Molecular cloning and characterization of a multidomain endoglucanase from Paenibacillus sp BP-23: evaluation of its performance in pulp refining

The gene celB encoding an endoglucanase from Paenibacillus sp. BP-23 was cloned and expressed in Escherichia coli. The nucleotide sequence of a 4161 bp DNA fragment containing the celB gene was determined, revealing an open reading frame of 2991 nucleotides that encodes a protein of 106,927 Da. Comparison of the deduced amino acid sequence of endoglucanase B with known β-glycanase sequences showed that the encoded enzyme is a modular protein and exhibits high homology to enzymes belonging to family 9 cellulases. The celB gene product synthesized in E. coli showed high activity on carboxymethyl cellulose and lichenan while low activity was found on Avicel. Activity was enhanced in the presence of 10 mM Ca²⁺ and showed its maximum at 53 °C and pH 5.5. The effect of the cloned enzyme in modifying the physical properties of pulp and paper from Eucalyptus was tested (CelB treatment). An increase in mechanical strength of paper and a decrease in pulp dewatering properties were found, indicating that CelB treatment can be considered as a biorefining. Treatment with CelB gave rise to an improvement in paper strength similar to that obtained with 1,000 revolutions increase in mechanical refining. Comparison with the performances of recently developed endoglucanase A from the same strain and with a commercial cellulase showed that CelB produced the highest refining effect. Received: 25 February 2000 / Received revision: 4 July 2000 / Accepted: 9 July 2000     

Purification and some properties of a novel microbial lactate oxidase

 Geotrichum candidum was found to produce a lactate oxidase. The enzyme was purified by gel filtration and ion-exchange chromatography. The purified lactate oxidase showed a molecular mass of 50 kDa under denaturing and about 400 kDa under non-denaturing conditions. Transmission electron micro-scopy analysis confirmed an octameric structure. FMN was found to be a cofactor for this enzyme. Polarographic studies confirmed an oxygen uptake by the lactate oxidase. The enzyme showed specificity towards the L isomer of lactate and did not oxidise pyruvate, fumarate, succinate, maleate and ascorbate. It was stable at alkaline pH and also for 15 min at 45°C. The addition of glycerol and dextran 500 000 to the enzyme sample enhanced storage stability. Received: 28 September 1995/Received revision: 10 January 1996/Accepted: 15 January 1996     

Molecular cloning,purification and characterization of two endo-1,4-β-glucanases from Aspergillus oryzae KBN616

Two endo-1,4-β-glucanase genes, designated celA and celB, from a shoyu koji mold Aspergillus oryzae KBN616, were cloned and characterized. The celA gene comprised 877 bp with two introns. The CelA protein consisted of 239 amino acids and was assigned to the cellulase family H. The celB gene comprised 1248 bp with no introns. The CelB protein consisted of 416 amino acids and was assigned to the cellulase family C. Both genes were overexpressed under the promoter of the A. oryzae taka-amylase A gene for purification and enzymatic characterization of CelA and CelB. CelA had a molecular mass of 31 kDa, a pH optimum of 5.0 and temperature optimum of 55 °C, whereas CelB had a molecular mass of 53 kDa, a pH optimum of 4.0 and temperature optimum of 45 °C. Received: 3 July 1996 / Accepted: 15 July 1996     

Cloning of the cyclodextrin glucanotransferase gene from alkalophilic Bacillus sp. A2-5a and analysis of the raw starch-binding domain

The cyclodextrin glucanotransferase (CGTase) gene of alkalophilic Bacillus sp. A2-5a was cloned and expressed in Bacillus subtilis ANA-1 as a host. The DNA region included an open reading frame encoding a 704-amino-acid polypeptide with a typical raw starch-binding motif in its C-terminal region. The CGTase purified from Bacillus sp. A2-5a bound to raw starch as strongly as porcine pancreas α-amylase, as expected from the sequence motif. A chromosomal region (a DNA fragment of about 14.1 kbp) including the CGTase gene was also cloned and the nucleotide sequence was determined. Possible cyclodextrinase and putative cyclodextrin-binding protein genes were found in the flanking region of the CGTase gene, which implied that the novel starch-degradation pathway postulated for a gram-negative bacterium [Klebsiella oxytoca; Fiedler et al. (1996) J Mol Biol 256: 279–291] also exists in a gram-positive bacterium i.e. Bacillus. Received: 6 August 1999 / Received last revision: 8 October 1999 / Accepted: 22 October 1999     

Effect of pH on transfructosylation and hydrolysis by β-fructofuranosidase from Aspergillus oryzae

 β-Fructofuranosidase was purified from commercial alkaline protease (Aspergillus oryzae origin). The optimal pH of its transfructosylating activity was more alkaline (pH 8) than that of its hydrolyzing activity (pH 5). In the case of a 24-h reaction with sucrose, the hydrolysis and transfructosylation reaction were optimal at pH 4–5 and pH 8, respectively. In the reaction at pH 8 1-kestose and nystose were the main fructooligosaccharides produced. The transfer ratio was hardly different between pH 5 and pH 8 early in the reaction, but the transfer products (1-kestose and nystose) were decreased at pH 5 as the reaction proceeded because of their hydrolysis. Received: 18 January 1995/Received last revision: 23 August 1995/Accepted: 13 September 1995     

Use of an industrial effluent as a carbon source for denitrification of a high-strength wastewater

Denitrification of a high-strength synthetic wastewater (150 g NO^- ₃ l^-1) was carried out using a wine distillery effluent as an example of an industrial carbon source (22.7 g chemical oxygen demand l^-1). Two configurations were tested: one consisted of an acidogenesis reactor followed by a denitrifying reactor and the other was a single reactor directly fed with the raw effluents. In both cases, denitrification was achieved at a nitrate load of 9.54 g NO^- ₃ l^-1 day^-1 (2.19 g N as NO^- ₃ l^-1 day^-1) with good specific reduction rates: 32.6 mg and 35.2 mg N as NO _x  g volatile suspended solids h^-1, calculated on a single day, for the two-step and the one-step process respectively. Dissimilatory nitrate reduction to ammonium did not occur, even in the one-step process. Received: 26 October 1995/Received revision: 15 February 1996/Accepted: 20 February 1996     

Long-term stability of a biofilter treating dimethyl sulphide

 The biofiltration of dimethyl sulphide (Me₂S) and other volatile sulphur compounds results in the accumulation of the metabolite sulphuric acid in the carrier material. Regeneration of an acidified (pH 4.7), Hyphomicrobium-MS3-inoculated compost biofilter degrading Me₂S was not possible by trickling tap water (days 0–28) or a KH₂PO₄/K₂HPO₄ buffer solution (1.26 g PO^3- ₄ l^-1, pH 7) (days 29–47) over the bioreactor at a superficial liquid flow rate of 34 lm^-2 day^-1. Since the protons produced displaced nutrient cations (Na⁺, K⁺, Ca²⁺, Mg²⁺, NH⁺ ₄) from the cation-exchange sites on the compost material, 95% of the SO^2- ₄ was leached as the corresponding sulphate salts and not as sulphuric acid. Concomitantly, the pH of the compost material decreased from 4.7 to 3.9 over the 47 days rinsing period. Moreover, the rinsing procedure resulted in the leaching of essential microbial nutrients from the compost material, such as NH⁺ ₄ (22.3% wash-out over the 47-day rinsing period) and PO^3- ₄ (39.3% washout over the 28-day tap-water rinsing period). However, mixing limestone powder into the Me₂S-degrading compost biofilter was a successful approach to controlling the pH in the optimal range for the inoculum Hyphomicrobium MS3 (pH 6–7). A stoichiometric neutralisation reaction (molar ratio CaCO₃/H₂SO₄=1.1) was observed between the CaCO₃ added and the metabolite of the Me₂S degradation, while high elimination capacities (above 100 g Me₂S m^-3 day^-1) were obtained over a prolonged (more than 100 days) period. Received: 1 December 1995/Received revision: 26 April 1995 Accepted: 29 April 1996     

Nucleotide Sequence of the Gene for an Alkaline Endoglucanase from an Alkalophilic Bacillus and Its Expression in Escherichia coli and Bacillus subtilis

The gene for an alkaline endoglucanase from the alkalophilic Bacillus sp. KSM-64 was cloned into the HindIII site of pBR322 and expressed in Escherichia coli HB101. The nucleotide sequence of a 4.1-kb region of the HindIII insert had two open reading frames, ORF-1 and ORF-2. The protein deduced from ORF-1 was composed of 244 amino acids with an M_r of 27,865. Subcloning analysis proved that the alkaline endoglucanase was encoded by ORF-2 (822 amino acids with an M_r of 91,040). Upstream from ORF-2, there were three consensus like sequences of the sigma A-type promoter of Bacillus subtilis, a putative Shine-Dalgarno sequence (AGGAGGT), and a catabolite repression operator-like sequence (TGTAAGC-GGTTAACC). The HindIII insert was subcloned into a shuttle vector, pHY300PLK, and the encoded alkaline endoglucanase gene was highly expressed both in E. coli and B. subtilis. One of the three promoter-like sequences in ORF-2 could be suitable for high levels of enzyme expression in both host organisms.     

Regulation of hydrogen metabolism in Butyribacterium methylotrophicum by substrate and pH

 Exogenous H₂/CO₂ and glucose were consumed simultaneously by Butyribacterium methylotrophicum when grown under glucose-limited conditions. CO₂ reduction to acetate was coupled to H₂ consumption. The addition of either H₂ or CO₂ to glucose batch fermentation resulted in an increase in cell density, hydrogenase (H₂-consuming and -producing) activities and fatty acid production by B. methylotrophicum as compared to when N₂ was the feed gas. Hydrogenase activities appeared to be tightly regulated and were produced at higher rates during the exponential phase when CO₂ was the feed gas as compared to H₂ or N₂. The increase in H₂-consuming activity and decrease in H₂-producing activity was correlated with an increase in butyrate synthesis. H₂-consuming and ferredoxin (Fd)–NAD reductase activities increased while H₂-producing and NADH–Fd reductase activities decreased in cells grown at pH 5.5 compared to those at pH 7.0. The molar ratio of butyrate/acetate was shifted from 0.35 at pH 7.0 to 1.22 at pH 5.5. The addition of exogenous H₂ did not decrease the butyrate/acetate ratio at pH 7.0 nor at pH 5.5. The results indicated that growth pH values regulated both hydrogenase and Fd–NAD oxidoreductase activities such that, at acid pH, more intermediary electron flow was directed towards butyrate synthesis than H₂ production. Received: 22 August 1995/Received revision: 18 December 1995/Accepted: 22 January 1996     

Cloning of a new endoglucanase gene from Bacillus sp. BP-23 and characterisation of the enzyme. Performance in paper manufacture from cereal straw

The gene celA, encoding an endoglucanase from the strain Bacillus sp. BP-23, was cloned and expressed in Escherichia coli. The nucleotide sequence of a 1867-bp DNA fragment containing the celA gene was determined, revealing an open reading frame of 1200 nucleotides that encodes a protein of 44 803 Da. The deduced amino acid sequence of the encoded enzyme shows high homology to those of enzymes belonging to subtype 4 of the family-A cellulases. The celA gene product synthesized in E. coli showed activity on carboxymethylcellulose and lichenan but no activity was found on Avicel. Activity was enhanced in the presence of 10 mM Mg²⁺ and Ca²⁺ and showed its maximum at 40 °C and pH 4.0. Study of the performance of CelA on paper manufacture from agricultural fibres showed that treatment with the enzyme improved the properties of the pulp and the quality of paper. CelA treatment enhanced the physical properties (stretch and tensile index) of paper from wheat straw, while dewatering properties were slightly diminished. Electron-microscope analysis showed that the surface of straw fibres was modified by CelA. Received: 11 February 1998 / Received revision: 20 March 1998 / Accepted: 20 March 1998     

Characterisation of a complex restriction/modification system detected in a Bifidobacterium longum strain

 Two type-II restriction endonucleases, BloI and BloII, have been detected in a Bifidobacterium longum strain. BloI is influenced by dam methylation: it cleaves dam ^- but not dam ⁺ DNA. It shows a temperature and pH optimum of 45°C and pH 7.5. Restriction analysis and cloning experiments showed that the recognition sequence is RGATCY and that the enzyme cuts 5′ to the guanine residue. It is an isoschizomer of commercial enzymes, BstYI and XhoII. The second activity is not inhibited by dam methylation. It has a temperature optimum between 25°C and 30°C and shows a broad pH optimum between 4.5 and 7.0. The activity is thermolabile and can be heat-killed by a 5 min incubation at 60°C. Cloning and sequencing experiments revealed that its recognition sequence is CTGCAG and that it cuts 5′ to the second guanine residue in the sequence. This enzyme is the first described isoschizomer of PstI. Received: 22 May 1995/Accepted: 26 July 1995     

Detoxification of cassava by Aspergillus niger B-1

 The effects of fermentation of cassava by Aspergillus niger B-1 β-glucosidase on its cyanide and protein content, and the optimal conditions for this enzyme’s activity, were examined. This fermentation process reduced the cyanide content of cassava by 95% to 2 mg/kg, and increased its total protein content by 50%, thereby improving its nutritional value. A significant decrease in cyanogenic glycosides was detected after 3 days of fermentation. The optimal pH for A. nigerβ-glucosidase activity on the cyanogenic glycoside linamarin was determined to be 3, the optimal temperature 55 °C, and its K _m 0.3 mM. The findings presented here will facilitate the development of an improved method for detoxification of cassava and for enhancement of its nutritional value. Received: 17 August 1995/Received revision: 27 October 1995/Accepted: 30 October 1995     

A β-1,4-endoglucanase-encoding gene from Cellulomonas pachnodae

 A gene library of Cellulomonas pachnodae was constructed in Escherichia coli and was screened for endoglucanase activity. Five endoglucanase-positive clones were isolated that carried identical DNA fragments. The gene, designated cel6A, encoding an endoglucanase enzyme, belongs to the glycosyl hydrolase family 6 (cellulase family B). The recombinant Cel6A had a molecular mass of 53 kDa, a pH optimum of 5.5, and a temperature optimum of 50–55 °C. The recombinant endoglucanase Cel6A bound to crystalline cellulose and beech litter. Based on amino acid sequence similarity, a clear cellulose-binding domain was not distinguished. However, the regions in the Cel6A amino acid sequence at the positions 262–319 and 448–473, which did not show similarity to any of the known family-6 glycosyl hydrolases, may be involved in substrate binding. Received: 14 January 1999 / Received revision: 29 March 1999 / Accepted: 6 April 1999     

Saccharomyces cerevisiae mutants selected for increased production of Trichoderma reesei cellulases

 Trichoderma reesei endoglucanase I (EGI) was used as a reporter enzyme for screening mutagenized yeast strains for increased ability to produce protein. Sixteen haploid Saccharomyces cerevisiae strains, transformed with a yeast multicopy vector pALK222, containing the EGI cDNA under the ADH1 promoter, produced EGI activity of 10^-5–10^-4 g/l. On the average 93% of the total activity was secreted into the culture medium. Two strains with opposite mating types were mutagenized, and several mutants were isolated possessing up to 45-fold higher EGI activity. The best mutants were remutagenized and a second-generation mutant, strain 2804, with an additional twofold increase in EGI activity was selected. The mutant strain 2804 grew more slowly and reached a lower final cell density than the parental strain. In the selective minimal medium, the 2804 strain produced 40 mg/l immunoreactive EGI protein, but only 2% was active enzyme. In the rich medium the secreted EGI enzyme stayed active, but without selection pressure the EGI production ceased after 2 days of cultivation, when the strain 2804 had produced 10 mg/l of EGI. A sevenfold difference was found between the parental and the 2804 strain in their total EGI production relative to cell density. The difference in favour of the mutant strain was also detected on the mRNA level. The 2804 mutant was found to be more active than the parental strain also in the production of T. reesei cellulases, cellobiohydrolase I, and cellobiohydrolase II. Received: 22 December 1995/Received revision: 26 February 1996/Accepted: 17 March 1996     

Cloning, sequencing and overexpression of a Rhodothermus marinus gene encoding a thermostable cellulase of glycosyl hydrolase family 12

A gene library from the thermophilic eubacterium Rhodothermus marinus, strain ITI 378, was constructed in pUC18 and transformed into Escherichia coli. Of 5400 transformants, 3 were active on carboxymethylcellulose. Three plasmids conferring cellulase activity were purified and were all found to contain the same cellulase gene, celA. The open reading frame for the celA gene is 780 base pairs and encodes a protein of 260 amino acids with a calculated molecular mass of 28.8 kDa. The amino acid sequence shows homology with cellulases in glycosyl hydrolase family 12. The celA gene was overexpressed in E. coli when the pET23, T7 phage RNA polymerase system was used. The enzyme showed activity on carboxymethylcellulose and lichenan, but not on birch xylan or laminarin. The expressed enzyme had six terminal histidine residues and was purified by using a nickel nitrilotriacetate column. The enzyme had a pH optimum of 6–7 and its highest measured initial activity at 100 °C. The heat stability of the enzyme was increased by removal of the histidine residues. It then retained 75% of its activity after 8 h at 90 °C. Received: 5 August 1997 / Received revision: 6 November 1997 / Accepted: 7 November 1997     

Growth of Corynebacterium glutamicum in ammonium- and potassium-limited continuous cultures under high osmotic pressure

 In order to determine the possible effect of nutrient limitations on the response of Corynebacterium glutamicum to a saline osmotic up-shock, the bacteria were grown in continuous cultures, at osmotic pressures of 0.4 osmol/kg and 1.2 osmol/kg, under ammonia and potassium limitation. At the low osmolality of 0.4 osmol/kg, the glutamate and proline levels of 15 mg/g and 5 mg/g dry weight respectively were lower than previously reported in glucose-limited continuous cultures (50 mg/g and 10 mg/g dry weight respectively). On the other hand, the internal trehalose pool was much higher at 40 mg/g dry weight. When the medium osmolality was increased to 1.2 osmol/kg by NaCl addition, under ammonia limitation, the proline content rose from 5 mg/g to 20 mg/g dry weight and the trehalose content from 40 mg/g to 70 mg/g dry weight, whereas the intracellular pool of glutamate remained essentially constant. An increase in the internal sodium content was also observed. Similar results were found for the internal pool of glutamate, proline and trehalose when C. glutamicum was grown under potassium limitations at an osmolality of 1.2 osmol/kg. There were also higher levels of sodium ions, glutamine and alanine. According to the present results, whereas proline was previously reported to be the dominantly accumulated osmoprotectant in C. glutamicum grown under glucose limitations, under ammonia and potassium limitations trehalose represented the dominantly synthesized metabolite. Received: 19 December 1995/Received revision: 9 April 1996/Accepted: 15 April 1996     

Microbial sensors for naphthalene using Sphingomonas sp. B1 or Pseudomonas fluorescens WW4

 Amperometric biosensors for naphthalene were developed using either immobilized Sphingomonas sp. B1 or Pseudomonas fluorescens WW4 cells. The microorganisms were immobilized within a polyurethane-based hydrogel, which was used for a microbial biosensor for the first time. Both strains were shown to be equally suited for the quantification of naphthalene in aqueous solutions. The biosensors were tested in a flow-through system and a stirred cell (batch method). In both systems a linear response down to the detection limit was obtained. Measurements in the flow-through system gave sensitivities of up to 1.2 nA mg⁻¹ l⁻¹ and a linear range from 0.03 mg/l to 2.0 mg/l. The response time (t ₉₅) was 2 min and the sample throughput six per hour; the repeatability was within ±5 %. With the batch method, sensitivities of between 3 nA mg⁻¹ l⁻¹ and 5 nA mg⁻¹l⁻¹ and a linear range of 0.01–3.0 mg/l were obtained; the response time was between 3 min and 5 min. The sensors reached an operational lifetime of up to 20 days. The sensitivity of both sensors for naphthalene was, in most cases, more than four times higher than for various other substrates. Received: 18 October 1995/Received revision: 22 December 1995/Accepted: 22 January 1996     

A new alkaline serine protease from alkalophilic Bacillus sp.: Cloning,sequencing, and characterization of an intracellular protease

To obtain a new serine protease from alkalophilic Bacillus sp. NKS-21, shotgun cloning was carried out. As a result, a new protease gene was obtained. It encoded an intracellular serine protease (ISP-1) in which there was no signal sequence. The molecular weight was 34,624. The protease showed about 50% homology with those of intracellular serine proteases (ISP-1) from Bacillus subtilis, B. polymyxa, and alkalophilic Bacillus sp. No. 221. The amino acid residues that form the catalytic triad, Ser, His and Asp, were completely conserved in comparison with subtilisins (the extracellular proteases from Bacillus). The cloned intracellular protease was expressed in Escherichia coli, and its purification and characterization were carried out. The enzyme showed stability under alkaline condition at pH 10 and tolerance to surfactants. The cloned ISP-1 digested well nucleoproteins, clupein and salmin, for the substrates.The nucleotide sequence data reported in this paper will appear in the GSDB, DDBJ, EMBL, and NCBI nucleotide sequence databases with the accession number D37921.     

Large-scale production and characterization of Bacillus thuringiensis subsp. tenebrionis insecticidal protein from Escherichia coli

Bacillus thuringiensis subsp. tenebrionis insecticidal protein was produced in recombinant Escherichia coli and purified to near homogeneity to provide quantities of protein for safety-assessment studies associated with the registration of transgenic potato plants. The 68-kDa protein is produced naturally by Bacillus thuringiensis subsp. tenebrionis by translation initiation at an internal initiation site in the native DNA sequence. The gene sequence specific for this truncated protein was expressed in E. coli strain JM 101 and fermented at the 1000-l scale. The protein accumulated as insoluble inclusion bodies, and was purified by extraction at pH␣10.8 with carbonate buffer, selective precipitation at pH 9.0, and differential centrifugation. No chromatography steps were required to produce over 50 g purified protein as a lyophilized powder with a purity greater than 95 % and demonstrating full insecticidal activity against Colorado potato beetle larvae. The protein was further characterized to assure identity and suitability for use in safety-assessment studies. Received: 31 May 1996 / Received revision: 11 September 1996 / Accepted: 13 October 1996