Microbial biofilm formation creates a persistent and resistant environment in which microorganisms can survive, contributing to antibiotic resistance and chronic inflammatory diseases. Increasingly, biofilms are caused by multi-drug resistant microorganisms, which, coupled with a diminishing supply of effective antibiotics, is driving the search for new antibiotic therapies. In this respect, antimicrobial peptides (AMPs) are short, hydrophobic, and amphipathic peptides that show activity against multidrug-resistant bacteria and biofilm formation. They also possess broad-spectrum activity and diverse mechanisms of action. In this comprehensive review, 150 publications (from January 2020 to September 2023) were collected and categorized using the search terms \'polypeptide antibiotic agent\', \'antimicrobial peptide\', and \'biofilm\'. During this period, a wide range of natural and synthetic AMPs were studied, of which LL-37, polymyxin B, GH12, and Nisin were the most frequently cited. Furthermore, although many microbes were studied, Staphylococcus aureus and Pseudomonas aeruginosa were the most popular. Publications also considered AMP combinations and the potential role of AMP delivery systems in increasing the efficacy of AMPs, including nanoparticle delivery. Relatively few publications focused on AMP resistance. This comprehensive review informs and guides researchers about the latest developments in AMP research, presenting promising evidence of the role of AMPs as effective antimicrobial agents.

Millions of tons of citrus peel waste are produced every year as a byproduct of the juice industry. Citrus peel is rich in pectin and xyloglucan, but while the pectin is extracted for use in the food industry, the xyloglucan is currently not valorized. To target hydrolytic degradation of citrus peel xyloglucan into oligosaccharides, we have used bioinformatics to identify three glycoside hydrolase 12 (GH12) endoxyloglucanases (EC 3.2.1.151) from the citrus fruit pathogens Penicillium italicum GL-Gan1 and Penicillium digitatum Pd1 and characterized them on xyloglucan obtained by alkaline extraction from citrus peel. The enzymes displayed pH-temperature optima of pH 4.6-5.3 and 35-37°C. PdGH12 from P. digitatum and PiGH12A from P. italicum share 84% sequence identity and displayed similar kinetics, although kcat was highest for PdGH12. In contrast, PiGH12B from P. italicum, which has the otherwise conserved Trp in subsite -4 replaced with a Tyr, displayed a 3 times higher KM and a 4 times lower kcat/KM than PiGH12A, but was the most thermostable enzyme of the three Penicillium-derived endoxyloglucanases. The benchmark enzyme AnGH12 from Aspergillus nidulans was more thermally stable and had a higher pH-temperature optimum than the enzymes from Penicillum spp. The difference in structure of the xyloglucan oligosaccharides extracted from citrus peel xyloglucan and tamarind xyloglucan by the new endoxyloglucanases was determined by LC-MS. The inclusion of citrus peel xyloglucan demonstrated that the endoxyloglucanases liberated fucosylated xyloglucan oligomers, implying that these enzymes have the potential to upgrade citrus peel residues to produce oligomers useful as intermediates or bioactive compounds.

OBJECTIVE: Staphylococcus epidermidis is one of the most common Gram-positive cocci in nosocomial infection, which could adhere to the surface of medical apparatus and causes biofilm-related infections. In the present study, we aim to explore the antimicrobial effects of GH12 and SAAP-148 against Staphylococcus epidermidis.
METHODS: Micro-dilution methods were used to detect the minimal inhibitory/bactericidal concentration of peptides on Staphylococcus epidermidis. Biofilm formation positive type strain was used to determine the antibiofilm effects of the peptides. Biofilms were built on the cover slides and fluorescent dye SYTO9 and laser confocal microscope were used to observe the effects of peptides on the three-dimensional structure of Staphylococcus epidermidis biofilms. The cell membrane permeability of Staphylococcus epidermidis was detected by flow cytometry. Expressions of icaA and icaDgenes were analyzed by real-time reverse transcription PCR.
RESULTS: The minimal inhibitory concentrations of GH12 and SAAP-148 against Staphylococcus epidermidis were 8 and 16 μg/mL, respectively, and the minimal bactericidal concentration was 64 μg/mL. GH12 and SAAP-148 significantly inhibited the biofilm formation of Staphylococcus epidermidis at the concentration of 8 μg/mL (t=7.193, P<0.05) and 16 μg/mL (t=7.808, P<0.05), respectively. Similarly, the GH12 and SAAP-148 significantly eradicated the pre-formed biofilms at the concentration of 16 μg/mL (t=5.369, P<0.05) and 32 μg/mL (t=4.474, P<0.05) in a dose-response manner, respectively. Meanwhile, the two peptides broke the structure of biofims and reduce the total biomass. GH12 and SAAP-148 at the concentration of minimal inhibitory concentration significantly disrupted the cell membrane of Staphylococcus epidermidis. The expressions of icaA and icaDgenes were significantly inhibited by antimicrobial peptides at the 1×minimal inhibitory concentration.
CONCLUSIONS: GH12 and SAAP-148 show significantly antimicrobial and anti-biofilm effects against Staphylococcus epidermidis by disruption of cell membrane and inhibition of icaAand icaDgene expression.
目的: 表皮葡萄球菌是院内感染常见的革兰氏阳性球菌之一，它亦黏附于医疗器械表面导致生物膜相关感染。本研究拟探讨两种人工合成的抗菌肽GH12和SAAP-148对表皮葡萄球菌浮游菌生长及其生物膜的影响。方法: 通过微量肉汤稀释法检测抗菌肽GH12和SAAP-148的最低抑菌浓度和最低杀菌浓度；利用生物膜成膜能力阳性标准菌株在微孔板中构建生物膜，分别检测这两种抗菌肽对表皮葡萄球菌生物膜的抑制和分散作用；在盖玻片上构建表皮葡萄球菌生物膜，通过荧光染料SYTO9染色和激光共聚焦显微镜观察，检测两种抗菌肽对表皮葡萄球菌生物膜三维结构的破坏作用；通过流式细胞仪检测抗菌肽对表皮葡萄球菌细胞膜的破坏作用；采用实时反转录聚合酶链反应分析表皮葡萄球菌黏附相关基因icaA和icaD的表达情况。结果: GH12和SAAP-148对表皮葡萄球菌的最低抑菌浓度分别为8和16 μg/mL，最低杀菌浓度均为64 μg/mL；GH12和SAAP-148的质量浓度分别为8 μg/mL(t=7.193，P<0.05)和16 μg/mL(t=7.808，P<0.05)时均可显著抑制表皮葡萄球菌RP62A生物膜的形成，且均呈现出一定的剂量依赖性；GH12和SAAP-148的质量浓度分别为16 μg/mL(t=5.369，P<0.05)和32 μg/mL(t=4.474，P<0.05)时可显著分散RP62A已形成的生物膜，亦呈现剂量依赖性；通过激光共聚焦显微镜观察，GH12和SAAP-148可显著破坏生物膜的三维结构，使生物膜的总量显著减少；流式细胞仪检测发现最低抑菌浓度的GH12和SAAP-148还能显著破坏表皮葡萄球菌的细胞膜；1×最低抑菌浓度的抗菌肽能够显著抑制表皮葡萄球菌黏附相关基因icaA和icaD的表达。结论: 抗菌肽GH12和SAAP-148通过破坏细胞膜对表皮葡萄球菌浮游菌生长具有显著的抑制作用，并能通过抑制黏附相关基因抑制和分散其生物膜。.

The cellulases from Glycoside Hydrolyses family 12 (GH12) play an important role in cellulose degradation and plant cell wall deconstruction being widely used in a number of bioindustrial processes. Aiming to contribute toward better comprehension of these class of the enzymes, here we describe a high-yield secretion of a endoglucanase GH12 from Aspegillus terreus (AtGH12), which was cloned and expressed in Aspergillus nidulans strain A773. The purified protein was used for complete biochemical and functional characterization. The optimal temperature and pH of the enzyme were 55°C and 5.0 respectively, which has high activity against β-glucan and xyloglucan and also is active toward glucomannan and CMC. The enzyme retained activity up to 60°C. AtGH12 is strongly inhibited by Cu2+, Fe2+, Cd2+, Mn2+, Ca2+, Zn2+ and EDTA, whereas K+, Tween, Cs+, DMSO, Triton X-100 and Mg2+ enhanced the enzyme activity. Furthermore, SAXS data reveal that the enzyme has a globular shape and CD analysis demonstrated a prevalence of a β-strand structure corroborating with typical β-sheets fold commonly found for other endoglucanases from GH12 family.

The β-glucanase Cel12A gene from Stachybotrys atra has been cloned and heterologously expressed in Aspergillus nidulans and Saccharomyces cerevisiae. The recombinant strains constructed, contained the exonic sequence of cel12A including its own signal peptide coding sequence. SDS-PAGE and zymography revealed that recombinant Cel12A has a molecular mass of 24 kDa which agrees with that deduced from its amino acid sequence, indicating that it is expressed in the non-glycosylated active form. Recombinant A. nidulans showed about eightfold greater activity yield than S. cerevisiae recombinant strain, namely 0.71 and 0.09 β-glucanase Units/ml of culture, respectively. In both host strains most of the activity was secreted to the extracellular media, evidencing the functionality of Cel12A signal peptide in yeast and fungi. This novel signal peptide might facilitate the expression and efficient secretion of other recombinant proteins difficult to secrete.

Fungal GH12 enzymes are classified as xyloglucanases when they specifically target xyloglucans, or promiscuous endoglucanases when they exhibit catalytic activity against xyloglucan and β-glucan chains. Several structural and functional studies involving GH12 enzymes tried to explain the main patterns of xyloglucan activity, but what really determines xyloglucanase specificity remains elusive. Here, three fungal GH12 enzymes from Aspergillus clavatus (AclaXegA), A. zonatus (AspzoGH12), and A. terreus (AtEglD) were studied to unveil the molecular basis for substrate specificity. Using functional assays, site-directed mutagenesis, and molecular dynamics simulations, we demonstrated that three main regions are responsible for substrate selectivity: (i) the YSG group in loop 1; (ii) the SST group in loop 2; and (iii) loop A3-B3 and neighboring residues. Functional assays and sequence alignment showed that while AclaXegA is specific to xyloglucan, AtEglD cleaves β-glucan, and xyloglucan. However, AspzoGH12 was also shown to be promiscuous contrarily to a sequence alignment-based prediction. We find that residues Y111 and R93 in AtEglD harbor the substrate in an adequate orientation for hydrolysis in the catalytic cleft entrance and that residues Y19 in AclaXegA and Y30 in AspzoGH12 partially compensate the absence of the YSG segment, typically found in promiscuous enzymes. The results point out the multiple structural factors underlying the substrate specificity of GH12 enzymes. Biotechnol. Bioeng. 2016;113: 2577-2586. © 2016 Wiley Periodicals, Inc.

Enzymes that cleave the xyloglucan backbone at unbranched glucose residues have been identified in GH families 5, 7, 12, 16, 44, and 74. Fungi produce enzymes that populate 20 of 22 families that are considered critical for plant biomass deconstruction. We searched for GH12-encoding genes in 27 Eurotiomycetes genomes. After analyzing 50 GH12-related sequences, the conserved variations of the amino acid sequences were examined. Compared to the endoglucanases, the endo-xyloglucanase-associated YSG deletion at the negative subsites of the catalytic cleft with a SST insertion at the reducing end of the substrate-binding crevice is highly conserved. In addition, a highly conserved alanine residue was identified in all xyloglucan-specific enzymes, and this residue is substituted by arginine in more promiscuous glucanases. To understand the basis for the xyloglucan specificity displayed by certain GH12 enzymes, two fungal GH12 endoglucanases were chosen for mutagenesis and functional studies: an endo-xyloglucanase from Aspergillus clavatus (AclaXegA) and an endoglucanase from A. terreus (AtEglD). Comprehensive molecular docking studies and biochemical analyses were performed, revealing that mutations at the entrance of the catalytic cleft in AtEglD result in a wider binding cleft and the alteration of the substrate-cleavage pattern, implying that a trio of residues coordinates the interactions and binding to linear glycans. The loop insertion at the crevice-reducing end of AclaXegA is critical for catalytic efficiency to hydrolyze xyloglucan. The understanding of the structural elements governing endo-xyloglucanase activity on linear and branched glucans will facilitate future enzyme modifications with potential applications in industrial biotechnology.