Showing papers by "Novozymes published in 2002"

A new non-linear normalization method for reducing variability in DNA microarray experiments

Abstract: Microarray data are subject to multiple sources of variation, of which biological sources are of interest whereas most others are only confounding. Recent work has identified systematic sources of variation that are intensity-dependent and non-linear in nature. Systematic sources of variation are not limited to the differing properties of the cyanine dyes Cy5 and Cy3 as observed in cDNA arrays, but are the general case for both oligonucleotide microarray (Affymetrix GeneChips) and cDNA microarray data. Current normalization techniques are most often linear and therefore not capable of fully correcting for these effects. We present here a simple and robust non-linear method for normalization using array signal distribution analysis and cubic splines. These methods compared favorably to normalization using robust local-linear regression (lowess). The application of these methods to oligonucleotide arrays reduced the relative error between replicates by 5-10% compared with a standard global normalization method. Application to cDNA arrays showed improvements over the standard method and over Cy3-Cy5 normalization based on dye-swap replication. In addition, a set of known differentially regulated genes was ranked higher by the t-test. In either cDNA or Affymetrix technology, signal-dependent bias was more than ten times greater than the observed print-tip or spatial effects. Intensity-dependent normalization is important for both high-density oligonucleotide array and cDNA array data. Both the regression and spline-based methods described here performed better than existing linear methods when assessed on the variability of replicate arrays. Dye-swap normalization was less effective at Cy3-Cy5 normalization than either regression or spline-based methods alone.

Munumbicins, wide-spectrum antibiotics produced by Streptomyces NRRL 30562, endophytic on Kennedia nigriscans.

Abstract: Munumbicins A, B, C and D are newly described antibiotics with a wide spectrum of activity against many human as well as plant pathogenic fungi and bacteria, and a Plasmodium sp. These compounds were obtained from Streptomyces NRRL 3052, which is endophytic in the medicinal plant snakevine (Kennedia nigriscans), native to the Northern Territory of Australia. This endophyte was cultured, the broth was extracted with an organic solvent and the contents of the residue were purified by bioassay-guided HPLC. The major components were four functionalized peptides with masses of 1269.6, 1298.5, 1312.5 and 1326.5 Da. Numerous other related compounds possessing bioactivity, with differing masses, were also present in the culture broth extract in lower quantities. With few exceptions, the peptide portion of each component contained only the common amino acids threonine, aspartic acid (or asparagine), glutamic acid (or glutamine), valine and proline, in varying ratios. The munumbicins possessed widely differing biological activities depending upon the target organism. For instance, munumbicin B had an MIC of 2.5 microg x ml(-1) against a methicillin-resistant strain of Staphylococcus aureus, whereas munumbicin A was not active against this organism. In general, the munumbicins demonstrated activity against Gram-positive bacteria such as Bacillus anthracis and multidrug-resistant Mycobacterium tuberculosis. However, the most impressive biological activity of any of the munumbicins was that of munumbicin D against the malarial parasite Plasmodium falciparum, having an IC(50) of 4.5+/-0.07 ng x ml(-1). This report also describes the potential of the munumbicins in medicine and agriculture.

Pep-13, a plant defense-inducing pathogen-associated pattern from Phytophthora transglutaminases.

Abstract: Innate immunity, an ancient form of defense against microbial infection, is well described for animals and is also suggested to be important for plants. Discrimination from self is achieved through receptors that recognize pathogen-associated molecular patterns (PAMPs) not found in the host. PAMPs are evolutionarily conserved structures which are functionally important and, thus, not subject to frequent mutation. Here we report that the previously described peptide elicitor of defense responses in parsley, Pep-13, constitutes a surface-exposed fragment within a novel calcium-dependent cell wall transglutaminase (TGase) from Phytophthora sojae. TGase transcripts and TGase activity are detectable in all Phytophthora species analyzed, among which are some of the most destructive plant pathogens. Mutational analysis within Pep-13 identified the same amino acids indispensable for both TGase and defense-eliciting activity. Pep-13, conserved among Phytophthora TGases, activates defense in parsley and potato, suggesting its function as a genus-specific recognition determinant for the activation of plant defense in host and non-host plants. In summary, plants may recognize PAMPs with characteristics resembling those known to trigger innate immune responses in animals.

Promoters for expressing genes in a fungal cell

Abstract: The present invention relates to methods for producing a polypeptide, comprising: (a) cultivating a fungal host cell in a medium conducive for the production of the polypeptide, wherein the fungal host cell comprises a first nucleic acid sequence encoding the polypeptide operably linked to a second nucleic acid sequence comprising a promoter foreign to the nucleic acid sequence, wherein the promoter comprises a sequence selected from the group consisting of nucleotides 1 to 3949 of SEQ ID NO. 1, nucleotides 1 to 938 of SEQ ID NO. 2, and nucleotides 1 to 3060 of SEQ ID NO. 3, and a subsequence thereof; and mutant, hybrid, and tandem promoters thereof; and (b) isolating the polypeptide from the cultivation medium. The present invention also relates to the isolated promoter sequences and to constructs, vectors, and fungal host cells comprising the promoter sequences operably linked to nucleic acid sequences encoding polypeptides.

Synthetic shuffling expands functional protein diversity by allowing amino acids to recombine independently

Abstract: We describe synthetic shuffling, an evolutionary protein engineering technology in which every amino acid from a set of parents is allowed to recombine independently of every other amino acid. With the use of degenerate oligonucleotides, synthetic shuffling provides a direct route from database sequence information to functional libraries. Physical starting genes are unnecessary, and additional design criteria such as optimal codon usage or known beneficial mutations can also be incorporated. We performed synthetic shuffling of 15 subtilisin genes and obtained active and highly chimeric enzymes with desirable combinations of properties that we did not obtain by other directed-evolution methods.

Microarray analysis of the Bacillus subtilis K-state: genome-wide expression changes dependent on ComK.

Abstract: Summary In Bacillus subtilis, the competence transcription factor ComK activates its own transcription as well as the transcription of genes that encode DNA transport proteins. ComK is expressed in about 10% of the cells in a culture grown to competence. Using DNA microarrays representing ≈ 95% of the protein-coding open reading frames in B. subtilis, we compared the expression profiles of wild-type and comK strains, as well as of a mecA mutant (which produces active ComK in all the cells of the population) and a comK mecA double mutant. In these comparisons, we identified at least 165 genes that are upregulated by ComK and relatively few that are downregulated. The use of reporter fusions has confirmed these results for several genes. Many of the ComK-regulated genes are organized in clusters or operons, and 23 of these clusters are preceded by apparent ComK-box promoter motifs. In addition to those required for DNA uptake, other genes that are upregulated in the presence of ComK are probably involved in DNA repair and in the uptake and utilization of nutritional sources. From this and previous work, we conclude that the ComK regulon defines a growth-arrested state, distinct from sporulation, of which competence for genetic transformation is but one notable feature. We suggest that this is a unique adaptation to stress and that it be termed the ‘K-state’.

Effects of Papain and a Microbial Enzyme on Meat Proteins and Beef Tenderness

Abstract: The relative effects of an aspartic proteinase (AP) and papain on meat proteins and beef tenderness were evaluated by measuring release of hydroxyproline in collagen, and breakdown of myofibrillar proteins. Tenderness was objectively measured by Warner-Bratzler shear. AP showed self-limiting hydrolysis of myofibrillar proteins resulting in 25 to 30% improvement in meat tenderness and was not adversely affected by pH, salt, phosphate, and ascorbate concentrations often encountered in meat processing. Like papain, its tenderizing effect was expressed primarily during cooking and caused no ignificant changes (p > 0.05) in tenderness during frozen or refrigerated storage. It was also inactivated at cooking temperatures in excess of 60 °C, therefore eliminating any undesirable side effects that may be associated with residual protease activity.

Polypeptides having cellobiase activity and polynucleotides encoding same

Abstract: The present invention relates to polypeptides having cellobiase activity and polynucleotides having a nucleotide sequence which encodes for the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid constructs as well as methods for producing and using the polypeptides, such as for producing ethanol from biomass or for shuffling of DNA.

Dependence of morphology on agitation intensity in fed-batch cultures of Aspergillus oryzae and its implications for recombinant protein production.

Abstract: We previously reported that, although agitation conditions strongly affected mycelial morphology, such changes did not lead to different levels of recombinant protein production in chemostat cultures of Aspergillus oryzae (Amanullah et al., 1999). To extend this finding to another set of operating conditions, fed-batch fermentations of A. oryzae were conducted at biomass concentrations up to 34 g dry cell weight/L and three agitation speeds (525, 675, and 825 rpm) to give specific power inputs between 1 and 5 kWm(-3). Gas blending was used to control the dissolved oxygen level at 50% of air saturation except at the lowest speed where it fell below 40% after 60-65 h. The effects of agitation intensity on growth, mycelial morphology, hyphal tip activity, and recombinant protein (amyloglucosidase) production in fed-batch cultures were investigated. In the batch phase of the fermentations, biomass concentration, and AMG secretion increased with increasing agitation intensity. If in a run, dissolved oxygen fell below approximately 40% because of inadequate oxygen transfer associated with enhanced viscosity, AMG production ceased. As with the chemostat cultures, even though mycelial morphology was significantly affected by changes in agitation intensity, enzyme titers (AGU/L) under conditions of substrate limited growth and controlled dissolved oxygen of >50% did not follow these changes. Although the measurement of active tips within mycelial clumps was not considered, a dependency of the specific AMG productivity (AGU/g biomass/h) on the percentage of extending tips was found, suggesting that protein secretion may be a bottle-neck in this strain during fed-batch fermentations.

Endo-beta-1,4-glucanase

Abstract: The present invention relates to an enzyme exhibiting endo-beta-1,4-glucanase activity (EC 3.2.1.4), which is a) a polypeptide encoded by the DNA sequence of positions 1 to 2322 of SEQ ID NO: 1; b) a polypeptide produced by culturing a cell comprising the sequence of SEQ ID NO: 1 under conditions wherein the DNA sequence is expressed; c) an endo-beta-1,4-glucanase enzyme having a sequence of at least 97% identity to the amino acid sequence of position 1 to position 773 of SEQ ID NO: 2; and fragments thereof exhibiting endo-beta-1,4-glucanase activity, and d) a polypeptide having endo-beta-1,4-glucanase activity that is encoded by a polynucleotide that hybridizes with the nucleotide sequence shown in positions 1–2322 of SEQ ID NO: 1, is useful for detergent and textile applications.

Enzymatic degradation of carboxymethyl cellulose hydrolyzed by the endoglucanases Cel5A, Cel7B, and Cel45A from Humicola insolens and Cel7B, Cel12A and Cel45Acore from Trichoderma reesei.

Abstract: Enzymatic hydrolysis of carboxymethyl cellulose (CMC) has been studied with purified endoglucanases Hi Cel5A (EG II), Hi Cel7B (EG I), and Hi Cel45A (EG V) from Humicola insolens, and Tr Cel7B (EG I), Tr Cel12A (EG III), and Tr Cel45Acore (EG V) from Trichoderma reesei. The CMC, with a degree of substitution (DS) of 0.7, was hydrolyzed with a single enzyme until no further hydrolysis was observed. The hydrolysates were analyzed for production of substituted and non-substituted oligosaccharides with size exclusion chromatography (SEC) and with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF-MS). Production of reducing ends and of nonsubstituted oligosaccharides was determined as well. The two most effective endoglucanases for CMC hydrolysis were Hi Cel5A and Tr Cel7B. These enzymes degraded CMC to lower molar mass fragments compared with the other endoglucanases. The products had the highest DS determined by MALDI-TOF-MS. Thus, Hi Cel5A and Tr Cel7B were less inhibited by the substituents than the other endoglucanases. The endoglucanase with clearly the lowest activity on CMC was Tr Cel45Acore. It produced less than half of the amount of reducing ends compared to Tr Cel7B; furthermore, the products had significantly lower DS. By MALDI-TOF-MS, oligosaccharides with different degree of polymerization (DP) and with different number of substituents could be separated and identified. The average oligosaccharide DS as function of DP could be measured for each enzyme after hydrolysis. The combination of techniques for analysis of product formation gave information on average length of unsubstituted blocks of CMC.

Engineering of phytase for improved activity at low pH.

Abstract: For industrial applications in animal feed, a phytase of interest must be optimally active in the pH range prevalent in the digestive tract. Therefore, the present investigation describes approaches to rationally engineer the pH activity profiles of Aspergillus fumigatus and consensus phytases. Decreasing the negative surface charge of the A. fumigatus Q27L phytase mutant by glycinamidylation of the surface carboxy groups (of Asp and Glu residues) lowered the pH optimum by ca. 0.5 unit but also resulted in 70 to 75% inactivation of the enzyme. Alternatively, detailed inspection of amino acid sequence alignments and of experimentally determined or homology modeled three-dimensional structures led to the identification of active-site amino acids that were considered to correlate with the activity maxima at low pH of A. niger NRRL 3135 phytase, A. niger pH 2.5 acid phosphatase, and Peniophora lycii phytase. Site-directed mutagenesis confirmed that, in A. fumigatus wild-type phytase, replacement of Gly-277 and Tyr-282 with the corresponding residues of A. niger phytase (Lys and His, respectively) gives rise to a second pH optimum at 2.8 to 3.4. In addition, the K68A single mutation (in both A. fumigatus and consensus phytase backbones), as well as the S140Y D141G double mutation (in A. fumigatus phytase backbones), decreased the pH optima with phytic acid as substrate by 0.5 to 1.0 unit, with either no change or even a slight increase in maximum specific activity. These findings significantly extend our tools for rationally designing an optimal phytase for a given purpose.

Metabolic engineering of the morphology of Aspergillus oryzae by altering chitin synthesis.

Abstract: Morphology and α-amylase production during submerged cultivation were examined in a wild-type strain (A1560) and in strains of Aspergillus oryzae in which chitin synthase B (chsB) and chitin synthesis myosin A (csmA) have been disrupted (ChsB/G and CM101). In a flowthrough cell, the growth of submerged hyphal elements was studied online, making it possible to examine the growth kinetics of the three strains. The average tip extension rates of the CM101 and ChsB/G strains were 25 and 88% lower, respectively, than that of the wild type. The branching intensity in the CM101 strain was 25% lower than that in the wild type, whereas that in the ChsB/G strain was 188% higher. During batch cultivation, inseparable clumps were formed in the wild-type strain, while no or fewer large inseparable clumps existed in the cultivations of the ChsB/G and CM101 strains. The α-amylase productivity was not significantly different in the three strains. A strain in which the transcription of chsB could be controlled by the nitrogen source-regulated promoter niiA (NiiA1) was examined during chemostat cultivation, and it was found that the branching intensity could be regulated by regulating the promoter, signifying an important role for chsB in branching. However, the pattern of branching responded very slowly to the change in transcription, and increased branching did not affect α-amylase productivity. α-Amylase residing in the cell wall was stained by immunofluorescence, and the relationship between tip number and enzyme secretion is discussed.

Characterization of a cell-wall acid phosphatase (PhoAp) in Aspergillus fumigatus.

Abstract: In the filamentous fungus Aspergillus fumigatus, the vast majority of the cell-wall-associated proteins are secreted proteins that are in transit in the cell wall. These proteins can be solubilized by detergents and reducing agents. Incubation of a SDS/β-mercaptoethanol-treated cell-wall extract with various recombinant enzymes that hydrolyse cell-wall polysaccharides resulted in the release of a unique protein in minute amounts only after incubation of the cell wall in the presence of 1,3-β-glucanase. Sequence analysis and biochemical studies showed that this glycoprotein, with an apparent molecular mass of 80 kDa, was an acid phosphatase (PhoAp) that was active on both phosphate monoesters and phosphate diesters. PhoAp is a glycosylphosphatidylinositol-anchored protein that was recovered in the culture filtrate and cell-wall fraction of A. fumigatus after cleavage of its anchor. It is also a phosphate-repressible acid phosphatase. The absence of PhoAp from a phosphate-rich medium was not associated with a reduction in fungal growth, indicating that this cell-wall-associated protein does not play a role in the morphogenesis of A. fumigatus.

Transcriptome analysis documents induced competence of Bacillus subtilis during nitrogen limiting conditions

Abstract: DNA microarrays were used to analyze the changes in gene expression in Bacillus subtilis strain 168 when nitrogen limiting (glutamate) and nitrogen excess (ammonium plus glutamate) growth conditions were compared. Among more than 100 genes that were significantly induced during nitrogen starvation we detected the comG, comF, comE, nin-nucA and comK transcription units together with recA. DNA was added to B. subtilis grown in minimal medium with glutamate as the sole nitrogen source and it was demonstrated that the cells were competent. Based on these observations we propose a simplification of previously designed one-step transformation procedures for B. subtilis strain 168.

0etergent compositions comprising bacillus subtilis pectate lyases

Abstract: Detergent composition comprising a surfactant and a pectate lyase (EC 4.2.2.2) enzyme en-coded by a DNA sequence endogeneous to a strain of Bacillus subtilis or a stabilized variant thereof provides superior cleaning and stain removal.

Methods for producing hyaluronan in a recombinant host cell

Abstract: The present invention relates to methods for producing a hyaluronic acid, comprising: (a) cultivating a Bacillus host cell under conditions suitable for production of the hyaluronic acid, wherein the Bacillus host cell comprises a nucleic acid construct comprising a hyaluronan synthase encoding sequence operably linked to a promoter sequence foreign to the hyaluronan synthase encoding sequence; and (b) recovering the hyaluronic acid from the cultivation medium. The present invention also relates to an isolated nucleic acid sequence encoding a hyaluronan synthase operon comprising a hyaluronan synthase gene and a UDP-glucose 6-dehydrogenase gene, and optionally one or more genes selected from the group consisting of a UDP-glucose pyrophosphorylase gene, UDP-N-acetylglucosamine pyrophosphorylase gene, and glucose-6-phosphate isomerase gene. The present invention also relates to isolated nucleic acid sequences encoding a UDP-glucose 6-dehydrogenase, UDP-glucose pyrophosphorylase, and UDP-N-acetylglucosamine pyrophosphorylase.

Modified subtilisins and detergent compositions containing same

Abstract: The present invention relates to enzymes produced by mutating the genes for a number of subtilisin proteases and expressing the mutated genes in suitable hosts are presented. The enzymes exhibit improved wash performance in comparison to their wild type parent enzymes. The enzymes are well-suited for use in detergent compositions.

Antimicrobial polypeptides from pseudoplectania nigrella

Abstract: The present invention relates to polypeptides having antimicrobial activity and polynucleotides having a nucleotide sequence which encodes for the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the nucleic acid constructs as well as methods for producing and using the polypeptides.

Effects of increased impeller power in a production-scale Aspergillus oryzae fermentation.

Abstract: The goal in this study was to determine how increased impeller power affects enzyme expression in large-scale (80 m 3 ), fed-batch Aspergillus oryzae fermentations. An approximate 50% increase in average impeller power was achieved by increasing impeller diameter approximately 10%, while operating at slightly reduced speed. Measured decreases in terminal (95%) mixing time show increased power improved bulk mixing. However, batches operated at increased power had lower recombinant enzyme productivity. Biomass assays and image analysis tests showed no significant difference between “high power” and control batches, suggesting that slower growth, altered morphology, or increased hyphal fragmentation were not the cause of reduced productivity. Off-line tests on the shear-thinning, highly viscous broth show oxygen limitation occurred after transport through the air-liquid interface and imply the limitation may involve bulk mixing. Specifically, oxygen transfer may be limited to a small zone surrounding each impeller. When this is the case, oxygen mass transfer will be determined by both impeller shear and fluid circulation, which have been characterized with the energy dissipation/circulation function (EDCF). EDCF values during control fermentations were approximately constant at 25 kW m -3 s -1 , while EDCF values during “high power” batches fell linearly from 40 to 15 kW m -3 s -1 . The point at which “high power” EDCF values drop below those in control fermentations corresponds almost exactly with the point at which product titer stops increasing. Thus, our findings suggest oxygen mass transfer was less efficient during the latter half of “high power” fermentations because of reductions in impeller speed and subsequent decreases in EDCF values. This observation has clear implications during the scale-up of viscous fungal fermentations, implying that not only is the level of impeller power important, but also relevant is how this power is applied.

Estimation of hyphal tensile strength in production-scale Aspergillus oryzae fungal fermentations.

Abstract: Fragmentation of filamentous fungal hyphae depends on two phenomena: hydrodynamic stresses, which lead to hyphal breakage, and hyphal tensile strength, which resists breakage. The goal of this study was to use turbulent hydrodynamic theory to develop a correlation that allows experimental data of morphology and hydrodynamics to be used to estimate relative (pseudo) tensile strength (sigma(pseudo)) of filamentous fungi. Fed-batch fermentations were conducted with a recombinant strain of Aspergillus oryzae in 80 m(3) fermentors, and measurements were made of both morphological (equivalent hyphal length, L) and hydrodynamic variables (specific power input, epsilon; kinematic viscosity, v). We found that v increased over 100-fold during these fermentations and, hence, Kolmogorov microscale (lambda) also changed significantly with time. In the impeller discharge zone, where hyphal fragmentation is thought to actually take place, lambda was calculated to be 700-3500 microm, which is large compared to the size of typical fungal hyphae (100-300 microm). This result implies that eddies in the viscous subrange are responsible for fragmentation. Applying turbulent theory for this subrange, it was possible to calculate sigma(pseudo)from morphological and hydrodynamic measurements. Pseudo tensile strength was not constant but increased to a maximum during the first half and then decreased during the second half of each fermentation, presumably due to differences in physiological state. When a literature correlation for hyphal fragmentation rate (k(frag)) was modified by adding a term to account for viscosity and tensile strength, the result was better qualitative agreement with morphological data. Taken together, these results imply hyphal tensile strength can change significantly over the course of large-scale, fed-batch fungal fermentations and that existing fragmentation and morphology models may be improved if they accounted for variations in hyphal tensile strength with time.

Polypeptides having protease activity and nucleic acids encoding same

Abstract: The invention relates to isolated proteases homologous to a protease derived from a strain of Thermoascus aurantiacus and DNA encoding the same. A homologous polypeptide is derived from Aspergillus oryzae. The proteases can be used in animal feed. The Thermoascus protease is a metalloprotease with an HEFTH motif. The optimum temperature is around 70 °C, the optimum pH is around pH6, and the protease is stable over a broad pH range of pH3 to pH10.