Asp 718 from a non-pathogenic species of the genus Achromobacter: a KpnI isoschizomer generating DNA-fragments with 5'-protruding ends

A new type II restriction endonuclease Asp7/8% has been isolated from the non-pathogenic species of Achromobacter 718. This novel enzyme, an isoschizomer of Kpnl, recognizes and cleaves specifically within the nucleotide sequence: 5'-G/GTAC-C-3' 3'-C-CATG/G-5'.In contrast to Kpnl, Asp718 generates fragments with 5'-protruding single-stranded ends. These 5'-terminal extensions of the Asp718 fragments may be efficiently labeled with T4 poly nucleotide kinase, whereas the recessed 3'-ends are suitable substrates for the terminal labeling reaction applying Klenow enzyme. The presence of only one restriction activity in this Achromobacter strain facilitates the preparation of Asp718 free of other contaminating site-specific nucleases which could interfere with the in vitro digestion of DNA.Restriction endonuclease KpnI isoschizomer5'-Protruding terminusT4 polynucleotide kinaseKlenow enzyme     

蒙古高原绣线菊属植物演化系统的研究

利用最小平行进化法对蒙古高原绣线菊属植物进行了分支分类研究,将本属植物分为4个组、6个系。其组的进化顺序为：Sect.Spiraea→Sect.Calospira→Sect.Glomerati→Sect.Chamaedryon。本属的进化趋势为冬芽芽鳞由数枚减少到2枚,花序由总状圆锥花序→复伞房花序→伞房花序→伞形花序。研究认为绣线菊属是泛北极起源。     

The xylanolytic enzyme system from the genus Penicillium

In nature, there are numerous microorganisms that efficiently degrade xylan, a major component of lignocellulose. In particular, filamentous fungi have demonstrated a great capability for secreting a wide range of xylanases, being the genus Aspergillus and Trichoderma the most extensively studied and reviewed among the xylan-producing fungi. However, an important amount of information about the production and genetics of xylanases from fungi of the genus Penicillium has accumulated in recent years. A great number of Penicillia are active producers of xylanolytic enzymes, and the use of xylanases from these species has acquired growing importance in biotechnological applications. This review summarizes our current knowledge about the properties, genetics, expression and biotechnological potential of xylanases from the genus Penicillium.     

Subunit structure of Achromobacter collagenase.

The highly active form of collagenase (EC 3.4.24.3) from Achromobacter iophagus (specific activity 2 microkat/mg) has a molecular weight of 70,000 and the sedimentation coefficient s20,2 = 4.4 S. It is composed of two subunits of molecular weight 35,000 and s20,w of 2.9 S. The dissociation of the dimer under different conditions resulted in the complete and irreversible loss of enzymic activity. A unique N-terminal sequence Thr-Ala-Ala-Asp-Leu-Glu-Ala-Leu-Val- indicates that the two subunits are identical, at least in the N-terminal part of the polypeptide chain. Reduction and pyridylethylation of the subunit change neither molecular weight nor amino acid composition: therefore each subunit of molecular weight 35,000 consists of a single polypeptide chain. Another active and homogeneous form of Achromobacter collagenase (specific activity 1.64 microkat/mg) gives a value for the apparent molecular weight of 80,000 on sodium dodecyl sulphate-polyacrylamide electrophoresis. It is also a dimer in which each of the two subunits of molecular weight 35,000 binds non-covalently a peptide of molecular weight 5000. The dissociation of this form of collagenase is also accompanied by irreversible loss of enzymic activity. The amino acid composition of the subunits which were isolated from both 70,000 and 80,000 collagenases is the same. The role of dimer-monometer equilibrium in the biological function of collagenase is discussed.     

Dissimilation of Methionine by Achromobacter starkeyi

Methionine was decomposed by some bacteria which were isolated from soil. The sulfur of the methionine was liberated as methanethiol, and part of this became oxidized to dimethyl disulfide. Detailed studies with one of these cultures, Achromobacter starkeyi, indicated that the first step in methionine decomposition was its oxidadative deamination to α-keto-γ-methyl mercaptobutyrate by a constitutive amino acid oxidase. The following steps were carried out by inducible enzymes, the synthesis of which was inhibited by chloramphenicol. α-Keto-γ-methyl mercaptobutyrate was split producing methanethiol and α-keto butyrate which was oxidized to propionate. The metabolism of propionate was similar to that described for animal tissues; the propionate was carboxylated to succinate via methyl malonyl coenzyme A, and the succinate was metabolized through the Krebs cycle.     

Chemical modifications of Achromobacter collagenase and their influence on the enzymic activity

A study of the influence of chemical modifications on the activity of Achromobacter iophagus collagenase (EC 3.4.24.8) has led to the following conclusions: a modification of 4 out of 80 COOH groups with carbodiimide led to 90% loss of enzymic activity. A 70% inactivation was found after modification of two tyrosines out of 30 with tetranitromethane. The modification of four to six tryptophans out of 16 with 2-hydroxy-5-nitrobenzyl bromide decreased enzyme activity to 36%. This inactivation is accelerated in the presence of collagen. An increase of reagent/enzyme molar ratio led to a modification of 16 tryptophan residues and denaturation of Acahromobacter collagenase. A modification of two arginines out of 18 with 1,2-cyclohexanedione and eight NH2 groups out of 24 with 2,3-dimethyl maleic anhydride does not change the collagenolytic activity. All NH2 groups become available for 2,3-dimethyl maleic anhydride after dissociation of the dimer. A possible analogy of hydrolytic site of collagenase with that of two other known bacterial metalloproteinases (thermolysin and Bacillus subtilis neutral proteinase (EC 3.4.24.4)) is discussed.     

Cell-surface collagen-binding protein in the procaryote Achromobacter iophagus

Collagen and its high-molecular-weight fragments specifically induce an extracellular collagenase (EC 3.4.24.8) in the Gram-negative Achromobacter iophagus. During the induction process the inducer is concentrated on the bacterial outer membrane. Two-dimensional electrophoresis of 125I-labelled outer membrane proteins has shown that, in particular, the amount of one protein which is already present on the surface of non-induced bacteria increases quantitatively when the inducer is added. After 125I-labelling of the cell membrane and its solubilization, the same protein is retained selectively on a gelatin-Sepharose column. It has isoelectric point of 4.9-5.1 and molecular weight of 40000. This molecular weight is close to that of the 35000 of the collagenase subunit. However, their non-identity was proved in three independent ways: upon two-dimensional electrophoresis, only those proteins in the range corresponding to the collagenase dimer (Mr 70000-80000) react with fluorescent anticollagenase antibody system, whereas the spot of the collagen-binding protein (mr 40000) is negative; the solubilized collagen-binding protein is not retained by anticollagenase-Sepharose affinity chromatography; in vivo, it is not protected by anti-collagenase antibodies against lactoperoxidase iodination. A hypothesis for the possible role of the collagen-binding protein in the induction of collagenase is proposed.