Ib Groth Clausen

The complete genome of the crenarchaeon Sulfolobus solfataricus P2

The genome of the crenarchaeon Sulfolobus solfataricus P2 contains 2,992,245 bp on a single chromosome and encodes 2,977 proteins and many RNAs. One-third of the encoded proteins have no detectable homologs in other sequenced genomes. Moreover, 40% appear to be archaeal-specific, and only 12% and 2.3% are shared exclusively with bacteria and eukarya, respectively. The genome shows a high level of plasticity with 200 diverse insertion sequence elements, many putative nonautonomous mobile elements, and evidence of integrase-mediated insertion events. There are also long clusters of regularly spaced tandem repeats. Different transfer systems are used for the uptake of inorganic and organic solutes, and a wealth of intracellular and extracellular proteases, sugar, and sulfur metabolizing enzymes are encoded, as well as enzymes of the central metabolic pathways and motility proteins. The major metabolic electron carrier is not NADH as in bacteria and eukarya but probably ferredoxin. The essential components required for DNA replication, DNA repair and recombination, the cell cycle, transcriptional initiation and translation, but not DNA folding, show a strong eukaryal character with many archaeal-specific features. The results illustrate major differences between crenarchaea and euryarchaea, especially for their DNA replication mechanism and cell cycle processes and their translational apparatus.

Two different and highly organized mechanisms of translation initiation in the archaeon Sulfolobus solfataricus

Abstract The translational starts of 144 Sulfolobus solfataricus genes have been determined by database comparison. Half the genes lie inside operons and the other half are at the start of an operon or single genes. A Shine–Dalgarno sequence is found upstream of the genes inside operons, but not for the first gene in an operon or isolated genes; this indicates that two different mechanisms are used for translation initiation in S. solfataricus. A box A transcriptional signal is found for the genes starting an operon or isolated genes, but not for the genes inside an operon. The box A signal is located about 27 nt upstream of the start codon, which implies that little or no upstream sequence is available for translation initiation for this group of genes. This finding is discussed.

LIPASES FROM RHIZOMUCOR MIEHEI AND HUMICOLA LANUGINOSA : MODIFICATION OF THE LID COVERING THE ACTIVE SITE ALTERS ENANTIOSELECTIVITY

The homologous lipases fromRhizomucor miehei andHumicola lanuginosa showed approximately the same enantioselectivity when 2-methyldecanoic acid esters were used as substrates. Both lipases preferentially hydrolyzed theS-enantiomer of 1-heptyl 2-methyldecanoate (R. miehei:ES=8.5;H. lanuginosa:ES=10.5), but theR-enantiomer of phenyl 2-methyldecanoate (ER=2.9). Chemical arginine specific modification of theR. miehei lipase with 1,2-cyclohexanedione resulted in a decreased enantioselectivity (ER=2.0), only when the phenyl ester was used as a substrate. In contrast, treatment with phenylglyoxal showed a decreased enantioselectivity (ES=2.5) only when the heptyl ester was used as a substrate. The presence of guanidine, an arginine side chain analog, decreased the enantioselectivity with the heptyl ester (ES=1.9) and increased the enantioselectivity with the aromatic ester (ER=4.4) as substrates. The mutation, Glu 87 Ala, in the lid of theH. lanuginosa lipase, which might decrease the electrostatic stabilization of the open-lid conformation of the lipase, resulted in 47% activity compared to the native lipase, in a tributyrin assay. The Glu 87 Ala mutant showed an increased enantioselectivity with the heptyl ester (ES=17.4) and a decreased enantioselectivity with the phenyl ester (ER=2.5) as substrates, compared to native lipase. The enantioselectivities of both lipases in the esterification of 2-methyldecanoic acid with 1-heptanol were unaffected by the lid modifications.

[19] Protein engineering of microbial lipases of industrial interest

Publisher Summary This chapter shows that protein engineering can be successfully used to produce new, commercially interesting products. An understanding of lipase function in general and under specific application conditions is mandatory to make the correct decisions in a protein-engineering strategy. Lipases have a number of potential industrial applications, such as production of esters and specialty fats, removal of resins from pulp, cleaning of hard surfaces, and use in detergents. Enzymes showing lipolytic activity can in some cases also act as esterases, phospholipases, cholesterolesterases, thioesterases, and cutinases. The specificities of lipases are broad, but each enzyme has a preference. The selectivity for a specific activity of the lipases can be improved by protein engineering. At present, most of the industrially relevant efforts in protein engineering of lipases have been to improve the hydrolytic efficiency, peracid generation, and detergent and protease stability, all aiming at applications for detergents.

Trp89 in the lid ofHumicola lanuginosa lipase is important for efficient hydrolysis of tributyrin

To determine whether Trp89 located in the lid of the lipase (EC 3.1.1.3) fromHumicola lanuginosa is important for the catalytic property of the enzyme, site-directed mutagenesis at Trp89 was carried out. The kinetic properties of wild type and mutated enzymes were studied with tributyrin as substrate. Lipase variants in which Trp89 was changed to Phe, Leu, Gly or Glu all showed less than 14% of the activity compared to that of the wild type lipase. The Trp89Glu mutant was the least active with only 1% of the activity seen with the wild type enzyme. All Trp mutants had the same binding affinity to the tributyrin substrate interface as did the wild type enzyme. Wild type lipase showed saturation kinetics against tributyrin when activities were measured with mixed emulsions containing different proportions of tributyrin and the nonionic alkyl polyoxyethylene ether surfactant, Triton DF-16. Wild type enzyme showed a Vmax=6000±300 mmol·min−1·g−1 and an apparent Km=16±2% (vol/vol) for tributyrin in Triton DF-16, while the mutants did not show saturation kinetics in an identical assay. The apparent Km for tributyrin in Triton DF-16 was increased as the result of replacing Trp89 with other residues (Phe, Leu, Gly or Glu). The activities of all mutants were more sensitive to the presence of Triton DF-16 in the tributyrin substrate than was wild type lipase. The activity of the Trp89Glu mutant was decreased to 50% in the presence of 2 vol% Triton DF-16 compared to the activity seen with pure tributyrin as substrate. Wild type lipase and all mutants except Trp89Glu had the same affinity for the substrate interface formed by 15.6 vol% tributyrin in Triton DF-16. The Trp89Glu mutant showed a lower affinity than all the other lipase variants for the interface of 15.6 vol% tributyrin in Triton DF-16. The study showed that Trp89 located in the lid ofH. lanuginosa lipase is important for the efficient hydrolysis of tributyrin and that this residue plays a role in the catalytic steps after adsorption of the lipase to the substrate interface.

Completing the sequence of the Sulfolobus solfataricus P2 genome

Abstract The Sulfolobus solfataricus P2 genome collaborators are poised to sequence the entire 3-Mbp genome of this crenarchaeote archaeon. About 80% of the genome has been sequenced to date, with the rest of the sequence being assembled fast. In this publication we introduce the genomic sequencing and automated analysis strategy and present intial data derived from the sequence analysis. After an overview of the general sequence features, metabolic pathway studies are explained, using sugar metabolism as an example. The paper closes with an overview of repetitive elements in S. solfataricus.

PROBING A FUNCTIONAL ROLE OF GLU87 AND TRP89 IN THE LID OF HUMICOLA LANUGINOSA LIPASE THROUGH TRANSESTERIFICATION REACTIONS IN ORGANIC SOLVENT

To reveal the functional role of Glu87 and Trp89 in the lid ofHumicola lanuginosa lipase, site-directed mutagenesis at Glu87 and Trp89 was carried out. The catalytic performance of wild-type and mutated lipases was studied in transesterification reactions in cyclohexane at a controlled water activity. Two different acyl donors were used in the investigation: tributyrin, a natural substrate for a lipase, and vinyl butyrate, an activated ester suitable for fast and efficient lipase-catalyzed transformations in preparative organic synthesis. As acyl acceptor 1-heptanol was used. The Glu87Ala mutation decreased theVmax,app value with tributyrin and vinyl butyrate by a factor of 1.5 and 2, respectively. TheKm,app for tributyrin was not affected by the Glu87Ala mutation, but theKm,app for vinyl butyrate increased twofold compared to the wild-type lipase. Changing Trp89 into a Phe residue afforded an enzyme with a 2.7- and 2-fold decreasedVmax,app with the substrates tributyrin and vinyl butyrate, respectively, compared to the wild-type lipase. No significant effects on theKm,app values for tributyrin or vinyl butyrate were seen as a result of the Trp89Phe mutation. However, the introduction of a Glu residue at position 89 in the lid increased theKm,app for tributyrin and vinyl butyrate by a factor of >5 and 2, respectively. The Trp89Glu mutated lipase could not be saturated with tributyrin within the experimental conditions (0–680 mM) studied here. With vinyl butyrate as a substrate theVmax,app was only 6% of that obtained with wild-type enzyme.

Effect of mutation in non-consensus sequence Thr-X-Ser-X-Gly of Candida antarctica lipase B on lipase specificity, specific activity and thermostability

Abstract Non-consensus residue threonine in Candida antarctica B lipase was exchanged with glycine residue using site specific mutation. The effect of the mutation on the thermostability was investigated by measuring residual activity after heat treatment of the lipase and the mutant. A significant increase in thermostability was found for the mutant lipase. Specific activity of the mutant lipase was determined using tributyrin as substrate which showed a twofold decrease in specific activity. To investigate the effect of mutation on the specificity and activity in ester synthesis, both the mutant lipase and the native lipases were immobilized on a solid support. Ester synthesis using decanol as alcohol with three different fatty acids was carried out. The activity and specificity of the mutant lipase was unaltered in the ester synthesis as compared with the native lipase.

Aspects in lipase screening

Abstract Lipases have become increasingly important for the industry over the last 10 years, and heterologous gene expression has played a key role. Screening of the biological diversity that Nature provides in order to identify lipases suitable for industrial use is a key issue. This paper will discuss some of the aspects to consider when carrying out a lipase screening program.