Proteins in Salinity Tolerant Callus and Cell Suspension Cultures of Brassica oleracea L.

Salt tolerant callus and cell suspension cultures of Brassica oleracea L. var. botrytis were obtained by the selection of cells from cultures growing in medium supplemented with 85, 170, and 255 mM NaCl. Salt adapted calli and cell suspensions differed in their RNA and protein concentrations. These concentrations tend to diminish in calli and increase in cell suspensions, both at one or three weeks periods of growth in NaCl. Contents of sucrose and reducing sugars, however, accumulate similarly both in calli and cell suspensions after NaCl treatments. The activity of sucrose synthase was higher in salt adapted cells than in controls. Calli exposed to 255 mM NaCl for six months synthesized a 27 kDa polypeptide, while a 13 kDa polypeptide present in control conditions was absent under salinity. Several high molecular mass polypeptides (> 200 kDa) were visualized in control calli and at moderate salt concentrations, when conditions of the gel were modified.     

Screening and optimization of protease production from a halotolerant Bacillus licheniformis isolated from saltern sediments

Protease producing halotolerant bacterium was isolated from saltern pond sediment (Tuticorin) and identified as Bacillus licheniformis (TD4) by 16S rRNA gene sequencing. Protease production was enhanced by optimizing the culture conditions. The nutritional factors such as carbon and nitrogen sources, NaCl and also physical parameters like incubation time, pH, agitation, inoculum size were optimized for the maximum yield of protease. Studies on the effect of different carbon and nitrogen sources revealed that xylose and urea enhances the enzyme production. Thus, with selected C–N sources along with 1 M NaCl the maximum protease production (141.46 U/mg) was obtained in the period of 24 h incubation at pH 8 under 250 rpm compared to the initial enzyme production (89.87 U/mg).     

Effect of ph, temperature and salinity on the production of gamma aminobutyric acid (GABA) from amines by marine bacteria

Abstract Bacteria isolated from sea-water grew on putrescine and spermidine as the sole carbon and nitrogen source, but not on cadaverine. Cell suspensions of one isolate (PU-8) produced gamma aminobutyric acid (GABA) from putrescine in 0.02 M phosphate buffer (pH 7.6) containing 0.33 M NaCl and 15 mM MgCl₂, and three other isolates produced the inducer when gabaculine (a natural inhibitor of GABA metabolism) was added. None of the isolates produced GABA from spermidine either in the absence or presence of gabaculine. Yields of GABA from putrescine were low in the suspension fluid and near stoichiometric quantities could only be obtained by extraction of incubations with methanol. Decreased NaCl (< 0.05 M) or increased pH resulted in an increase of GABA released into the suspension fluid during incubations, although in growth cultures only pH appeared to have a substantial effect. GABA release was not influenced by temperature in the range 17 to 32°C. Replacement of the normal concentration of NaCl (0.33 M) with equivalent LiCl, sodium glucuronate, or sucrose in cell suspensions did not result in increased GABA in the suspension fluid, indicating non-involvement of a sodium or chloride ion-dependent transport system in GABA release. The results show that marine bacteria can produce GABA, an inducer of marine invertebrate larval settlement, and indicate that extenal changes in osmotic pressure and pH which influence GABA release may be important factors to consider in the production of this inducer.     

Production of extracellular alkaline lipase by a new thermophilicBacillus sp

A thermophilic soil isolate—Bacillus sp. RS-12, grew optimally at 50°C and not below 40°C. Production of an extracellular lipase by this organism was substantially enhanced when the type and concentration of carbon and nitrogen sources and initial pH of the culture medium were consecutively optimized. The lipase production was found to be growth-associated with maximum secretion in the late exponential growth phase,i.e. 15h of incubation. The enzyme activity as high as 0.98 nkat/mL was obtained under optimum conditions. Tween 80 (0.5%) and yeast extract (0.5%) were found to be the best carbon and nitrogen sources inducing maximum enzyme yield with initial pH 8.0 at 50°C. The kinetic characteristics of the crude lipase indicated the highest activity at 50–55°C and pH 8.0. It had a half life of 60, 18 and 15 min at 65, 70 and 75°C, respectively.     

Nucleic acid reduction from yeast activation of intracellular rnase

The study of a heat-shock process for RNA reduction was carried out for different yeast strains. Different results were obtained from each of them. Candida utilis NRRL Y-660 shows its best performance after a 8-s. heat-shock in the presence of 3% NaCl. For commercial baker's yeast Saccharomyces cerevisiae and Kluyveromyces fragilis L-1930, similar results were obtained with only 1% of NaCl. The latter needed longer heat-shock periods. e.g. 15s. to give such an RNA reduction. Biomass recovery ranged from 60 to 75%, being higher for C. utilis and K. fragilis while excessive losses were observed in S. cerevisiae cells. No significant protein deterioration was obtained in the best performance samples. The aminoacid profile appears to be improved in comparison to the starting material in these strains after RNA reduction.     

Removal of RNA by heat treatment

Summary Eight bacterial strains were subjected to a discontinuous heat shock treatment aimed at causing a degradation of RNA. The treatment involved a 10 s to 10 min exposure to 65°C and then an incubation period of up to 3 h at 50°C. At intervals the cells were analyzed for RNA, DNA and protein. Whereas the contents of protein and DNA were not affected, RNA was degraded. An almost complete degradation of RNA occurred inAlcaligenes eutrophus H 16 — PHB^–4 andEscherichia coli K 12; only about 50% of the cellular RNA were degraded inPseudomonas putida andP.flava GA; inCorynebacterium autotrophicum 7 C,Nocardia opaca 1 b and coryneform strains 11 X and 30.1 b RNA degradation occurred only to a small extent.A continuous flow system for the treatment of cell suspensions by heat shock followed by incubation at an elevated temperature was developed. The results confirmed those obtained by batch-wise heat treatment.     

Cellulase from Bacillus licheniformis and Brucella sp. isolated from mangrove soils of Mahanadi river delta,Odisha, India

In the present study, two cellulose-degrading bacteria (CDB-5 and CDB-12) were isolated from mangrove soils of Mahanadi river delta, based on halo zone formation in Congo red agar medium and evaluation for cellulase production in CMC broth medium. Based on morphological, biochemical and 16S rRNA gene sequencing, the two strains, CDB-5 and CDB-12, were identified as Brucella sp. and Bacillus licheniformis, respectively. The gene bank accession number of the strains CDB-5 and CDB-12 are KR632646 and KR632645, respectively. The strain Brucella sp. and B. licheniformis showed an enzyme activity of 96.37?U/ml and 98.25?U/ml, respectively, after 72?h of incubation period. Enzyme production was optimized under different growth conditions such as pH, temperature, agitation rate, carbon source, sodium chloride (NaCl), and nitrogen sources. Maximum cellulase production by both the strains was obtained in the same parameter condition such as pH (7.0), rpm (150), and NaCl (2%, w/v) which varies for other parameters. The strain, CDB-5, produced maximum cellulase at 35?°C temperature, maltose as a carbon source, and yeast extract as a nitrogen source where as the strain CDB-12 produces maximum cellulase at 45?°C temperature, carboxyl methyl cellulose (CMC) as carbon source and trypton as a nitrogen source. The bacterial crude enzyme was purified by ammonium sulfate precipitation followed by overnight dialysis. SDS-PAGE analysis of the partially purified cellulase enzyme exhibited band sizes of approximately 55 and 72?kDa.     

Xylanase production using inexpensive agricultural wastes and its partial characterization from a halophilic <Emphasis Type="Italic">Chromohalobacter</Emphasis> sp. TPSV 101

A halophilic and alkali-tolerant Chromohalobacter sp. TPSV 101 with an ability to produce extracellular halophilic, alkali-tolerant and moderately thermostable xylanase was isolated from solar salterns. Identification of the bacterium was done based upon biochemical tests and 16S rRNA sequence. The culture conditions for higher xylanase production were optimized with respect to NaCl, pH, temperature, substrates and metal ions and additives. Maximum xylanase production was achieved in the medium with 20% NaCl, pH-9.0 at 40°C supplemented with 1% (w/v) sugarcane bagasse and 0.5% feather hydrolysate as carbon and nitrogen sources. Sugarcane bagasse (250 U/ml) and wheat bran (190 U/ml) were the best inducer of xylanase when used as carbon source as compared to xylan (61 U/ml). The xylanase that was partially purified by protein concentrator had a molecular mass of 15 kDa approximately. The xylanase from Chromohalobacter sp. TPSV 101 was active at pH 9.0 and required 20% NaCl for optimal xylanolytic activity and was active over a broad range of temperature 40–80°C with 65°C as optimum. The early stage hydrolysis products of sugarcane bagasse were xylose and xylobiose, after longer periods of incubation only xylose was detected.     

Investigation of lipase production by a new isolate of Aspergillus sp.

Fungi isolated from soil were screened for exogenous lipolytic activity. The highest lipase activity was found in a new soil isolate of Aspergillus sp. Some optimal cultural parameters influencing the growth and production of extracellular lipase from this Aspergillus sp. were investigated. The lipase yield was maximum on day 4 of incubation of the culture at pH 5.5 and 30 °C. When the medium was prepared using olive oil as carbon source and peptone as a nitrogen source, better lipase yields were obtained. Aeration enhanced growth and lipase production.     

Thermoalkalophilic lipase from an extremely halophilic bacterial strain Bacillus atrophaeus FSHM2: Purification,biochemical characterization and application

The present study was designed to isolate and identify an extremely halophilic lipase-producing bacterial strain, purify and characterize the related enzyme and evaluate its application for ethyl and methyl valerate synthesis. Among four halophilic isolates, the lipolytic ability of one isolate (identified as Bacillus atrophaeus FSHM2) was confirmed. The enzyme (designated as BaL) was purified using three sequential steps of ethanol precipitation and dialysis, Q-Sepharose XL anion-exchange chromatography and SP Sepharose cation-exchange chromatography with a final yield of 9.9% and a purification factor of 31.8. The purified BaL (Mw～85?kDa) was most active at 70?°C and pH 9 in the presence of 4 M NaCl and retained 58.7% of its initial activity after 150?min of incubation at 80?°C. The enzyme was inhibited by Cd²⁺ (35.6?±?1.7%) but activated by Ca²⁺ (132.4?±?2.2%). Evaluation of BaL's stability in the presence of organic solvents showed that xylene (25%) enhanced the relative activity of the enzyme to 334.2?±?0.6% after 1?h of incubation. The results of esterification studies using the purified BaL revealed that maximum ethyl valerate (88.5%) and methyl valerate (67.5%) synthesis occurred in the organic solvent medium (xylene) after 48?h of incubation at 50?°C.     

THE BOOK CORNER

In order to increase the yield of prothrombin complex concentrates (PCCs) and to reduce their associated thrombotic risks, the influence of washing conditions on the yield, purity, and balance of coagulation factors (FII, FVII, FIX, and FX), and inhibitor proteins (PC, PS, PZ, and AT [antithrombin]) in PCCs was investigated by orthogonal testing, in which three variables (sodium citrate, NaCl, and pH) and their three levels were selected. It was found that AT yield and purity were extraordinarily low, and at lower NaCl content, the general yield, purity, and balance were higher, lower, and better, respectively; however, the results became contrary at higher NaCl. Moreover, within the investigated levels, NaCl was the first determinant for the yield except AT and the purity except FVII, PC, PS, and AT. Sodium citrate was the first determinant for AT yield and FVII, PS, and AT purity. The yield except FII, PS, and AT decreased and the purity except PC increased with increase of sodium citrate content. Just for PC purity, pH was the first determinant. The effect with pH fluctuation on the yield and purity was characteristically unobvious. The outcome undoubtedly supplies the guidance to further improve PCCs.     

Bacterial growth on N,N-dimethylformamide: implications for the biotreatment of industrial wastewater

The degradation of N,N-dimethylformamide (DMF) by bacterial consortia was investigated under aerobic, fermentative and nitrate-reducing conditions and a variety of salt concentrations (0.2%, 4% and 7% NaCl w/v) and pH values (5 and 7). Optimization of degradation conditions was studied to provide information and recommendations for large-scale biological treatment processes. Under aerobic conditions, mineralization of DMF (200 mg l⁻¹, 2.7 mM) was achieved under all combinations of salinity and pH. The rate of bacterial growth decreased with increasing salinity. Changes in the salt concentration and pH still resulted in mineralization and unchanged yield of bacterial cells. At 0.2% NaCl and either pH 5 or 7, growth occurred on DMF in the range 0.2–1 g l⁻¹. However, cell yield decreased with increasing concentrations of DMF. Under conditions of 0.2% NaCl, pH 7 and 4% NaCl, pH 5, growth on DMF at 5 g l⁻¹ resulted in the production of an intermediate that was detected using gas chromatography (GC). It is proposed that the intermediate was dimethylamine, and its persistence in growth media was attributed to suppressed growth as a result of an increase in pH. A culture capable of degrading DMF under nitrate-reducing conditions was obtained at 0.2% NaCl and pH 7, but not at more saline and acidic conditions. Growth and degradation of DMF were considerably slower under these conditions compared with aerobic conditions. Fermentative degradation of DMF was not observed. Journal of Industrial Microbiology & Biotechnology (2000) 25, 8–16. Received 14 July 1999/ Accepted in revised form 30 March 2000