Biomedical subjects

R Fagerström

Publications and source records attributed to R Fagerström.

At least 19 recordsLinked to original sources

PubMed2000

Endoxylanase II from Trichoderma reesei has several isoforms with different isoelectric points.

Two minor xylanases present in Trichoderma reesei Rut C30 cultivation broth were purified as a mixture using ion-exchange, hydrophobic-interaction and gel chromatography. The purified enzyme preparation contained two active xylanases with pI values of 7.1 and 8.1. Both components had a molecular mass of 20 kDa. The purified xylanase preparation exhibited properties very similar to those of the previously isolated XYL II (pI 9.0) of T. reesei Rut C30. The activity and stability properties, apparent kinetic parameters as well as the titration curve forms were similar. The major difference in enzymic properties was the significantly lower specific activity of the pI-7.1+8.1 xylanase mixture (3350 nkat/mg) compared with the specific activity of XYL II (13500 nkat/mg). Amino acid sequences of tryptic peptides (34% of the total amino acid sequence was determined) were identical to the amino acid sequence of XYL II. Furthermore, in vitro modification of the pI-9.0 form of XYL II to pI-8.1 and pI-7.1 forms was demonstrated. Thus the purified xylanase preparation most probably contained two modified forms of XYL II. The primary amino acid sequence of XYL II contains 28 glutamine and asparagine residues and theoretically deamination of one of them lowers the pI to 8.06 and deamination of two amino acids lowers the pI to 7.02.

Amino Acid Sequence↗

PubMed1996

Protein purification, and cloning and characterization of the cDNA and gene for xylose isomerase of barley.

The first eukaryotic xylose isomerase protein was purified from barley Hordeum vulgare. The enzyme requires Mn2+ for its activity and is fairly thermostable, with the optimum temperature being 60 degrees C. It showed maximum activity over a broad pH range (7.0-9.0). The molecular mass of the monomer was about 50,000 Da based on the SDS/PAGE, and the calculated value from the cDNA-deduced polypeptide sequence was 53,620 Da. A relative mass estimation of 100,000 Da was obtained from the Superose 12 chromatography, suggesting that the barley enzyme is a dimer. The cloned corresponding cDNA sequence of 1710 nucleotides encoded a polypeptide of 480 amino acids. The genomic sequence of 4473 nucleotides, revealed that the isomerase gene contained 20 introns, all starting with GT and ending with AG. One large intron was located in the 5'untranslated region. The barley isomerase has an insertion of about 40 residues at its amino terminus when compared to the prokaryotic cluster (family) II isomerases; cluster (family) I and cluster (family) II isomerases vary from the former in an insertion of around 50 residues at their amino termini. Comparison of the barley protein with the prokaryotic isomerases shows that the conserved catalytic and metal binding regions are also well conserved in barley.

Aldose-Ketose Isomerases↗

PubMed1995

Characterization, subsite mapping and partial amino acid sequence of glucoamylase from the filamentous fungus Trichoderma reesei.

The pH optimum, temperature-dependence, thermal stability, substrate specificity and subsite affinities of the 66 kDa, pI 4.0 glucoamylase of the filamentous fungus Trichoderma reesei were determined. It had a pH optimum of 5.5 and a temperature optimum (5 min reaction time) of 70 degrees C with soluble starch as substrate. Thermal-inactivation studies revealed that the glucoamylase is relatively thermostable up to 60 degrees C. Metal ions and EDTA tested at 5 mM concentrations had no significant effect, and beta-cyclodextrin only slightly inhibitory effects, on the digestion of soluble starch. Estimated Km and kcat. values for soluble starch where 0.11 mg.ml-1 and 28.5 s-1 respectively. Hydrolysis of pullulan (Km 14 mg.ml-1 and kcat. = 6.6 s-1) indicated substantial activity towards 1,6-O-glucosidic bonds. From ratios of kinetic parameters of malto- and isomalto-oligosaccharides, it was apparent that the glucoamylase showed approx. 3-fold higher selectivity towards isomalto-oligosaccharides than most other reported fungal glucoamylases. Substrate binding affinities were calculated from kinetic data for the linear series of malto- and isomalto-oligosaccharides. The results were in good agreement with other reported glucoamylases. The main difference was that subsite 1 showed a slightly negative free energy of binding with malto-oligosaccharides, whereas most other glucoamylases show a positive free energy at this subsite. A set of peptides obtained from purified glucoamylase by tryptic digestion where sequenced. They covered approx. 17% of the total amino acid sequence as estimated from molecular mass on SDS/PAGE. Some of the sequences were tentatively aligned to known glucoamylase sequences. They showed about 60% identity with the extensively studied Aspergillus glucoamylase.

Amino Acid Sequence↗

PubMed1994

Purification and specificity of recombinant Hormoconis resinae glucoamylase P and endogenous glucoamylase from Trichoderma reesei.

Hormoconis resinae glucoamylase P of high debranching activity was purified from a recombinant Trichoderma reesei strain. Four different purified fractions were obtained. Three had the same amino terminal sequence as the wild-type enzyme and about the same specific activity, and yielded the same single band on SDS-PAGE after deglycosylation. Presumably they resulted from different glycosylation patterns of the recombinant glucoamylase P. One fraction had a much lower specific activity and yielded tryptic peptides that identified it as the host cellobiohydrolase I contaminated with glucoamylase P. The different glycosylation patterns of recombinant glucoamylase P had only minor effects on its thermal inactivation. During purification of the recombinant glucoamylase, a protein with lower debranching activity was found and purified by chromatofocusing to homogeneity as assessed by SDS-PAGE. It had a pI of about 4.0 and a ratio of pullulan- to starch-degrading activity of 15%. Its amino terminal sequence showed 60% identity to the amino terminal sequence of glucoamylases P and S from Hormoconis resinae. Presumably this enzyme is the endogenous glucoamylase of Trichoderma reesei.

Amino Acid Sequence↗

PubMed1994

Evidence for a polysaccharide-binding domain in Hormoconis resinae glucoamylase P: effects of its proteolytic removal on substrate specificity and inhibition by beta-cyclodextrin.

The hydrolysis of soluble starch, raw starch and pullulan with recombinant glucoamylase P from Hormoconis resinae was competitively inhibited by beta-cyclodextrin with apparent Ki values of 190 microM, 13 microM and 1.4 microM, respectively. Inhibition of dextran hydrolysis was partial: a maximum inhibition of 22% was achieved with a dextran concentration of 0.3 x Km and up to 4 mM beta-cyclodextrin. Hydrolysis of short oligosaccharides was not inhibited by beta-cyclodextrin at levels up to 20 mM. The enzyme bound to raw starch at pH 4.3 and 4 degrees C with an association constant of 3.4 x 10(5) M-1. Sequence alignment studies showed raw-starch-binding consensus amino acids in the C-terminal part of glucoamylase P. Partial hydrolysis with papain resulted in degradation of deglycosylated glucoamylase P into three fragments of 53, 51 and 14 kDa, respectively, as estimated by SDS-PAGE. The amino-terminal sequences of the 51 and 53 kDa fragments were identical with that of native glucoamylase P. The amino terminus of the 14 kDa fragment (Ser-Ser-X-Gln-Val-Ser-), corresponded to the sequence starting at residue 474 of intact glucoamylase P. Kinetic measurements of truncated glucoamylase P showed changes in the Km values of larger polysaccharides, but no changes in kcat values compared to the intact enzyme. It was concluded that glucoamylase P contains a catalytic core domain and a raw-starch-binding domain involved in inhibition of polysaccharide hydrolysis by beta-cyclodextrin.

Amino Acid Sequence↗

PubMed1994

Correlation between depression and vision in aged patients before and after cataract operations.

To investigate the association between depression and vision, 100 cataract operation patients (25 were men and 75 women) aged 71 to 76 years were studied. One day before the operation and 3 mo. after, the patients' depression was tested with the short form of the Beck Depression Inventory and their personalities with Mini-Mult MMPI. The cataract operation restored visual acuity sufficient for reading (minimum E-test value 0.40) to 79% of the subjects. The analysis indicated that their depression was significantly correlated with vision only after the cataract operation. Depression increased with weakened visual acuity and diminished with improved visual acuity. Part of the postoperation depression was, however, associated with glaucoma and serious somatic diseases (asthma, cerebrovascular disorders, and heart diseases).

Aged↗

PubMed1993

Cloning, sequencing and enhanced expression of the Trichoderma reesei endoxylanase II (pI 9) gene xln2.

The Trichoderma reesei xln2 gene coding for the pI9.0 endoxylanase was isolated from the wild-type strain QM6a. The gene contains one intron of 108 nucleotides and codes for a protein of 223 amino acids in which two putative N-glycosylation target sites were found. Three different T. reesei strains were transformed by targeting a construct composed of the xln2 gene, including its promoter, to the endogenous cbh1 locus. Highest overall production levels of xylanase were obtained using T. reesei ALKO2721, a genetically engineered strain, as a host. Integration into the cbh1 locus was not required for enhanced expression under control of the xln2 promoter.

Amino Acid Sequence↗

PubMed1993

Fear of a cataract operation in aged persons.

Self-reports of fear from 100 patients (25 men and 75 women 71 to 76 years old) having two cataract operations were investigated. 33% of the patients reported having fear and 32% felt tension about the operation performed on the first eye. Women feared the operation significantly more than men. Fears were significantly associated with hypochondriasis, hysteria, and hypomania (unadjusted) as indicated by correlations with scores on the Mini-Mult MMPI. The cataract operation restored sufficient visual acuity for reading (minimum E-test value 0.40 or 1.8-cm high letters at a distance of 6 meters) to 79% of the subjects. The experience of a good operation result on the first eye significantly reduced the fear of the cataract operation on the second eye and at the same time the fear of becoming blind. Other factors reducing fear included positive experiences of a safe and painless cataract operation.

Adaptation, Psychological↗

PubMed1992

Correlations of memory and learning with vision in aged patients before and after a cataract operation.

The connection between memory and learning with vision was investigated by studying 100 cataract operation patients, aged 71 to 76 years, 25 of them being men and 75 women. The cataract operation restored sufficient acuity of vision for reading (minimum E-test value 0.40) to 79% of the subjects. Short-term memory was studied with series of numbers, homogenic and heterogenic inhibition, and long sentences. Learning was tested with paired-associate learning and word learning. Psychological symptoms were measured on the Brief Psychiatric Rating Scale and personality on the Mini-Mult MMPI. Memory and learning improved significantly when vision was normalized after the cataract operation. Poor memory and learning scores correlated with monocular vision before the operation and with defects in the field of vision, due to glaucoma and exceeding 20%, postsurgery. Monocular vision and defects in the visual field caused a continuous sense of abnormalness, which impaired old people's ability to concentrate on tasks of memory and learning. Cerebrovascular disturbances, beginning dementia, and moderate psychological symptoms obstructed memory and learning on both test rounds. Depression was the most important psychological symptom contributing to poor memory and learning scores after the cataract operation. The memory and learning defects mainly reflected disturbances in memorizing.

Aged↗

PubMed1991

Correlation between psychic and somatic symptoms and vision in aged patients before and after a cataract operation.

The connection between psychic and somatic symptoms with vision was investigated by studying 100 cataract operation patients, aged 71 to 76 years, 25 of them being men and 75 women. The investigations were conducted one day before the operation and three months afterwards. The cataract operation restored sufficient acuity of vision for reading (minimum E-test value 0.40) to 79% of the old people. Psychic symptoms were tested with the Brief Psychiatric Rating Scale, Mini-Mult MMPI, and direct questions. Somatic symptoms were studied through questionnaires. Psychic symptoms showed a statistically significant correlation with vision before the cataract operation but not afterwards. Psychic symptoms increased with deterioration of the acuity of vision and diminished when the acuity of vision improved. Somatic symptoms did not show similar association with vision but the symptoms were significantly alleviated after the cataract operation. Restoration of vision through the cataract operation normalized the old people's psychic condition and reduced their somatic symptoms to correspond with their prior chronic diseases.

Adaptation, Psychological↗

PubMed1990

Comparison of two glucoamylases from Hormoconis resinae.

Two extracellular glucoamylases (EC 3.2.1.3), glucoamylase P and glucoamylase S, were purified to homogeneity from the culture medium of Hormoconis resinae (ATCC 20495; formerly Cladosporium resinae) by a new method. Their apparent molecular masses (71 kDa glucoamylase P; 78 kDa glucoamylase S) and catalytic properties agreed well with those previously reported in the literature. Heat inactivation studies suggested that the high debranching (1,6-glycosidic) activity of glucoamylase P preparations (measured with pullulan) may reside in the same protein molecule as its 1,4-glycosidic activity (measured with soluble starch). Although glucoamylase S had virtually no debranching activity, it cross-reacted with polyclonal antibodies raised against glucoamylase P, and the two enzymes had very similar amino acid compositions. However, peptide mapping and amino-terminal sequencing studies of the peptides showed that the two enzymes have different sequences and must be encoded by different genes.

Amino Acid Sequence↗

PubMed1988