Purification of two chitinases from Rhizopus oligosporus and isolation and sequencing of the encoding genes

Abstract

Two chitinases were purified from Rhizopus oligosporus, a filamentous fungus belonging to the class Zygomycetes, and designated chitinase I and chitinase II. Their N-terminal amino acid sequences were determined, and two synthetic oligonucleotide probes corresponding to these amino acid sequences were synthesized. Southern blot analyses of the total genomic DNA from R. oligosporus with these oligonucleotides as probes indicated that one of the two genes encoding these two chitinases was contained in a 2.9-kb EcoRI fragment and in a 3.6-kb HindIII fragment and that the other one was contained in a 2.9-kb EcoRI fragment and in a 11.5-kb HindIII fragment. Two DNA fragments were isolated from the phage bank of R. oligosporus genomic DNA with the synthetic oligonucleotides as probes. The restriction enzyme analyses of these fragments coincided with the Southern blot analyses described above and the amino acid sequences deduced from their nucleotide sequences contained those identical to the determined N-terminal amino acid sequences of the purified chitinases, indicating that each of these fragments contained a gene encoding chitinase (designated chi 1 and chi 2, encoding chitinase I and II, respectively). The deduced amino acid sequences of these two genes had domain structures similar to that of the published sequence of chitinase of Saccharomyces cerevisiae, except that they had an additional C-terminal domain. Furthermore, there were significant differences between the molecular weights experimentally determined with the two purified enzymes and those deduced from the nucleotide sequences for both genes. Analysis of the N- and C-terminal amino acid sequences of both chitinases and comparison of them with the amino acid sequences deduced from the nucleotide sequences revealed posttranslational processing not only at the N-terminal signal sequences but also at the C-terminal domains. It is concluded that these chitinases are synthesized with pre- and prosequences in addition to the mature enzyme sequences and that the prosequences are located at the C terminal.

Cloning and Heterologous Expression of the Antibiotic Peptide (ABP) Genes fromRhizopus oligosporusNBRC 8631

“…Furthermore the predicted cysteine content (20.4%) was consistent with that of the amino acid analysis (20.0%) of purified ABP. Southern analysis results also suggest that abp1 and abp2 are allelic genes, as in the case of chi1 and chi2 5) or chs1 and chs2.…”

Isolation and Characterization of Two Chitin Synthase Genes ofRhizopus oligosporus

“…The possibility that the two chitinase genes of R. oligmporus are allelic to each other was suggested in our laboratory. 13) Because these genes can elevate the chitin synthase activity, when over-expressed in S. eerevisiae, it was concluded that the two cloned genes encode functional chitin synthases.…”

“…After screening of the genomic DNA library of this strain in ..12001 13 ) probed with CHS2, 18 positive clones were obtained, which were classified into four groups according to restriction enzyme digestion patterns ( Fig. 1 B).…”

“…We have already reported the isolation and characterization of two chitinase genes from this organism. 13) The aim of the present investigation is to isolate chitin synthase genes and to analyze the functions of their gene products.…”

Purification and Characterization of the Chitinase (ChiA) fromEnterobactersp. G-l

“…Chitin is mainly derived from crustaceans such as shrimps and crab shells and has received attention as a useful biomass. Chitinases (EC 3.2.1.14), which hydrolyze the /3-1 ,4-N-acetyl-glucosaminidic linkages in the insoluble chitin polymer, have been characterized in various kinds of organisms such as yeast,1) fungi, 2) bacteria, 3,4) and plants. 5 7) Chitinolytic enzymes have been attracted biotechnological research, since the chitinase specific substrate, chitin, is one of the major components of fungal cell walls.…”