Assay

The procedure suggested by Mandels［1i］was used to assay CMC（30 min），filter paper（FP）（60 min）and cotton（24 h）cellulase at 50℃，except that in our early study CM cellulase was assayed at 40℃for 30 min in a system consisting of 2.0 ml 0.63%CMC solution and 0.5 ml diluted enzyme（see Tables 2，3，5 and 6；Figure 2—Figure 7 below）.The number of units（Cx）equals the inverse of the dilution to produce 0.5 mg glucose.FP cellulase，cotton cellulase and，in some cases，（see Table 4 and Figure 8 below）CM cellulase were expressed as mg glucose produced per ml enzyme in the above defined incubation time（60 min，24 h and 30 min，respectively）.Avicelase activity was assayed on the basis of glucose produced from 0.25 ml 0.5%Avicel as substrate incubated with 0.25 ml enzyme solution at 50℃for 4 h.［9］Cellulase activity on phosphoric acidswollen cellulose was determined by incubating 0.5 ml enzyme solution with 0.5 ml 0.1 M acetate buffer（pH=4.5）and 4 ml 0.5%Walseth cellulose at 50℃for 2 h.［9］Cellobiase was estimated by glucose produced in the hydrolysis of 2.0 ml 10-3 M cellobiose（in 0.02 M acetate buffer，pH=5.0）with 0.5 ml culture filtrate at 40℃for 2 h.β-Glucosidase was determined by incubating 0.2 ml diluted enzyme solution with 0.1 ml 0.5%p-nitrophenyi-β-glucoside（PNPG）in 0.1 M acetate buffer（pH=4.5）at 50℃for 30 min.Theβ-glucosidase activity was expressed as μg of released p-nitrophenol/ml.Residual sugars were measured by the phenol-sulphuric acid method，［12］glycerol was measured by colorimetry［13］and protein was determined by the procedure of Lowry et al［14］RNA was measured by the orcinol reaction.［15］For the determination of amino acid content，4.07 mg purified C1cellulase was hydrolysed with 5.7 N[3]HCl at 110℃for 24 h and assayed with an Hitachi KLA-3B amino acid analyser.(https://www.daowen.com)