Substrate selectivity of various lipases in the esterification of cis- and trans-9-octadecenoic acid

The substrate selectivity of numerous commercially available lipases from microorganisms, plants and animal tissue towards 9-octadecenoic acids with respect to the cis/trans configuration of the CC double bond was examined by the esterification of cis- and trans-9-octadecanoic acid (oleic and elaidic acid respectively) with n-butanol in n-hexane. A great number of lipases studied, e.g. those from Pseudomonas sp., porcine pancreas or Carica papaya, were unable to discriminate between the isomeric 9-octadecenoic acids. However, lipases from Candida cylindracea and Mucor miehei catalysed the esterification of oleic acid 3–4 times faster than the corresponding reaction of elaidic acid and therefore have a high preference for the cis isomer. Of all biocatalysts examined, only recombinant lipases from Candidaantarctica favoured elaidic acid as substrate. While the preference of Candida antarctica lipase B for the trans isomer was quite low, Candida antarctica lipase A had an extraordinary substrate selectivity and its immobilized enzyme preparation [Chirazyme L-5 (3) from Boehringer] esterified elaidic acid about 15 times faster than oleic acid. Received: 29 October 1998 / Received revision: 18 December 1998 / Accepted: 21 December 1998     

Cloning and overexpression in Escherichia coli of the gene encoding dihydroxyacetone kinase isoenzyme I from Schizosaccharomyces pombe, and its application to dihydroxyacetone phosphate production

The gene dak1 encoding a dihydroxyacetone kinase (DHAK) isoenzyme I, one of two isoenzymes in the Schizosaccharomyces pombe IFO 0354 strain, was cloned and sequenced. The dak1 gene comprises 1743 bp and encodes a protein of 62 245 Da. The deduced amino acid sequence showed a similarity to a putative DHAK of Saccharomyces cerevisiae and DHAK of Citrobacter freundii. The dak1 gene was expressed at a high level in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized. The acetone powder of recombinant E. coli cells was used to produce dihydroxyacetone phosphate. Received: 25 August 1998 / Received revision: 22 September 1998 / Accepted: 11 October 1998     

Competition of plasmid-bearing Pseudomonas putida strains catabolizing naphthalene via various pathways in chemostat culture

Plasmid-carrying Pseudomonas putida strains degrade naphthalene through different biochemical pathways. The influence of various combinations of host bacteria and plasmids on growth characteristics and competitiveness of P. putida strains was studied in chemostat culture at a low dilution rate (D=0.05 h⁻¹) with naphthalene as the sole source of carbon and energy. Under naphthalene limitation, the plasmid-bearing strains degrading naphthalene that use catechol 1,2-dioxygenase for catechol oxidation (ortho pathway), were the most competitive. The strains bearing plasmids that control naphthalene catabolism via catechol 2,3-dioxygenase (meta pathway), were less competitive. Under these conditions the strain carrying plasmid pBS4, which encodes for naphthalene catabolism via gentisic acid, was the least competitive. Received: 24 February 1997 / Received revision: 22 May 1997 / Accepted: 25 May 1997     

Modelling the influence of pH, temperature, glucose and lactic acid concentrations on the kinetics of lactic acid production by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour

A kinetic model of the fermentative production of lactic acid from glucose by Lactococcus lactis ssp. lactis ATCC 19435 in whole-wheat flour has been developed. The model consists of terms for substrate and product inhibition as well as for the influence of pH and temperature. Experimental data from fermentation experiments under different physical conditions were used to fit and verify the model. Temperatures above 30 °C and pH levels below 6 enhanced the formation of by-products and d-lactic acid. By-products were formed in the presence of maltose only, whereas d-lactic acid was formed independently of the presence of maltose although the amount formed was greater when maltose was present. The lactic acid productivity was highest between 33 °C and 35 °C and at pH 6. In the concentration interval studied (up to 180 g l⁻¹ glucose and 89  g l⁻¹ lactic acid) simulations showed that both substances were inhibiting. Glucose inhibition was small compared with the inhibition due to lactic acid. Received: 28 October 1997 / Received revision: 3 February 1998 / Accepted: 6 February 1998     

Cloning and characterization of the gene encoding a repressible acid phosphatase (PHO1) from the methylotrophic yeast Hansenula polymorpha

A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with a calculated M _r of 49400. The␣encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression. Received: 15 January 1998 / Received revision: 2 March 1998 / Accepted: 4 March 1998     

Molecular cloning and nucleotide sequence of the pyruvate kinase gene of an actinomycete Microbispora thermodiastatica

The gene for the thermostable pyruvate kinase of Microbispora thermodiastatica IFO 14046, a moderate thermophilic actinomycete, was cloned in Escherichia coli. This gene consists of an open reading frame of 1422 nucleotides and encodes a protein of 474 amino acids with molecular mass of 50 805 Da. The open reading frame was confirmed as the pyruvate kinase gene by comparison with the N-terminal amino acid sequence of the purified pyruvate kinase from M. thermodiastatica. Received: 19 May 1997 / Received last revision: 22 September 1997 / Accepted: 14 October 1997     

Cloning of the transketolase gene and the effect of its dosage on aromatic amino acid production in Corynebacterium glutamicum

Transketolase is a key enzyme of the nonoxidative pentose phosphate pathway. The effect of its overexpression on aromatic amino acid production was investigated in Corynebacterium glutamicum, a typical amino-acid-producing organism. For this purpose, the transketolase gene of the organism was cloned on the basis of its ability to complement a C. glutamicum transketolase mutant with pleiotropically shikimic-acid-requiring, ribose- and gluconic-acid-negative phenotype. The gene was shown by deletion mapping and complementation analysis to be located in a 3.2-kb XhoI-SalI fragment of the genome. Amplification of␣the gene by use of low-, middle-, and high-copy-number vectors in a C. glutamicum strain resulted in overexpression of transketolase activities as well as a␣protein of approximately 83kDa in proportion to the copy numbers. Introduction of the plasmids into a tryptophan and lysine co-producer resulted in copy-dependent increases in tryptophan production along with concomitant decreases in lysine production. Furthermore, the presence of the gene in high copy numbers enabled tyrosine, phenylalanine and tryptophan producers to accumulate 5%–20% more aromatic amino acids. These results indicate that overexpressed transketolase activity operates to redirect the glycolytic intermediates toward the nonoxidative pentose phosphate pathway in vivo, thereby increasing the intracellular level of erythrose 4-phosphate, a precursor of aromatic biosynthesis, in the aromatic-amino-acid-producing C. glutamicum strains. Received: 27 July 1998 / Received last revision: 12 October 1998 / Accepted: 24 October 1998     

High-level expression of the angiotensin-converting-enzyme-inhibiting peptide, YG-1, as tandem multimers in Escherichia coli

To produce a large quantity of the angiotensin-converting-enzyme(ACE)-inhibiting peptide YG-1, which consists of ten amino acids derived from yeast glyceraldehyde-3-phosphate dehydrogenase, a high-level expression was explored with tandem multimers of the YG-1 gene in Escherichia coli. The genes encoding YG-1 were tandemly multimerized to 9-mers, 18-mers and 27-mers, in which each of the repeating units in the tandem multimers was connected to the neighboring genes by a DNA linker encoding Pro-Gly-Arg for the cleavage of multimers by clostripain. The multimers were cloned into the expression vector pET-21b, and expressed in E. coli BL21(DE3) with isopropyl β-d-thiogalactopyranoside induction. The expressed multimeric peptides encoded by the 9-mer, 18-mer and 27-mer accumulated intracellularly as inclusion bodies and comprised about 67%, 25% and 15% of the total proteins in E. coli respectively. The multimeric peptides expressed as inclusion bodies were cleaved with clostripain, and active monomers were purified to homogeneity by reversed-phase high-performance liquid chromatography. In total, 105 mg pure recombinant YG-1 was obtained from 1 l E. coli culture harboring pETYG9, which contained the 9-mer of the YG-1 gene. The recombinant YG-1 was identical to the natural YG-1 in molecular mass, amino acid sequence and ACE-inhibiting activity. Received: 6 January 1998 / Received revision: 23 February 1998 / Accepted: 24 February 1998     

Simultaneous degradation of chloro- and methyl-substituted aromatic compounds: competition between Pseudomonas strains using the ortho and meta pathway or the ortho pathway exclusively

Pseudomonas sp. D7-4 and Pseudomonas sp. B13 FR1(pFRC20P) degraded mixtures of chloro- and methyl-substituted benzoates exclusively via an extended ortho pathway, whereas in Pseudomonas putida WR201 both ortho and meta fission were induced by mixtures of 3-chloro- and 3-methylbenzoate or even by 3-chlorobenzoate alone. The competition behaviour of these strains was compared in batch and in chemostat cultures. Despite misrouting of metabolites, strain WR201 was competitive, in a lot of the competition experiments, with mixtures of these substrates. Only in a narrow range of the mixing ratio of chloro- and methylbenzoate was the presence of both the meta and ortho pathways a disadvantage for competitiveness. Outside these ranges other attributes, such as high growth rates or short lag periods, of a respective strain were even more essential for one strain to outcompete another. Received: 13 February 1998 / Received revision: 28 April 1998 / Accepted: 30 April 1998     

Reversal of the inhibitory effect of surfactants upon germination and growth of a consortium of two strains of Bacillus

Anionic, cationic, amphoteric and non-ionic surfactants inhibited spore germination and subsequent growth of a mixture of two Bacillus strains at surfactant concentrations ranging from 1 ppm to 50 ppm. Germination appeared to be more affected than cell growth by the presence of surfactants, the inhibitory thresholds being largely increased when media were inoculated with vegetative cells. The bacterial species forming the consortium were incapable of growing on liquid and agar-solidified media prepared with non-diluted domestic wastewater. Addition of hydrolases (protease, cellulase, α-amylase and lipase) to the wastewater medium allowed the germination of spores and their vegetative growth. Received: 9 July 1998 / Received revision: 26 October 1998 / Accepted: 30 October 1998     

Detection of ferulic acid esterase production by Bacillus spp. and lactobacilli

The production of feruloyl esterase activity by Bacillus spp. and lactobacilli can be detected in an agar-plate assay. The assay involves the substitution of the main carbon source in specific agar with ethyl ferulate. A number of Bacillus spp., predominantly B. subtilis strains, were found to exhibit feruloyl esterase activity by this method. Of the examined lactobacilli, Lb. fermentum (NCFB 1751) showed the highest level of ferulic acid esterase activity. The enzyme was released from harvested cells by sonication and showed pH and temperature optima of 6.5 and 30 °C respectively. Received: 2 February 1998 / Received revision: 20 April 1998 / Accepted: 27 April 1998     

Construction of L-lysine-overproducing strains of Brevibacterium lactofermentum by targeted disruption of the hom and thrB genes

The mobilization of plasmids from gram-negative Escherichia coli to gram-positive Brevibacterium lactofermentum, mediated by P-type transfer functions, was used to construct disrupted mutants blocked specifically in the homoserine branch of the aspartate pathway. The mutant strain B. lactofermentum R31 showed an efficiency of conjugal transfer two to three orders of magnitude higher than that of the wild-type strain B.␣lactofermentum ATCC 13869. The hom- and thrB- disrupted mutants of B. lactofermentum ATCC 13869 were lysine overproducers. B. lactofermentum R31 mutants do not overproduce lysine because R31 is an alanine-overproducing strain and channels the pyruvate needed for lysine biosynthesis to the production of alanine. Received: 23 January 1996 / Received last revision: 28 July 1996 / Accepted: 5 August 1996     

Monitoring nitric oxide from immobilised denitrifying bacteria, Pseudomonas stutzeri, by the use of chemiluminescence

A chemiluminescence detector was used to measure the production of nitric oxide, NO, from the denitrifying bacteria Pseudomonas stutzeri. NO is an intermediate when P. stutzeri converts nitrate into nitrogen gas. The reaction between NO and ozone is selective and sensitive in generating chemiluminescence. Calibrations were made down to 1 nM, with a signal-to-noise ratio of 3. Bacteria were immobilised in alginate beads. Denitrification experiments were made in an anaerobic non-growth medium by adding nitrate to a certain concentration in the reactor. The bacteria were exposed to nitrate in the concentration range 1 pM–5 mM. The lowest concentration to give a measurable NO response was 100 nM. Received: 16 October 1997 / Received revision: 20 January 1998 / Accepted: 24 January 1998     

A genetically engineered strain of Saccharopolyspora erythraea that produces 6,12-dideoxyerythromycin A as the major fermentation product

The erythromycin producer, Saccharopolyspora erythraea ER720, was genetically engineered to produce 6,12-dideoxyerythromycin A, a novel erythromycin derivative, as the major macrolide in the fermentation broth. Inspection of the biosynthetic pathway for erythromycin would suggest that production of this compound could be achieved simply through the disruption of two genes, that encoding the erythromycin C-6 hydroxylase (eryF ) and that encoding the erythromycin C-12 hydroxylase (eryK ). The double mutant, however, was found to produce a mixture of 6,12-dideoxyerythromycin A and the precursor, 6-deoxyerythromycin D. Complete conversion to the desired product (to the limit of detection by TLC) was achieved by inserting an additional copy of the eryG gene, encoding the erythromycin 3′′-O-methyltransferase and driven by the ermE* promoter, into the S. erythraea chromosome. Received: 6 October 1997 / Received revision: 27 January 1998 / Accepted: 24 February 1998     

Cloning and expression of a gene encoding cyanidase from Pseudomonas stutzeri AK61

The gene coding for cyanidase, which catalyzes the hydrolysis of cyanide to formate and ammonia, was cloned from chromosomal DNA of Pseudomonas stutzeri AK61 into Escherichia coli. The cyanidase gene consisted of an open reading frame of 1004 bp, and it was predicted that cyanidase was composed of 334 amino acids with a calculated molecular mass of 37 518 Da. The amino acid sequence of cyanidase showed a 35.1% and 26.4% homology to aliphatic nitrilase from Rhodococcus rhodochrous K22 and cyanide hydratase from Fusarium lateritium, respectively. A unique cysteine residue of aliphatic nitrilase, which was suggested to play an essential role in the catalytic activity, was conserved in cyanidase. The active form of cyanidase was successfully expressed by a DNA clone containing the cyanidase gene in E.coli. Its productivity was approximately 230 times larger than that of P. stutzeri AK61. The characteristics of the expressed cyanidase, including optimum pH, optimum temperature, Michaelis constant (K _m) for cyanide and specific activity, were similar to those of the native enzyme from P. stutzeri AK61. Received: 24 October 1997 / Received last revision: 17 March 1998 / Accepted: 20 March 1998     

Optimal conditions for bioconversion of ferulic acid into vanillic acid by Pseudomonas fluorescens BF13 cells

Pseudomonas fluorescens BF13 is especially capable of promoting the formation of vanillic acid during ferulic acid degradation. We studied the possibility of enhancing the formation of this intermediary metabolite by using suspensions of cells at high density. The bioconversion of ferulic into vanillic acid was affected by several parameters, such as the concentration of the biomass, the amount of ferulic acid that was treated, the carbon source on which the biomass was grown. The optimal yield of vanillic acid was obtained with 6 mg/ml cells pre-grown on p-coumaric acid and 2 mg/ml ferulic acid. Under these conditions the bioconversion rate was 95% in 5 h. Therefore BF13 strain represents a valid biocatalyst for the preparative synthesis of vanillic acid. Received: 1 July 1997 / Received revision: 28 October 1997 / Accepted: 16 November 1997     

Production of high yields of arachidonic acid in a fed-batch system by Mortierella alpina ATCC 32222

Of six strains of Mortierella tested, Mortierella alpina ATCC 32222 produced the highest yields of arachidonic acid. Supplementation of soy flour (1% w/v) and vegetable oils (1% v/v) significantly increased the biomass, lipid content and arachidonic acid level. Replacement of NaNO₃ with corn steep liquor (1% w/v) also improved arachidonic acid production. A fed-batch culture system at 25 °C, producing a high biomass (52.4 g/l) and arachidonic acid content (9.1 g/l) in 8␣days, was developed. A fed-batch system at low temperature (15 °C) gave even higher arachidonic acid levels (11.1 g/l) in 11 days. Received: 28 October 1996 / Received revision: 3 March 1997 / Accepted: 7 March 1997     

Repression of the expression of genes encoding xylanolytic enzymes in Aspergillusoryzae by introduction of multiple copies of the xynF1 promoter

A xylanase gene, xynF1, was cloned and characterized from a shoyu koji mould Aspergillus oryzae KBN616. The xynF1 gene was found to be comprised of 1484 bp with ten introns. The deduced amino acid sequence encodes a protein consisting of 327 amino acids (35,402 Da) which is very similar to the fungal family F xylanases such as Aspergillus nidulans XlnC, Aspergillus kawachii XynA and Penicillium chrysogenum XylP. The intron/exon organization of xynF1 is very similar to that of the fungal family F xylanase genes. Plasmid pXPR64, which contains 64 copies of the xynF1 promoter region (PxynF1) in the same direction, was constructed and introduced into A. oryzae. This led to reduced expression of both xylanase and β-xylosidase genes in the transformants. Received: 18 May 1998 / Received revision: 7 July 1998 / Accepted: 9 July 1998     

Biotechnology of succinic acid production and markets for derived industrial products

Succinic acid, derived from fermentation of agricultural carbohydrates, has a specialty chemical market in industries producing food and pharmaceutical products, surfactants and detergents, green solvents and biodegradable plastics, and ingredients to stimulate animal and plant growth. As a carbon-intermediate chemical, fermentation-derived succinate has the potential to supply over 2.7 × 10⁸ kg industrial products/year including: 1,4-butanediol, tetrahydrofuran, γ-butyrolactone, adipic acid, n-methylpyrrolidone and linear aliphatic esters. Succinate yields as high as 110 g/l have been achieved from glucose by the newly discovered rumen organism Actinobacillus succinogenes. Succinate fermentation is a novel process because the greenhouse gas CO₂ is fixed into succinate during glucose fermentation. New developments in end-product recovery technology, including water-splitting electrodialysis and liquid/liquid extraction have lowered the cost of succinic acid production to U.S. $ 0.55/kg at the 75 000 tonne/year level and to $ 2.20/kg at the 5000 tonne/year level. Research directions aimed at further improving the succinate fermentation economics are discussed. Received: 27 October 1998 / Received revision: 22 January 1999 / Accepted: 22 January 1999     

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