Effect of salicin on induction and carbon catabolite repression of endoxylanase synthesis in Penicillium janthinellum MTCC 10889

Amongst various carbon sources, xylan was found to be the sole inducer of endoxylanase production by Penicillium janthinellum MTCC 10889 in submerged cultivation. Endoxylanase synthesis by a xylan induced culture was initially repressed after a simultaneous addition of xylose, probably by the inducer exclusion mechanism, but it was resumed and achieved its highest level at a much later stage of growth (at 120 h). Xylose added after 30 h of growth cannot exert its full repressive effect. Although glucose was proved to be a more potent repressor than xylose, supplementation of salicin, an alcoholic β-glycoside containing d-glucose, with pure xylan resulted in an about 3.22 fold increase in the enzyme synthesis at 72 h followed by constant high production of the enzyme at least until the 144th h of growth. Inducing capacity of salicin in a xylan induced culture was significantly reduced when it was added after 30 h of growth. Addition of salicin and xylan help to partially overcome the repressive effect of xylose and glucose. Failure of salicin in recovering the endoxylanase synthesis in actinomycin D and cyclohexamide inhibited the xylan induced culture indicating that salicin cannot initiate the de novo synthesis of the enzyme.     

Production, purification, and characterization of a low-molecular-mass xylanase from Aspergillus sp. and its application in baking

An extracellular xylanase produced by a Mexican Aspergillus strain was purified and characterized. Aspergillus sp. FP-470 was able to grow and produce extracellular xylanases on birchwood xylan, oat spelt xylan, wheat straw, and corncob, with higher production observed on corncob. The strain also produced enzymes with cellulase, amylase, and pectinase activities on this substrate. A 22-kDa endoxylanase was purified 30-fold. Optimum temperature and pH were 60°C and 5.5, respectively, and isoelectric point was 9.0. The enzyme has good stability from pH 5.0 to 10.0 retaining >80% of its original activity within this range. Half-lives of 150 min at 50°C and 6.5 min at 60°C were found. K _m and activation energy values were 3.8 mg/mL and 26 kJ/mol, respectively, using birch wood xylan as substrate. The enzyme showed a higher affinity for 4-O-methyl-d-glucuronoxylan with a K _m of 1.9 mg/mL. The enzyme displayed no activity toward other polysaccharides, including cellulose. Baking trials were conducted using the crude filtrate and purified enzyme. Addition of both preparations improved bread volume. However, addition of purified endoxylanase caused a 30% increase in volume over the crude extract.     

Detection of major xylanase-containing cellulose-binding domain from Penicillium verruculosum by combination of chromatofocusing and limited proteolysis

Adsorption on microcrystalline cell ulose of enzyme components of cellulase complex from Penicillium verruculosum was studied by chromatofocusing on a Mono P column. The most strongly adsorbed and major component was identified as xylanase (XYN) with MW 65 k Da and pl 4.5. The high adsorption degree of XYN on cellulose indicated the possible presence of a cellulose-binding domain in the molecular sturcture. Limited proteolysis of XYN with papain was carried out. Kinetics of proteolysis was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and measuring activities toward insoluble xylan and 4-methylumbelliferyl-β-d-lactoside (MUF-LAC). During the proteolysis, formation of two polypeptides with MW 51 and 14k Da was observed. No loss of activity toward thesolu blesubstrate was observed, wherease the activity toward xylan decreased rapidly. Adsorption distribution coefficient (K _d) of the core protein separated by gel-filtration was found to be 15 times lower than the K _d for the initial nondigested XYN (0.02 and 0.29 L/g, respectively). The activity of core protein toward insoluble xylan was close to zero, whereas the activity toward MUF-LAC was close to that exhibited by the original enzyme. The results presented indicate a bifunctional organization of XYN, where one domain acts as a binding anchor for insoluble substrates and the other, localized in the core protein, contains the active site.     

Xylanase production by the thermophilic mold Humicola lanuginosa in solid-state fermentation

Among the lignocellulosic substrates tested, wheat bran supported a high xylanase (EC 3.2.1.8) secretion by Humicola lanuginosa in solid-state fermentation (SSF). Enzyme production reached a peak in 72 h followed by a decline thereafter. Enzyme production was very high (7832 U/g of dry moldy bran) when wheat bran was moistened with tap water at a substrate-to-moistening agent ratio of 1:2.5 (w/v) and an inoculum level of 3 × 10⁶ spores/10 g of wheat bran at a water activity (a _w ) of 0.95. Cultivation of the mold in large enamel trays yielded a xylanase titer comparable with that in flasks. Parametric optimization resulted in a 31% increase in enzyme production in SSF. Xylanase production was approx 23-fold higher in SSF than in submerged fermentation (SmF). A threshold constitutive level of xylanase was secreted by H. lanuginosa in a medium containing glucose as the sole carbon source. The enzyme was induced by xylose and xylan. Enzyme synthesis was repressed beyond 1.0% (w/v) xylose in SmF, whereas it was unaffected up to 3.0% (w/w) in SSF, suggesting a minimization of catabolite repression in SSF.     

A β-glucosidase from Sclerotinia sclerotiorum

The filamentous fungus Sclerotinia sclerotiorum, grown on a xylose medium, was found to excrete one β-glucosidase (β-glu x). The enzyme was purified to apparent homogeneity by ammonium sulfate precipitation, gel filtration, anion-exchange chromatography, and high-performance liquid chromatography (HPLC) gel filtration chromatography. Its molecular mass was estimated to be 130 kDa by HPLC gel filtration and 60 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, suggesting that β-glu x may be a homodimer. For p-nitrophenyl β-d-glucopyranoside hydrolysis, apparent K _m and V _max values were found to be 0.09 mM and 193 U/mg, respectively, while optimum temperature and pH were 55–60°C and pH 5.0, respectively. β-Glu x was strongly inhibited by Fe²⁺ and activated about 35% by Ca²⁺. β-Glu x possesses strong transglucosylation activity in comparison with commercially available β-glucosidases. The production rate of total glucooligosaccharides (GOSs) from 30% cellobiose at 50°C and pH 5.0 for 6 h with 0.6 U/mL of enzyme preparation was 80 g/L. It reached 105 g/L under the same conditions when using cellobiose at 350 g/L (1.023 M). Finally, GOS structure was determined by mass spectrometry and ¹³C nuclear magnetic resonance spectroscopy.     

Morphology and size-controlled synthesis of silver nanoparticles in aqueous surfactant polymer solutions

We have employed a number of reducing and capping agents to obtain Ag(0) metallic nanoparticles of various sizes and morphologies. The size and morphology were tuned by selecting reducing and capping agents. Spherical particles of 15 and 43 nm diameter were obtained when 1 wt% aqueous starch solution of AgNO₃ precursor salt was reduced by d(+)-glucose and NaOH, respectively, on heating at 70 °C for 30 min. Smaller size particles obtained in the case of d(+)-glucose reduction has been attributed to the slow reduction rate by mild reducing agent d(+)-glucose compared to strong NaOH. Conducting the reduction at ambient temperature of silver salt in liquid crystalline pluronic P123 and L64 also gave spherical particles of 8 and 24 nm, respectively, without the addition of any separate reducing agent. NaOH reduction of salt in ethylene glycol (11 g)/polyvinyl pyrolidone (PVP; 0.053 g) mixture produced large self-assembled cubes of 520 nm when smaller (26–53 nm) star-shaped sharp-edged structures formed initially aggregated on heating the preparation at 190 °C for 1 h. Increasing the amount of PVP (0.5 g) in ethylene glycol (11 g) and heating at 70 °C for 30 min yielded a mixture of spherical and non-spherical (cubes, hexagons, pentagons, and triangle) particles without the addition of an extra reducing agent. Addition of 5 wt% PVP to 1 wt% aqueous starched solution resulted in the formation of a mixture of spherical and anisotropic structures when solution heated at 70 °C for 1 h. Homogeneous smaller sized (29 nm) cubes were synthesized by NaOH reduction of AgNO₃ in 12.5 wt% of water-soluble polymer poly(methyl vinyl ether) at ambient temperature in 30 min reaction time.     

Enhanced Production of an Extracellular β-<Emphasis Type="SmallCaps">d</Emphasis>-Fructofuranosidase Fructohydrolase from a 2-Deoxy-<Emphasis Type="SmallCaps">d</Emphasis>-glucose Stabilized Mutant of <Emphasis Type="Italic">Candida utilis</Emphasis>

The enzyme β-d-fructofuranosidase fructohydrolase (FFH) cleaves the α-1,4 glycosidic linkage between α-d-glucose and β-d-fructose molecules of sucrose, releasing monosaccharides by hydrolysis. In the present study, FFH production in Candida utilis GC-46, a lipolytic wild yeast strain was improved by exposure to N-methyl N-nitro N-nitroso guanidine (NG) and 2-deoxy-d-glucose (2dg) at various levels. The mutant strain NG-5 was obtained after exposure to 0.06 mg/ml of NG for 20 min. NG-5 offers improved extracellular FFH production (34 ± 2.6 U/ml/min) when compared to the wild strain (1.15 ± 0.01 U/ml/min). A 40-fold increase of FFH (45.65 ± 2.0 U/ml/min) was achieved when the process parameters, including incubation period (48 h), sucrose concentration (5.0 g/l), initial pH (6.0), inoculum size (2.0% v/v, 16 h old), and urea concentration (0.2%, w/v) were identified using Plackett–Burman design. The kinetic parameters viz. Q _p (0.723 U/g/h), Y _p/s (2.036 U/g), and q _p (0.091 U/g yeast cells/h) indicate that NG-5 is a hyperproducer of extracellular FFH with a concomitant increase in growth rate. The volumetric productivity of NG-5 was over sixfold improved over the parental strain. The enzyme production improvement is highly significant (HS, LSD 0.042, p ≤ 0.05), indicating commercial utility.     

Production of extracellular xylanases byPenicillium janthinellum

Xylanase production byPenicillium janthinellum using 10–100 mM of 2,2-dimethylsuccinate (DMS) buffer, in a range of pH 4.5-6.0 was studied. The enzyme activity was enhanced using oat xylan as the carbon source. Under these conditions a culture produced 1.14 Μmol/ min (11.4 U/mL or 84.4 U/mg) of Β-xylanase after 5 d of growth in a 10-mM buffer solution at pH 4.5. Protease was absent in the DMS buffer except when 100 mM phosphate buffer at pH 6.0 was used (4 U/mL). Β-Xylosidase was only found at a pH of 4.5 in all the buffer concentrations. At a 50 mM DMS buffer concentration at pH 4.5 Β- xylanases were induced by both oat and birch xylans, having a greater effect with oat spelt xylans. Electrophoretic analyses showed that the birchwood xylan induction exhibited different proteins profiles. No Β-xylosidase or Β- glucosidase was induced until d 5. The Β-xylanases were rapidly inactivated at 50‡C, however, birch xylanase appeared to be more stable than oat xylanase. Using oat xylan as an inductor, theΒ-xylosidase andΒ-glucosidase were 85 and 91 U/L, respectively, on d 7. The xylanase produced by induction from sugar cane bagasse hydrolyzate was used for pulp biobleaching. A 20% decrease on the Kappa value in Kraft pulp using the culture extract was obtained. These selective growth conditions led us to modulate the xylanase production for pulp delignification.     

Mechanism of the oxidation of d-glucose onto colloidal MnO2 surface in the absence and presence of TX-100 micelles

Aqueous colloidal manganese dioxide (MnO₂) was prepared via titration by using potassium permanganate and sodium thiosulphate in aqueous neutral medium. The kinetics of oxidation of d-glucose onto the surface of colloidal MnO₂ have been studied spectrophotometrically. The results show that the rate of initial stage (nonautocatalytic path) increases with increasing the [d-glucose], [H⁺], and temperature and also upon addition of nonionic surfactant Triton X-100 (TX-100), which indicates that the surfactant enhances the concentration of d-glucose at the surface of the colloidal MnO₂. Hydrogen bonding interaction seemingly arises between –OH groups of d-glucose and oxygen of the ether linkages of polyoxyethylene chain of TX-100. A possible mechanism of the oxidative degradation of d-glucose is discussed in terms of d-glucose/TX-100 and colloidal MnO₂ interaction.     

d-glucose Enhanced 5-Aminolevulinic Acid Production in Recombinant Escherichia coli Culture

In this study, we introduced a new strategy, feeding d-glucose, to overproduce extracellular 5-aminolevulinic acid (ALA) in the recombinant Escherichia coli. We investigated that the d-glucose concentration is dependent on extracellular ALA production. The results indicated that increasing d-glucose concentration in bacteria culture enhanced final cell density and ALA yield and simultaneously decreased the activities of ALA synthase (ALAS) and ALA dehydratase (ALAD); then, the inhibitory effect of d-glucose on ALAS activity was relieved with the metabolism of d-glucose. when 4.0 g/L d-glucose was added at late exponential phase; 1.46 g/L ALA was achieved in shaking culture, which is 47% or 109% higher than the ALA yields with 30 mM levulinic acid of ALAD inhibitor or no inhibitor. In jar fermenter, final extracellular ALA concentration reached 3.1 g/L by feeding with d-glucose.     

Production of chitinolytic enzymes with Trichoderma longibrachiatum IMI 92027 in solid substrate fermentation

Thirty Trichoderma strains representing 15 species within the genus were screened for extracellular production of chitinolytic enzymes in solid substrate fermentation. Trichoderma longibrachiatum IMI 92027 (ATCC 36838) gave the highest yield (5.0 IU/g of dry matter of substrate) after 3 d of fermentation on wheat bran-crude chitin (9:1 mixture) medium. The optimal moisture content (66.7%), chitin content (20%), initial pH of the medium (2.0–5.0), and time course (5 d) of solid substrate fermentation were determined for strain IMI 92027. Cellulase, xylanase, α-amylase, and β-xylosidase activities were also detected. The pH and temperature optima of the chitinase complex of T. longibrachiatum IMI 92027 were 4.5 and 55°C, respectively. The enzyme totally lost its activity at 70°C in 5 min in the absence of the substrate but retained about 15% of its initial activity even at 70°C after a 60-min incubation in the presence of solid substrate fermentation solids. Purification of protein extract from the solid substrate fermentation material revealed high chitinolytic activities between pI 5.9 and 4.8, where N-acetyl-β-d-hexosaminidase and chitinase peaks have been found in the same pI range. Two chitinases of 43.5 and 30 kDa were purified at acidic pI.     

Steady-state measurements of lactic acid production in a wild-type and a putative d-lactic acid dehydrogenase-negative mutant of zymomonas mobilis

This work represents a continuation of our investigation into environmental conditions that promote lactic acid synthesis by Zymomonas mobilis. The characteristic near theoretical yield of ethanol from glucose by Z. mobilis can be compromised by the synthesis of d- and l-lactic acid. The production of lactic acid is exacerbated by the following conditions: pH 6.0, yeast extract, and reduced growth rate. At a specific growth rate of 0.048/h, the average yield of dl-lactate from glucose in a yeast extract-based medium at pH 6.0 was 0.15 g/g. This represents a reduction in ethanol yield of about 10% relative to the yield at a growth rate of 0.15/h. Very little lactic acid was produced at pH 5.0 or using a defined salts medium (without yeast extract) Under permissive and comparable culture conditions, a tetracycline-resistant, d-ldh negative mutant produced about 50% less lactic acid than its parent strain Zm ATCC 39676. d-lactic acid was detected in the cell-free spent fermentation medium of the mutant, but this could be owing to the presence of a racemase enzyme. Under the steady-state growth conditions provided by the chemostat, the specific rate of glucose consumption was altered at a constant growth rate of 0.075/h. Shifting from glucose-limited to nitrogen-limited growth, or increasing the temperature, caused an increase in the specific rate of glucose catabolism. There was good correlation between an increase in glycolytic flux and a decrease in lactic acid yield from glucose. This study points to a mechanistic link between the glycolytic flux and the control of end-product glucose metabolism. Implications of reduced glycolytic flux in pentose-fermenting recombinant Z. mobilis strains, relative to increased byproduct synthesis, is discussed.     

Bioconversion of <Emphasis Type="SmallCaps">d</Emphasis>-glucose into <Emphasis Type="SmallCaps">d</Emphasis>-glucosone by Glucose 2-oxidase from <Emphasis Type="Italic">Coriolus versicolor</Emphasis> at Moderate Pressures

Glucose 2-oxidase (pyranose oxidase, pyranose:oxygen-2-oxidoreductase, EC 1.1.3.10) from Coriolus versicolor catalyses the oxidation of d-glucose at carbon 2 in the presence of molecular O₂ producing d-glucosone (2-keto-glucose and d-arabino-2-hexosulose) and H₂O₂. It was used to convert d-glucose into d-glucosone at moderate pressures (i.e. up to 150 bar) with compressed air in a modified commercial batch reactor. Several parameters affecting biocatalysis at moderate pressures were investigated as follows: pressure, [enzyme], [glucose], pH, temperature, nature of fluid and the presence of catalase. Glucose 2-oxidase was purified by immobilized metal affinity chromatography on epoxy-activated Sepharose 6B-IDA-Cu(II) column at pH 6.0. The rate of bioconversion of d-glucose increased with the pressure since an increase in the pressure with compressed air resulted in higher rates of conversion. On the other hand, the presence of catalase increased the rate of reaction which strongly suggests that H₂O₂ acted as inhibitor for this reaction. The rate of bioconversion of d-glucose by glucose 2-oxidase in the presence of either nitrogen or supercritical CO₂ at 110 bar was very low compared with the use of compressed air at the same pressure. The optimum temperature (55°C) and pH (5.0) of d-glucose bioconversion as well as kinetic parameters for this enzyme were determined under moderate pressure. The activation energy (E _a) was 32.08 kJ mol⁻¹ and kinetic parameters (V _max, K _m, K _cat and K _cat/K _m) for this bioconversion were 8.8 U mg⁻¹ protein, 2.95 mM, 30.81 s⁻¹ and 10,444.06 s⁻¹ M⁻¹, respectively. The biomass of C. versicolor as well as the cell-free extract containing glucose 2-oxidase activity were also useful for bioconversion of d-glucose at moderate pressures. The enzyme was apparently stable at moderate pressures since such pressures did not affect significantly the enzyme activity.     

Use of steam explosion liquor from sugar cane bagasse for lignin peroxidase production by Phanerochaete chrysosporium

The possibility of using two by-products of the sugar cane industry, molasses and bagasse steam explosion liquor (SEL), for lignin peroxidase (LiP) production by Phanerochaete chrysosporium was investigated. For comparison, the fungus was initially cultivated in synthetic media containing either glucose, sucrose, xylose, or xylan as sole carbon sources. The effect of veratryl alcohol (VA) was also investigated in relation to the enzyme activity levels. Results showed that sucrose was not metabolized by this fungus, which precluded the use of molasses as a carbon source. Glucose, xylose, and xylan promoted equivalent cell growth. Enzyme levels in the absence of VA were lower than 28 UI/L and in the presence of VA reached 109 IU/L with glucose and 85 IU/L with xylose or xylan. SEL was adequate for P. chrysosporium LiP production as LiP activity reached 90 IU/L. When VA was added to this medium, enzyme concentration increased to 155 IU/L.     

Properties of the Macrophomina phaseolina endoglucanase (EGL 1) gene product in bacterial and yeast expression systems

Functional expression of a β-d-1,4 glucanase-encoding gene (egl1) from a filamentous fungus was achieved in both Escherichia coli and Saccharomyces cerevisiae using a modified version of pRS413. Optimal activity of the E. coli-expressed enzyme was found at incubation temperatures of 60°C, whereas the enzyme activity was optimal at 40°C when expressed by S. cerevisiae. Enzyme activity at different pH levels was similar for both bacteria and yeast, being highest at 5.0. Yeast expression resulted in a highly glycosylated protein of approx 60 kDa, compared to bacterial expression, which resulted in a protein of 30 kDa. The hyperglycosylated protein had reduced enzyme activity, indicating that E. coli is a preferred vehicle for production scale-up.     

Improvement of inulin hydrolysis yeast cell reactor by mutants selection

In a previous publication, we described a continuous production ofd-fructose from enzymatic hydrolysis of inulin with immobilized permeabilized cells (1).Kluyveromyces fragilis ATCC 12424 have been shown to possess inulase activity. The half life of the reactor was at least 1300 h, but productivity was relatively low (around 40 g/L/day). A selection of 50 mutants was tested on liquid medium for a possible increase of productivity. In relation to the improvement of the reactor, the most important factor is intracellular inulase activity, and this activity was increased with the KF 28 mutant. Productivity reached 2000 g/L/day with the increase (of the productivity), proportional to the increase of intracellular inulase activity.     

Production of laccase by immobilized cells of Agaricus sp.

Laccase was produced in the supernatant of culture of a local isolate of Agaricus sp. obtained from decaying Ficus religiosa wood. The enzyme was produced at a constitutive level when growing the fungus in a nitrogenlimited medium supplemented with either glycerol, glucose, fructose, mannitol, arabinose, maltose, sacch arose, cellulose, or cellobiose. Atwo-to sixfold increase in enzyme specific activity was observed when growing the strain in the presence of straw, xylan, xylose, lignosulfonate, veratryl alcohol, and ferulic and veratric acid. Experimentsare consistent with the existence of an induction control on laccase and the absence of a form of carbon catabolite repression mediated by noninducing carbon sources. Immobilization of the Agaricus sp. on several supports, including polyurethane foam, textilestrips, and straw, resulted in an increase of enzyme production as compared to cultivation in liquid medium.     

Structure and Function of a Multidomain Alkaline Xylanase from Alkaliphilic Bacillus Sp. Strain 41M-1

Xylanase is an enzyme that catalyzes the hydrolysis of xylan, a -1,4-linked xylose polymer. Alkaliphilic Bacillus sp. strain 41M-1 secretes a xylanase (xylanase J) that has an alkaline pH optimum. Xylanase J is a multidomain enzyme and consists of two functional domains: a family 11/G catalytic domain and a non-catalytic xylan-binding domain. The xylan-binding domain bound to xylan and enhanced catalytic activity of the adjacent catalytic domain. Mutational analyses revealed some amino acid residues that contribute to catalytic activity, alkaliphily and xylan-binding activity of xylanase J.     

Cellulases and Hemicellulases from Endophytic <Emphasis Type="Italic">Acremonium</Emphasis> Species and Its Application on Sugarcane Bagasse Hydrolysis

The aim of this work was to have cellulase activity and hemicellulase activity screenings of endophyte Acremonium species (Acremonium zeae EA0802 and Acremonium sp. EA0810). Both fungi were cultivated in submerged culture (SC) containing l-arabinose, d-xylose, oat spelt xylan, sugarcane bagasse, or corn straw as carbon source. In solid-state fermentation, it was tested as carbon source sugarcane bagasse or corn straw. The highest FPase, endoglucanase, and xylanase activities were produced by Acremonium sp. EA0810 cultivated in SC containing sugarcane bagasse as a carbon source. The highest β-glucosidase activity was produced by Acremonium sp. EA0810 cultivated in SC using d-xylose as carbon source. A. zeae EA0802 has highest α-arabinofuranosidase and α-galactosidase activities in SC using xylan as a carbon source. FPase, endoglucanase, β-glucosidase, and xylanase from Acremonium sp. EA0810 has optimum pH and temperatures of 6.0, 55 °C; 5.0, 70 °C; 4.5, 60 °C; and 6.5, 50 °C, respectively. α-Arabinofuranosidase and α-galactosidase from A. zeae EA0802 has optimum pH and temperatures of 5.0, 60 °C and 4.5, 45 °C, respectively. It was analyzed the application of Acremonium sp. EA0810 to hydrolyze sugarcane bagasse, and it was achieved 63% of conversion into reducing sugar and 42% of conversion into glucose.     

Efficient production of poly-γ-glutamic acid by bacillus subtilis ZJU-7

A strain with high poly-γ-glutamic acid (γ-PGA) production was isolated from fermented bean curd, a traditional Chinese food. The strain was named Bacillus subtilis ZJU-7 according to 16s rDNA sequencing and its taxonomic characters. The culture conditions for γ-PGA production were evaluated. The most suitable carbon and nitrogen sources were sucrose and tryptone, respectively. Exogenous l-glutamic acid was necessary for γ-PGA production, and the production of γ-PGA increased on the addition of l-glutamic acid to the medium. In the medium containing 60 g/L of sucrose, 60 g/L of tryptone, 80 g/L of l-glutamic acid, and 10 g/L of NaCl, the yield of γ-PGA reached 54.4 g/L after cultivation at 37°C for 24h, which was the highest γ-PGA production compared with values reported in the literature. The average molecular mass of γ-PGA produced was about 1.24×10⁶ Daltons. B. subtilis ZJU-7 is genetically stable and can synthesize levan instead of γ-PGA without the addition of l-glutamic acid to the medium.