High-temperature production of protein-enriched feed from cassava by fungi.

A simple, nonaseptic, low-cast process for the conversion of cassava, a starchy tropical root crop, into microbial protein for use as animal feed was sought. Screening tests culminated in the isolation of a thermotolerant, amylase-producing mold, designated I-21, which was identified as Aspergillus fumigatus. The optimum pH for protein synthesis was 3-5, but the optimum temperature was less than the desired temperature (larger than or equal to 45 C) required for a nonaseptic fermentation. A. fumigatus I-21 and its asporogenous mutant I-21A grew equally well in a medium prepared from whole cassava roots with a mean protein doubling time at 45 C and pH 3.5 of 3.5 h. In batch culture, approximately 4% carbohydrate, supplied as whole cassava, could be feremented in 20 h, giving a final yield of 24 g of dry product, containing 36.9% crude protein, per liter. The conversion of carbohydrate used to crude protein was 22.1%. When determined as amino acids, the protein content of the product, which contained cassava bark and other unfermented residues, was 27.1%. With urea as the nitrogen source, no pH control was necessary. Preliminary data indicated that medium prepared from whole cassava roots was inhibitory to the mold unless the cassava pulp was heated to 70 C immediately after being ground. Heating to 70 C was required to gelatinize the starch and permit its complete utilization.     

Two-stage fermentation method for production of protein-enriched feed from cassava

Studies have been carried out into the production of microbial protein from cassava using Trichoderma reesei and yeast. In monoculture studies, T. reesei was grown on whole cassava medium to give 0.74g dry cell/g cassava. The dry material contained 42% protein. The culture filtrate contained 5.8 g/l glucose, which supported the growth of yeast. Mixed culture fermentation was also carried out with the two microorganisms. Besides accelerating the rate of degradation and conversion of cassava to cells (0.85g cell/g cassava) the yeast boosted the protein content of the growth product to 51%.     

Stimulation of Ligninolytic Peroxidase Activity by Nitrogen Nutrients in the White Rot Fungus Bjerkandera sp. Strain BOS55

Bjerkandera sp. strain BOS55, a newly isolated wild-type white rot fungus, produced lignin peroxidase (LiP) in nitrogen (N)-sufficient glucose-peptone medium, whereas no LiP was detectable in N-limited medium. The production of LiP was induced by the peptide-containing components of this medium and also by soy bean protein. Furthermore, the production of manganese-dependent peroxidase was stimulated by organic N sources, although lower production was also evident in N-limited medium. Further research showed that the induction of LiP depended on the combination of pH and the type of N source. An amino acid mixture and ammonium induced LiP only at either pH 6 or 7.3, respectively. Peptone induced LiP activity at all pH values tested; however, the highest activity was observed at pH 7.3. The results presented here indicate that Bjerkandera spp. are distinct from the model white rot fungus, Phanerochaete chrysosporium, which produces ligninolytic peroxidases in response to N limitation.     

Formulating rations with cassava meal to promote high live weight gain in crossbred Limousin bulls

Formulating rations with high energy and protein feeds such as cassava and locally sourced protein meals is an important strategy to increase live weight gain (LWG) of crossbred bulls in Indonesia. Current systems of production for Indonesian smallholders fatten bulls using cut and carry. Formulating a diet for an optimal combination of available feeds will increase production and potential profitability for smallholders. An experiment was conducted to evaluate the effect of using cassava meal in the diet at levels of 70C, 60C, 50C, 40C and 30C% (with most of the remainder being the protein meals) on the LWG of Limousin × Ongole bulls so as to determine the optimum combination of cassava meal and protein meals for LWG. Thirty bulls were allocated in a completely randomized block design with 6 blocks based on initial live weight (LW) and 5 treatments based on level of cassava meal. The combination of cassava meal (with 2% urea) and protein meals significantly affected LWG with the highest (1.35 kg/day) recorded at 40C (40% cassava meal, 40% protein meals and 20% maize stover). The LWG and nutrient intake increased curvilinearly with decreasing cassava meal and increasing protein meals (P < 0.05). Measured cassava meal inclusion in the final ration as a consequence of the changes in intake was 60, 56, 47, 37 and 28% for the designated 70C, 60C, 50C, 40C and 30C treatments, respectively. Dry matter intake reached 96 g/kg^0.75 per day or equal to 2.24% LW at this 40% level of inclusion. At the 70C treatment with 60% cassava meal and 9% protein meals, DM digestibility (69.1%) was lowest and that value increased as the proportion of cassava meal decreased and was highest at the 40C treatment (75.8%). Feed treatments significantly affected rumen pH, ammonia N (NH₃N) and volatile fatty acid concentrations (P < 0.05). There was no significant effect on protozoal population (P > 0.05). Rumen pH ranged from 6.3 to 6.9. It was concluded that a combination of 40% dried cassava meal and 40% protein meals with roughage (20%) maximized intake and LWG and beyond that level of cassava meal inclusion, LWG and intake decreased markedly.     

Factors Affecting Yield and Safety of Protein Production from Cassava by Cephalosporium eichhorniae

The properties of Cephalosporium eichhorniae 152 (ATCC 38255) affecting protein production from cassava carbohydrate, for use as an animal feed, were studied. This strain is a true thermophile, showing optimum growth at 45° to 47°C, maximum protein yield at 45°C, and no growth at 25°C. It has an optimum pH of about 3.8 and is obligately acidophilic, being unable to sustain growth at pH 6.0 and above in a liquid medium, or pH 7.0 and above on solid media. The optimum growth conditions of pH 3.8 and 45°C were strongly inhibitive to potential contaminants. It rapidly hydrolyzed cassava starch. It did not utilize sucrose, but some (around 16%) of the small sucrose component of cassava was chemically hydrolyzed during the process. Growth with cassava meal (50 g/liter [circa 45 g/liter, glucose equivalent]) was complete in around 20 h, yielding around 22.5 g/liter (dry biomass), containing 41% crude protein (48 to 50% crude protein in the mycelium) and 31% true protein (7.0 g/liter). Resting and germinating spores (10⁶ to 10⁸ per animal) injected by various routes into normal and γ-irradiated 6-week-old mice and 7-day-old chickens failed to initiate infections.     

Effect of carbohydrates on the growth ofRhizoctonia solani Kühn

The growth OfRhizoctonia solani in different carbohydrates was studied. The rate of growth of the fungus was traced by taking the dry weights of mycelia obtained from the carbohydrate medium at regular intervals and shifts in the pH were recorded. Different carbohydrate sources had different effects on the growth of the organism. The exoenzymes from the organism were capable of cleaving carbohydrates irrespective of whether the fungus grew in them or not.     

Responses of Cultured Tobacco Cells to Cryptogein, a Proteinaceous Elicitor from Phytophthora cryptogea: Possible Plasmalemma Involvement

In culture, the phytopathogenic fungus Phytophthora cryptogea secretes a protein which elicits hypersensitive-like necroses and protects tobacco plants against invasion by the pathogen Phytophthora parasitica var. nicotianae. This protein, named cryptogein, has been purified and its amino acid sequence determined. In this work, we studied the effect of cryptogein on tobacco cell suspension cultures. Cryptogein was lethal at about 0.10 micromolar. When added at sublethal doses, it elicited the production of ethylene and phytoalexins. It also induced a rapid increase in pH and conductivity of the extracellular medium without affecting the integrity of the plasma membrane. Cryptogein reduced the fusicoccin-induced acidification of the extracellular medium. The concentration which inhibited the fusicoccin response by 50% was 0.8 nanomolar, while 1 micromolar erythrosine B, an ATPase inhibitor, was needed to produce the same inhibition. However, cryptogein did not inhibit the activity of a purified plasma membrane ATPase. Results of binding studies with whole cells suggested the presence of elicitor-binding sites with a high affinity for cryptogein. The involvement of the plasma membrane during the initial interaction between elicitor and cells is discussed.     

A novel xylanase from Streptomyces sp. FA1: Purification,characterization, identification,and heterologous expression

A xylanase (XynA) was purified from the culture medium of Streptomyces sp. FA1, which was previously isolated from a bamboo retting system. XynA had a molecular mass of 43 kDa, displayed maximal activity at pH 5.5, retained 41% of its maximal activity at pH 11.0, and was stable over a wide pH range (3.0 ～ 11.0). Purified XynA was subjected to peptide mass fingerprinting, which led to the cloning of the xynA gene. The xynA gene, which encodes a mature protein of 436 amino acids, was heterologously expressed in E. coli BL21(DE3). The activity in the culture medium could reach 213.5 U/mL, which was 11.2-fold higher than that produced by Streptomyces sp. FA1. BLAST searching revealed that full-length XynA shares less than 90% identity with most of its homologues, whereas amino acids 48-436 of the enzyme share 97% identity with an open reading frame encoding a putative full-length mature xylanase from Streptomyces tendae. The truncated xynA gene, xynA ^48-436 , was cloned and expressed in E. coli, however, no xylanase activity could be detected in the culture medium. Based on these results, it is suggested that XynA is a new member of glycoside hydrolases family10 with exceptional catalytic efficiency at alkaline pH.     

松材线虫携带一株荧光假单胞菌分泌毒素的初步研究

本文研究了离体情况下松材线虫携带的致病菌一株荧光假单胞菌（Pseudomonas fluorescens GcM5-1A）在LB、NB和PD三种培养基中的毒性,以及产生的毒素对黑松（Pinus thunbergii）切根苗和悬浮细胞的效应。结果显示,菌体在LB和NB培养液的毒性较高,其中LB培养液的毒性最高,且培养液的pH值为7时比pH值为5时毒性高,而该菌EPD培养基中几乎不产毒。细菌培养液经硫酸铵分级沉淀,得到了主要含有50kDa蛋白的蛋白组分,该蛋白组分对黑松悬浮细胞和切根苗均有较高的毒性,并能改变黑松悬浮细胞细胞膜的透性,导致胞内可溶性糖和游离氨基酸外渗。     

松材线虫携带一株荧光假单胞菌分泌毒素的初步研究

本文研究了离体情况下松材线虫携带的致病菌一株荧光假单胞菌(Pseudomonas fluorescens GcM5-1A)在LB、NB和PD三种培养基中的毒性, 以及产生的毒素对黑松(Pinus thunbergii)切根苗和悬浮细胞的效应。结果显示, 菌体在LB和NB 培养液的毒性较高, 其中 LB培养液的毒性最高, 且培养液的pH值为7时比pH值为5时毒性高, 而该菌在PD培养基中几乎不产毒。细菌培养液经硫酸铵分级沉淀, 得到了主要含有50 kDa蛋白的蛋白组分, 该蛋白组分对黑松悬浮细胞和切根苗均有较高的毒性, 并能改变黑松悬浮细胞细胞膜的透性, 导致胞内可溶性糖和游离氨基酸外渗。     

Self-assembling, protein-based intracellular bacterial organelles: emerging vehicles for encapsulating, targeting and delivering therapeutical cargoes

Background

Optimization of conditions during recombinant protein production for improved yield is a major goal for protein scientists. Typically this is achieved by changing single crucial factor settings one at a time while other factors are kept fixed through trial-and-error experimentation. This approach may introduce larger bias and fail to identify interactions between the factors resulting in failure of finding the true optimal conditions.

Results

In this study we have utilized design of experiments in order to identify optimal culture conditions with the aim to improve the final yield of the anti-keratin 8 scFv TS1-218, during expression in P. pastoris in shake flasks. The effect of: pH, temperature and methanol concentration on the yield of TS1-218 using buffered minimal medium was investigated and a predictive model established. The results demonstrated that higher starting pH and lower temperatures during induction significantly increased the yield of TS1-218. Furthermore, the result demonstrated increased biomass accumulation and cell viability at lower temperatures which suggested that the higher yield of TS1-218 could be attributed to lower protease activity in the culture medium. The optimal conditions (pH 7.1, temperature 11°C and methanol concentration 1.2%) suggested by the predictive model yielded 21.4 mg TS1-218 which is a 21-fold improvement compared to the yield prior to optimization.

Conclusion

The results demonstrated that design of experiments can be utilized for a rapid optimization of initial culture conditions and that P. pastoris is highly capable of producing and secreting functional single-chain antibody fragments at temperatures as low as 11°C.     

Biosurfactant production by Pseudomonas fluorescens 378: growth and product characteristics

Summary An isolate of Pseudomonas fluorescens, strain 378 was shown to produce a novel surface active compound (code name AP-6). The compound is unique in being a high molecular weight compound but has, in some aspects, properties of a low molecular weight surfactant. The product is extracellular and its formation appeared to be partly growth-associated. Using a semisynthetic medium, fermentor cultivations were performed in the pH range 6.8–8.4. The product yield was optimal at pH 8.0 and gave a final concentration of 210 times critical micelle dilution. At higher pH, specific growth rate, final biomass and product concentration decreased. It consists mainly of carbohydrates and protein, the molecular weight is 1×10⁶ and the isoelectric point is pH 9.1.The surface tension of an aqueous solution reached 27 mN/m which is a very low value even compared to other surfactants of considerably lower size and the critical micelle concentration was less than 10 mg/l in 0.9% (w/v) NaCl. The kinetics of the adsorption process at the air-water interface was studied using the drop volume technique, and the reaction was found to be rapid, considering the size of the molecule. A concentration as low as 0.025 g/l reached a surface tension of 30 mN/m within 70 s.     

Production of Solvents by Clostridium acetobutylicum Cultures Maintained at Neutral pH

The formation of acetone and n-butanol by Clostridium acetobutylicum NCIB 8052 (ATCC 824) was monitored in batch culture at 35°C in a glucose (2% [wt/vol]) minimal medium maintained throughout at either pH 5.0 or 7.0. At pH 5, good solvent production was obtained in the unsupplemented medium, although addition of acetate plus butyrate (10 mM each) caused solvent production to be initiated at a lower biomass concentration. At pH 7, although a purely acidogenic fermentation was maintained in the unsupplemented medium, low concentrations of acetone and n-butanol were produced when the glucose content of the medium was increased (to 4% [wt/vol]). Substantial solvent concentrations were, however, obtained at pH 7 in the 2% glucose medium supplemented with high concentrations of acetate plus butyrate (100 mM each, supplied as their potassium salts). Thus, C. acetobutylicum NCIB 8052, like C. beijerinckii VPI 13436, is able to produce solvents at neutral pH, although good yields are obtained only when adequately high concentrations of acetate and butyrate are supplied. Supplementation of the glucose minimal medium with propionate (20 mM) at pH 5 led to the production of some n-propanol as well as acetone and n-butanol; the final culture medium was virtually acid free. At pH 7, supplementation with propionate (150 mM) again led to the formation of n-propanol but also provoked production of some acetone and n-butanol, although in considerably smaller amounts than were obtained when the same basal medium had been fortified with acetate and butyrate at pH 7.     

Utilization of cellulose from waste paper by Myrothecium verrucaria

Extensive screening studies on cellulolytic bacteria and fungi led to the selection of Myrothecium verrucaria as the organism producing the maximum rate of protein biosynthesis from ball-milled newspaper. Studies in aerated stirred-jar fermentors were carried out to determine the conditions for maximum protein synthesis rate and maximum final protein concentration. The optimum aeration rate was 250 to 374 mM of oxygen at 300 to 400 rpm stirring rate. The pH optimum was broad, from 3.9 to 6.5. Urea at 0.03% and yeast autolysate at 0.1% stimulated growth rate and protein production. The maximum rate of protein biosynthesis and the maximum protein yield were 0.3 g/liter/day and 1.42 g/liter, respectively, from medium G3 with 4% ball-milled newspaper. The final product, obtained by evaporation of the total culture, was 33.7 g from one liter of medium which originally contained 40 g of ball-milled newspaper and 11.3 g of other dissolved materials. The protein content of this final product was 3.3 g, calculated from total organic N × 6.25 or 1.42 g calculated from the biuret method. Both the synthesis rate and the final cell yield are below those obtainable by growing Fungi Imperfecti, yeasts or bacteria on soluble materials such as glucose.     

Products of enzymatic hydrolysis of cellulose and its derivatives

Cellobiose and glucose were determined in a mixture of the two carbohydrates by methods involving the use of glucose oxidase and of β-glucosidase.Paper-partition chromatography is used as a confirmatory method in the identification of the hydrolysis products and in the detection of the various constituents.The cellulolytic organisms studied produce large amounts of the enzyme Cx, which diffuses into the medium. Only small amounts of β-glucosidase are found outside the cell. Cellobiose resulting from Cx activity can enter the cells as rapidly as can glucose.The role of cellobiose as a principal product in the hydrolysis of cellulose is confirmed. It is hypothesized that the principal final product of Cx activity is cellobiose, and that the presence of cellobiase in the medium is not a prerequisite to utilization of cellobiose by the organism. This is a correction of the hypothesis previously published stating that glucose appeared to be the final product of Cx activity.     

Direct evidence for the presence of beta-hydroxyphenylalanine and beta-hydroxytyrosine in cutinase from Fusarium solani pisi

Extracellular cutinase induced by cutin hydrolysate in glucose-grown Fusarium solani f. pisi was isolated in electrophoretically homogeneous form. This enzyme was similar to cutinase I generated by cutin-grown cells in its catalytic properties such as pH optimum, substrate specificity, and inactivation by “active serine”-directed reagents. Its molecular weight was 26,300 and this enzyme had a much larger content of serine and threonine residues than that found in cutinase from the cutin-grown cells. The hydrolysate-induced enzyme was a glycoprotein containing 6% carbohydrates. Alkaline NaB³H₄ treatment of the protein generated labeled protein and labeled carbohydrates. Analyses of the hydrolysates of these labeled products showed that alanine, α-aminobutyrate, phenylalanine, and tyrosine in the protein were labeled strongly suggesting that serine, threonine, β-hydroxyphenylalanine, and β-hydroxytyrosine were involved in O-glycosidic linkages in this protein. The protein hydrolysate also contained labeled gulonic acid, suggesting that d-glucuronic acid was attached to the protein via a base stable linkage, presumably an amide linkage at the N-terminus. The labeled reduced carbohydrates were identified by ion-exchange, thin-layer, gas-liquid, and high-performance liquid chromatographic techniques as mannitol, arabitol, gulonic acid, and 2-aminosorbitol. Thus mannose, arabinose, glucuronic acid, and glucosamine (possibly N-acetylated) were attached O-glycosidically to the hydroxyamino acids. Induction of cutinase by cutin hydrolysate in the presence of tritiated phenylalanine gave labeled cutinase. Cleavage of the O-glycosidically attached carbohydrates by anhydrous HF, followed by enzymatic hydrolysis of the labeled protein, gave rise to labeled amino acids, which upon analysis with an amino acid analyzer revealed four radioactive components. Two of them were identified as phenylalanine and tyrosine, while the other two cochromatographed with authentic β-hydroxyphenylalanine and β-hydroxytyrosine not only by the amino acid analyzer but also upon thin-layer chromatography. These results constitute the first direct evidence for the presence of the novel β-hydroxyaromatic amino acids in a protein.     

Diauxic growth of Penicillium camembertii on glucose and arginine

The growth of Penicillium camembertii during batch culture in a synthetic medium containing glucose and arginine was examined. The diauxic growth observed can be well characterized. Indeed, in a first phase, glucose and arginine were, respectively, assimilated as carbon and nitrogen sources, with an acidification of the medium (until 3.5), since arginine was taken up in exchange for protons. During this phase of growth, arginine, in addition to glucose, was also assimilated as an energy source, resulting in the release of the arginine carbon content as CO₂. Then, in a second phase, characterized by reduced growth rates after glucose depletion, arginine was assimilated as a carbon and nitrogen source, as well as an energy source, resulting in ammonium release which raised the pH (final pH 6.3), despite the amino acid/H⁺ exchange, since amino acids contain excess nitrogen in relation to their carbon content for fungi.     

Use of a Cellulase-Derepressed Mutant of Cellulomonas in the Production of a Single-Cell Protein Product from Cellulose

A cellulase-derepressed mutant of a Cellulomonas species was used to produce single-cell protein from crystalline cellulose. In preliminary tests, maximum yield of single-cell protein was obtained at 30°C (pH 7.0) with urea as the nitrogen source. A continuous-flow foam flotation procedure was developed for rapid and efficient separation of bacteria from the culture liquid and cellulose residue. A pH of 4.5 was optimum for foam flotation of this organism. In preliminary trials, recovery was 85% of the cells with the flotation procedure. Cellulomonas was 68% true protein and had an essential amino acid profile featuring a high lysine content (6.5% of protein). The Cellulomonas product was evaluated nutritionally with weanling rats. The net protein utilization value for the protein supplemented with methionine was 50.4% Weight gain of rats on the Cellulomonas diet was similar to that of rats fed a casein diet.     

Raw cassava starch-digestive glucoamylase of Aspergillus sp. N-2 isolated from cassava chips

One hundred and eighty strains of black aspergilli isolated from cassava fields and factories in Thailand were screened for the activity of raw cassava starch-digestive glucoamylase. Aspergillus sp. N-2 was selected as the best producer and its extracellular glucoamylase production was investigated. Conditions for the production were optimized for both liquid and solid cultures, and solid culture was found to be approximately three times more efficient than liquid culture. The culture filtrate showed strong glucoamylase activity at low pH (pH 2.0) and high temperature (55°C), and could digest high concentration raw cassava starch. The glucoamylase activity was separated to four fractions (A, B, C and D) by DEAE-Sephacel column chromatography. Fraction C was obtained in a homogeneous state with a molecular weight of 92,000. Each fraction was characterized in terms of the properties of the glucoamylase activity and the efficiency of digestion of cooked and raw cassava starch.     

Chemostat optimization of biomass production of a mixed bacterial culture utilizing methanol

Summary A continuous culture technique was used to optimize the medium composition and growth conditions of a mixed bacterial culture utilizing methanol. The improved medium resulted in satisfactory growth, high-yield coefficients and gave a product containing reduced polysaccharide concentrations. Optimal growth and biomass yields occurred at pH 6.8 a temperature of 37° C and dissolved oxygen at >20% saturation. The maximum growth rate was 0.58 h^–1 and maximum biomass yield 0.48 g g^–1. The protein content of the product ranged between 81%–83%, and nucleic acid content between 10%–12%, increasing with growth rate. The amino acid profile of the mixed culture product met and, in some cases, exceeded the UN Food and Agricultural Organization standard, indicating a good source of feed protein.Offprint requests to: A. S. Abu-Ruwaida