Features of aspergillus japonicus cellobiase formation and optimization of its production

We studied some mechanisms of cellobiase formation in Asp. Japonicus VKM F-2092 aiming at the subsequent development of controlled cultivation to produce the enzyme. Formation of cellobiase was found to be of noninducible constitutive character. The enzyme was predominantly localized in the cytosole and the periplasm, its minor portion being cell-wall bound. The release of the enzyme into the medium was due to autolytic processes. The two-phaseness of cellobiase formation in Asp. Japonicus grown on a mineral medium with 0.5% glucose was shown to be determined by the following mechanisms:

• 1 Catabolic repression. We established glucose concentrations causing the repression and found it to be dependent on carbon sources in the growth medium.
• 2 Catabolic inhibition. Cellobiase activity was found to be inhibited by assumed glucose metabolites in the presence of nitrate nitrogen in the medium.

. Practical recommendations to optimize the process of cellobiase production by Asp. Japonicus are given.     

Characterization of a bacterial xylanase resistant to repression by glucose and xylose

Summary Bacillus subtilis CD4, when grown in nutrient broth or minimal medium in presence of xylan, produced extracellular xylanase that hydrolyzed xylan optimally at pH 5. The enzyme was induced by xylan, xylose and glucose. Addition of xylose or glucose in xylan containing medium did not affect enzyme production. The structural gene encoding xylanase was cloned and expressed in E. coli. The recombinant enzyme exhibited similar properties like that of native enzyme including resistance to repression by xylose and glucose.     

Induction and glucose repression of xylanase from a color variant strain ofAureobasidum pullulans

Summary Xylose, xylobiose and arabinose were identified as natural and direct inducers of xylanase from a color variant strain ofAureobasidium pullulans. Arabinose, in contrast to xylose, xylobiose and xylan, induced only the major isozyme of xylanase. Xylanase induction was subject to glucose repression.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U. S. Department of Agriculture over other firms or similar products not mentioned.     

Growth,plasmid stability and α-amylase production in batch fermentations using a recombinantBacillus subtilis strain

Summary Fermentation time-courses for a recombinantBacillus subtilis strain grown in starch and glucose modia were investigated. The results indicate the absence of glucose catabolite repression controlling expression of the -amylase genes in the recombinant but not in the donor strain. Plasmid instability was associated largely with the post-exponential phases of growth and decline.     

Fermentation capabilities ofZymomonas mobilis glycolytic enzymes

Summary Cell-free extracts ofZymomonas mobilis were capable of fermenting glucose to ethanol and CO₂ when stimulated by arsenate to act as an ATP uncoupler. 2M glucose was completely converted resulting in a final concentration of 16.5 % w/v ethanol. 1 M glucose was completely converted at temperatures up to 50°C. The results demonstrate that the glycolytic enzymes are more resistant to temperature and ethanol than are the living cells.     

Glucose and glycerol repression of α-amylase in Streptomyces kanamyceticus and isolation of deregulated mutants

Summary Glucose and glycerol at concentrations of 2 % negatively affected amylase synthesis in plate and submerged Streptomyces kanamyceticus cultures. This microorganism was insensitive to growth inhibition by glucose analogs and deregulated mutants were identified by a clearing zone around colonies grown on starch and glycerol or glucose, and selected. Three kinds of mutants were obtained: one insensitive to glucose (Mutant 41), another insensitive to glycerol repression (Mutant E) and the last (Mutant 29) an amylase-hyperproducing mutant, albeit regulated by glucose or glycerol like the wild type. The levels of glucokinase, an enzyme involved in catabolite regulation of Enterobacteria, were determined and results showed no differences between the parental strain and the mutants.     

Construction of multiple herbicide resistant ammonia excreting strains of cyanobacteriumNostoc muscorum

Summary Machete resistant (Mat ^r), basalin resistant (Bas ^r), 3(3,4 dichlorophenyl)-1,1-dimethyl urea resistant (DCMU ^r), atrazine resistant (Atr ^r) and propanil resistant (Prp ^r) phenotypes ofGloeocapsa sp. were cotransformed toNostoc muscorum at high frequency. Spontaneously occurring mutants of the multiple herbicide resistant transformant containing L-methionine-DL-sulfoximine resistant (Msx ^r), ethylene diamine resistant (Eda ^r), or phosphinothricin resistant (Ppt ^r) glutamine synthetase (GS) showed extracellular liberation of ammonia resulting from fixation of N₂ under photosynthetic conditions. Results suggest a definite role of GS activity in regulation of extracellular ammonia.     

Xylose catabolism by a mutant ofStreptomyces chrysomallus altered in the regulation of D-glucose isomerase

Summary A mutant ofS. chrysomallus with a D-glucose isomerase inducible by D-glucose or its catabolites was characterized. In contrast to the wild-type strain it showed a decreased catabolite repression by D-glucose of D-xylose consumption and a constitutive pentose phosphate pathway as well. A hypothesis concerning altered induction pattern of its D-glucose isomerase is discussed.     

Some approaches to adaptive control of fed-batch culture of the cellobiase producer aspergillus japonicus

Biosynthesis of cellobiase by an Aspergillus japonicus culture under various modes of cultivation was studied using a fermenter-computer system. Two modes of fed-batch cultivation were developed: temperate and intensive. Both modes used the double algorithm of glucose supply based on the process control by the preset profile of CO₂ concentration in exhaust gas and consumption of the pH stabilizing titrant. Intensive fed-batch proved the most efficient mode: it maintained conditions for derepressed cellobiase Liosynthesis and decreased the carbon and energy limit for enzyme production. Intensive fed-batch is, thus, the most adapted to cellobiase biosynthesis conditions and nutrient requirements of the producing culture.     

Production and characterization of a highly active cellobiase from Aspergillus niger grown in solid state fermentation

Summary Aspergillus niger produced extracellular cellobiase when grown on different lignocellulosic substrates in solid state fermentation. The enzyme activity and yield were variable according to the carbon source. In Vogel’s medium, the cellobiase productivity was significantly higher on wheat bran, followed by Leptochloa fusca (kallar grass) straw augmented with corn steep liquor. Maximum yield of cellobiase/g wheat bran was significantly higher than the values reported on other potent fungi, bacteria and recombinants, harboring heterologous gene for cellobiase. This enzyme in the presence and absence of Trichoderma reesei and celluloclast, saccharified the biomass and the percentage saccharification as well as glucose yield from lignocellulosic biomass was doubled in its presence. The partially purified enzyme was thermotolerant as evidenced by melting temperature, activation energy demand for active catalysis, enthalpy and entropy of activation for reversible or irreversible thermal inactivation.     

Dominant expression of a gene amplification-related herbicide resistance in medicago cell hybrids

Protoplasts from phosphinotricin resistant M. sativa and M. varia cell lines carrying an amplified glutamine synthethase gene were fused with leaf protoplasts of kanamycin resistant M. varia transformants. The dominant nature of both PPT and kanamycin resistant traits was shown by the double resistant phenotype of the intra- and interspecific cell hybrids obtained. The presence of amplified GS gene in the hybrid genomes and the expression of chimeric neomycin phosphotransferase II gene was detected. The highly embryogenic character of the M. varia parent was not expressed after cell fusion. All hybrid cell lines with the double resistant phenotype showed non-morphogenic growth similarly to the PPT resistant parent. The possible role of GS gene amplification and other factors in the dominant behaviour of unorganized cell growth in alfalfa somatic hybrids is discussed.     

Preparation of Mutants Resistant to Catabolite Repression of Trichoderma reesei

The development of agar plate screening techniques has allowed the isolation of mutants of Trichoderma reesei capable of synthesizing cellulase under the conditions of a high concentration of glucose. Mutants resistant to catabolite repression by glycerol or glucose were isolated on Walseth’s cellulose (WC) agar plates containing 5% glycerol or 5% glucose, respectively. Mutants resistant to catabolite repression by glycerol were not derepressed enough for the production of cellulase on WC agar plates containing 5% glucose or in flask cultures with a mixture of 1% Avicel and 3% glucose. On the contrary, two mutant strains resistant to catabolite repression by glucose (KDD-10 and DGD-16) produced large clearing zones on WC agar plates containing 5% glucose. Both strains could begin to produce CMCase even in the presence of residual glucose and finally produced 1.5 times the CMCase activity, in flask cultures on 1% Avicel and 3% glucose, than that with 1% Avicel alone. These results suggest that KDD-10 and DGD-16 are comparatively derepressed by glucose for cellulase production.     

A one step process for the conversion of cellulose to ethanol using anaerobic microorganisms in mono- and co-culture

Summary The reducing sugars, glucose, and ethanol produced during growth of the anaerobes Clostridium thermocellum and Acetivibrio cellulolyticus on cellulose were assayed. Zymomonas mobilis was grown under similar conditions and could ferment glucose to ethanol. The ethanol production by the cellulolytic bacteria alone and in co-culture with Zymomonas is described. Approximately 27% of a 1% cellulose substrate could be converted to ethanol by this co-culture.     

Characterization of extracellular substrate specific glucose and xylose isomerases of Chainia

Summary Specific glucose and xylose isomerases have been identified in cell-free culture filtrates of a Chainia species. Treatment with DEAE-cellulose selectively adsorbed xylose isomerase activity while only the glucose isomerase was adsorbed on CM-cellulose. Glucose isomerase was completely inhibited by xylose at 1.3 × 10^-4 M concentration. The differential identity of the extracellular glucose and xylose isomerases, unique to Chainia, is discussed.(NCL Communication 3562)     

Enhanced cellulase production using Solka Floc in a fed-batch fermentation

Summary Use of a fed-batch mode of cultivation of T. reesei has permitted high concentrations of substrate to be consumed. This has resulted in the production of high titre cellulase preparations around 30 FPU/ml at high volumetric productivities (177 IU/L.hr).Perhaps the most obvious area for major improvement in the process of cellulose utilization is the production of cellulase enzyme for hydrolysis of wood and agricultural residues. It has been estimated that some 50% of the cost of producing glucose from cellulosic material is attributable to enzyme production alone (Perez, et al., 1980). Improvements in the area would therefore have a dramatic impact, and are of paramount importance if economical hydrolysis processes are to be realized. The first major thrust in the area has been the development of improved mutant strains of T. reesei, free from catabolite repression and capable of constitutive cellulase production (Montenecourt and Eveleigh, 1977; Gallo, 1982).While this effort continues to develop further high yielding mutants, improvement must also come from developments in fermentation techniques. A major advance is the use of fed-batch cultivation, which provides a means of avoiding the agitation and aeration difficulties, as well as repression effects encountered with high substrate concentration batch fermentation. This report briefly compares batch and fed-batch operation over a range of substrate concentrations.     

Increased enzyme secretion by 2-deoxy-d-glucose in presence of succinate by suppression of metabolic enzymes in Termitomyces clypeatus

Regulatory mode of secretion of proteins was detected for the industrial glycosidase, cellobiase, under secreting conditions (in presence of TCA cycle intermediates like succinate etc.) in the filamentous fungus Termitomyces clypeatus. The titers of key metabolic enzymes were investigated under secreting and non-secreting conditions of growth and compared to the corresponding production of intra and extracellular levels of cellobiase. Results were compared in presence of 2-deoxy-d-glucose, a potent glycosylation inhibitor in the secreting media. Inclusion of 2-deoxy-d-glucose in presence of succinate caused about 10 to 100 times decrease in titers of the metabolic enzymes hexokinase, fructose-1,6-bisphosphatase, isocitrate lyase and malate dehydrogenase leading to increased secretion of cellobiase by more than 100 times. The intracellular concentration of cAMP (86-fold decrease in presence of 2-deoxy-d-glucose under secreting conditions) and turnover rate of proteins also dropped significantly. In this suppressed metabolic state, a 10-fold increase in the titer of the secreted cellobiase was noticed. The results indicated elucidation of carbon catabolite repression like phenomenon in the fungus under secreting conditions which was more pronounced by 2-deoxy-d-glucose. The interdependence between secretion and regulation of metabolic enzymes will help in better understanding of the physiology of these highly adapted organisms for increasing their secretion potential of glycosidases like cellobiase with high industrial value.     

Production of citric acid from sugars present in wood hemicellulose usingAspergillus niger andSaccharomycopsis lipolytica

Summary One strain each of the fungus,Aspergillus niger, and the yeast,Saccharomycopsis lipolytica, were investigated for their ability to produce citric acid from the sugars present in hemicellulose hydrolysates.S. lipolytica produced citric acid as efficiently from mannose as from glucose, but failed to assimilate xylose, arabinose or galactose.A. niger readily assimilated mannose, xylose and arabinose, and produced citric acid from these sugars although the yields were lower than from glucose. A possible inhibitory effect of arabinose on citric acid production from other sugars was observed usingA. niger.     

Enhancement of ethanol production in the presence of metabolic inhibitors inClostridium thermocellum strain SS8

Summary Clostridium thermocellum strain SS8 produced 0.25g of ethanol and 0.24g of acetic acid per g cellulose consumed. Enhancement in ethanol production upto 0.39g/g substrate was observed in the presence of 0.15mM concentration of sodium azide and 7% polyethyleneglycol along with significant repression in acetic acid formation.     

Effects of the physiological state of five yeast species on H+-ATPase-related processes

Effects of starvation and glucose preincubation on membrane potential, ATPase-mediated acidification and glutamic acid transport were studied in yeast speciesSaccharomyces cerevisiae, Schizosaccharomyces pombe, Dipodascus magnusii, Lodderomyces elongisporus andRhodotorula gracilis. Themembrane potential was highest after preincubation with glucose in all species butL. elongisporus andR. gracilis. In all cases the membranes were depolarized in the presence of 20 mmol/L KCl and hyperpolarized with 50 μmol/L diethylstilbestrol (DES). Theextracellular acidification caused by addition of glucose was highest after preincubation with glucose in all cases except inR. gracilis where there was none. In all cases except inR. gracilis addition of KCl caused a marked increase in the acidification rate. Addition of DES with glucose caused a large decrease in rate inS. cerevisiae but had much less effect on the other species.Transport of glutamic acid was clearly increased after pretreatment with glucose inS. cerevisiae, S. pombe andD. magnusii (mainly due to enhanced synthesis of the carrier) but actually decreased inR. gracilis andL. elongisporus. Addition of DES had an inhibitory effect in all species but much more pronounced inS. cerevisiae andS. pombe than in others. In general, both the acidification and the transport of glutamate were enhanced after preincubation with glucose but much more so in the semianaerobic species, such asS. cerevisiae, than in the strict aerobes (R. gracilis) where the effect was occasionally negative. There was no relationship between the ATPase-mediated acidification and the membrane potential.     

High Cellobiase and Xylanase Production by Sclerotium rolfsii UV-8 Mutant in Submerged Culture

Sclerotium rolfsii UV-8 mutant secretes high levels of cellobiase and xylanase in addition to having high cellulase production. The apparent K_m and V_max of cellobiase (grown in NM-2 + 2% corn steep liquor medium) with cellobiose as a substrate were 5.6 mM and 22.2 μmol of glucose liberated per min per ml of culture filtrate, respectively. The addition of 2% corn steep liquor to NM-2 medium increased endo-β-glucanase, cellobiase, and xylanase yields by approximately 1.5-fold.