Ci enzymes

In this section, we summarize information about five Ci enzymes involved in metabolism of 5-formyl-THF (5-CHO-THF) and formate, whose existence was predicted from genomics data. 5-Formyltetrahydrofolate cycloligase (5-FCL) and S-formylglutathione hydrolase have since been shown to catalyze the anticipated reactions the other three enzymes (10-formyl-THF deformylase, formamidase, and sarcosine oxidase) are still putative. 

Since essentially all of the protein in the cellulose-grown culture or the sophorose-incubation mixture can be accounted for by the three principal proteins and the / -glucosidase, it seems reasonable to conclude that the multiplicity of enzymes seen after several days of culture growth are caused by degradation. This may be attributable either to inherent instability (3) or to the presence of hydrolytic enzymes (16). There seems to be no evidence for the Ci enzyme as a nonhydrolytic or affinity factor, since all known enzymes of the system are hydrolytic. That the sophorose-stimulated system (with no possible loss of the Ci ... 

In 1971 and 1972 Halliwell and co-workers (21, 57) separated the T. viride cellulase into four fractions—Ci, C2, CM-cellulase, and cello-biase. It was suggested that the Ci enzyme was a cellobiohydrolase since the principal product of its action was cellobiose. Addition of cellobiase with the Cl enzyme permitted 70% solubilization of cotton after 21 days. No evidence of the enzyme purity was presented. 

This mechanism could be unique for these anaerobic bacteria, however, the major findings reported in the literature are compatible with this proposal. Ci enzyme (3, 4) requires the addition of Cx, a hydrolytic factor, in order to effectively demonstrate activity. It may be that the Ci component is an affinity factor that binds the hydrolytic factor, Cx, to the insoluble cellulose. 

Since the enzyme which we consider the Ci enzyme has not been completely purified yet, I will not stress our results too much, but I think the possibility for the mechanism of breakdown of cellulose I have postulated here makes sense, and I hope that it soon will be possible for us to confirm these results. ... 

In 1950 Reese and his co-workers (43) introduced their Ci, Cx hypothesis. It was postulated that truly cellulolytic microorganisms are equipped with both Ci and Cx enzymes, while microorganisms able to hydrolyze only modified cellulose lack the Ci enzyme. 

However, cell-free enzyme preparations from many, according to the above definition, truly cellulolytic microorganisms fail to solubilize native cotton completely unless this is first converted into a reactive form by swelling in acidic or basic milieu (14,41). Thus, these organisms do not seem to produce a Ci enzyme. The most well-known example is culture filtrate from Myrothecium verrucaria (26, 55). 

The Ci, Cx concept has recently been extensively studied by a number of workers (16,23, 24, 27, 31, 32,46), for two species of Trichoderma, namely T. viride and T. koningi. It was shown that cell-free culture solutions of these fungi were able to solubilize cotton fibers. The solubilization of cotton fibers is also a way of measuring the activity of the Ci enzyme. In the most recent of these publications (46) Selby and Maitland were able to isolate the Ci enzyme from culture filtrates of T. viride. The enzyme was shown not to act upon cellobiose or carboxymethyl cellulose and to lose its ability to solubilize cotton in the absence of the Cx component. The mechanism of action of the Ci enzyme is thus still obscure although many different hypotheses have been presented (34). 

Specific Adsorption on Different Cellulose Columns. Purification of cellulolytic enzymes, including the Ci enzyme, has been carried out by Li et al. (32) by specific adsorption. By passing the crude enzyme through a column of Avicel the Ci fraction was retained by the column while 95% of the cellulase activity could be eluted. The cellulase fraction was then further purified by passing a column of alkali-swollen cellulose where the cellulase was retained. The enzyme could, however, be eluted by buffer in a high yield from the column. The adsorption on an alkali-swollen cellulose column was then repeated. The yield of enzyme obtained with the specific adsorption method is surprisingly high if compared with the yield on Sephadex and on polyacrylamide gel columns. 

In an excellent chapter under the headline Criteria for Characterization of Cellulases, Whitaker (55) has reviewed the results of work on this subject up to 1961. This chapter dealt with works comprising characterization of cellulases both as enzymes and as proteins. As far as known to the present author no essentially new methods have been presented since Whitakers review concerning characterization of cellulases as enzymes. Characterization of the Ci enzyme has failed so far though a considerable amount of work has been carried out lately on the Ci enzyme from two species of Trichoderma, namely T. viride and T. koningi. 

---