Comparison of different strains ofCellulomonas for production of cellulolytic and xylanolytic enzymes from biomass produced on saline lands

A comparison of the rate of carboxymethyl cellulase (CMCase), avicelase, xylanase, -glucosidase and -xylosidase production and rates of growth by 4 different strains ofCellulomonas revealed a wide range of behaviour; with some strains producing more CMCase, avicelase, xylanase, -glucosidase and -xylosidase from complex lignocellulosic (LC) biomass (from saline land) and CMC while some others producing small amounts of these enzymes. One strain,C. biazotea, was better with respect to enzyme production potential and growth behaviour than most of the other strains and has been chosen as a starting strain for genetic improvement for producing enzymes of the cellulase complex.     

O-Glycosylhydrolases of Marine Filamentous Fungi: β-1,3-Glucanases of Trichoderma aureviride

The ability to produce extracellular O-glycosylhydrolases was studied in 14 strains of marine filamentous fungi sampled from the bottom sediments of the South China Sea. The following activities were detected in the culture liquids of the fungi: N-acetyl--D-glucosaminidase, -D-glucosidase, -D-galactosidase, -1,3-glucanase, amylase, and pustulanase. -1,3-Glucanases were isolated by ultrafiltration, hydrophobic interaction chromatography, and ion exchange chromatography, and their properties were studied. Data on products of enzymatic digestion of laminaran, absence of transglycosylation activity, and the pattern of action of natural inhibitors confirmed that -1,3-glucanase belonged to the exo type. Inhibitor analysis demonstrated the role of a thiol group and tryptophan and tyrosine residues in the catalytic activity.     

Regulation of β-xylosidase formation by xylose in Trichoderma reesei

The soft-rot fungus Trichoderma reesei forms -xylosidase (EC 3.2.1.37) activity during cultivation on xylan and xylose, but not on glucose. When mycelia precultivated on glycerol were washed and transferred to fresh medium without a carbon and nitrogen source, -xylosidase formation was induced by xylan, xylobiose and xylose. A supply of 4 mm xylose and a pH of 2.5 provided optimal conditions for induction. -Xylosidase accounted for the major portion of total extracellular protein under these conditions, and could be purified to physical homogeneity by a single anion exchange chromatography step. A recombinant strain of T. reesei that carries multiple copies of the homologous xylanase II-encoding gene has a six-fold increased xylanase activity, but forms comparable -xylosidase activities. This shows that the rate of xylan hydrolysis has no effect on the induction of -xylosidase. Methyl--d-xyloside inhibited -xylosidase competitively and was a weak -xylosidase inducer. The induction by xylobiose and xylan was strongly enhanced by the simultaneous addition of methyl--d-xylosidese and xylan or xylobiose. The results suggest that a slow supply of xylose is a trigger for -xylosidase induction.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Resolution of Clostridium stercorarium cellulase by fast protein liquid chromatography (FPLC)

Summary A fast and efficient separation procedure for the analysis of the cellulase components of the thermophilic anaerobe Clostridium stercorarium was developed. Culture respernatants were concentrated without loss of cellulase activity by tangential flow ultrafiltration. Resolution of the cellulase system was achieved by fast protein liquid chromatography (FPLC) on a Mono Q anion exchange column. Enzyme fractions were assayed for hydrolysis of Avicel, carboxymethylcellulose (CMC), -nitrophenyl--d-cellobioside, and p-nitrophenyl--d-glucoside. Two Avicelases, two -cellobiosidases, and one -glucosidase were identified and characterized by SDS-polyacrylamide electrophoresis and isoelectric focusing. On the basis of their activities towards CMC, Avicelase I was classified as endo--glucanase and Avicelase II as exo--glucanase. Efficient hydrolysis of microcrystalline cellulose was shown to result from the combined action of both Avicelases.     

Very stable enzymes from extremely thermophilic archaebacteria and eubacteria

Summary Thirty-six thermophilic archaebacteria and nine extremely thermophilic eubacteria have been screened on solid media for extracellular amylase, protease, hemicellulase (xylanase), cellulase, pectinase and lipase activities. Extracellular enzymes were detected in 14 archaebacteria belonging to three different orders. Twelve of these were able to degrade starch and casein and the two Thermofilum strains were able to degrade starch, xylan and carboxymethylcellulose. Three of the eubacteria could degrade only starch. The other six (including four Thermotoga strains) all had activity against starch, xylan and carboxymethylcellulose, and one also had activity against casein. Some of the amylolytic archaebacteria released -glucosidase, -glucosidase, amylase and transglucosylase activities into liquid media containing starch or maltose. Thermotoga strain FjSS3B.1 released amylase, xylanase, cellulase and -glucosidase activities into the medium when grown in the presence of substrates. When the partially purified enzymes from Thermotoga and some of the archaebacteria were compared with known thermostable enzymes the majority were found to be the most thermostable of their type. The -glucosidase, xylanase and cellulase from Thermotoga and two -glucosidases, a -glucosidase, an amylase and a pullulanase from archaebacteria all have half-lives of at least 15 min at 105°C.     

Large scale preparation of PA-oligosaccharides from glycoproteins using an improved extraction method

We have developed a new method for the large scale preparation of pyridylaminated (PA-) oligosaccharides from glycoproteins. Phenol/chloroform extration was adapted for the removal of protein and excess 2-aminopyridine, improving the efficiency of preparation. From a 2.5 g sample of human apo-transferrin, 25–30 mol of agalacto biantennary PA-oligosaccharide could be obtained. By increasing the concentration of PA-oligosaccharide substrate, we were able to detect a very low level ofN-acetylglucosaminlytransferase IV activity in CHO cell extracts.Abbreviations PA 2-aminopyridine - SDS sodium dodecyl sulfate - GlcNAc N-acetylglucosamine - GnT N-acetylglucosaminyltransferase - Gn,Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-tri-PA GlcNAc1-2(GlcNAc1-4)Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-trí-PA GlcNAc1-2Man1-3({GlcNAc1-2(GlcNAc1-6)Man1-6})Man1-4GlcNac1-4GlcNAc-2-aminopyridine - Gn,(Gn),Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-4)(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine     

Testis-specific β2 tubulins are identical in Drosophila melanogaster and D. hydei but differ from the ubiquitous β1 tubulin

In Drosophila as in many organisms tubulins are encoded by a gene family. We have determined the complete nucleotide sequences coding for the 1 and 2 tubulins of Drosophila melanogaster and the 2 tubulin of D. hydei, and found these insect tubulins to be highly conserved and like tubulins of other organisms. This is discussed with reference to the possible functional domains of these proteins. — The 1 tubulin gene of Drosophila is constitutively expressed, whereas the 2 tubulin is expressed specifically in the testes. In D. melanogaster the amino acid sequences of these proteins are 95% homologous, differing at only 25 positions. In the testes the 2 tubulin participates in different microtubules as shown by genetic analysis (Kemphues et al. 1982). Interestingly, all of the amino acids characteristic of the testis-specific 2 tubulin are also present in the corresponding gene of D. hydei. Of special interest is the high degree of conservation of the carboxy-terminal domain in these functionally equivalent tubulins.     

Polymorphism of human liver alcohol dehydrogenase: Identification of ADH2 2-1 and ADH2 2-2 phenotypes in the Japanese by isoelectric focusing

Liver homogenate-supernatants from most Japanese exhibit an atypical pH optimum for ethanol oxidation at pH 8.8 instead of 10.5, the typical pH-activity optimum. It has been proposed that atypical livers contain alcohol dehydrogenase isozymes with ₂ subunits while typical livers contain isozymes with ₁ subunits, both produced by the ADH ₂ gene. Because it is difficult to differentiate the atypical ADH₂ 2-2 phenotype from the ADH₂ 2-1 phenotype by starch gel electrophoresis, an agarose isoelectric focusing procedure was developed that clearly separated the atypical Japanese livers into two groups, A1 and A2. The isozymes in A1 and A2 livers were purified. Type A1 livers contained a single isozyme with an atypical pH-rate profile; it was designated ₂₂. Three isozymes were isolated from A2 livers, two of which corresponded to ₁₁ and ₂₂. A third, absent from the typical and the atypical A1 livers, had an intermediate mobility; it was designated ₂₁. Type A1 livers are, therefore, the homozygous ADH₂ 2-2 phenotype, and type A2 livers, the heterozygous ADH₂ 2-1 phenotype. The ADH₂ 2-2 phenotype was found in 53% of 194 Japanese livers, and the ADH₂ 2-1 phenotype, in 31%. Accordingly, the frequency of ADH ₂ ² was 0.68.This study was supported by U.S. Public Health Service Grant AA 02342.     

The mechanism of object fixation and its relation to spontaneous pattern preferences in Drosophila melanogaster

The orientation behavior of walking flies, Drosophila melanogaster, towards a single 6° wide black vertical stripe (elementary stripe) can be explained by use of the turning tendency function H(). This function is characterized by maximal values at an angular distance of =25° from the stable zero position (=orienting direction), a sharp decline from this maximum to =60°, and a very slow approach to the unstable zero position (Horn and Wehner, 1975). The shape of this function is influenced by both translatory and rotatory components of movement. If the translatory component is minimized by measuring the turning function W() (see 2.3) at a distance of 10 mm (C1) from the center of the arena, a change in the strength of this decline is caused. But with increasing translatory component, i.e. at a greater distance from the center of the arena, W() approximates the heuristical function H() (Fig. 12). The turning functions W() are pattern-specific; the angular positions of the maximum responses shift to greater angles with increasing width of the patterns (Fig. 2). In the twopattern configuration with double or single stripes, there is always a coincidence between the stable zero positions of W (), the mean of the frequency distributions P() of the flies' positions and n _g() of the straight courses, and the stable zero positions of H () obtained from an additive superposition of two or more angular shifted turning tendency functions H() (Fig. 5, 7). Therefore, the mean positions of the flies in a multi-stripe experiment composed of elementary stripes can be predicted from the addition of many angular shifted turning tendency functions H(). Between H() and the frequency distribution P() of the flies' positions , the following formula holds: P() =C·H()d (Fig. 13). With this equation, the spontaneous preference of the broader of two double stripes can be explained presuming lateral interactions between the components of the patterns (Fig. 8, 10). The strength x _i ^* of this lateral interaction depends on the width of the double stripes. The greater , the smaller is x _i ^* . x _i ^* is a pattern-specific value (Table 1, 2).Supported by the Deutsche Forschungsgemeinschaft, Ho 664/2     

Effects of supplied cinnamic acids and biosynthetic intermediates on the anthocyanins accumulated by wild carrot suspension cultures

Anthocyanins isolated and characterized from the wild carrot suspension cultures used here were 3-O--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D<-galactopyranosylcyanidin (1), 3-O-[-D- xylopyranosyl-(12)--D-galactopyranosyl]cyanidin (2), 3-O-(6-O-sinapoyl)--D-glucopyranosyl-(16)-[-D- xylopyranosyl-(12)-]-D-galactopyranos ylcyanidin (3), 3-O-(6-O-feruoyl)--D-glucopyranosyl-(16)-[- D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (4), 3-O-(6-O-coumaroyl)--D-glucopyranosyl-(16)- [-D-xylopyranosyl-(12)-]-D-galactopyrano sylcyanidin (5), 3-O-[6-O-(3,4,5-trimethoxycinnamoyl)]-- D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (6), 3-O-[6-O-(3,4-dime- thoxycinnamoyl)]--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (7), 3-O-[(6-O-sinapoyl)--D-glucopyranosyl-(16)--D-galactopyranosyl]cyanidin (8), and 3-O-(-D-galactopyranosyl)cyanidin (9). Except when cinnamic acids were provided in the culture medium, the major anthocyanin present in the two clones examined was 2. When the naturally occurring and some non-naturally occurring cinnamic acids were provided individually in the medium, 1 and 2 were minor components and the anthocyanin acylated with the supplied cinnamic acid, namely 3, 4, 5, 6, or 7 was the major anthocyanin present in the tissue. When caffeic acid was provided the major anthocyanin in the tissue was 4, thereby suggesting that the caffeic acid was methylated before its use in anthocyanin biosynthesis. Other cinnamic acids supplied had limited effects on the anthocyanins accumulated and appeared not to result in the accumulation of new anthocyanins by the tissue. Thus the tissue can use some but not all analogues of sinapic acid to acylate anthocyanins. Additional anthocyanins were detected in extracts of the wild carrot tissue cultures using mass spectrometry (both MS/MS and HPLC/MS). The additional compounds detected have also been found in cultures of black carrot, an Afghan cultivar of Daucus carota ssp. sativa and the flowers of wild carrot giving no evidence for qualitative differences in the anthocyanins synthesized by subspecies, cell cultures from subspecies, or clones from cell cultures. There are major differences in the amounts of individual anthocyanins found in cultures from different subspecies and in different clones from cell cultures. Here anthocyanins without acyl groups were usually found in the tissues and their accumulation is discussed. On the basis of the structures of the isolated anthocyanins, a likely pathway from cyanidin to the accumulated anthocyanins is proposed and discussed.Abbreviations Sin sinapoyl - Fer feruoyl - 4-Coum. 4-coumaroyl - 3,4-MeO₂Cin 3,4-dimethoxyeinnamoyl - 3,4,5-MeO₃Cin 3,4,5-trimethoxycinnamoyl - Cya cyanidin     

Studies of two strains of Actinopolyspora halophila,an extremely halophilic actinomycete

Actinopolyspora halophila is an extremely halophilic actinomycete requiring a minimum salt concentration of 12% (w/v) for growth in liquid media at 37°C. During antibiotic sensitivity testing of the organism, an erythromycin-resistant phenotypic variant designated A. halophila ER, was isolated. This strain was approximately 60-fold more resistant to erythromycin than the wild-type strain, and, moreover, was capable of growth in 6% sodium chloride. Optimal growth of both strains in liquid media was observed in 20% salt.After resuspension in water for up to 20 days, the organism exhibited good growth in fresh salt-containing media. Cultures resuspended in salt-containing media, however, were subject to extensive cellular autolysis.In addition to resistance to -lactam antibiotics which was mediated by cellular and exocellular -lactamases, the organism was insensitive to all other tested antibiotics except amakacin, chloramphenicol, kanamycin, gentamycin, naladixic acid, streptomycin, and tetracycline. No plasmids were detected in either strain. A. halophila possessed high cellular levels of catalase, -lactamase, and -glucosidase. A variety of exocellular enzymes including protease, -lactamase, xylanase, and carboxymethyl cellulase, were secreted maximally into growth media containing 15% NaCl.Issued as NRCC 25148     

Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases

The primary structure of the bglA gene region encoding a -glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51545 Da. The T, maritima -glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15–20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity -glucosidase and as a member of the -glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family -glucosidases, a -xylosidase, -1,4-glycanases of the enzyme family F (mostly xylanases), and other families of -1,4-glycosyl hydrolases. This result indicates that BGA -glucosidases may comprise one enzyme family within a large enzyme order of retaining -glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.     

Fromβ-lactams toα- andβ-amino acid derived peptides

Summary The potential of-lactams as intermediates for the access to- and-amino acid-derived peptides is shortly reviewed, with major focus on the technologies developed in our group. The two general strategies lie, on one side, in the oxidative ring expansion of 3-hydroxy-lactams toN-carboxy-amino acid anhydrides or Leuch's anhydrides and subsequent coupling with-amino acid esters and, on the other side, in the nucleophilic ring opening ofN-Boc--lactams. Both approaches have been successfully applied to the synthesis of,-diamino acid,-amino--hydroxy acid, polyhydroxylated-amino acid,,-disubstituted-amino acid,-amino acid,-amino--hydroxy acid and,-disubstituted-amino acid derived peptides. Because of the mild reaction conditions needed for the above transformations and the highly stereoselective procedures employed for the construction of the starting-lactam ring, the whole process allows the production of optically pure final products.     

Molecular cloning of the genes for xylan degradation of Bacillus pumilus and their expression in Escherichia coli

Summary The 7.7 Mdal PstI fragment of Bacillus pumilus IPO containing genes for xylan degradation, xylanase, and -xylosidase was inserted at the PstI site of pBR322 and cloned in E. coli C600. The hybrid plasmid thus formed was named pOXN29. The amount of xylanase and -xylosidase expressed in E. coli harboring pOXN29 was about 6% and 20% of the activity produced by the donor, B. pumilus. The reverse orientation of the inserted fragment resulted respectively in 5 times and 50 times increases in xylanase and -xylosidase productivities. Both enzymes expressed in E. coli transformants were shown to be indistinguishable from those of B. pumilus by immunological and chemical criteria. Digestion of pOXN29 with BglII produced two fragments; one was 6.7 Mdal in size and contained the whole pBR322 and the -xylosidase gene, and the other was 3.7 Mdal and coded for xylanase. Analysis of enzymes expressed in the transformant cells indicated that neither enzyme was secreted into the culture medium, periplasm nor membrane bound, although xylanase but not -xylosidase, was secreted into the medium in a B. pumilus culture.     

Purification and properties of recombinant β-glucosidase of the hyperthermophilic bacterium Thermotoga maritima

A -glucosidase of the hyperthermophilic bacterium Thermotoga maritima has been purified from a recombinant Escherichia coli clone expressing the corresponding gene. The enzyme was found to be a dimer with an apparent molecular mass of approximately 95 kDa as determined by size exclusion chromatography. It was composed of two apparently identical subunits of about 47 kDa (determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis). The enzyme had a bbroadsubstrate specificity and attacked -glucoside, -galactoside, -fucoside, and, to a very small extent, also -xyloside substrates. -Glycosidic bonds were not hydrolysed. Kinetic measurement of the hydrolysis of o-nitrophenyl--d-glucopyranoside (oNPGlc) and o-nitrophenyl--d-galactopyranoside (oNPGal) in the concentration ranges 0.05–20 mm and 0.1–10 mm, respectively, at 75°C resulted in non-linear Lineweaver-Burk and Eadie-Hofstee 3lots whereas cellobiose and lactose did not induce this type of effect. Lactose caused substrate inhibition above 350 mm. The enzyme was optimally active at about pH 6.1. The T. maritima -glucosidase represents the most thermostable -glucosidase described to date. In 50 mm sodium phosphate buffer, pH 6.2, at an enzyme concentration of 50 g/ml, the pure enzyme without additives retained more than 60% of its initial activity after a 6-h incubation at 95°C. Correspondence to: W. Liebl     

T-cell receptor V Tβ genes in natural populations of mice

The composition of 15 V _T gene subfamilies has been examined by Southern hybridization among a broad spectrum of colony bred rat and mouse species extending phylogenetically from Rattus to Mus musculus domesticus. Most mouse species contain a similar content of V _T genes as determined by the number of hybridizing restriction fragment (RF) bands. Furthermore, the extent of restriction fragment length polymorphism (RFLP) appears to be limited. Some V _T gene families, however, are missing from Rattus (VT7, V _T12) and M. shortridgei (V _T9, V _T16). Extension of the V _T survey to a panel of 38 wild-caught mice reveals that nearly a third lack specific hybridization to the V _T5 probe. Previous reports have established that the mouse inbred strains SJL, C57BR, C57L, and SWR lack 50% of their V _T repertoire, including V _T5 (Behlke et al. 1985). This study demonstrates that natural populations of mice also carry a significantly reduced V _T gene repertoire.     

Bacillus subtilis bacteriophages SP beta c1 is a deletion mutant of SP beta

Summary The restriction fragment patterns of two mutant forms of the temperate Bacillus subtilis bacteriophage SP have been examined. The DNA of a heat-inducible mutant, SPc2, which has a molecular size of 128 kilobases (kb), yields the same restriction pattern as the wild type SPc⁺ DNA. The DNA of a clear-plaque mutant, SPc1, has a molecular size of 117 kb, and is deleted for an 11 kb region of phage DNA. Neither SPc1 nor SPc2 DNA is cleaved by the endonuclease HaeIII.     

Presence of hybridizing DNA sequences homologous to bovine acidic and basicβ-crystallins in all classes of vertebrates

Summary The eye lens-crystallins in cow and chicken are encoded by a family of at least six genes. In order to assess the distribution of the corresponding genes among other vertebrates we hybridized -crystallin sequences (A2, A3/A1, A4, B1, B2, B3), isolated from a bovine lens cDNA library, to Southern blots on whichEcoR1-digested chromosomal DNA was blotted from different vertebrate species. These included human, chimpanzee, calf, rat, pigeon, duck, monitor lizard, toad, trout, and lamprey. Positive hybridization signals were found in the representatives of virtually all classes of vertebrates. The basic B-crystallins gave hybridization signals in more species than the acidic A ones. In monitor lizard and toad the weakest hybridization signals for basic crystallin probes were found. For acidic crystallin probes the distribution pattern was more simple; among cold-blooded vertebrates a signal for A2 was found in trout and lamprey, for A4 in trout, and for A3/A1 only in toad. The results demonstrate that the duplications leading to the -crystallin gene family occurred before or during the earliest stages of vertebrate evolution.