Overexpression and molecular characterization of Aga50D from Saccharophagus degradans 2-40: an exo-type β-agarase producing neoagarobiose

β-Agarases are mostly categorized into three glycoside hydrolase (GH) families 16, 50, and 86. Recent genomic analysis of Saccharophagus degradans 2–40 revealed the presence of five agarase genes belonging to these GH families. Among the five agarases, Aga50D (a member of GH50) had neither been functionally characterized nor overexpressed. In this report, we present soluble overexpression and molecular characterization of Aga50D. Aga50D was expressed in an active form resulting in a single major product from agarose without intermediates. While known GH50 agarases have both endo-lytic and exo-lytic activities, which produce neoagarobiose as a final product through the intermediate, neoagaro-oligosaccharides, identification and analysis of the reaction product by mass spectrometry and ¹³C NMR showed that Aga50D had unique exo-lytic activity and was able to produce neoagarobiose directly from agarose. The optimum pH and temperature for the activity were 7.0 and 30°C, respectively. The K _m and V _max for agarose were 41.9 mg/ml (4.2 mM) and 17.9 U/mg, respectively.     

Crystal structure of a key enzyme in the agarolytic pathway, α-neoagarobiose hydrolase from Saccharophagus degradans 2-40

In agarolytic microorganisms, α-neoagarobiose hydrolase (NABH) is an essential enzyme to metabolize agar because it converts α-neoagarobiose (O-3,6-anhydro-alpha-l-galactopyranosyl-(1,3)-d-galactose) into fermentable monosaccharides (d-galactose and 3,6-anhydro-l-galactose) in the agarolytic pathway. NABH can be divided into two biological classes by its cellular location. Here, we describe a structure and function of cytosolic NABH from Saccharophagus degradans 2–40 in a native protein and d-galactose complex determined at 2.0 and 1.55 Å, respectively. The overall fold is organized in an N-terminal helical extension and a C-terminal five-bladed β-propeller catalytic domain. The structure of the enzyme–ligand (d-galactose) complex predicts a +1 subsite in the substrate binding pocket. The structural features may provide insights for the evolution and classification of NABH in agarolytic pathways.     

Cloning, expression and characterization of a thermostable exo-β-D-glucosaminidase from the hyperthermophilic archaeon Pyrococcus horikoshii

An exo-β-d-glucosaminidase gene (PH0511) was cloned from the hyperthermophilic archaeon, Pyrococcus horikoshii, and expressed in Escherichia coli. The purified protein showed a strong exo-β-d-glucosaminidase activity by TLC analysis. DTT (50 mM) had little effect on its homodimeric structure during SDS-PAGE. The enzyme was optimally active at 90°C (over 20 min) and pH 6. It had a half-life of 9 h at 90°C and is the most thermostable glucosaminidase described up to now. The activity was not inhibited by ethanol, 2-propanol, DMSO, PEG-400, denaturing agents SDS (5%, w/v), urea, guanidine hydrochloride (5 M) and Mg²⁺, Mn²⁺, Co²⁺, Ca²⁺, Sr²⁺, Ni²⁺ (at up to 10 mM).     

Plasminogen Substrate Recognition by the Streptokinase-Plasminogen Catalytic Complex Is Facilitated by Arg253, Lys256, and Lys257 in the Streptokinase ��-Domain and Kringle 5 of the Substrate

Streptokinase (SK) conformationally activates the central zymogen of the fibrinolytic system, plasminogen (Pg). The SK·Pg* catalytic complex binds Pg as a specific substrate and cleaves it into plasmin (Pm), which binds SK to form the SK·Pm complex that propagates Pm generation. Catalytic complex formation is dependent on lysine-binding site (LBS) interactions between a Pg/Pm kringle and the SK COOH-terminal Lys⁴¹⁴. Pg substrate recognition is also LBS-dependent, but the kringle and SK structural element(s) responsible have not been identified. SK mutants lacking Lys⁴¹⁴ with Ala substitutions of charged residues in the SK β-domain 250-loop were evaluated in kinetic studies that resolved conformational and proteolytic Pg activation. Activation of [Lys]Pg and mini-Pg (containing only kringle 5 of Pg) by SK with Ala substitutions of Arg²⁵³, Lys²⁵⁶, and Lys²⁵⁷ showed decreases in the bimolecular rate constant for Pm generation, with nearly total inhibition for the SK Lys²⁵⁶/Lys²⁵⁷ double mutant. Binding of bovine Pg (BPg) to the SK·Pm complex containing fluorescently labeled Pm demonstrated LBS-dependent assembly of a SK·labeled Pm·BPg ternary complex, whereas BPg did not bind to the complex containing the SK Lys²⁵⁶/Lys²⁵⁷ mutant. BPg was activated by SK·Pm with a K_m indistinguishable from the K_D for BPg binding to form the ternary complex, whereas the SK Lys²⁵⁶/Lys²⁵⁷ mutant did not support BPg activation. We conclude that SK residues Arg²⁵³, Lys²⁵⁶, and Lys²⁵⁷ mediate Pg substrate recognition through kringle 5 of the [Lys]Pg and mini-Pg substrates. A molecular model of the SK·kringle 5 complex identifies the putative interactions involved in LBS-dependent Pg substrate recognition.Streptokinase (SK)⁶ activates the human fibrinolytic system by activating plasminogen (Pg) through a unique mechanism that is responsible for the use of SK as a thrombolytic drug and its role as a key pathogenicity factor in Group A streptococcal infection (1, 2). The crystal structure of SK bound to the catalytic domain of plasmin (μPm) shows that SK consists of three β-grasp, tightly folded domains, α, β, and γ, linked by flexible segments (3). In solution, SK is highly flexible and behaves hydrodynamically like three beads on a string (4). When bound to μPm, SK assumes a highly ordered structure resembling a three-sided crater surrounding the catalytic site that provides an exosite(s) for binding the catalytic domain of Pg as a substrate (3, 5). In the first step of the SK-mediated Pg activation pathway, SK binds the catalytic domain of the Pg zymogen in a rapid equilibrium process and inserts its NH₂-terminal Ile¹ residue into the NH₂-terminal binding cleft of Pg, activating the catalytic site nonproteolytically (6–10). Although structural proof is lacking, SK Ile¹ presumably forms a critical salt bridge with Asp^740(194) (plasminogen numbering; chymotrypsinogen numbering is in parentheses) that initiates conformational activation of the substrate binding site and oxyanion hole required for proteolytic activity (6, 8–10). The activated SK·Pg* complex binds a second molecule of Pg as a specific substrate and cleaves it at Arg^561(15)-Val^562(16) to form the fibrin-degrading proteinase, plasmin (Pm) (10–14). Proteolytic generation of Pm is propagated by formation of a high affinity SK·Pm complex that converts the remaining free Pg into Pm (5, 11).[Glu]Pg, the full-length form of Pg circulating in blood, consists of an NH₂-terminal PAN (Pg/Apple/Nematode (15, 16)) module, followed by five kringle domains (K1–K5), and the trypsin-like serine proteinase catalytic domain (17). Formation of the SK·Pg* and SK·Pm catalytic complexes and Pg substrate binding are inhibited by the lysine analog, 6-aminohexanoic acid (6-AHA), which binds to lysine-binding sites (LBS) located primarily in kringles K1, K4, and K5 of Pg and Pm (10, 11, 18–23). Cleavage of the Lys⁷⁷-Lys⁷⁸ peptide bond in [Glu]Pg by Pm releases the PAN module and generates the truncated form, [Lys]Pg. Formation of [Lys]Pg is accompanied by a conformational change of [Glu]Pg from a compact, closed α-conformation to a partially extended β-conformation with expression of higher affinity LBS for 6-AHA (24, 25). The fourth kringle module mediates a second conformational change, from the β-conformation to the extended γ-conformation (25).Binding of SK to [Glu]Pg is independent of LBS, with a dissociation constant of 100–150 nm, whereas formation of SK·[Lys]Pg is LBS-dependent with a 13–20-fold higher affinity that is reduced to that of [Glu]Pg by saturating concentrations of 6-AHA (10, 21). Activation of the catalytic domain in [Lys]Pm increases affinity for SK about 830-fold, which is reduced 11–20-fold by 6-AHA (5, 21). Interaction of the COOH-terminal Lys⁴¹⁴ residue of SK with a Pg/Pm kringle domain is responsible for the LBS-dependent enhancement of the affinity of SK·[Lys]Pg* and SK·Pm catalytic complex formation (22). Recent rapid reaction kinetic studies of the SK·Pm binding pathway demonstrated that interaction of Lys⁴¹⁴ with a Pm kringle enhances formation of an initial rapid equilibrium SK·Pm encounter complex, succeeded by two sequential, tightening conformational changes, to achieve an overall dissociation constant of ∼12 pm (26). The Pg/Pm kringle domain responsible for the enhancement of SK·Pg* and SK·Pm complex formation is not known. Productive interaction of Pg as a substrate of the SK·Pg*/Pm complexes is also greatly inhibited by saturating 6-AHA (11). Kinetic and equilibrium binding studies of SK-mediated Pm formation resolved the conformational activation process from the coupled proteolytic generation of Pm (10, 11). The kinetic approach demonstrated that Lys⁴¹⁴ deletion reduced the affinity of formation of the SK·Pg* catalytic complex specifically, whereas the subsequent LBS-dependent proteolytic formation of Pm was unaffected, indicating that Pg substrate recognition is mediated by a structurally distinct region of SK and an unknown kringle (22).Previous structure-function studies have yielded diverse interpretations and conclusions regarding the structural basis of LBS-dependent Pg substrate recognition (23, 27–34). Each of the three domains of SK has been implicated in this regard (29, 30, 35, 36), and binding of two Pg molecules to the residue 1–59 sequence of the α-domain has been reported (36). In particular, segments 16–36, 41–48, 48–59, and 88–97 of the SK α-domain have been concluded to play a role in Pg substrate recognition (32, 33, 37, 38). For several SK mutants, a complex mixture of functional effects on their binding to [Glu]Pg and its conformational and proteolytic activation has been reported (28, 31, 33). Some of these effects may result from the inherent flexibility of SK when bound to Pg or Pm (39), and others may be due to the use of kinetic approaches that do not clearly discriminate between conformational and proteolytic activation.Some observations implicate a protruding hairpin loop called the 250-loop (residues Ala²⁵¹–Ile²⁶⁴) in the SK β-domain in Pg substrate recognition (27, 28, 31, 34). This loop is disordered in the structure of the SK·μPm complex but is ordered in the structure of the isolated β-domain (3, 40). Deletion of the 250-loop, Ala substitution of Lys²⁵⁶ and Lys²⁵⁷ at the apex of the loop, and substitution of multiple residues near and within the loop resulted in disparate effects on K_m and k_cat for [Glu]Pg activation (27, 28, 31). The conclusions of these studies were that Lys²⁵⁶ and Lys²⁵⁷ are involved in SK binding and conformational activation of [Glu]Pg in addition to proteolytic processing of Pg as a substrate. Some of these studies are problematic because the natural NH₂-terminal Ile¹ residue necessary for conformational activation is preceded either by an additional methionine (27, 31) or maltose-binding protein (28) in the recombinant SK species used.Because of the diverse conclusions regarding the functional properties of the 250-loop mutations and the possibility of other potential Pg substrate binding sites, the present studies were undertaken to resolve the function of residues in the 250-loop in LBS-dependent Pg substrate recognition by the SK·Pg* complex. The kringle domain of Pg involved in Pg substrate recognition has not been clearly identified but has been suggested to be K5 (27) on the basis that the isolated β-domain bound Pg (30) and K5 (29) in an LBS-dependent manner. Given the general specificity of Pg kringles for COOH-terminal Lys residues and zwitterionic ligands, such as 6-AHA, and the internal sequence of the 250-loop, it appeared possible that a pseudolysine motif on SK was involved. In the binding of a 30-residue peptide from plasminogen binding Group A streptococcal M-like protein (PAM), VEK-30, to K2 of Pg, Castellino and co-workers (41, 42) showed by crystallography and mutagenesis that residues with cationic (Arg and His) and anionic side chains (Glu) arranged spatially on a helix constituted a pseudolysine structure similar to 6-AHA that binds specifically to the LBS of K2. Additional evidence for pseudolysine structures in Pg binding comes from studies of α-enolase from Streptococcus pneumoniae, which has a 9-residue internal binding site for Pg containing essential basic (two Lys residues) and acidic (Asp and Glu residues) located on a surface loop (43, 44).To determine whether a similar SK structure is involved in [Lys]Pg substrate recognition, anionic and cationic residues in the 250-loop were substituted with Ala and characterized in kinetic studies using methods that resolve conformational and proteolytic activation. Studies with [Lys]Pg and mini-Pg, which contains only K5 and the catalytic domain, showed that Arg²⁵³, Lys²⁵⁶, and Lys²⁵⁷ facilitate LBS-dependent substrate recognition through interactions with K5. The absence of evidence for a pseudolysine structure in the 250-loop is compatible with the established atypical specificity of K5 for cationic ligands, such as benzamidine, N^α-acetyl-Lys-methyl ester, 6-aminohexane, and 5-aminopentane, in addition to zwitterionic ligands (19, 45–47). The studies resolve for the first time the structural features of SK that mediate the LBS-dependent interactions that enhance affinity of SK·Pg* and SK·Pm catalytic complex formation and those that facilitate binding of Pg as a substrate of these complexes.     

d-Xylose Isomerase from a Marine Bacterium, Vibrio sp. Strain XY-214, and d-Xylulose Production from β-1,3-Xylan

The xylA gene from a marine bacterium, Vibrio sp. strain XY-214, encoding d-xylose isomerase (XylA) was cloned and expressed in Escherichia coli. The xylA gene consisted of 1,320-bp nucleotides encoding a protein of 439 amino acids with a predicted molecular weight of 49,264. XylA was classified into group II xylose isomerases. The native XylA was estimated to be a homotetramer with a molecular mass of 190 kDa. The purified recombinant XylA exhibited maximal activity at 60°C and pH 7.5. Its apparent K _m values for d-xylose and d-glucose were 7.93 and 187 mM, respectively. Furthermore, we carried out d-xylulose production from β-1,3-xylan, a major cell wall polysaccharide component of the killer alga Caulerpa taxifolia. The synergistic action of β-1,3-xylanase (TxyA) and β-1,3-xylosidase (XloA) from Vibrio sp. strain XY-214 enabled efficient saccharification of β-1,3-xylan to d-xylose. d-Xylose was then converted to d-xylulose by using XylA from the strain XY-214. The conversion rate of d-xylose to d-xylulose by XylA was found to be approximately 40% in the presence of 4 mM sodium tetraborate after 2 h of incubation. These results demonstrated that TxyA, XloA, and XylA from Vibrio sp. strain XY-214 are useful tools for d-xylulose production from β-1,3-xylan. Because d-xylulose can be used as a source for ethanol fermentation by yeast Saccharomyces cerevisiae, the present study will provide a basis for ethanol production from β-1,3-xylan.     

Hydrolysis of phosphohydroxypyruvate and β-glycerophosphate by a phosphatase preparation from beef brain

A phosphatase preparation has been partially purified from beef brain which catalyzes the hydrolysis of phosphohydroxypyruvic acid, β-glycerophosphate, glucose-6-phosphate, and fructose-6-phosphate. The pH optimum for the first two substrates is in the range of 7.5. Magnesium ions are required for optimal activity, and the reaction is inhibited by fluoride and manganese. The enzyme preparation has negligible activity with phosphoserine, p-nitrophenylphosphate, 5′-mononucleotides, 3-phosphoglycerate, and phosphoethanolamine. The K_m values for phosphohydroxypyruvate and dl-β-glycerophosphate are 7 × 10⁻³M and 6 × 10⁻⁴M, respectively.     

A new systematic strategy for the isolation of proteins, illustrated by the purification of a mammalian exo-β-N-acetyl-d-glucosaminidase

The ;B' form of the exo-beta-N-acetyl-d-glucosaminidase from pig epididymis was purified by a new systematic strategy to yield a preparation with a specific activity at least equal to that obtainable by an existing empirically derived procedure. The new strategy is essentially a sequence of three carefully chosen steps consisting of an initial fractionation of the constituent proteins according to molecular size, followed by an ion-exchange step designed to select out proteins with closely similar electric-charge properties to those of the protein of interest, and a final high-resolution step dependent on the isoelectric points of the residual proteins. Gel isoelectric focusing itself, or as an element in the technique described by Leaback & Robinson (1974) for the separate display of the molecular size and electric-charge characteristics of proteins, played an important part in the choice of the experimental conditions used in the new strategy, and also in monitoring the progress of the purification.     

Characterization of a Glucose-, Xylose-, Sucrose-, and d-Galactose-Stimulated ��-Glucosidase from the Alkalophilic Bacterium Bacillus halodurans C-125

The gene (Bhbgl) encoding a β-glucosidase from the alkalophilic bacterium Bacillus halodurans C-125 was synthesized chemically via the PCR-based two-step DNA synthesis (PTDS) method and expressed in Escherichia coli. Bhbgl contained an open reading frame (ORF) of 1359 bp encoding a 453-amino acid protein belonging to glycoside hydrolase family 1 (GHF1), and the deduced molecular mass of recombinant Bhbgl (52,488 Da) was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited a high specific activity with o-nitrophenyl-β-D-glucopyranoside (oNPGlu) and an apparent K (m) value of 0.32 mM. With oNPGlu as the substrate, Bhbgl displayed pH and temperature optima of ~7.0 and 50°C, respectively. The enzyme was relatively stable under alkaline conditions and >50% activity was retained after incubation at pH 9.5 for 24 h at 4°C. Recombinant Bhbgl activity was inhibited by 5 mM Zn(2+), Fe(3+), or Cd(2+), but was enhanced by 1 mM Mg(2+) and other metal ions. Enzyme activity was also stimulated by at least four sugars (sucrose, D-galactose, xylose, glucose) at concentrations ranging from 50 to 800 mM.     

Hydrolysis of natural and synthetic substrates by α-l-fucosidase, β-d-glucuronidase and β-n-acetylhexosaminidase purified from molluscs

• 1.1. Several mollusc glycosidases have been studied for their activities towards natural substrates. α-l-Fucosidases from Chamelea gallina, Tapes rhomboideus and Mytilus edulis hydrolyze oligosaccharides (di, tri and pentasaccharides) with α1 → 2, α1 → 3 and α1 → 4 bonds, fucose-containing glycopeptides from bovine thyroglobulin and the porcine submandibular mucin (devoid of sialic acid); α-l-fucosidase from Littorina littorea hydrolyzes fucose-containing glycopeptides from bovine thyroglobulin.
• 2.2. β-d-Glucuronidase from L. littorea hydrolyzes hyaluronic acid, chondroitin 4-sulfate and heparin with a very low activity; however, it is much more active on oligosaccharides (from the above-mentioned macromolecules) containing non-reducing terminal glucuronyl residues.
• 3.3. β-N-Acetylhexosaminidase from Helicella ericetorum acts mainly with an endo-hydrolase activity on β1 → 4N-acetylhexosamine linkages of ovalbumin, ovomucoid, chitin, hyaluronic acid and chondroitin
• 4.4-sulfate; it has also a secondary exo-hydrolase activity on these substrates.

     

Phylogenetic and Kinetic Characterization of a Suite of Dehydrogenases from a Newly Isolated Bacterium,Strain SG61-1L,That Catalyze the Turnover of Guaiacylglycerol-β-Guaiacyl Ether Stereoisomers

Lignin is a complex aromatic polymer found in plant cell walls that makes up 15 to 40% of plant biomass. The degradation of lignin substructures by bacteria is of emerging interest because it could provide renewable alternative feedstocks and intermediates for chemical manufacturing industries. We have isolated a bacterium, strain SG61-1L, that rapidly degrades all of the stereoisomers of one lignin substructure, guaiacylglycerol-β-guaiacyl ether (GGE), which contains a key β-O-4 linkage found in most intermonomer linkages in lignin. In an effort to understand the rapid degradation of GGE by this bacterium, we heterologously expressed and kinetically characterized a suite of dehydrogenase candidates for the first known step of GGE degradation. We identified a clade of active GGE dehydrogenases and also several other dehydrogenases outside this clade that were all able to oxidize GGE. Several candidates exhibited stereoselectivity toward the GGE stereoisomers, while others had higher levels of catalytic performance than previously described GGE dehydrogenases for all four stereoisomers, indicating a variety of potential applications for these enzymes in the manufacture of lignin-derived commodities.     

Molecular Basis for the Recognition and Cleavages of IGF-II, TGF-α, and Amylin by Human Insulin-Degrading Enzyme

Insulin-degrading enzyme (IDE) is involved in the clearance of many bioactive peptide substrates, including insulin and amyloid-β, peptides vital to the development of diabetes and Alzheimer's disease, respectively. IDE can also rapidly degrade hormones that are held together by intramolecular disulfide bond(s) without their reduction. Furthermore, IDE exhibits a remarkable ability to preferentially degrade structurally similar peptides such as the selective degradation of insulin-like growth factor (IGF)-II and transforming growth factor-α (TGF-α) over IGF-I and epidermal growth factor, respectively. Here, we used high-accuracy mass spectrometry to identify the cleavage sites of human IGF-II, TGF-α, amylin, reduced amylin, and amyloid-β by human IDE. We also determined the structures of human IDE-IGF-II and IDE-TGF-α at 2.3 Å and IDE-amylin at 2.9 Å. We found that IDE cleaves its substrates at multiple sites in a biased stochastic manner. Furthermore, the presence of a disulfide bond in amylin allows IDE to cut at an additional site in the middle of the peptide (amino acids 18-19). Our amylin-bound IDE structure offers insight into how the structural constraint from a disulfide bond in amylin can alter IDE cleavage sites. Together with NMR structures of amylin and the IGF and epidermal growth factor families, our work also reveals the structural basis of how the high dipole moment of substrates complements the charge distribution of the IDE catalytic chamber for the substrate selectivity. In addition, we show how the ability of substrates to properly anchor their N-terminus to the exosite of IDE and undergo a conformational switch upon binding to the catalytic chamber of IDE can also contribute to the selective degradation of structurally related growth factors.     

Identification and Characterization of a Halotolerant,Cold-Active Marine Endo-β-1,4-Glucanase by Using Functional Metagenomics of Seaweed-Associated Microbiota

A metagenomic library was constructed from microorganisms associated with the brown alga Ascophyllum nodosum. Functional screening of this library revealed 13 novel putative esterase loci and two glycoside hydrolase loci. Sequence and gene cluster analysis showed the wide diversity of the identified enzymes and gave an idea of the microbial populations present during the sample collection period. Lastly, an endo-β-1,4-glucanase having less than 50% identity to sequences of known cellulases was purified and partially characterized, showing activity at low temperature and after prolonged incubation in concentrated salt solutions.     

β-cyclodextrin production by the cyclodextrin glucanotransferase from Paenibacillus illinoisensis ZY-08: cloning, purification, and properties

The gene encoding the cyclodextrin glucanotransferase (CGTase, EC2.4.1.19) of Paenibacillus illinoisensis was isolated, cloned, sequenced and expressed in Escherichia coli. Sequence analysis showed that the mature enzyme (684 amino acids) was preceded by a signal peptide of 34-residues. The deduced amino acid sequence of the CGTase from P. illinoisensis ZY-08 exhibited highest identity (99 %) to the CGTase sequence from Bacillus licheniformis (P14014). The four consensus regions of carbohydrate converting domain and Ca²⁺ binding domain could be identified in the sequence. The CGTase was purified by using cold expression vector, pCold I, and His-tag affinity chromatography. The molecular weight of the purified enzyme was about 74 kDa. The optimum temperature and pH of the enzyme were 40 °C and pH 7.4, respectively. The enzyme activity was increased by the addition of Ca²⁺ and inhibited by Ba²⁺, Cu²⁺, and Hg²⁺. The K _m and V _max values calculated were 0.48 mg/ml and 51.38 mg of β-cyclodextrin/ml/min. The ZY-08 and recombinant readily converted soluble starch to β-cyclodextrin but ZY-08 did not convert king oyster mushroom powder and enoki mushroom powder. However the recombinant CGTase converted king oyster mushroom powder and enoki mushroom powder to β-cyclodextrin.     

Inhibition of penicillium digitatum and penicillium italicum in vitro and in planta with Panomycocin, a novel exo-β-1,3-glucanase isolated from Pichia anomala NCYC 434

Panomycocin, a novel exo-beta 1,3 glucanase, was tested as an antifungal agent against green and blue mold diseases, the most important causes of post harvest decay in citrus fruits. All tested isolates of Penicillium digitatum and Penicillium italicum were susceptible to panomycocin in vitro. Effective panomycocin concentrations for 50% growth inhibition (MIC-2) for P. digitatum and P. italicum were 2 and 1 μg ml⁻¹, respectively. Complete (MIC-0) growth inhibition of all isolates observed at a panomycocin concentration of 16 μg ml⁻¹. Treatment of spores with panomycocin at values lower than the MIC-0 led to slower germ tube elongation and mycelium growth. In tests on fruit, panomycocin at concentrations equal to in vitro MIC-0 value protected lemon fruit from decay.