Molecular cloning of the β-cyclodextrin synthetase gene from an alkalophilic Bacillus and its expression in Escherichia coli and Bacillus subtilis

Summary The gene for -CGTase from an alkalophilic bacterium, Bacillus sp. #1011, was cloned in an Escherichia coli phage D69 and recloned in an E. coli plasmid pBR322 and a B. subtilis plasmid pUB110. An E. coli recombinant plasmid pTUE202 and a B. subtilis plasmid pTUB703 were selected from ten plasmids, because the transformants by each of the two plasmids produced the highest amount of extracellular -CGTase in each strain. The plasmids were stably maintained and expressed in each bacterial strain. A common DNA region of approximately 2.5 kb was defined in the ten plasmids, and the enzymatic activity was lost when a part of the common region was deleted. The major product of hydrolysis from starch by the -CGTases of E. coli [pTUB202] and B. subtilis [pTUB703] was -CD as in the case of the enzyme of the parental Bacillus sp. #1011.Abbreviations -CGTase -cyclodextrin synthetase - -CD -cyclodextrin - -CD -cyclodextrin - -CD -cyclodextrin - [] designates plasmid-carrier state     

Purification and properties of the Endo-1,4-β-glucanase from Ruminococcus albus and its gene product in Escherichia coli

Endoglucanases, EGI and EgI, were produced from the same Ruminococcus albus gene in R. albus and recombinant Escherichia coli, respectively. EGI was purified from R. albus culture supernatant and EgI was extracted from the transformant E. coli (JM101/pURA1) and purified. The purified enzymes EGI and EgI revealed maximum endoglucanase activity at a same pH of 6.8 and a temperature of 37°C. Both enzymes were stable at temperatures below 30°C. In addition, about 10% of their original activities were conserved even after boiling for 10 min. Amino acid sequences of both enzymes at the N-terminal (Ala-Ala-Asp-Glu-Ser-Glu-Thr-Glu-Asn-Val-Pro-Val-Ser-Gln-Thr-His--) were consistent with each other. The antiserum against EgI reacted with both EgI and EGI, indicating that both their protein moieties were the same immunologically. However, the molecular size of EGI (43,000) was larger than that of EgI (39,000) due to the presence of sugar moiety. The specific activity (54 units/mg) of EGI was almost double that (27 units/mg) of EgI. EGI was immunologically different from the endoglucanase purified in the previous paper [Ohmiya et al.: Carbohydrate Res., 166, 145–155 (1987)].     

Molecular cloning and expression of a Micromonospora chalcae β-glucosidase encoding gene in Escherichia coli

A Sau3A I genomic library from the actinomycete Micromonospora chalae was constructed in Escherichia coli using the expression vector pUC18. Using the chromogenic substrate 5-bromo-4-chloro-3-indolyl--glucoside (X-glu), a number of positive recombinant colonies were identified. One of those exhibiting the strongest phenotype contained a recombinant plasmid, pANNA1 which harboured a 4.2kb DNA insert. Using restriction endonuclease site mapping and subcloning strategies a 2.3kb DNA fragment encoding the -glucosidase activity was identified. Characterization of the strongly expressed recombinant enzyme demonstrated that it had a dramatically increased thermal stability at 50 °C. The Km values obtained for the recombinant enzyme and that from M. chalcae using the substrate p-nitrophenyl--D-glucoside were 0.19mM and 0.25mM, respectively.     

The gene for indole-3-acetyl-L-aspartic acid hydrolase from Enterobacter agglomerans : molecular cloning, nucleotide sequence, and expression in Escherichia coli

A 5.5-kb DNA fragment containing the indole-3-acetyl-aspartic acid (IAA-asp) hydrolase gene (iaaspH) was isolated from Enterobacter agglomerans strain GK12 using a hybridization probe based on the N-terminal amino acid sequence of the protein. The DNA sequence of a 2.4-kb region of this fragment was determined and revealed a 1311-nucleotide ORF large enough to encode the 45-kDa IAA-asp hydrolase. A 1.5-kb DNA fragment containing iaaspH was subcloned into the Escherichia coli expression plasmid pTTQ8 to yield plasmid pJCC2. Extracts of IPTG-induced E. coli cultures containing the pJCC2 recombinant plasmid showed IAA-asp hydrolase levels 5 to 10-fold higher than those in E. agglomerans extracts. Homology searches revealed that the IAA-asp hydrolase was similar to a variety of amidohydrolases. In addition, IAA-asp hydrolase showed 70% sequence identity to a putative thermostable carboxypeptidase of E. coli.     

Cloning of β-1,3-1,4-glucanase gene from Bacillus licheniformis EGW039 (CGMCC 0635) and its expression in Escherichia coli BL21 (DE3)

β-1,3-1,4-Glucanase has been applied in the brewing and animal feed additive industry. It can effectively improve digestibility of barley-based diets and reduce enteritis. It also reduces viscosity during mashing for high-quality brewers malt. The aim of this work is to clone β-1,3-1,4-glucanase-encoding gene and express it heterogeneously. The gene was amplified by polymerase chain reaction using Bacillus licheniformis genomic DNA as the template and ligated into the expression vector pET28a. The recombinant vector was transformed into Escherichia coli. The estimated molecular weight of the recombinant enzyme with a six-His tag at the N terminus was about 28 kDa, and its activities in cell lysate supernatant were 1,286 and 986 U ml⁻¹ for 1% (w/v) barley β-glucan and 1% (w/v) lichenan, respectively. Accordingly, the specific activities were 2,479 and 1,906 U mg⁻¹ for these two substrates. The expression level of recombinant β-1,3-1,4-glucanase was about 60.9% of the total protein and about 12.5% of the total soluble protein in crude cell lysate supernatant. Acidity and temperature optimal for this recombinant enzyme was pH 5.6 and 40°C, respectively.     

Cloning and expression of β-galactosidase gene from Bifidobacterium infantis into Escherichia coli

Bifidobacterium infantis HL96 produces three -galactosidases (-gal I, II and III). A genomic bank of B. infantis was constructed in E. coli by using pBR322 as a cloning vector. Two E. coli transformants, BIG1 and BIG4, possessing -galactosidase activity, were selected from X-gal plates. They contained two different recombinant plasmids with insert DNA fragments of approx. 4.6 and 4.4 kb, respectively. The restriction maps of pBIG1 and pBIG4 were constructed. -Galactosidases from crude cell-free extracts of B. infantis and of two E. coli recombinants were analyzed by native PAGE and characterized by activity staining. pBIG1 and pBIG4 were shown to carry the genes for -gal I and -gal III, respectively. Optimal pH and temperature for hydrolytic activity of the native enzyme were 7.5 and 40°C, while those for recombinant BIG1 and BIG4 were 7.5, 50°C and 8.0, 40°C, respectively. © Rapid Science Ltd. 1998     

Molecular cloning,nucleotide sequence and expression in Escherichia coli of the β-cyclodextrin glycosyltransferase gene from Bacillus circulans strain no. 8

Summary The -cyclodextrin glycosyltransferase (-CGTase) gene was isolated from a -library prepared from Bacillus circulans strain no. 8. It was subcloned into plasmid pTZ and expressed by its endogenous regulatory sequences in Escherichia coli JM 103. The structural gene was sequenced and showed an open reading frame for a polypeptide of 718 amino acid residues. The recombinant -CGTase had the same enzymatic properties as the extracellular CGTase (684 amino acid residues, corresponding to a mol. wt. of 74416) produced by B. circulans strain no. 8. The amino acid sequence showed the highest homology (74.6% identical amino acids) with the CGTase of B. circulans strain F-2, which had been erroneously described as an amylase. The homology with the enzyme from the alkalophilic Bacillus sp. strain no. 1011 was 71.4%. The amino acid sequence derived will be used for elucidating the three-dimensional structure of the enzyme. Offprint requests to: H. Bender     

Molecular cloning and expression in Escherichia coli of a thermophilic Bacillus sp. PDV endo-β-1,4-glucanase gene

The gene encoding endoglucanase in thermophilic Bacillus sp. PDV was cloned in Escherichia coli strain TB1 using pUC 8 as vector. The cloned 3.1 kb PstI DNA fragment was found to express the endoglucanase activity in either orientation. The deletion analysis of pSD 81 suggested that the Bacillus endoglucanase gene expressed in E. coli under the control of its own natural promoter, contained putatively in the 0.2 kb HindIII fragment at the 5′ end of the insert. The relative level of endoglucanase expression in E. coli was about three times higher than that in parent Bacillus sp. PDV. The cloned organism secreted about 84% of the total synthesized CMCase into the culture medium. The CMCase was stable up to 60°C and in the pH range of 4–10.     

Cloning and expression of a β -1,3-glucanase gene from Bacillus circulans KCTC3004 in Escherichia coli

Summary A new gene encoding the -1,3-glucanase(laminarinase) of Bacillus circulans KCTC3004 was cloned into Escherichia coli using pUC19 as a vector. The gene localized in the 5.3 kb PstI DNA fragment was expressed independently of its orientation in the cloning vector showing enzyme activity about 33 times greater than that produced by the original B. circulans. The optimum pH and temperature of the cloned enzyme were pH 5.4 and 50°C, respectively. The molecular weight of the enzyme was about 38,000 and the processing of the enzyme molecule within the E. coli cell was not observed. The enzyme hydrolyzed laminarin to produce laminaritriose, laminaribiose, and glucose as main products, but it was inactive for lichenan, CMC, or xylan.     

Purification,gene cloning,and characterization of γ-butyrobetainyl CoA synthetase from Agrobacterium sp. 525a

The report is the first of purification, overproduction, and characterization of a unique γ-butyrobetainyl CoA synthetase from soil-isolated Agrobacterium sp. 525a. The primary structure of the enzyme shares 70–95% identity with those of ATP-dependent microbial acyl-CoA synthetases of the Rhizobiaceae family. As distinctive characteristics of the enzyme of this study, ADP was released in the catalytic reaction process, whereas many acyl CoA synthetases are annotated as an AMP-forming enzyme. The apparent K_m values for γ-butyrobetaine, CoA, and ATP were, respectively, 0.69, 0.02, and 0.24 mM.     

Molecular cloning,characterization and expression of an elongation factor 1α gene in maize

A cDNA (zmEF1A) and the corresponding genomic clone (zmgEF1A) of a member of the gene family encoding the subunit of translation elongation factor 1 (EF-1) have been isolated from maize. The deduced amino acid sequence is 447 residues long interrupted by one intron. Southern blot analysis reveals that the cloned EF-1 gene is one member out of a family consisting of at least six genes. As shown by northern hybridizations in leaves the mRNA level increases at low temperature whereas time-course experiments over 24 h at 5°C show that in roots the overall mRNA level of EF-1 is transiently decreased. These results indicate that the expression of EF-1 is differently regulated in leaves and roots under cold stress.     

Secretory expression and enzymatic characterization of recombinant Agarivorans albus β-agarase in Escherichia coli

Agarase catalyzes the hydrolysis of agar, which is primarily used as a medium for microbiology, various food additives, and new biomass materials. In this study, we described the expression of the synthetic gene encoding β-agarase from Agarivorans albus (Aaβ-agarase) in Escherichia coli. The synthetic β-agarase gene was designed based on the biased codons of E. coli to optimize its expression and extracellular secretion in an active, soluble form. The synthesized agarase gene, including its signal sequence, was cloned into the pET-26 expression vector, and the pET-Aaβ-agarase plasmid was introduced into E. coli BL21-Star (DE3) cells. The E. coli transformants were cultured for high-yield secretion of recombinant Aaβ-agarase in Luria-Bertani broth containing 0.6?mM isopropyl β-D-1-thiogalactopyranoside for 9?h at 37°C. The expressed recombinant Aaβ-agarase was purified by ammonium sulfate precipitation and diethylaminoethyl-sepharose column chromatography, yielding ～10?mg/L Aaβ-agarase. The purified recombinant Aaβ-agarase exhibited optimal activity at pH 7 and 40°C, and its activity was strongly inhibited by Cu²⁺, Mn²⁺, Zn²⁺, and Al³⁺ ions. Furthermore, the K_M and k_cat values for purified Aaβ-agarase were ～0.02?mM and ～45/s, respectively. These kinetic values were up to approximately 15–100-fold lower than the K_M values reported for other agarases and approximately 7–30-fold higher than the k_cat/K_M values reported for other agarases, indicating that recombinant Aaβ-agarase exhibited good substrate-binding ability and high catalytic efficiency. These results demonstrated that the E. coli expression system was capable of producing recombinant Aaβ-agarase in an active form, at a high yield, and with attributes useful in the relevant industries.     

Cloning, sequencing, and expression of a β-1,4-endoxylanase gene from Indonesian Bacillus licheniformis strain I5 in Escherichia coli

The gene encoding an endo-β-1,4-xylanase from an Indonesian indigenous Bacillus licheniformis strain I5 was amplified using PCR, cloned, and expressed in Escherichia coli. The nucleotide sequence of a 642 bp DNA fragment was determined, revealing one open reading frame that encoded a xylanase. Based on the nucleotide sequence, calculated molecular mass of the enzyme was 23 kDa. This xylanase has a predicted typical putative signal peptide; however, in E. coli, the active protein was located mainly in intracellular form. Neither culture supernatant of recombinant E. coli nor periplasmic fraction has significantly detectable xylanase activity. The deduced amino acid of the gene has 91% identity with that of Bacillus subtilis endoxylanase. Optimal activity of the recombinant enzyme was at pH 7 and 50°C     

High-level expression of extracellular secretion of a β-xylosidase gene from Paecilomyces thermophila in Escherichia coli

A novel β-xylosidase gene (designated as PtXyl43) from thermophilic fungus Paecilomycesthermophila was cloned and extracellularly expressed in Escherichia coli. PtXyl43 belonging to glycoside hydrolase (GH) family 43 has an open reading frame of 1017 bp, encoding 338 amino acids without a predicted signal peptide. No introns were found by comparison of the PtXyl43 genomic DNA and cDNA sequences. The recombinant β-xylosidase (PtXyl43) was secreted into the culture medium in E. coli with a yield of 98.0 U mL(-1) in shake-flask cultures. PtXyl43 was purified 1.2-fold to homogeneity with a recovery yield of 61.5% from the cell-free culture supernatant. It appeared as a single protein band on SDS-PAGE with a molecular mass of approx 52.3 kDa. The enzyme exhibited an optimal activity at 55 °C and pH 7.0, respectively. This is the first report on the cloning and expression of a GH family 43 β-xylosidase gene from thermophilic fungi.     

Expression and Purification of an Antimicrobial Peptide, Bovine Lactoferricin Derivative LfcinB-W10 in Escherichia coli

Antimicrobial peptides (AMPs) are extremely attractive candidate for therapeutic agents due to their wide spectrum of antimicrobial activity and action mechanism different from antibiotics. In this study, a method using genetic engineering for obtaining an antimicrobial peptide, bovine lactoferricin derivative peptide LfcinB-W10, has been developed. According to the coden usage of Escherichia coli, a gene encoding the peptide was synthesized and a recombinant vector of E. coli expression pGEX-EN-LFW was constructed. The LfcinB-W10 peptide fused with glutathione S-transferase (GST) was successfully expressed and about 20 mg fusion protein with 90% purity was obtained from 1 l culture. The recombinant LfcinB-W10 (rLfcinB-W10) was released from fusion protein by the enterokinase digestion, and about the LfcinB-W10 yield reached 300 μg per 1 l culture. The purified rLfcinB-W10 was found to have growth inhibition activity against Staphylococcus aureus (S. aureus) ATCC25923.     

The orotidine-5′-phosphate decarboxylase gene (URA3) of a methylotrophic yeast,Candida boidinii: Nucleotide sequence and its expression in Escherichia coli

Previously, we cloned a DNA fragment from a genomic library of a methylotrophic yeast, Candida boidinii. This 3.5-kb SalI fragment was capable of complementing the pyrF mutation in Escherichia coli. In this report, we identify this fragment as that harboring an orotidine-5′-phosphate decarboxylase (ODCase) gene (C. boidinii URA3); we have also determined the complete DNA sequence of the C. boidinii URA3 gene. The deduced amino acid sequence of the gene showed homology to ODCase genes from other sources, and it could complement the ura3 mutation of Saccharomyces cerevisiae. The DNA fragment, which harbored the C. boidinii URA3 gene, was able to express ODCase activity in the E. coli pyrF mutant strain without an exogenous E. coli promoter. From nested-deletion analysis, both the 5′-(136 bp) and 3′-(58 bp) flanking regions were shown to be required for pyrF-complementation of the E. coli mutant. The 5′-flanking region had sequences homologous to E. coli promoter consensus sequences (−35 and −10 regions) which may function in the expression of the C. boidinii URA3 gene in E. coli.     

Cloning and expression of a thermostable exo-α-1,4-glucosidase gene from Bacillus stearothermophilus ATCC12016 in Escherichia coli

The gene coding for a thermostable exo--1,4-glucosidase (-glucoside glucohydrolase: EC 3.2.1.20) of Bacillus stearothermophilus ATCC 12016 was cloned within a 2.8-kb AvaI fragment of DNA using the plasmid pUC19 as a vector and Escherichia coli JM109 as a host. E. coli with the hybrid plasmid accumulated exo--1,4-glucosidase mainly in the cytoplasm. The level of enzyme production was about sevenfold higher than that observed for B. stearothermophilus. The cloned enzyme coincided absolutely with the B. stearothermophilus enzyme in its relative molecular mass (62 000), isoelectric point (5.0), amino-terminal sequence of 15 residues (Met-Lys-Lys-Thr-Trp-Trp-Lys-Glu-Gly-Val-Ala-Tyr-Gln-Ile-Tyr-), the temperature dependency of its activity and stability, and its antigenic determinants.Correspondence to: Y. Suzuki