In silico mining and identification of a novel lipase from Paenibacillus larvae: Rational protein design for improving catalytic performance.

Lipases play a vital role in various biological processes, from lipid metabolism to industrial applications. However, the ever-evolving challenges and diverse substrates necessitate the continual exploration of novel high-performance lipases. In this study, we employed an in silico mining approach to search for lipases with potential high sn-1,3 selectivity and catalytic activity. The identified novel lipase, PLL, from Paenibacillus larvae subsp. larvae B-3650 exhibited a specific activity of 111.2 ± 5.5 U/mg towards the substrate p-nitrophenyl palmitate (pNPP) and 6.9 ± 0.8 U/mg towards the substrate olive oil when expressed in Escherichia coli (E. coli). Computational design of cysteine mutations was employed to enhance the catalytic performance of PLL. Superior stability was achieved with the mutant K7C/A386C/H159C/K108C (2M3/2M4), showing an increase in melting temperature (Tm) by 1.9°C, a 2.05-fold prolonged half-life at 45°C, and no decrease in enzyme activity. Another mutant, K7C/A386C/A174C/A243C (2M1/2M3), showed a 4.9-fold enhancement in specific activity without compromising stability. Molecular dynamics simulations were conducted to explore the mechanisms of these two mutants. Mutant 2M3/2M4 forms putative disulfide bonds in the loop region, connecting the N- and C-termini of PLL, thus enhancing overall structural rigidity without impacting catalytic activity. The cysteines introduced in mutant 2M1/2M3 not only form new intramolecular hydrogen bonds but also alter the polarity and volume of the substrate-binding pocket, facilitating the entry of large substrate pNPP. These results highlight an efficient in silico exploration approach for novel lipases, offering a rapid and efficient method for enhancing catalytic performance through rational protein design.

Effect of microbial biostimulants on the antioxidant profile, antioxidant capacity and activity of enzymes influencing the quality level of raspberries (Rubus idaeus L.).

The influence of four microbial biostimulants containing various strains of Bacillus subtilis and/or Paenibacillus sp. on the quality of raspberries cv. Delniwa, Poemat, and Enrosadira cultivated in two consecutive seasons was investigated. The biostimulants influenced the antioxidant level, antioxidant capacity, phenolic acids and flavonoids profiles, enzymatic activity, and the degree of methylation and acetylation of the pectin in the raspberry fruits. The biostimulants had the greatest effect on the antioxidant content (16% - 20% increase) and capacity in the Delniwa raspberry fruits from the first season. A positive correlation was found between the activity of the ß-galactosidase enzyme and ferric reducing power. In the second season, a decrease in the activity of pectin esterase and α-L-arabinofuranosidase and an increase in the degree of methylation of pectin were noted. Our results suggest that the changes in raspberry quality were related to the type of biostimulant applied.

In vitro effects of alginate lyase SG4 + produced by Paenibacillus lautus alone and combined with antibiotics on biofilm formation by mucoid Pseudomonas aeruginosa.

Alginate is a major extra polymeric substance in the biofilm formed by mucoid Pseudomonas aeruginosa. It is the main proven perpetrator of lung infections in patients suffering from cystic fibrosis. Alginate lyases are very important in the treatment of cystic fibrosis. This study evaluated the role of standalone and in conjugation, effect of alginate lyase of SG4 + isolated from Paenibacillus lautus in enhancing in vitro bactericidal activity of gentamicin and amikacin on mucoid P. aeruginosa. Using Response Surface Methodology (RSM) alginate lyase SG4 + production was optimized in shake flask and there 8.49-fold enhancement in enzyme production. In fermenter, maximum growth (10.15 mg/ml) and alginate lyase (1.46 International Units) production, 1.71-fold was increased using Central Composite Design (CCD). Further, fermentation time was reduced from 48 to 20 h. To the best of our knowledge this is the first report in which CCD was used for fermenter studies to optimize alginate lyase production. The Km and Vmax of purified enzyme were found to be 2.7 mg/ml and 0.84 mol/ml-min, respectively. The half-life (t 1/2) of purified alginate lyase SG4 + at 37 °C was 180 min. Alginate lyase SG4 + in combination with gentamicin and amikacin eradiated 48.4- 52.3% and 58- 64.6%, alginate biofilm formed by P. aeruginosa strains, respectively. The study proves that alginate lyase SG4 + has excellent exopolysaccharide disintegrating ability and may be useful in development of potent therapeutic agent to treat P. aeruginosa biofilms.

In Vitro Characterization of an O-Specific Glycosyltransferase Involved in Flagellin Glycosylation.

Glycosyltransferases play a fundamental role in the biosynthesis of glycoproteins and glycotherapeutics. In this study, we investigated protein glycosyltransferase FlgGT1, belonging to the GT2 family. The GT2 family includes cysteine S-glycosyltransferases involved in antimicrobial peptide biosyntheses, sharing conserved catalytic domains while exhibiting diverse C-terminal domains. Our in vitro studies revealed that FlgGT1 recognizes structural motifs rather than specific amino acid sequences when glycosylating the flagellin protein Hag. Notably, FlgGT1 is selective for serine or threonine O-glycosylation over cysteine S-glycosylation. Molecular dynamics simulations provided insights into the structural basis of FlgGT1's ability to accommodate various sugar nucleotides as donor substrates. Mutagenesis experiments on FlgGT1 demonstrated that truncating the relatively large C-terminal domain resulted in a loss of flagellin glycosylation activity. Our classification based on sequence similarity network analysis and AlphaFold2 structural predictions suggests that the acquisition of the C-terminal domain is a key evolutionary adaptation conferring distinct substrate specificities on glycosyltransferases within the GT2 family.

Highly efficient bioconversion of icariin to icaritin by whole-cell catalysis.

BACKGROUND: Icaritin is an aglycone of flavonoid glycosides from Herba Epimedii. It has good performance in the treatment of hepatocellular carcinoma in clinical trials. However, the natural icaritin content of Herba Epimedii is very low. At present, the icaritin is mainly prepared from flavonoid glycosides by α-L-rhamnosidases and ß-glucosidases in two-step catalysis process. However, one-pot icaritin production required reported enzymes to be immobilized or bifunctional enzymes to hydrolyze substrate with long reaction time, which caused complicated operations and high costs. To improve the production efficiency and reduce costs, we explored α-L-rhamnosidase SPRHA2 and ß-glucosidase PBGL to directly hydrolyze icariin to icaritin in one-pot, and developed the whole-cell catalytic method for efficient icaritin production. RESULTS: The SPRHA2 and PBGL were expressed in Escherichia coli, respectively. One-pot production of icaritin was achieved by co-catalysis of SPRHA2 and PBGL. Moreover, whole-cell catalysis was developed for icariin hydrolysis. The mixture of SPRHA2 cells and PBGL cells transformed 200 g/L icariin into 103.69 g/L icaritin (yield 95.23%) in 4 h in whole-cell catalysis under the optimized reaction conditions. In order to further increase the production efficiency and simplify operations, we also constructed recombinant E. coli strains that co-expressed SPRHA2 and PBGL. Crude icariin extracts were also efficiently hydrolyzed by the whole-cell catalytic system. CONCLUSIONS: Compared to previous reports on icaritin production, in this study, whole-cell catalysis showed higher production efficiency of icaritin. This study provides promising approach for industrial production of icaritin in the future.

Differential ligand-selective control of opposing enzymatic activities within a bifunctional c-di-GMP enzyme.

Cyclic dimeric guanosine monophosphate (c-di-GMP) serves as a second messenger that modulates bacterial cellular processes, including biofilm formation. While proteins containing both c-di-GMP synthesizing (GGDEF) and c-di-GMP hydrolyzing (EAL) domains are widely predicted in bacterial genomes, it is poorly understood how domains with opposing enzymatic activity are regulated within a single polypeptide. Herein, we report the characterization of a globin-coupled sensor protein (GCS) from Paenibacillus dendritiformis (DcpG) with bifunctional c-di-GMP enzymatic activity. DcpG contains a regulatory sensor globin domain linked to diguanylate cyclase (GGDEF) and phosphodiesterase (EAL) domains that are differentially regulated by gas binding to the heme; GGDEF domain activity is activated by the Fe(II)-NO state of the globin domain, while EAL domain activity is activated by the Fe(II)-O2 state. The in vitro activity of DcpG is mimicked in vivo by the biofilm formation of P. dendritiformis in response to gaseous environment, with nitric oxide conditions leading to the greatest amount of biofilm formation. The ability of DcpG to differentially control GGDEF and EAL domain activity in response to ligand binding is likely due to the unusual properties of the globin domain, including rapid ligand dissociation rates and high midpoint potentials. Using structural information from small-angle X-ray scattering and negative stain electron microscopy studies, we developed a structural model of DcpG, providing information about the regulatory mechanism. These studies provide information about full-length GCS protein architecture and insight into the mechanism by which a single regulatory domain can selectively control output domains with opposing enzymatic activities.

Simple stain-free screening method for pectinolytic microorganisms under alkalophilic conditions.

OBJECTIVES: To develop a simple pectin-degrading microorganism screening method. RESULTS: We developed a method utilizing the phenomenon whereby cooling an alkaline agar medium containing pectin causes the agar to become cloudy. This highly simplified method involves culturing the microorganisms on pectin-containing agar medium until colony formation is observed, and subsequent overnight cooling of the agar medium to 4 °C. Using this simple procedure, we successfully identified pectin-degrading microorganisms by observing colonies with halos on the clouded agar medium. We used alkaline pectinase and Bacillus halodurans, which is known to secrete alkaline pectinase, to establish the screening method. We demonstrated the screening of pectin-degrading microorganisms using the developed method and successfully isolated pectin-degrading microorganisms (Paenibacillus sp., Bacillus clausii, and Bacillus halodurans) from a soil sample. CONCLUSIONS: The developed method is useful for identifying pectin-degrading microorganisms.

Structural determination of the sheath-forming polysaccharide of Sphaerotilus montanus using thiopeptidoglycan lyase which recognizes the 1,4 linkage between α-d-GalN and ß-d-GlcA.

Sphaerotilus natans is a filamentous sheath-forming bacterium commonly found in activated sludge. Its sheath is assembled from a thiolic glycoconjugate called thiopeptidoglycan. S. montanus ATCC-BAA-2725 is a sheath-forming member of stream biofilms, and its sheath is morphologically similar to that of S. natans. However, it exhibits heat susceptibility, which distinguishes it from the S. natans sheath. In this study, chemical composition and solid-state NMR analyses suggest that the S. montanus sheath is free of cysteine, indicating that disulfide linkage is not mandatory for sheath formation. The S. montanus sheath was successfully solubilized by N-acetylation, allowing solution-state NMR analysis to determine the sugar sequence. The sheath was susceptible to thiopeptidoglycan lyase prepared from the thiopeptidoglycan-assimilating bacterium, Paenibacillus koleovorans. The reducing ends of the enzymatic digests were labeled with 4-aminobenzoic acid ethyl ester, followed by HPLC. Two derivatives were detected, and their structures were determined. We found that the sheath has no peptides and is assembled as follows: [→4)-ß-d-GlcA-(1→4)-ß-d-Glc-(1→3)-ß-d-GalNAc-(1→4)-α-d-GalNAc-(1→4)-α-d-GalN-(1→]n (ß-d-Glc and α-d-GalNAc are stoichiometrically and substoichiometrically 3-O-acetylated, respectively). Thiopeptidoglycan lyase was thus confirmed to cleave the 1,4 linkage between α-d-GalN and ß-d-GlcA, regardless of the peptide moiety. Furthermore, vital fluorescent staining of the sheath demonstrated that elongation takes place at the tips, as with the S. natans sheath.

Utilization of Seafood Processing By-Products for Production of Proteases by Paenibacillus sp. TKU052 and Their Application in Biopeptides' Preparation.

Microbial fermentation of by-products is a renewable and efficient technique in the development of a range of useful products. In this study, protease synthesis by Paenibacillus sp. TKU052 was carried out on culture media containing some common seafood processing by-products (SPBPs) as the sole source of carbon and nitrogen (C/N). The most suitable C/N nutrition source for the production of proteases was found to be 3.0% (w/v) demineralized crab shells powder (deCSP) and maximal enzyme activity of 4.41 ± 0.16 U/mL was detected on the third day of the culture. Two proteases (P1 and P2) with a similar molecular weight of 31 kDa were successfully isolated and purified from the 3-day deCSP-containing medium. Both P1 and P2 exhibited the highest activity of gelatin hydrolysis at pH 6 and 60 °C. The gelatin hydrolysates catalyzed by Paenibacillus TKU052 proteases were evaluated for biological activities, including 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, angiotensin-I converting enzyme (ACE) inhibition, and prebiotic activities. The gelatin hydrolysates expressed 31.76-43.95% DPPH radical scavenging activity and 31.58-36.84% ACE inhibitory activity, which was higher than those from gelatin. Gelatin hydrolysates also showed the growth-enhancing effect on Bifidobacterium bifidum BCRC 14615 with an increase to 135.70-147.81%. In short, Paenibacillus sp. TKU052 could be a potential strain to utilize crab shell wastes to produce proteases for bio-active peptides' preparation.

Paenibacillus algicola sp. nov., a novel alginate lyase-producing marine bacterium.

A Gram-stain-variable, facultatively anaerobic, endospore-forming, rod-shaped bacterium, designated HB172198T, was isolated from brown alga collected at Qishui Bay, Hainan, PR China. Phylogenetic analysis of 16S rRNA gene sequences indicated that strain HB172198T belonged to the genus Paenibacillus, and the closest phylogenetically related species was Paenibacillus lemnae NBRC 109972T (97.6% similarity). The other 16S rRNA gene sequence similarities were under 97.0%. The whole genome average nucleotide identity value between strain HB172198T and the closest type strain was 75.3% and the in silico DNA-DNA hybridization value was 20.2%. The predominant isoprenoid quinone was menaquinone 7 and the major fatty acids were anteiso-C15:0, C16:0, anteiso-C17:0, iso C16:0 and C16:1 ω11c. The combined phylogenetic relatedness, phenotypic and genotypic features supported the conclusion that strain HB172198T represents a novel species of the genus Paenibacillus, for which the name Paenibacillus algicola sp. nov. is proposed. The type strain is HB172198T (=CGMCC 1.13583T=JCM 32683T).

Extracellular alpha/beta-hydrolase from Paenibacillus species shares structural and functional homology to tobacco salicylic acid binding protein 2.

An alpha/ beta hydrolase annotated as a putative salicylate esterase within the genome of a species of Paenibacillus previously identified from differential and selective growth on Kraft lignin was structurally and functionally characterised. Feruloyl esterases are key to the degradation of lignin in several bacterial species and although this activity was investigated, no such activity was observed. The crystal structure of the Paenibacillus esterase, here denoted as PnbE, was determined at 1.32 Å resolution, showing high similarity to Nicotiana tabacum salicylic acid binding protein 2 from the protein database. Structural similarities between these two structures across the core domains and key catalytic residues were observed, with superposition of catalytic residues giving an RMSD of 0.5 Å across equivalent Cα atoms. Conversely, the cap domains of PnbE and Nicotiana tabacum SABP2 showed greater divergence with decreased flexibility in the PnbE cap structure. Activity of PnbE as a putative methyl salicylate esterase was supported with binding studies showing affinity for salicylic acid and functional studies showing methyl salicylate esterase activity. We hypothesise that this activity could enrich Paenibacillus sp. within the rhizosphere by increasing salicylic acid concentrations within the soil.

Whole genome mining reveals a diverse repertoire of lanthionine synthetases and lanthipeptides among the genus Paenibacillus.

AIM: Lanthionine or methyllanthionine-containing lanthipeptides belongs to ribosomally synthesized and post-translationally modified peptides (RiPPs) family. Recent revolution in sequencing has made available huge genome sequence dataset of micro-organisms. In this study, we performed genome mining of the complete and partial genome sequences of 479 bacteria of the genus Paenibacillus to determine the diversity and distribution of lanthipeptide gene clusters. METHODS AND RESULTS: All genome sequences were annotated by RAST and subsequently analysed by BAGEL and antiSMASH. A total of 221 lanthipeptide gene clusters were identified in 127 strains of the genus Paenibacillus. One hundred and fifty gene clusters were found associated with the production of class I lanthipeptides while 58 and 13 gene clusters were related to class II and class IV lanthipeptide production respectively. Frequency of strains whose genomes encode putative lanthipeptide precursors was 26·5%. CONCLUSIONS: The results of lanthionine synthetases analysis suggested that diversity of lanthipeptides is much more than anticipated, while lanthionine synthetases must have been co-evolved among various species of the genus Paenibacillus. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report showing diversity and distribution of different classes of lanthipeptides among various species of the genus Paenibacillus. This study also reveals the novel lanthipeptide sequences which may be further developed as potential antimicrobials for therapeutic applications.

Combined Drug Resistance Mutations Substantially Enhance Enzyme Production in Paenibacillus agaridevorans.

This study shows that sequential introduction of drug resistance mutations substantially increased enzyme production in Paenibacillus agaridevorans The triple mutant YT478 (rsmG Gln225→stop codon, rpsL K56R, and rpoB R485H), generated by screening for resistance to streptomycin and rifampin, expressed a 1,100-fold-larger amount of the extracellular enzyme cycloisomaltooligosaccharide glucanotransferase (CITase) than the wild-type strain. These mutants were characterized by higher intracellular S-adenosylmethionine concentrations during exponential phase and enhanced protein synthesis activity during stationary phase. Surprisingly, the maximal expression of CITase mRNA was similar in the wild-type and triple mutant strains, but the mutant showed greater CITase mRNA expression throughout the growth curve, resulting in enzyme overproduction. A metabolome analysis showed that the triple mutant YT478 had higher levels of nucleic acids and glycolysis metabolites than the wild type, indicating that YT478 mutant cells were activated. The production of CITase by the triple mutant was further enhanced by introducing a mutation conferring resistance to the rare earth element, scandium. This combined drug resistance mutation method also effectively enhanced the production of amylases, proteases, and agarases by P. agaridevorans and Streptomyces coelicolor This method also activated the silent or weak expression of the P. agaridevorans CITase gene, as shown by comparisons of the CITase gene loci of P. agaridevorans T-3040 and another cycloisomaltooligosaccharide-producing bacterium, Paenibacillus sp. strain 598K. The simplicity and wide applicability of this method should facilitate not only industrial enzyme production but also the identification of dormant enzymes by activating the expression of silent or weakly expressed genes.IMPORTANCE Enzyme use has become more widespread in industry. This study evaluated the molecular basis and effectiveness of ribosome engineering in markedly enhancing enzyme production (>1,000-fold). This method, due to its simplicity, wide applicability, and scalability for large-scale production, should facilitate not only industrial enzyme production but also the identification of novel enzymes, because microorganisms contain many silent or weakly expressed genes which encode novel antibiotics or enzymes. Furthermore, this study provides a new mechanism for strain improvement, with a consistent rather than transient high expression of the key gene(s) involved in enzyme production.

Structural and functional characterisation of multi-copper oxidase CueO from lignin-degrading bacterium Ochrobactrum sp. reveal its activity towards lignin model compounds and lignosulfonate.

The identification of enzymes responsible for oxidation of lignin in lignin-degrading bacteria is of interest for biotechnological valorization of lignin to renewable chemical products. The genome sequences of two lignin-degrading bacteria, Ochrobactrum sp., and Paenibacillus sp., contain no B-type DyP peroxidases implicated in lignin degradation in other bacteria, but contain putative multicopper oxidase genes. Multi-copper oxidase CueO from Ochrobactrum sp. was expressed and reconstituted as a recombinant laccase-like enzyme, and kinetically characterized. Ochrobactrum CueO shows activity for oxidation of ß-aryl ether and biphenyl lignin dimer model compounds, generating oxidized dimeric products, and shows activity for oxidation of Ca-lignosulfonate, generating vanillic acid as a low molecular weight product. The crystal structure of Ochrobactrum CueO (OcCueO) has been determined at 1.1 Å resolution (PDB: 6EVG), showing a four-coordinate mononuclear type I copper center with ligands His495, His434 and Cys490 with Met500 as an axial ligand, similar to that of Escherichia coli CueO and bacterial azurin proteins, whereas fungal laccase enzymes contain a three-coordinate type I copper metal center. A trinuclear type 2/3 copper cluster was modeled into the active site, showing similar structure to E. coli CueO and fungal laccases, and three solvent channels leading to the active site. Site-directed mutagenesis was carried out on amino acid residues found in the solvent channels, indicating the importance for residues Asp102, Gly103, Arg221, Arg223, and Asp462 for catalytic activity. The work identifies a new bacterial multicopper enzyme with activity for lignin oxidation, and implicates a role for bacterial laccase-like multicopper oxidases in some lignin-degrading bacteria. DATABASE: Structural data are available in the PDB under the accession number 6EVG.

Dual substrate-controlled kinase activity leads to polyphosphorylated lasso peptides.

Lasso peptides are characterized by their peculiar lariat knot-like structure. Except for maturation of this fold, post-translational modifications of lasso peptides are rare. However, we recently delineated the biosynthetic pathway of a post-translationally phosphorylated lasso peptide, paeninodin. In this study, further investigation of two kinases revealed their ability to transfer multiple phosphate groups onto precursor peptide substrates, ultimately leading to polyphosphorylated lasso peptides. We found that this polyphosphorylating activity depended on the identity of the phosphate donor and the sequence of the precursor peptide. Our investigations provide new insight into the remarkable strategies for chemical diversification employed by the lasso peptide biosynthetic machinery.

Purification and biochemical characterization of novel acidic chitinase from Paenicibacillus barengoltzii.

The novel chitinase (PbChi67) from the marine bacterium Paenicibacillus barengoltzii CAU904 was purified and biochemically characterized. PbChi67 was purified to apparent homogeneity with 10.2 fold purification and 8.0% recovery yield. The molecular mass of the enzyme was 67.0kDa by SDS-PAGE and 67.9kDa by gel filtration, respectively. PbChi67 was most active at pH 3.5 and was stable within pH 3.0-9.0. The optimal temperature of PbChi67 was 60°C and it was stable up to 55°C with a thermal denaturing half-life of 43min at 65°C. The enzyme exhibited strict substrate specificity towards colloidal chitin and glycol chitin but showed no or trace activities towards other tested substrates. The Km and Vmax values of PbChi67 for colloidal chitin and glycol chitin were 3.35mg/mL and 17.1µmol/min/mg, and 2.66mg/mL and 15.0µmol/min/mg, respectively. PbChi67 hydrolyzed colloidal chitin to yield N-acetyl chitooligosaccharides (COSs) with degree of polymerization (DP) of 2-4 at the initial hydrolysis stage, indicating that it is an endo-type chitinase. These properties make the enzyme as a good candidate for recycling of chitin materials.

Paenibacillus puernese sp. nov., a ß-glucosidase-producing bacterium isolated from Pu'er tea.

A Gram-staining-positive, endospore-forming, aerobic, rod-shaped bacterium, designated as DCY97(T), was isolated from ripened Pu'er tea and was identified by using a polyphasic approach. 16S rRNA gene sequence analysis showed that strain DCY97(T) was closely related to Paenibacillus dongdonensis KUDC0114(T) (98.0 %), Paenibacillus oceanisediminis L10(T) (97.7 %), and Paenibacillus barcinonensis BP-23(T) (97.2 %). The phenotypic and chemotaxonomic characteristics of strain DCY97(T) matched with the characteristics of members belonging to the genus Paenibacillus. The major identified polar lipids included phosphatidylglycerol, phosphatidylethanolamine, and diphosphatidylglycerol. The predominant quinone was MK-7. The major fatty acids were anteiso-C15:0 (35.1 %), anteiso-C16:0 (19.0 %), and iso-C16:0 (13.9 %). The peptidoglycan cell wall was composed of meso-diaminopimelic acids, alanine, and D-glutamic acid. The genomic DNA G + C content was determined to be 46.7 mol%. The DNA-DNA relatedness between strain DCY97(T) and Paenibacillus dongdonensis KCTC 33221(T), Paenibacillus oceanisediminis KACC 16023(T), Paenibacillus barcinonensis KCTC 13019(T) were 27, 19, and 10 %, respectively. Based on the genotypic, phenotypic, and chemotaxonomic characteristics, strain DCY97(T) is considered as a novel species of the genus Paenibacillus, for which the name Paenibacillus puernese sp. nov. is proposed. The type strain is DCY97(T) (=KCTC 33596(T) = JCM 140369(T)).

A novel esterase from Paenibacillus sp. PBS-2 is a new member of the ß-lactamase belonging to the family VIII lipases/esterases.

Screening of a gene library from Paenibacillus sp. PBS-2 generated in Escherichia coli led to the identification of a clone with lipolytic activity. Sequence analysis showed an open reading frame encoding a polypeptide of 378 amino acid residues with a predicted molecular mass of 42 kDa. The esterase displayed 69% and 42% identity with the putative ß-lactamases from Paenibacillus sp. JDR-2 and Clostridium sp. BNL1100, respectively. The esterase contained a Serx- x-Lys motif that is conserved among all ß-lactamases found to date. The protein PBS-2 was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at 18°C. The enzyme is a serine protein and was active against p-nitrophenyl esters of C2, C4, C8, and C10. The optimum pH and temperature for enzyme activity were pH 9.0 and 30°C, respectively. Relative activity of 55% remained at up to 5°C with an activation energy of 5.84 kcal/mol, which indicates that the enzyme is cold-adapted. Enzyme activity was inhibited by Cd(2+), Cu(2+), and Hg(2+) ions. As expected for a serine esterase, activity was inhibited by phenylmethylsulfonyl fluoride. The enzyme was remarkably active and stable in the presence of commercial detergents and organic solvents. This cold-adapted esterase has potential as a biocatalyst and detergent additive for use at low temperatures.

Recombinant production and characterization of full-length and truncated ß-1,3-glucanase PglA from Paenibacillus sp. S09.

BACKGROUND: ß-1,3-Glucanases catalyze the hydrolysis of glucan polymers containing ß-1,3-linkages. These enzymes are of great biotechnological, agricultural and industrial interest. The applications of ß-1,3-glucanases is well established in fungal disease biocontrol, yeast extract production and wine extract clarification. Thus, the identification and characterization of novel ß-1,3-glucanases with high catalytic efficiency and stability is of particular interest. RESULTS: A ß-1,3-glucanase gene designated PglA was cloned from a newly isolated strain Paenibacillus sp. S09. The gene PglA contained a 2631-bp open reading frame encoding a polypeptide of 876 amino acids which shows 76% identity with the ß-1,3-glucanase (BglH) from Bacillus circulans IAM1165. The encoded protein PglA is composed of a signal peptide, an N-terminal leader region, a glycoside hydrolase family 16 (GH16) catalytic domain and a C-terminal immunoglobulin like (Ig-like) domain. The Escherichia coli expression system of PglA and five truncated derivatives containing one or two modules was constructed to investigate the role of catalytic and non-catalytic modules. The pH for optimal activity of the enzymes was slightly affected (pH 5.5-6.5) by the presence of different modules. However, the temperature for optimal activity was strongly influenced by the C-terminal domain and ranged from 50 to 60°C. Deletion of C-terminal domain resulted in obviously enhancing enzymatic thermostability. Specific activity assay indicated that PglA specifically hydrolyzes ß-1,3-glucan. Insoluble ß-1,3-glucan binding and hydrolysis were boosted by the presence of N-and C-terminal domains. Kinetic analysis showed that the presence of N-and C-terminus enhances the substrate affinity and catalytic efficiency of the catalytic domain toward laminarin. Carbohydrate-binding assay directly confirmed the binding capabilities of the N-and C-terminal domains. CONCLUSIONS: This study provides new insight into the impacts of non-catalytic modules on enzymatic properties of ß-1,3-glucanase. Activity comparison of full-length PglA and truncated forms revealed the negative effect of C-terminal region on thermal stability of the enzyme. Both the N-and C-terminal domains exerted strong binding activity toward insoluble ß-1,3-glucan, and could be classified into CBM families.

Cooperative degradation of chitin by extracellular and cell surface-expressed chitinases from Paenibacillus sp. strain FPU-7.

Chitin, a major component of fungal cell walls and invertebrate cuticles, is an exceedingly abundant polysaccharide, ranking next to cellulose. Industrial demand for chitin and its degradation products as raw materials for fine chemical products is increasing. A bacterium with high chitin-decomposing activity, Paenibacillus sp. strain FPU-7, was isolated from soil by using a screening medium containing α-chitin powder. Although FPU-7 secreted several extracellular chitinases and thoroughly digested the powder, the extracellular fluid alone broke them down incompletely. Based on expression cloning and phylogenetic analysis, at least seven family 18 chitinase genes were found in the FPU-7 genome. Interestingly, the product of only one gene (chiW) was identified as possessing three S-layer homology (SLH) domains and two glycosyl hydrolase family 18 catalytic domains. Since SLH domains are known to function as anchors to the Gram-positive bacterial cell surface, ChiW was suggested to be a novel multimodular surface-expressed enzyme and to play an important role in the complete degradation of chitin. Indeed, the ChiW protein was localized on the cell surface. Each of the seven chitinase genes (chiA to chiF and chiW) was cloned and expressed in Escherichia coli cells for biochemical characterization of their products. In particular, ChiE and ChiW showed high activity for insoluble chitin. The high chitinolytic activity of strain FPU-7 and the chitinases may be useful for environmentally friendly processing of chitin in the manufacture of food and/or medicine.