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The Serratia marcescens serine protease (SSP; 66 kDa) is synthesized as a precursor (preproSSP; 112 kDa) composed of the NH2-terminal signal peptide of 27 amino acids, the mature protease part and a large COOH-terminal domain. When the SSP gene is expressed in Escherichia coli under the control of the tac promoter, the mature enzyme is excreted into the medium through the outer membrane, whereas preproSSP and two proteins, C-1 (40 kDa) and C-2 (38 kDa), processed from the COOH-terminal domain, are accumulated in the membrane fraction. Although treatment of the intact cells with trypsin caused slight truncation of C-1 and C-2, the main parts of C-1 and C-2, both of which are detected in the outer membrane, were resistant to trypsin, even after the cells had been osmotically shocked. Consistent with this, a high content of beta-sheet structure in C-2 was suggested by marked heat-modifiability, as determined by their electrophoretic mobilities on SDS-polyacrylamide gel. These findings suggest rigid integration of C-1 and C-2 in the outer membrane. Upon induction of the tac promoter, rapid excretion of SSP into the medium was first accompanied by the accumulation of C-1 in the outer membrane, which was followed by conversion of C-1 to C-2. PreproSSP was not detected during the accumulation of SSP in the medium, but it was gradually accumulated after the accumulation of SSP had reached a plateau. In addition, preproSSP still containing the intact NH2-terminal signal peptide was completely digested with trypsin when added to osmotically shocked cells.(ABSTRACT TRUNCATED AT 250 WORDS)

The Serratia marcescens serine protease gene encoding a 1,045-amino-acid precursor protein of 112 kDa directs excretion of the mature protease of ca. 58 kDa through the outer membrane of Escherichia coli. A typical signal peptide of 27 amino acids and a large COOH-terminal domain of the precursor are both functionally essential for the excretion of the mature protease into the medium. Sequence analysis of the fragment peptides of the mature protease as well as site-directed mutagenesis indicated that the COOH-terminus of the mature enzyme was Asp645. By using the polyclonal antibody against the 112-kDa precursor protein, not only the intact precursor but also two proteins, C-1 (40 kDa) and C-2 (38 kDa), corresponding to the processed COOH-terminal domains were detected in the insoluble fraction of E. coli cells. Further fractionation by sucrose density gradient centrifugation showed that C-1 and C-2 were localized in the outer membrane. The NH2-terminal residues of C-1 and C-2 were determined to be Ala702 and Phe717, respectively. All these data suggest that the precursor is cleaved at three positions, between Asp645-Ser646, Glu701-Ala702, and Gly716-Phe717, probably by the self-processing activity in the normal excretion pathway through the outer membrane.

Mutarotation of products from p-nitrophenyl beta-D-cellobioside and cellopentaitol by two different types of exo-cellulases from Trichoderma viride was investigated. It was found that an exo-cellulase of glucosidase type produced from the former substrate D-glucose which was mutarotated in a downward direction, while another exo-cellulase of Avicelase type produced from the latter substrate cellobiose which was mutarotated in an upward direction.

An enzyme extract from Cellulase-Onozuka, a commercial product of Trichoderma viride, was fractionated by Amberlite CG-50 column chromatography into three cellulase [EC 3.2.1.4] groups, peaks I to III. A noval enzyme, which has both beta-glucosidase [EC 3.2.1.21] and exo-carboxymethyl-cellulase (exo-CMCase) properties was obtained from peak III by extensive purification throuh consecutive column chromatography. The enzyme was homogeneous on ultracentrifugation, SDS-gel and cellulose acetate film electrophoreses and molecular sieve chromatography on Bio-Gel P-150. The molecular weight of this enzyme was estimated to be 53,000. The enzyme appeared to release cellobiose residues one by one from the nonreducing end of higher cellooligosaccharides and CM-cellulose (CMC), but to release glucosyl residues from reduced cellotriose and beta-cellobioside, resembling a beta-glucosidase in this respect. Furthermore, this exo-CMCase also attacked xylan exo-wise to produce xylobiose moleculaes one by one, but it scarcely attacked insoluble cellulose, except for a cellodextrin apparently rich in amorphous structure.