ANCUT2, a Thermo-alkaline Cutinase from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> and Its Potential Applications

Biochemical characterization of purified ANCUT2 cutinase from Aspergillus nidulans is described. The identified amino acid sequence differs from that predicted in Aspergillus genomic databases in amino acids not relevant for catalysis. The enzyme is thermo-alkaline, showing its maximum activity at pH 9 and 60 °C, and it retains more than 60% of its initial activity after incubation for 1 h at 60 °C for pH values between 6 and 10. ANCUT2 is more active towards long-chain esters and it hydrolyzes cutin; however, it also hydrolyzes short-chain esters. Cutinase is inhibited by metal ions, PMSF, SDS, and EDTA (10 mM). It retains 50% of its activity in most of the solvents tested, although it is more stable in hydrophobic solvents. According to its found biochemical properties, preliminary assays demonstrate its ability to synthesize methyl esters from sesame oil and the most likely application of this enzyme remains in detergent formulations.     

Production Optimization and Characterization of Recombinant Cutinases from Thermobifida fusca sp. NRRL B-8184

Two genes, cut1 and cut2, of Thermobifida fusca NRRL B-8184 with cutin-hydrolyzing activity were cloned and expressed in Escherichia coli BL21 (DE3) separately. Enhanced expression was achieved after screening of six different media, optimization of the culture conditions and medium components. Among the screened media, modified Terrific Broth was found to be the best for maximum production of recombinant cutinases in E. coli BL21 (DE3). Under optimal conditions, the production of recombinant Cut1 and Cut2 (cutinases) were found to be 318?±?0.73 and 316?±?0.90 U/ml, respectively. The production of recombinant cutinases was increased by 11-fold as compared with T. fusca NRRL B-8184 wild-type strain. Both the recombinant cutinases were purified to homogeneity. They were found to be thermostable, organic solvent, and surfactant tolerant. Both the cutinase were active in a broad range of temperature (40–80 °C) and pH (6.8–9) with optimum activity at pH 8.0 and 55 °C.     

Direct enzymatic esterification of cotton and Avicel with wild-type and engineered cutinases

In this work, the surface of cellulose, either Avicel or cotton fabric, was modified using cutinases without any previous treatment to swell or to solubilise the polymer. Aiming further improvement of cutinase ester synthase activity on cellulose, an engineered cutinase was investigated. Wild-type cutinase from Fusarium solani and its fusion with the carbohydrate-binding module N1 from Cellulomonas fimi were able to esterify the hydroxyl groups of cellulose with distinct efficiencies depending on the acid substrate/solvent system used, as shown by titration and by ATR-FTIR. The carbonyl stretching peak area increased significantly after enzymatic treatment during 72 h at 30 °C. Cutinase treatment resulted in relative increases of 31 and 9 % when octanoic acid and vegetable oil were used as substrates, respectively. Cutinase-N1 treatment resulted in relative increases of 11 and 29 % in the peak area when octanoic acid and vegetable oil were used as substrates, respectively. The production and application of cutinase fused with the domain N1 as a cellulose ester synthase, here reported for the first time, is therefore an interesting strategy to pursuit.     

Structural Stability and Unfolding Properties of Cutinases from Thermobifida fusca

A comparative analysis of the structural and functional aspects along with equilibrium unfolding of two homologous cutinases, Cut1 and Cut2, from Thermobifida fusca was carried out. The CD and fluorescence profile at different pH in the range of 6 to 9 showed no structural variations for both cutinases, indicating their stability to a wide range of pH. Tryptophan quenching studies suggested that all the four Trp residues in the protein are in inaccessible hydrophobic pockets. Further, near-UV CD analysis of tertiary structure revealed a dissimilar distribution of aromatic amino acid on the surface of these two enzymes. Denaturation profiles obtained in aqueous solutions of the guanidine hydrochloride revealed different tolerance levels for unfolding of the two cutinases, with Cut2 showing higher resistivity to unfolding in comparison to Cut1. Both cutinases retained all the structural parameters even in the presence of 8 M urea, indicating the protein to be highly resistant to urea-induced unfolding. Structural study by homology modeling revealed a high resemblance of secondary structure between the two cutinases; however, their tertiary structure, hydrophobicity, and surface electrostatic properties were very different, which contributed to the difference in the structural stability of these two cutinases.     

The computer-assisted carboxypeptidase method for C-terminal sequencing of proteins and polypeptides

On further study of the computer-assisted carboxypeptidase method for sequencing the C-terminals of proteins or peptides, two computer programs—DPS and CPA were successfully developed on VAX-11/780 computer and tested with some synthetic peptides and the degradation fragment of the natural protein as model substrates. The C-terminal sequence of CB-3, one of the CNBr degradation fragments of trichosanthin, had first been determined by this method as: -Ser-Ala-Ser-Ala-Ala-Leu-Hse-OH, which was later confirmed by other ways of sequencing. This method is not only able to extend the C-terminal sequencing up to 7 amino acid residues, but also useful for determining the C-terminal sequence with repeating amino acid residues.     

Synthesis,and Helix or Hairpin‐Turn Secondary Structures of ‘Mixed’ α/β‐Peptides Consisting of Residues with Proteinogenic Side Chains and of 2‐Amino‐2‐methylpropanoic Acid (Aib)

Twelve peptides, 1 – 12 , have been synthesized, which consist of alternating sequences of α‐ and β‐amino acid residues carrying either proteinogenic side chains or geminal dimethyl groups (Aib). Two peptides, 13 and 14 , containing 2‐methyl‐3‐aminobutanoic acid residues or a ‘random mix’ of α‐, β²‐, and β³‐amino acid moieties were also prepared. The new compounds were fully characterized by CD (Figs. 1 and 2), and ¹H‐ and ¹³C‐NMR spectroscopy, and high‐resolution mass spectrometry (HR‐MS). In two cases, 3 and 14 , we discovered novel types of turn structures with nine‐ and ten‐membered H‐bonded rings forming the actual turns. In two other cases, 8 and 11 , we found 14/15‐helices, which had been previously disclosed in mixed α/β‐peptides containing unusual β‐amino acids with non‐proteinogenic side chains. The helices are formed by peptides containing the amino acid moiety Aib in every other position, and their backbones are primarily not held together by H‐bonds, but by the intrinsic conformations of the containing amino acid building blocks. The structures offer new possibilities of mimicking peptide–protein and protein–protein interactions (PPI).     

Ring‐Closing and Cross‐Metathesis with Artificial Metalloenzymes Created by Covalent Active Site‐Directed Hybridization of a Lipase

A series of Grubbs‐type catalysts that contain lipase‐inhibiting phosphoester functionalities have been synthesized and reacted with the lipase cutinase, which leads to artificial metalloenzymes for olefin metathesis. The resulting hybrids comprise the organometallic fragment that is covalently bound to the active amino acid residue of the enzyme host in an orthogonal orientation. Differences in reactivity as well as accessibility of the active site by the functionalized inhibitor became evident through variation of the anchoring motif and substituents on the N‐heterocyclic carbene ligand. Such observations led to the design of a hybrid that is active in the ring‐closing metathesis and the cross‐metathesis of N,N‐diallyl‐p‐toluenesulfonamide and allylbenzene, respectively, the latter being the first example of its kind in the field of artificial metalloenzymes.     

Synthesis and Conformational Analysis of Hybrid α/β‐Dipeptides Incorporating S‐Glycosyl‐β2,2‐Amino Acids

We synthesized and carried out the conformational analysis of several hybrid dipeptides consisting of an α‐amino acid attached to a quaternary glyco‐β‐amino acid. In particular, we combined a S‐glycosylated β^2,2‐amino acid and two different types of α‐amino acid, namely, aliphatic (alanine) and aromatic (phenylalanine and tryptophan) in the sequence of hybrid α/β‐dipeptides. The key step in the synthesis involved the ring‐opening reaction of a chiral cyclic sulfamidate, inserted in the peptidic sequence, with a sulfur‐containing nucleophile by using 1‐thio‐β‐D ‐glucopyranose derivatives. This reaction of glycosylation occurred with inversion of configuration at the quaternary center. The conformational behavior in aqueous solution of the peptide backbone and the glycosidic linkage for all synthesized hybrid glycopeptides was analyzed by using a protocol that combined NMR experiments and molecular dynamics with time‐averaged restraints (MD‐tar). Interestingly, the presence of the sulfur heteroatom at the quaternary center of the β‐amino acid induced θ torsional angles close to 180° (anti). Notably, this value changed to 60° (gauche) when the peptidic sequence displayed aromatic α‐amino acids due to the presence of CH–π interactions between the phenyl or indole ring and the methyl groups of the β‐amino acid unit.     

Cloning,Expression, and Characterization of a Novel (<Emphasis Type="Italic">S</Emphasis>)-Specific Alcohol Dehydrogenase from <Emphasis Type="Italic">Lactobacillus kefir</Emphasis>

A gene encoding a novel (S)-specific NADH-dependent alcohol dehydrogenase (LK-ADH) was isolated from the genomic DNA of Lactobacillus kefir DSM 20587 by thermal asymmetric interlaced-polymerase chain reaction. The nucleotide sequence of (S)-LK-ADH gene (adhS) was determined, which consists of an open reading frame of 1,044 bp, coding for 347 amino acids with a molecular mass of 37.065 kDa. After a BLAST similarity search in GenBank database, the amino acid sequence of (S)-LK-ADH showed some homologies to several zinc containing medium-chain alcohol dehydrogenases. This novel gene was deposited into GenBank with the accession number of EU877965. adhS gene was subcloned into plasmid pET-28a(+), and recombinant (S)-LK-ADH was successfully expressed in E. coli BL21(DE3) by isopropyl-β-d-1-thiogalactopyranoside induction. Purified enzyme showed a high enantioselectivity in the reduction of acetophenone to (S)-phenylethanol with an ee value of 99.4%. The substrate specificity and cofactor preference of recombinant (S)-LK-ADH were also tested.     

Human milk lysozyme: unpublished data concerning the establishment of the complete primary structure; comparison with lysozymes of various origins

Details concerning the establishment of the complete primary structure of human milk lysozyme (previously published in a preliminary note) are presented. The chymotryptic peptides obtained from the reduced alkylated enzyme were purified and their amino acid sequences determined chiefly by the ‘Edman-dansylation’; procedure, and in two cases by partial acid or peptic hydrolyses. The tryptic peptides are alined into a single chain containing 129 amino acid residues, on the basis of overlapping peptides. Two labile glutamine residues easily converted into glutamic acid residues were characterized. Human milk lysozyme is compared with other human lysozymes (from normal and leuchaemic individuals) prepared by our group. The structure proposed is identical with the sequence of human leuchaemia lysozyme (from the urine of a patient with chronic monocytic leuchaemia) reported by Canfield. Human milk lysozyme is also near by related to several bird egg-white lysozymes (and bovine α-lactalbumin): Identical positions of Cys and Trp residues and of the residues essential for the catalytic activity or involved in some hydrogen bonds; several identical regions, especially in the β-sheet region; between 71 and 77 identical amino acid residues. It is suggested that by an insertion and a deletion in the sequence of human milk lysozyme, sequences homologous to those of bird lysozymes can be obtained.     

L‐Valine assisted distinction between the stereo‐isomers of D‐hexoses by positive ion ESI tandem mass spectrometry

The binary mixtures of 7 hexoses and 20 amino acids were investigated by electrospray ionization ion trap mass spectrometry (ESI‐ITMS). The adduct ions of the amino acid and the hexose were detected for 12 amino acids but not for the other 8 amino acids which are basic acidic amino acids and amides. The ions of amino acid–hexose complexes were further investigated by tandem mass spectrometry (MS/MS), and some of them just split easily into two parts whereas the others gave rich fragmentation, such as the complex ions of isoleucine, phenylalanie, tyrosine, and valine. We found that hexoses could be complexed by two molecules of valine but only by one molecule of the other amino acids. Among seven kinds of valine–hexose complexes coordinated by potassium ion, the MS² spectra of the ion at m/z 453 yielded unambiguous differentiation. And the fragmentation ions are sensitive to the stereochemical differences at the carbon‐4 of hexoses in the complexes, as proved by the MS². Copyright © 2010 John Wiley & Sons, Ltd.     

Nucleo‐β‐Amino Acids: Synthesis and Oligomerization to β‐Homoalanyl‐PNA

The synthesis of thyminyl‐, uracilyl‐, cytosinyl‐, and guaninyl‐β³‐amino acids and the oligomerization of the cytosinyl‐ and guaninyl‐β³‐amino acids to β‐homoalanyl‐PNA are presented. The pyrimidinyl nucleobases were connected to the γ‐position of β‐homoalanine by Mitsunobu reaction with a β‐homoserine derivative or by nucleophilic substitution of methanesulfonates. For the preparation of the guaninyl‐β³‐amino acid, a β‐lactam route was established that might be of interest also for the synthesis of other β³‐amino acid derivatives. The cytosinyl and guaninyl building blocks were oligomerized to hexamers. They form quite stable self‐pairing complexes in H₂O as indicated by temperature dependent UV and CD spectroscopy.     

Les β-cétonitriles groupes protecteurs de la fonction amine. Préparation d'amino-alcools

β-Ketonitrile-Derived Protecting Groups of the Amino Function. Synthesis of Amino Alcohols The amino group of natural L -amino acid esters is protected by condensation with 2-oxocyclopentanenitrile ( 1 ) or 2-formyl-2-phenylacetonitrile ( 10 ). Only the ester group of the formed cyanoenamino esters 2 and 11 reacts with nucleophilic reagents such as organometallics (RMgX, RLi), borohydrides, or metal amides, whereas the cyanoenamino group is unchanged (Schemes 1 and 2). Cyanoenamino alcohols obtained by reduction of cyanoenamino esters 2 are hydrolyzed under acidic conditions to amino alcohols with retention of the configuration of the starting amino acid. This sequence of reactions allows to prepare derivatives of L -tyrosinol from (?)-L -tyrosine (see, e.g., Scheme 4). Cyanoenamino esters 11 are readily methylated at the N-atom to give N-methylated cyanoenamino esters (Scheme 3). This property is exploited on the way of a multistep procedure to obtain N-methylated amino alcohols homologous to natural (?)-(1R,2S)-ephedrine.     

Oxazoline chemistry part III. Synthesis and characterisation of [2‐(2′‐anilinyl)‐2‐oxazolines] and some related compounds

The syntheses and characterisation of a series of chiral and achiral 2‐(aminophenyl)‐2‐oxazolines and some related compounds is reported. All of the derivatives have been produced by a one‐step procedure involving the treatment of isatoic anhydride (i.e. [2H]‐3, 1‐benzoxazine‐[1H‐2,4‐dione: 1 ) or its 5‐chloro analogue with a slight excess of appropriate amino‐alcohols. In most cases, anhydrous ZnCl₂ is shown to be an effective Lewis acid catalyst for this reaction at reflux temperature in high boiling aromatic solvents (PhCl or PhMe). Oxazolines have been readily formed using rac‐2‐amino‐1‐butanol, (S)‐phenylglycinol, 2‐methyl‐2‐amino‐1‐propanol and (1S,2R) or (IR,2S)‐cis‐ 1 ‐amino‐2‐indanol; yields range from 85% to 22%. The use of aminoalcohols such as 2‐ethanolamine, (±)‐2‐amino‐1‐phenyl‐1‐propanol or 3‐amino‐1‐propanol (to give the corresponding 4,5‐dihydro‐1,3‐oxazine) results in poor yields. The use of other Lewis acid catalysts (silicic acid, Cd(acac)₂·2H₂O, CuCl₂·2H₂O, InCl₃) or higher temperatures did not improve the yields with these latter two substrates. Benzoxazoles and N‐substituted benzoxazoles can also be obtained in reasonable yields from 1 using 2‐aminophenol (36%) or 2‐amino‐3‐hydroxypyridine (45%).     

Cloning and Characterization of a Novel Aspartic Protease Gene from Marine-Derived <Emphasis Type="Italic">Metschnikowia reukaufii</Emphasis> and its Expression in <Emphasis Type="Italic">E. coli</Emphasis>

Metschnikowia reukaufii W6b isolated from marine environment was found to produce a cell-bound acid protease. The full-length cDNA (cDNASAP6 gene) of the acid protease (SAP6) from the marine-derived yeast M. reukaufii W6b was cloned. The insert was 1,755-bp long and contained an open reading frame of 1,527-bp encoding 508 amino acids. The deduced amino acid sequence included a signal peptide of 16 amino acids. The consensus motifs contained a VLLDTGSSDLRM active site and an ALLDSGTTITQF active site. The protein sequence deduced from the cDNASAP6 gene exhibited 12.9% overall identity with Cwp1 of Saccharomyces cerevisiae and a hydropathy profile characteristic of glycosylphosphatidylinositol cell-wall proteins. The cDNASAP6 gene without 48 bp encoding the signal peptide sequence was subcloned into an expression plasmid pET-24a (+) and fused with a 6-His Tag and transformed into Escherichia coli BL21 (DE3) for recombinant expression of the protease. The expressed fusion protein was found to have a unique band with molecular mass of about 54 kDa. The crude acid protease of the culture of the marine yeast strain W6b and the crude recombinant acid protease had milk clotting activity.     

Structural and catalytic aspects of cutinase in w/o microemulsions

 Structural and catalytic properties of cutinase were studied in bis(2-ethylhexyl) sodium sulfo-succinate (AOT)-isooctane microemulsion systems. The effect of the water content of the microemulsions on the cutinase activity on an esterification reaction of lauric acid with pentanol showed that cutinase followed a bell-shaped profile presenting a maximum at w _o=9, with w _o=[H₂O]/[AOT]. Kinetic studies allowed the determi-nation of the apparent parameters K _m and V _max. Electron paramagnetic resonance (EPR) spectroscopy studies of active site labeled cutinase in microemulsions with varying w _o values showed that in all microemulsions, the mobility of the label is higher than in the aqueous solution. Furthermore, it was found that the maximum of the enzyme activity did not correspond to a reduced active site mobility. Up to w _o=9 there was an increase of both activity and active site mobility. As the water content of the system became higher, the mobility of the bound spin label further increased whereas the enzymatic activity dropped considerably. Received: 20 December 1996 Accepted: 24 February 1997     

Cloning and Identification of a Novel P-II Class Snake Venom Metalloproteinase from <Emphasis Type="Italic">Gloydius halys</Emphasis>

Ahpfibrase was a new snake venom metalloproteinase (SVMP) which was cloned from Gloydius halys. The cDNA sequence with 1,891 base pairs encodes an open reading frame of 477 amino acids which includes a 17 amino acid signal peptide, plus a 171 amino acid segment of zymogen-like propeptide, a metalloproteinase domain of 200 amino acids, a spacer of 16 amino acids, and a disintegrin-like peptide of 73 amino acids. The metalloproteinase domain contained a conserved signature zinc-binding motif HEXXHXXGXXH in the catalytic region and a methionine-turn CIM. To determine the activity of ahpfibrase, the coding region including both the metalloproteinase domain and disintegrin region was amplified by PCR, inserted into the pET25b(+) vector, and expressed in Escherichia coli. The recombinant protein was recovered from inclusion bodies with 8 M urea and refolding was performed by fed-batch dilution method, and purified recombinant ahpfibrase showed the fibrinolytic activity and platelet aggregation–inhibition ability.     

Amino Acid Sequence of N-Terminal Peptide of Normal Human Serum Albumin

From the peptic digest of normal human serum albumin., the N-terminal peptide comprising 24 amino acid residues was obtained by means of peptide mapping. Combined uses of trypsin, α-chymotrypsin, thermolysin, carboxypeptidase A and Dansyl-Edman technique resulted in the elucidation of amino acid sequence of no. 1 to no. 24 as follows: NH₂-Asp-Ala-His-Lys-Ser-Glu-Val-Ala-His-Arg-Phe-Lys-Asp-Leu-GIy-Glu-Glu-Asn-Phe-Lys-Ala-Leu-Val-Leu-COOH These sequence results agree completely with those recently published by other workers.     

Purification, Characterization and 1H NMR Resonance Assignment of an a－Like Neurotoxin BmK 16 from the Venom of Chinese Scor－pion Buthus martensii Karsch

A natural scorpion toxin BmK 16 was purified for the first time from the venom of the Chinese scorpion Buthus martensii Karsch (BmK) by using combined gel-filtration, ion exchange and reversed phase chromatography. The sequence of the N-terminal 8 amino acid residues was determined by Edman degradation. Using the N-terminal sequence as a tag, the database searching revealed a hit in the scorpion cDNA Bank. The sequence for N-terminal 8 amino acid residues, molecular weight and amino acid compositions of BmK 16 were identical with the calculated values according to the first 64 residues‘ se-quence of the precursor peptide alpha-neurotoxin TX16 derived from the sequence of the cDNA AF156597 (EMBL). The se-quence-specific resonance assignment of BmK 16 was achieved and the intact sequence of BmK 16 was determined as follow-ings: VRDAY IAKPH NCVYE CARNE YCNDL CTKNG AKSGY CQWVG KYGNG CWCKE LPDNV PIRVP GKCH. Furthermore, the results from the sequence homology analysis and the toxicity assays indicated that BmK 16 was an α-likescorpion neurotoxin.     

Stapled Peptides with γ‐Methylated Hydrocarbon Chains for the Estrogen Receptor/Coactivator Interaction

“Stapled” peptides are typically designed to replace two non‐interacting residues with a constraining, olefinic staple. To mimic interacting leucine and isoleucine residues, we have created new amino acids that incorporate a methyl group in the γ‐position of the stapling amino acid S5. We have incorporated them into a sequence derived from steroid receptor coactivator 2, which interacts with estrogen receptor α. The best peptide (IC₅₀=89 nm ) replaces isoleucine 689 with an S‐γ‐methyl stapled amino acid, and has significantly higher affinity than unsubstituted peptides (390 and 760 nm ). Through X‐ray crystallography and molecular dynamics studies, we show that the conformation taken up by the S‐γ‐methyl peptide minimizes the syn‐pentane interactions between the α‐ and γ‐methyl groups.