Cloning, Sequencing and Heterologous Expression of the Gene Encoding Glucoamylase from Clostridium thermoamylolyticum and Biochemical Characterization of the Recombinant Enzyme

Abstract

Glucoamylase (1,4Dglucan glucohydrolase, E.C. 3.2.1.3, GA) catalyzes the release of glucose from the nonreducing end of maltodextrin chains. A number of glucoamylases have been isolated from a variety of eu-karyotic and prokaryotic organisms, and they are typified by previous reports. 16) All of them have been compiled in family 15 of classification of the glycoside hydrolase su-perfamily. 7) In this paper, we present the cloning using PCR by synthetic oligonucleotides from a nucleotide sequence of Clostridium sp. G0005 glucoamylase, 8) se-quencing, expression of a glucoamylase gene from Clos-tridium thermoamylolyticum (ATCC39251) and biochemical characterization of the recombinant enzyme. All chemicals were purchased from NACALAI TESQUE. DNA manipulations were performed in E. coli DH5 (Invitrogen) using standard procedures. Restriction enzymes were from TaKaRa BIOCHEMICALS. Polyme-rase chain reactions were carried out using LATaq poly-merase (TaKaRa BIOCHEMICALS) as recommended by the manufacturer. PCR products were sequenced in both orientations with a LiCOR 4000 automated DNA se-quencer (Aloka). Chromosomal DNA of C. thermoamy-lolyticum was prepared as described by Ausubel et al. 9) In the first round amplification, the C. thermoamylolyticum glucoamylase gene was amplified by using the above chromosomal DNA as a template with a set of following primers. Two oligonucleotide primers, GAs (5 GGG TAG CCG ATT GTA CTG TGG GGC ATT3) and GA as (5 TCG TAT TTA TCG CCG ATG TTG ATG TTT TGC3), were designed from the sequence of Clostri-dium sp. G0005 chromosomal DNA (DDBJ accession number D12818). The PCR amplimers were gelpurified and cloned into pCR2.1TOPO using TA cloning (Invitro-gen), yielding pGA, which contained a complete functional glucoamylase gene. The sequence data for the gene displayed in Fig. 1 has been deposited with DDBJ (acces-sion number AB115912). This glucoamylase gene consisted of 2133 bp that encoded a 710aminoacid protein with a molecular mass of 79,920 Da. The glucoamylase showed 84% identity and 90% similarity to an amino acid sequence of Clostridium sp. G0005 glucoamylase, 8) and showed 82% identity and 87% similarity to that of Ther-moanaerobacterium thermosaccharolyticum (DSM 572) glucoamylase. 10) The glucoamylase had low similarity, showing 22% amino acid sequence similarity to Ther-moactinomyces vulgaris R47 glucoamylase. 11) The puta-tive amino acid sequence of the C. thermoamylolyticum glucoamylase was compared with the primary structures of glucoamylases from various eukaryotes, 1215) and the five highly conserved regions, CR1, CR2, CR3, CR4 and CR5 (Fig. 1), that have been reported 16) were found in the glucoamylase. The glucoamylase had a primary structure quite different from the structures of eukaryotic gluco-Data deposition: The sequence reported in this paper has been deposited in the DDBJ nucleotide sequence database (accession number AB115912). Abstract: Clostridium thermoamylolyticum glucoamylase gene was overexpressed in Escherichia coli cells. This glucoamylase gene consisted of 2133 bp that encoded a 710-amino-acid protein with a molecular mass of 79,920 Da. The glucoamylase fell into glycoside hydrolase family 15, showing 84% identity and 90% similarity to an amino acid sequence of Clostridium sp. G0005 glucoamylase, and showing 82% identity and 87% similarity to that of Thermoanaerobacterium thermosaccharolyticum glucoamylase. The corresponding sequence to the mature protein was placed under the control of the T7 promoter as a strong and constitutive promoter. The recombinant glucoamylase was purified by a Ni-NTA column. The molecular mass of the mature gluco-amylase was 77 kDa by SDS-PAGE, and it was purified 10-fold with a recovery of 65%. The specific activity was determined to be 1.8 Umg for maltose. The value of Km for maltose was determined to be 5.4 mM, and the k0 was 7.1 s 1. The optimum pH of the enzyme was determined to be 4.5, and more than 80% of the enzyme activity remained between pH 3.5 and 9.0. The optimum temperature was 65 C, and more than 80% of the enzyme activity remained up to 65 C.