Changing the substrate specificity of P450cam towards diphenylmethane by semi-rational enzyme engineering.

A focused library comprising nine residues of the active site of P450cam monooxygenase resulting in ∼ 300,000 protein variants was screened for activity on diphenylmethane (DPM). The assay was based on the depletion of NADH by an in vitro reconstituted P450cam system in a 96-well scale. The throughput was increased by the parallel cultivation, purification and analysis of 20 variants per well (cluster screening).

Hyperthermostable acetyl xylan esterase.

An esterase which is encoded within a Thermotoga maritima chromosomal gene cluster for xylan degradation and utilization was characterized after heterologous expression of the corresponding gene in Escherichia coli and purification of the enzyme. The enzyme, designated AxeA, shares amino acid sequence similarity and its broad substrate specificity with the acetyl xylan esterase from Bacillus pumilus, the cephalosporin C deacetylase from Bacillus subtilis, and other (putative) esterases, allowing its classification as a member of carbohydrate esterase family 7. The recombinant enzyme displayed activity with p-nitrophenyl-acetate as well as with various acetylated sugar substrates such as glucose penta-acetate, acetylated oat spelts xylan and DMSO (dimethyl sulfoxide)-extracted beechwood xylan, and with cephalosporin C.

Genome sequence of the polysaccharide-degrading, thermophilic anaerobe Spirochaeta thermophila DSM 6192.

Spirochaeta thermophila is a thermophilic, free-living anaerobe that is able to degrade various α- and β-linked sugar polymers, including cellulose. We report here the complete genome sequence of S. thermophila DSM 6192, which is the first genome sequence of a thermophilic, free-living member of the Spirochaetes phylum.

Characterization and engineering of a novel pyrroloquinoline quinone dependent glucose dehydrogenase from Sorangium cellulosum So ce56.

A novel pyrroloquinoline quinone dependent glucose dehydrogenase like enzyme (PQQ GDH) was isolated from Sorangium cellulosum So ce56. The putative coding region was cloned, over expressed in E. coli and the resulting enzyme was characterized.

A nitrilase from a metagenomic library acts regioselectively on aliphatic dinitriles.

Several novel nitrilases were selected from metagenomic libraries using cinnamonitrile and a mixture of six different nitriles as substrates. The nitrilase gene nit1 was expressed in Escherichia coli and the resulting protein was further examined concerning its biochemical properties. Nit1 turned out to be an aliphatic nitrilase favoring dinitriles over mononitriles.

Indication for a new lipolytic enzyme family: isolation and characterization of two esterases from a metagenomic library.

We have isolated several novel esterase genes from a sheep rumen metagenomic library using the activity-based cluster screening approach as a highly efficient screening technology. The two most remarkable esterase genes, designated estGK1 and estZ3, were further examined. Sequence analysis of estGK1 and estZ3 revealed that they encoded proteins covering 322 and 317 amino acids, respectively.

Identification and characterization of a novel intracellular alkaline alpha-amylase from the hyperthermophilic bacterium Thermotoga maritima MSB8.

The gene for a novel alpha-amylase, designated AmyC, from the hyperthermophilic bacterium Thermotoga maritima was cloned and heterologously overexpressed in Escherichia coli. The putative intracellular enzyme had no amino acid sequence similarity to glycoside hydrolase family (GHF) 13 alpha-amylases, yet the range of substrate hydrolysis and the product profile clearly define the protein as an alpha-amylase. Based on sequence similarity AmyC belongs to a subgroup within GHF 57.

Structure of the novel alpha-amylase AmyC from Thermotoga maritima.

alpha-Amylases are essential enzymes in alpha-glucan metabolism and catalyse the hydrolysis of long sugar polymers such as amylose and starch. The crystal structure of a previously unidentified amylase (AmyC) from the hyperthermophilic organism Thermotoga maritima was determined at 2.2 Angstroms resolution by means of MAD.

AmyA, an alpha-amylase with beta-cyclodextrin-forming activity, and AmyB from the thermoalkaliphilic organism Anaerobranca gottschalkii: two alpha-amylases adapted to their different cellular localizations.

Two alpha-amylase genes from the thermophilic alkaliphile Anaerobranca gottschalkii were cloned, and the corresponding enzymes, AmyA and AmyB, were investigated after purification of the recombinant proteins. Based on their amino acid sequences, AmyA is proposed to be a lipoprotein with extracellular localization and thus is exposed to the alkaline milieu, while AmyB apparently represents a cytoplasmic enzyme. The amino acid sequences of both enzymes bear high similarity to those of GHF13 proteins.