Eva M. Kubicek-Pranz

Properties of a Conidial-bound Cellulase Enzyme System from Trichoderma reesei

SUMMARY: Conidia of Trichoderma reesei QM 9414 are able to degrade crystalline cellulose to glucose in the absence of protein synthesis, indicating the presence of a cellulase enzyme system. Measurement of enzyme activities revealed the presence of several glycanases and glycosidases. The cellulase enzyme system appeared irrespective of the conditions applied to induce conidiation. Removal of the endo-1,4-β-glucanase from the cellulase system could be achieved by treatment of conidia with β-octylglucoside or Triton X-100. Conidia treated in this way grew poorly on cellulose but well on glycerol. The endo-1,4-β-glucanase released from conidia by β-octylglucoside appeared as a single enzyme upon SDS-PAGE/immunoblotting with an apparent molecular mass of 68 kDa, an isoelectric point of 4.8-5.3, and a pH optimum between pH 4-6. These properties were similar to endo-1,4-β-glucanase I purified from culture filtrates of cellulose-grown T. reesei. Thermostability of the released enzyme was, however, lower than that of endo-1,4-β-glucanase I. Germination of T. reesei conidia in a medium containing glycerol as a carbon source led to a loss of endo-1,4-β-glucanase activity which paralleled the decrease in the number of ungerminated conidia.

Transformation of Trichoderma reesei with the cellobiohydrolase II gene as a means for obtaining strains with increased cellulase production and specific activity

Abstract Trichoderma reesei QM 9414 was shown to contain a single copy of both the cbh1 and cbh2 gene, coding for cellobiohydrolase I (CBH 1) and II (CBH II) synthesis. In the attempt to increase CBH II formation, multiple copies of the cbh2 gene were introduced by cotransformation with the Neurospora crassa pyr4 gene using a pyrG auxotrophic mutant of T. reesei QM 9414 ( T. reesei TU-6). Transformants with mitotically stable pyrG prototrophy, were shown to display both homologous as well as ectopic integration of the cbh2 gene. The cbh2 copy number varied between 10- and 20-fold. Secretion of CBH I and II by the transformants during growth on lactose as a carbon source leading to cellulase formation was quantified by means of monoclonal antibodies. Three out of 14 transformants exhibited a 2- to 4-fold increased CBH II titer. Hence, no correlation between the cbh2 copy number and the amount of secreted CBH II was observed. The same three transformants exhibited a roughly 1.5-fold increase in specific cellulase activity (FPU). The crude cellulase mixture of transformant 3, which displayed a 3- to 4-fold elevated CBH 11 formation, exhibited an increased specific activity against crystalline cellulose in vitro. It is therefore concluded that transformation with individual cellulase genes can he a useful and simple tool to alter the quantitative pattern of cellulolytic enzymes produced by T. reesei .

Cellobiohydrolase II is the main conidial-bound cellulase in Trichoderma reesei and other Trichoderma strains.

Monoclonal antibodies have been used to determine the presence of cellobiohydrolases I and II (CBH I and II), and endoglucanase I (EG I) on the surface of conidia from Trichoderma reesei QM 9414 and RUT C-30, and 8 other Trichoderma species. For this purpose, proteins were released from the conidial surface by treatment with a non-ionic detergent (Triton X-100 and β-octylglucoside), followed by SDS-PAGE/Western blotting and immunostaining. Both CBH I and II were clearly present, but — unlike in extracellular culture fluids from Trichoderma — CBH II was the predominant cellulase. In T. reesei EG I could not be detected. The higher producer strain T. reesei RUT C-30 exhibited a higher conidial level of CBH II than T. reesei QM 9414. In order to assess the importance of the conidial CBH II level for cellulase induction by cellulose, multiple copies of the chb2 gene were introduced into the T. reesei genome by cotransformation using PyrG as a marker. Stable multicopy transformants secreted the 2- to 4-fold level of CBH II into the culture medium when grown on lactose as a carbon source, but their CBH I secretion was unaltered. Upon growth on cellulose, both CBH I and CBH II secretion was enhanced. Those strain showing highest cellulase activity on cellulose also appeared to contain the highest level of conidial bound CBH II. CBH II was also the predominant conidial cellulase in various other Trichoderma sp. However, roughly the same amount of conidial bound CBH II was detected in all strains, although their cellulase production differed considerably.

Changes in the concentration of fructose 2,6-bisphosphate in Aspergillus niger during stimulation of acidogenesis by elevated sucrose concentration.

The presence of fructose 2,6-bisphosphate (Fru-2,6-P2) and phosphofructokinase 2 (PFK 2) were established in the citric-acid-producing filamentous fungus Aspergillus niger. Fru-2,6-P2 levels were around 3.0 (+/- 0.8) nmol per g dry weight during growth on sucrose, and half of this in mycelia grown on citrate as a carbon source. PFK 2 was detected with a specific activity of 150 mU/mg protein and a Km for fructose 6-phosphate of 40 microM. Induction of citric acid accumulation (acidogenesis) in A. niger by cultivation on high concentrations of sucrose, or replacement on 14% (w/v) sucrose correlated with an increase in the intracellular concentration of Fru-2,6-P2. A similar correlation was obtained when A. niger was cultivated on different carbon sources, which induced different rates of acidogenesis. The increase in Fru-2,6-P2 during transfer to 14% (w/v) sucrose was not correlated with the behaviour of mycelial concentrations of cyclic AMP, a potential regulator of Fru-2,6-P2 formation in other organisms, nor with that of Fru-6-P and ATP, the precursors of its formation. The extracellular addition of cyclic AMP and theophylline, an inhibitor of cellular cyclic AMP breakdown, increased both Fru-2,6-P2 concentration and acidogenesis in mycelia cultivated in 1% (w/v) sucrose medium. It is concluded that Fru-2,6-P2 controls citric acid accumulation by enabling increased rates of glucolysis, a prerequisite to acidogenesis.

NADPH-specific and NADH-specific xylose reduction is catalyzed by two separate enzymes in Pachysolen tannophilus

SummaryPachysolen tannophilus contains — in addition to an NADPH-linked xylose reductase — a separate NADH-linked one, in this respect differing from the yeast Pichia stipitis. Both enzyme proteins can conveniently be separated from each other by either ion exchange chromatography or chromatofocusing.

D-amino acid oxidase fromTrigonopsis variabilis: Comparison of enzyme specificity “in vivo” and “in vitro”

SummaryA method is described for permeabilization of intact cells of the yeastTrigonopsis variabilis with respect toin vivo measuring D-amino acid oxidase activity. The kinetic results so obtained differ from those obtained with the purified enzyme, pointing to the advantage of using the purified enzyme or the permeabilized cells in the oxidative deamination of different D-amino acids.

Regulation of 6-phosphofructo-2-kinase from the citric-acid-accumulating fungus Aspergillus niger

SummaryPhosphofructokinase 2 (PFK 2) was isolated from mycelia of the citric-acid-accumulating fungus Aspergillus niger, and partially purified by Trisacryl-Blue chromatography and Mono Q fast protein liquid chromatography. The appearance of a 96/94-kDa double band correlated with PFK 2 activity during purification. Purified PFK 2 had a half-life of 240 min at 4° C. The enzyme exhibited Michaelis-Menten type kinetics with respect to its substrates fructose-6-phosphate and ATP, required inorgaic phosphate for activity, and was only weakly inhibited by phospho(enol)pyruvate, AMP and citrate. The enzyme activity was not influenced by incubating partially purified PFK 2 preparations with ATP, MG2+ and the catalytic subunit of bovine heart protein kinase, although such treatment phosphorylated the 96/94-kDa protein. Consistently, treatment with alkaline phosphatase had no effect on PFK 2 activity. Also, no influence on PFK 2 activity was observed when cell-free extracts (containing A. niger protein kinases) from either glucose or citrate-grown mycelia were incubated with ATP and Mg2+ alone. It is concluded that, in A. niger, regulation of PFK 2 by phosphorylation/dephosphorylation does not occur, and this is related to the development of high glycolytic flow and citrate accumulation under conditions of supplying high sugar concentrations.