Discussion

Regulation of enzyme synthesis and gene expression in eukaryotes is one of the most important branches of modern molecular biology.The importance of the study of the synthesis and regulation of cellulase，a typical inducible-extracellular enzyme produced by the simplest eukaryotes（Trichoderma）should be emphasized for its potential significance in solving energy problems as well as for its being a convenient material in the study of gene expression in eukaryotes.Several reviews have dealt with this subject.［3，4，11，19，21-24］However，knowledge of cellulase synthesis and regulation is still limited.

Figure 11　Chromatography of total RNA of mycelium of Trichoderma N2-78 induced and non-induced by sophorose through oligo（dT）-Cellulose column.［9］●，Control；○，sophorose.A，0.01 MTris，0.5 M KCl，pH=7.5；B，H2O，pH=7.5

What is the true and natural inducer of cellulase？How does the mycelium of Trichoderma recognize the presence of cellulose substrate in the medium？Mandels and Reese，in their early work，［25］have shown that of various carbon sources tested，only cellulose，cellobiose，lactose and reagent glucose were able to induce cellulase production.Cellobiose，a hydrolysate of cellulose，was presumed to be the natural inducer，［26］The induction activity of cellobiose was low；more cellulase could be induced by cellobiose only at a high concentration of cellobiose（1%or more），by the decrease of metabolism，or the supply of surfactant.［27，28］Therefore，it is questionable whether cellobiose is the native inducer.On the other hand，Mandels and Reese［10，29］have demonstrated that sophorose as an impurity in reagent glucose could strongly induce cellulase production.This discovery is exciting and could contribute profoundly to the cellulase induction research.It prompted us to conduct a series of investrgations of cellulase synthesis and regulation since we had isolated two mutants of Trichoderma，EA3-867and N2-78，with high cellulase yield in 1973.

Since cellulase was induced by cellobiose only at higher concentration during growth，no cellulase induction was observed in the washed mycelium test，a standard and reliable test for inducer，it is more likely that some product of cellobiose conversion during mycelial growth，but not cellobiose itself，is the true inducer.Sophorose is more effective in cellulase induction，even at a concentration of 0.3 μg/ml（the ratio of sophorose to mycelium dry weight was 1∶5，000，with a lag of 2—3 h）.Moreover，the induction of cellulase by sophorose and cellulose was quite similar both in the extracellular or intracellular components and in their patterns of polyacrylamide gel electrophoresis.Therefore，sophorose might be a true inducer of cellulase，as proposed recently by others.［30］Sophorose was presumed to be formed by transglucosylation ofβ-glucoside from cellobiose in vivo，［6，30］a phenomenon similar toβ-glucosidase in E.coli，［31］in which the natural inducer allolactose is formed by a similar mechanism.It is not yet known why organisms have developed a mechanism to form the true inducer by modifying substrates（e.g.lactose）or more direct products（e.g.cellobiose from cellulose）.It may be needed for a more complicated control of metabolism.On the other hand，on a medium containing a rapidly utilizable carbon source such as glycerol or glucose，which are not inducers，Trichoderma EA3-867 produced traces of cellulase.Thus，the concept of constitutive cellulase was proposed.［6］The induction of cellulase by insoluble cellulose is presumed to occur as follows：constitutive cellulase，on the cell surface or released into the medium，hydrolyses cellulose to cellobiose which may enter the cell and be converted to sophorose in vivo by transglycosylation.Sophorose enters the nuclei，and activates the transcription process in nuclei whereby the mRNA is synthesized.The latter is transported to the cell membrane.As a consequence，a great amount of inducible cellulase is synthesized there and secreted into the extra-cellular environment.

The cellulase synthesis of Trichodermn is regulated not only by the induction system，but also by catabolite repression，a common phenomenon in other fungi and bacteria.［7，32-35］This is a self-regulation process by which the cells avoid wastage of resources and energy in synthesizing unnecessary cellulase in the presence of other rapidly utilizable carbon sources until the latter have been used.It is of interest that the catabobte repression of glucose could not be overcome by cyclic-AMP，indicating that cellulase regulation in the eukaryote Trichoderma is different from the enzyme regulation in prokaryotes，in which catabolite repression is due to the decrease in the c-AMP level，which acts as a necessary effector in the initiation process of the operon replication.［35］

The coordinate induction of a complete cellulase system（C1，Cx，andβ-glucosidase）induced by sophorose suggests that these three functionally related enzymes may be controlled by the same regulatory gene，although it is not necessary for three structural genes to be linked at the same chromosome.The result presented here is different from other reports，［30］in which no complete cellulase system was found to be induced by sophorose.It raises the interesting question of whether different species of Trichoderma have different genetic control mechanisms in cellulase synthesis.(https://www.daowen.com)

Cellulase synthesis in washed mycelium was inhibited by the inhibitors of nucleic acid metabolism（5-fluouracil，6-azauracil）and protein synthesis（actidione）.Thus，the metabolism of nucleic acid，especially the regulation of mRNA synthesis，plays an important role in cellulase synthesis.As yet，no information on mRNA in Trichoderma has been reported.In the higher eukaryotes，it has been found that mRNA molecules contain a polyARNA sequence at the 5'-end，which is the basis for isolation of mRNA by oligo（dT）-cellulose affrnity chromatography.In a microorganism，however，the short lifespan of mRNAs makes it difficult to isolate them.The data presented in our work indicate that most of the extracellular proteins induced by sophorose were cellulases.It is thus likely that the mRNA of cellulase should be enriched when induced by sophorose.By adding the inhibitor of nuclease，fast extraction at lower temperature，a polyA-RNA has been obtained in washed mycelium induced by sophorose.The ratio A260/A280 for polyA-RNA was 1.5 and the yield was 1%of total RNA.Analysis of polyacrylamide gel electrophoresis has shown that its molecular size was in the size range of mRNA.［2］It is important that the content of polyARNA in mycelium increased after the induction by sophorose.This increased polyA-RNA may be mRNA of cellulase，and its biological activity should be examined further.Furthermore，cell wall-bound ribosomes were observed in the sophorose-induced mycelium by electronmicroscopy（data not shown here）.The biological implication of this phenomenon is not yet clear.

Our knowledge of the regulation of cellulase synthesis has been used to increase cellulase production and genetic analysis of the mutants.Inducer or gratuitous inducers are often used to increase enzyme yields.Examples are xylose for glucose isomerase，［35］sucrose monopalmitate for invertase，［27］and starch，glycogen and maltotriose for amylase.［37］The use of sophorose as an inducer of cellulase has been considered impractical because of its high price.［22］However，the present study suggests that the waste glucose molasses may provide an available and inexpensive resource of sophorose.In our pilot fermenter（1000 litre），waste glucose molasses was effectively used to increase cellulase yields and to shorten the culture time to 60 h.

Two mutants of Trichoderma，EA3-867 and N2-78，were compared with their parents on the basis of regulation of cellulase synthesis.The cellulase in the mutants did not change either in kinetics or electrophoretic pattern compared with their parents except that their yields increased considerably，suggesting that only the regulatory gene，but not the structural gene，had changed in the mutants.This suggestion was supported by the discovery that the mutants showed increased sensitivity to inducer and decreased sensitivity to catabolite repression.One possible explanation for the increased sensitivity to inducer in the mutants is the change in the repressor structure which combines with the inducer more tightly as in lac operon mutants.Similarly，the cellulase in the mutants could be induced by much less sophorose than in the parent strains.Although the sensitivity of mutants EA3-867 and N2-78 to catabolite repression decreased，cellulase synthesis in the mutants was still repressed by glucose at high concentration（10-2 M）.Thus，the mutants are considered as partially derepressed strains.Mutations that effect catabolite repression may occur in the promotor，or the genes for CRP protein or adenyl cyclase.Moreover，the mutation of genes in the main metabolic pathway would indirectly affect the degree of catabolite repression.［38］From our results，the latter possibility may hold，as the mutants grew slowly and showed small colonies on synthetic medium，but grew as well as wild type strains on peptone-yeast extract medium.This suggests that the decreased catabolite repression may be due to the retardation of growth and the block in some metabolic reaction which synthesizes necessary growth factor（s）present in peptone and yeast extracts.

The study on cellulase synthesis and regulation in Trichoderma is not only helpful in understanding basic biological process such as gene expression in eukaryotes but it also has practical significance.The cellulases of EA3-867 and N2-78 are utilized to isolate protoplasts from plant cells，to increase alcohol production，to make medicines to aid the digestion of plant foods，to improve feed efficiencies in pig feeding，partially to replace alkaline treatment in textile production，and to improve quality in the food industry.［3］