Expression in Escherichia coli of a cloned β-glucanase gene from Bacillus amyloliquefaciens

Summary The recombinant phage G1 has been identified by screening 700 plaques of a Charon 4A library, containing DNA of Bacillus amyloliquefaciens, for phage clones directing the hydrolysis of lichenan in Escherichia coli, as indicated by haloes surrounding plaques on lichenan agar. The gene coding for an endo--1.3–1.4-glucanase was recloned within a 3.6 kb EcoRI fragment into the EcoRI site of plasmid pBR322, in both orientations.The location and extent of the bgl gene on the 3.6 kb Bacillus DNA insert was estimated by insertion mutagenesis with transposon Tn5 and restriction mapping of Tn5 insertions within or near to the bgl gene.The -glucanase synthesized by E. coli harbouring plasmids pEG1 or pEG2 was shown to accumulate mainly in the periplasmic space but -glucanase activities were also detected extracellulary and in the cytoplasm. The molecular weight of the enzyme synthesized in E. coli harbouring pEG1 was estimated by SDS-polyacrylamide gel electrophoresis to be about 24000. It was shown that the level of bgl gene expression in E. coli varies about 10-fold, depending on the orientation of the 3.6 kb DNA-fragment cloned within the EcoRI site of pBR322. After insertion of HindIII-DNA fragments from phage into the HindIII site of the -glucanase-high-expression plasmid pEG1, we obtained clones also showing an approximately 10-fold reduction in -glucanase activites. It was thus concluded that on plasmid pEG1 the leftward acting Ap^r (PI) promotor of plasmid pBR322 strongly increases the expression in E. coli of the cloned B. amyloliquefaciens bgl gene.Abbreviations Ap ampicillin, Km, kanamycin - kd kilodalton - kb kilobase pairs - moi multiplicity of infection - pfu plaque forming units - SDS sodium dodecylsulphate - Tc tetracycline     

Expression of a β-galactosidase gene from Lactobacillus sake in Escherichia coli

Summary A -galactosidase gene from Lactobacillus sake coding for lactose hydrolysis was cloned and expressed in Escherichia coli. Chromosomal DNA from L. sake was partially digested with the restriction enzyme Sau3AI, and the 3–6 Kb fragment was ligated to the cloning vector pSP72 digested with BamHI. One E. coli transformant expressing -galactosidase was isolated on X-gal plates. It contained a plasmid with an insertion of approx. 4 Kb. The restriction map of the recombinant plasmid was constructed. The characteristics of the recombinant -galactosidase were compared with those of the wild type. The optima pH and temperature for both enzymes was 6.5 and 50°C, respectively. Stability of the enzymes at different temperatures and activity on lactose were determined.     

The cloned β-mannanase gene from alkalophilic Bacillus sp. AM-001 produces two β-mannanases in Escherichia coli

The gene encoding -mannanase was cloned from alkalophilic Bacillus sp. AM-001 into Escherichia coli JM 101 by inserting HindIII-generated DNA fragments into the HindIII site of pUC19. A 2.0 kb XbaI-PstI fragment of the donor strain DNA was sufficient for -mannanase synthesis. The amount of -mannanase expressed in E. coli JM101 harboring pMAH3 (containing a 2.4 kb XbaI-HindIII fragment) was about 24% of the activity produced by the donor strain. E. coli JM101 harboring pMAH3 was found to produce two enzymatically active -mannanases (A and B). These two -mannanases were purified to electrophoretically homogenous states. The -mannanase A had enzymatic properties similar to those of the -mannanases M-I and M-II produced by alkalophilic Bacillus sp. AM-001, and the -mannanase B resembled its -mannanase M-III. In contrast to -mannanase production in the donor strain, that in E. coli was not inducible. The NH₂-terminal amino acid sequences from amino acid 1 (Asn) to 9 (Gln) of the three -mannanases purified from alkalophilic Bacillus sp. AM-001 coincide with those from amino acid 4 (Asn) to 12 (Gln) of the two -mannanases purified from E. coli transformant.     

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.     

Increased production of cloned β-galactosidase in two-stage culture of Bacillus amyloliquefaciens

Bacillus amyloliquefaciens harboring recombinant plasmid pHG5, which encodes B. stearothermophilus β-galactosidase, was cultured in a jar fermentor. By feeding lactose a considerable concentration of the enzyme was produced, but the cells stopped growing at an OD₆₆₀ of about 30. On the other hand, the microorganism grew to a very high cell concentration with an OD₆₆₀ of around 110 with glucose as a carbon source, but the enzyme specific activity was a half of the maximum value with lactose. Based on these facts, B. amyloliquefaciens was first grown using glucose, and the carbon source was then switched to lactose to induce β-galactosidase production. By this two-step culture method, both good cell growth and high enzyme productivity were obtained.     

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     

Cloning and expression in Escherichia coli of a Thermoanaerobacter cellulolyticus gene coding for heat-stable β-glucanase

Summary A 4.8 kb HindIII fragment of Thermoanaerobacter cellulolyticus DNA cloned in Escherichia coli was shown to direct the synthesis of -glucanase. The enzyme produced by the transformant was extremely heat-stable and the optimum temperature for the enzyme reaction was 80°C. The cloned enzyme could hydrolyse carboxymethyl cellulose and lichenan, but could not digest laminarin, xylan and cellobiose. Although T. cellulolyticus secreted cellulase(s) into the medium, most of the cloned enzyme activity was detected only in cytoplasm in the recombinant clone.     

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     

Extracellular production of a hybrid β-glucanase from Bacillus by Escherichia coli under different cultivation conditions in shaking cultures and bioreactors

Cultivation conditions for the extracellular production of a hybrid β-glucanase from Bacillus were established by using Escherichiacoli JM109 carrying the plasmid pLF3. This plasmid contained a novel secretion system consisting of the kil gene (killing protein) of plasmid ColE1 under the stationary-phase promoter of either the fic or the bolA gene, an omega interposon (Prentki and Krisch 1984) located upstream of the promoters and a hybrid β-glucanase gene of Bacillus. When controlled by the fic promoter, the kil gene led to a higher total production of β-glucanase and a higher protein secretion than when it was under control of the bolA promoter. When the effect of different distances between the stationary-phase promoters and the kil gene was investigated, a shorter distance was generally found to result in a higher secretion. With a complex growth medium, the kinetics of extracellular production of the enzyme depended on several operating variables, such as the salt concentration (NaCl) and the oxygen supply, which were varied by changing the culture volume and the shaking speed. In defined media the secretion of β-glucanase into the medium was increased significantly by the addition of glycerol as a carbon source and by prolonged cultivation. The strain with the highest production and secretion yield of β-glucanase [E. coli JM109(pLF3)] was tested on the fermenter scale. Received: 1 July 1996 / Received revision: 23 September 1996 / Accepted: 29 September 1996     

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.     

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.     

Improvement of culture conditions to overproduce β-galactosidase from Escherichia coli in Bacillus subtilis

The effect of some culture variables in the production of β-galactosidase from Escherichia coli in Bacillus subtilis was evaluated. The lacZ gene was expressed in B. subtilis using the regulatory region of the subtilisin gene aprE. The host contained also the hpr2 and degU32 mutations, which are known to overexpress the aprE gene. We found that, when this overproducing B. subtilis strain was grown in mineral medium supplemented with glucose (MMG), β-galactosidase production was partially growth-associated, as 40%–60% of the maximum enzyme activity was produced before the onset of the stationary phase. In contrast, when a complex medium was used, β-galactosidase was produced only at low levels during vegetative growth, whereas it accumulated to high levels during early stationary phase. Compared with the results obtained in complex media, a 20% increase in specific β-galactosidase activity in MMG supplemented with 11.6 g/l glucose was obtained. On the 1-l fermenter scale, a threefold increase in volumetric β-galactosidase activity was obtained when the glucose concentration was varied from 11 g/l to 26 g/l. In addition, glucose feeding during the stationary phase resulted in a twofold increase in volumetric enzyme activity as cellular lysis was prevented. Finally, we showed that oxygen uptake and carbon dioxide evolution rates can be used for on-line determination of the onset of stationary phase, glucose depletion and biomass concentration. Received: 18 April 1996 / Received revision: 27 August 1996 / Accepted: 6 September 1996     

Expression in Escherichia coli and characterization of β-xylosidases GH39 and GH-43 from Bacillus halodurans C-125

To develop xylosidases as tools for the hydrolysis of wheat bran arabinoxylans, two β-xylosidases from Bacillus halodurans C-125 have been cloned and expressed in Escherichia coli. The recombinant (His)₆-tagged enzymes, designated as XylBH39 and XylBH43, were efficiently purified using Ni²⁺-affinity chromatography. Determination of native molecular masses indicated that XylBH43 is dimeric in solution, whereas a similar analysis of XylBH39 did not allow differentiation between the dimeric and trimeric states. Both enzymes had similar pH and temperature optima (pH 7.5 and 55 °C for XylBH39 and pH 8 and 60 °C for XylBH43) and were relatively stable over the pH range of 3.5–8.5. In contrast, XylBH39 was more thermostable. At 60 °C, XylBH39 and XylBH43 displayed approximate half-life values of 2.40 and 0.05 h, respectively. The comparison of the ratio k _cat/K _M revealed that XylBH43 hydrolyzed p-nitrophenyl-β-d-xyloside more efficiently (4.6-fold) than XylBH39. Similarly, while XylBH43 was 18-fold less active on p-nitrophenyl-α-l-arabinofuranoside, XylBH39 was essentially inactive on this substrate. Using either p-nitrophenyl-β-d-xyloside or xylotriose, XylBH39 performed transglycosylation, while xylobiose proved to be a poor substrate for both hydrolysis and transglycosylation. The use of XylBH39 and XylBH43 for the posttreatment of endoxylanase-generated wheat bran hydrolysates revealed that XylBH43 efficiently produced xylose monomers (385 μg/ml after 330 min incubation). Its activity was improved by the simultaneous deployment of an α-l-arabinofuranosidase. Together, these enzymes were able to release 521 μg/ml of xylose after 330 min. This constitutes an approximate yield improvement of 35%.     

In silico characterization of a novel β-1,3-glucanase gene from Bacillus amyloliquefaciens—a bacterial endophyte of Hevea brasiliensis antagonistic to Phytophthora meadii

We report the molecular characterization of β-1,3-glucanase-producing Bacillus amyloliquefaciens—an endophyte of Hevea brasiliensis antagonistic to Phytophthora meadii. After cloning and sequencing, the β-1,3-glucanase gene was found to be 747 bp in length. A homology model of the β-1,3-glucanase protein was built from the amino acid sequence obtained upon translation of the gene. The target β-1,3-glucanase protein and the template protein, endo β-1,3-1,4-glucanase protein (PDB ID: 3o5s), were found to share 94 % sequence identity and to have similar secondary and tertiary structures. In the modeled structure, three residues in the active site region of the template—Asn52, Ile157 and Val158—were substituted with Asp, Leu and Ala, respectively. Computer-aided docking studies of the substrate disaccharide (β-1, 3-glucan) with the target as well as with the template proteins showed that the two protein-substrate complexes were stabilized by three hydrogen bonds and by many van der Waals interactions. Although the binding energies and the number of hydrogen bonds were the same in both complexes, the orientations of the substrate in the active sites of the two proteins were different. These variations might be due to the change in the three amino acids in the active site region of the two proteins. The difference in substrate orientation in the active site could also affect the catalytic potential of the β-1,3 glucanase enzyme.     

Cloning and Expression of a Gene for an 87-kDa β-1,3-Glucanase of Bacillus circulans IAM1165 in Escherichia coli K-12

Partially Sau3AI-digested fragments of chromosomal DNA from Bacillus circulans IAM1165, a high producer of β-1,3-glucanases able to lyse fungal cell walls, were inserted into a BamHI site of the plasmid vector pHSG399. A gene for the glucanase was cloned in Escherichia coli K-12 by the shotgun method. An 8-kb inserted DNA directed synthesis of an 87-kDa endo-β-1,3-n-glucanase in E. coli. The β-glucanase gene was in a 2.6-kb EcoRI-SmaI segment within the insert DNA. The enzyme activity was found mainly in the periplasmic fraction of E. coli carrying the gene.     

Expression and characterization of Kluyveromyces lactis β-galactosidase in Escherichia coli

Kluyveromyces lactis -galactosidase gene, LAC4, was expressed in Escherichia coli as a soluble His-tagged recombinant enzyme under the optimized culture conditions. The expressed protein was multimeric with a subunit molecular mass of 118 kDa. The dimeric form of the -galactosidase was the major fraction but had a lower activity than those of the multimeric forms. The purified enzyme required Mn²⁺ for activity and was inactivated irreversibly by imidazole above 50 mM. The activity was optimal at 37 and 40 °C for o-nitrophenyl--d-galactopyranoside (oNPG) and lactose, respectively. The optimum pH value is 7. The K _m and V _max values of the purified enzyme for oNPG were 1.5 mM and 560 mol min^–1 mg^–1, and for lactose 20 mM and 570 mol min^–1 mg^–1, respectively.     

Expression and characterization of Pichia etchellsii β-glucosidase in Escherichia coli

The β-glucosidase enzyme is important as the terminal enzyme involved in hydrolysis of cellobiose and short-chain cellodextrins generated during enzymatic cellulose degradation. Under controlled reaction conditions the enzyme also displays cello-oligosaccharide synthesizing ability (based on either the thermodynamic or kinetic approach). We present here the purification of the enzyme β-glucosidase (BGL) of Pichia etchellsii from recombinant pBG55 Escherichia coli clone. The kinetic parameters, substrate specificity and oligosaccharide synthesizing ability of the purified enzyme are also reported. The purified 200-kDa protein (tetramer of 50 kDa) was identified as a broad-substrate-specificity enzyme exhibiting increased temperature and glucose tolerance compared to the native yeast enzyme. Temperature directed substrate specificity for aryl β,1–4 linkage, and β(1–2), β(1–4), β(1–6) and β(2-1) linkages in various natural disaccharides was observed. Glycosylation of the enzyme was found to be unimportant for enzyme activity. With both cellobiose and glucose, oligosaccharide synthesis was detected. The implications of this information with regard to cellulose hydrolysis and oligosaccharide synthesis are discussed.     

Molecular characterization of a β-1,4-endoglucanase from an endophytic Bacillus pumilus strain

Endophytes comprise mainly microorganisms that colonize inner plant tissues, often living with the host in a symbiotic manner. Several ecological roles have been assigned to endophytic fungi and bacteria, such as antibiosis to phytopathogenic agents and plant growth promotion. Nowadays, endophytes are viewed as a new source of genes, proteins and biochemical compounds that may be used to improve industrial processes. In this study, the gene EglA was cloned from a citrus endophytic Bacillus strain. The EglA encodes a -1,4-endoglucanase capable of hydrolyzing cellulose under in vitro conditions. The predicted protein, EglA, has high homology to other bacterial cellulases and shows a modular structure containing a catalytic domain of the glycosyl hydrolase family 9 (GH9) and a cellulose-binding module type 3 (CBM3). The enzyme was expressed in Escherichia coli, purified to homogeneity, and characterized. EglA has an optimum pH range of 5–8, and remarkable heat stability, retaining more than 85% activity even after a 24-h incubation at pH 6–8.6. This characteristic is an important feature for further applications of this enzyme in biotechnological processes in which temperatures of 50–60°C are required over long incubation periods.