α-Mannosidase and ß-D-N-acetylhexosaminidase outside the wall: partner exoglycosidases involved in fruit ripening process.

Cell wall is a strong and complex net whose function is to provide turgor, pathogens attack protection and to give structural support to the cell. In growing and expanding cells, the cell wall of fruits is changing in space and time, because they are changing according to stage of ripening. Understand which mechanisms to produce significant could help to develop tools to prolong the fruit shelf life. Cell wall proteins (CWPs) with enzymatic activity on cell wall polysaccharides, have been studied widely. Another investigations take place in the study of N-glycosylations of CWPs and enzymes with activity on glycosidic linkages. α-mannosidase (α-Man; EC 3.2.1.24) and ß-D-N-acetylhexosaminidase (ß-Hex; EC 3.2.1.52), are enzymes with activity on mannose and N-acetylglucosamine sugar presents in proteins as part of N-glycosylations. Experimental evidence indicate that both are closely related to loss of fruit firmness, but in the literature, there is still no review of both enzymes involved fruit ripening. This review provides a complete state-of-the-art of α-Man and ß-Hex enzymes related in fruit ripening. Also, we propose a vesicular α-Man (EC 3.2.1.24) name to α-Man involved in N-deglycosylations of CWPs of plants.

An extracellular ß-N-acetylhexosaminidase of Medicago truncatula hydrolyzes chitooligosaccharides and is involved in arbuscular mycorrhizal symbiosis but not required for nodulation.

Establishment of symbiosis between plants and arbuscular mycorrhizal (AM) fungi depends on fungal chitooligosaccharides (COs) and lipo-chitooligosaccharides (LCOs). The latter are also produced by nitrogen-fixing rhizobia to induce nodules on leguminous roots. However, host enzymes regulating structure and levels of these signals remain largely unknown. Here, we analyzed the expression of a ß-N-acetylhexosaminidase gene of Medicago truncatula (MtHEXO2) and biochemically characterized the enzyme. Mutant analysis was performed to study the role of MtHEXO2 during symbiosis. We found that expression of MtHEXO2 is associated with AM symbiosis and nodulation. MtHEXO2 expression in the rhizodermis was upregulated in response to applied chitotetraose, chitoheptaose, and LCOs. M. truncatula mutants deficient in symbiotic signaling did not show induction of MtHEXO2. Subcellular localization analysis indicated that MtHEXO2 is an extracellular protein. Biochemical analysis showed that recombinant MtHEXO2 does not cleave LCOs but can degrade COs into N-acetylglucosamine (GlcNAc). Hexo2 mutants exhibited reduced colonization by AM fungi; however, nodulation was not affected in hexo2 mutants. In conclusion, we identified an enzyme, which inactivates COs and promotes the AM symbiosis. We hypothesize that GlcNAc produced by MtHEXO2 may function as a secondary symbiotic signal.

Genomic and transcriptomic analyses of chitin metabolism enzymes in Tenebrio molitor.

Chitin is of great importance in the cuticle and inner cuticular linings of insects. Chitin synthases (CHSs), chitin deacetylases (CDAs), chitinases (CHTs), and ß-N-acetylhexosaminidases (HEXs) are important enzymes required for chitin metabolism, and play essential roles in development and metamorphosis. Although chitin metabolism genes have been well characterized in limited insects, the information in the yellow mealworm, Tenebrio molitor, a model insect, is presently still unavailable. With the help of bioinformatics, we identified 54 genes that encode putative chitin metabolism enzymes, including 2 CHSs, 10 CDAs, 32 CHTs, and 10 HEXs in the genome of T. molitor. All these genes have the conserved domains and motifs of their corresponding protein family. Phylogenetic analyses indicated that CHS genes were divided into two groups. CDA genes were clustered into five groups. CHT genes were phylogenetically grouped into 11 clades, among which 1 in the endo-ß-N-acetylglucosaminidases group and the others were classified in the glycoside hydrolase family 18 groups. HEX genes were assorted into six groups. Developmental and tissue-specific expression profiling indicated that the identified chitin metabolism genes showed dynamical expression patterns concurrent with specific instar during molting period, suggesting their significant roles in molting and development. They were predominantly expressed in different tissues or body parts, implying their functional specialization and diversity. The results provide important information for further clarifying their biological functions using the yellow mealworm as an ideal experimental insect.

Aerosol fluorescence, airborne hexosaminidase, and quantitative genomics distinguish reductions in airborne fungal loads following major school renovations.

Fluorescent aerosol cytometry (FAC) was compared to concurrent recovery of airborne ß-N-acetylhexosaminidase (NAHA) and quantitative polymerase chain reaction (qPCR) for the respective ability of these methods to detect significant changes in airborne fungal loads in response to building renovations. Composite, site-randomized indoor aerosol samples for airborne fungi measurements were acquired from more than 70 occupied classrooms in 26 different public schools in the Colorado Rocky Mountain Front Range region of the United States. As judged by ANOVA and Pearson's correlation test, statistically significant associations were observed between real-time FAC and airborne NAHA levels, which detected significant reductions in airborne fungal loads immediately following building rehabilitations. With lower confidence, a statistically significant association was also resolved between fluorescing aerosols, NAHA levels, and the recovery of fungal 18S rRNA gene copies by qPCR from simultaneous, collocated aerosol samples. Quantitative differences encountered between the recovery of common genomic markers for airborne fungi and that of optical and biochemical methods are attributed to the variance in 18S rRNA target gene copies that different fungal species can host.

Mutation Hotspot for Changing the Substrate Specificity of ß-N-Acetylhexosaminidase: A Library of GlcNAcases.

ß-N-Acetylhexosaminidase from Talaromyces flavus (TfHex; EC 3.2.1.52) is an exo-glycosidase with dual activity for cleaving N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) units from carbohydrates. By targeting a mutation hotspot of the active site residue Glu332, we prepared a library of ten mutant variants with their substrate specificity significantly shifted towards GlcNAcase activity. Suitable mutations were identified by in silico methods. We optimized a microtiter plate screening method in the yeast Pichia pastoris expression system, which is required for the correct folding of tetrameric fungal ß-N-acetylhexosaminidases. While the wild-type TfHex is promiscuous with its GalNAcase/GlcNAcase activity ratio of 1.2, the best single mutant variant Glu332His featured an 8-fold increase in selectivity toward GlcNAc compared with the wild-type. Several prepared variants, in particular Glu332Thr TfHex, had significantly stronger transglycosylation capabilities than the wild-type, affording longer chitooligomers - they behaved like transglycosidases. This study demonstrates the potential of mutagenesis to alter the substrate specificity of glycosidases.

Synthesis of ureido thioglycosides as novel insect ß­N­acetylhexosaminidase OfHex1 inhibitors.

The insect ß-N-acetylhexosaminidase OfHex1 from Ostrinia furnacalis (one of the most destructive agricultural pests) has been considered as a promising pesticide target. In this study, a series of novel and readily available ureido thioglycosides were designed and synthesized based on the catalytic mechanism and the co-crystal structures of OfHex1 with substrates. After evaluation via enzyme inhibition experiments, thioglycosides 11c and 15k were found to have inhibitory activities against OfHex1 with the Ki values of 25.6 µM and 53.8 µM, respectively. In addition, all these ureido thioglycosides exhibited high selectivity toward OfHex1 over hOGA and HsHexB (Ki > 100 µM). Furthermore, to investigate the inhibitory mechanism, the possible binding modes of 11c and 15k with OfHex1 were deduced based on molecular docking analysis. This work may provide useful structural starting points for further rational design of potent inhibitors of OfHex1.

Converting a ß-N-acetylhexosaminidase into two trans-ß-N-acetylhexosaminidases by domain-targeted mutagenesis.

We have recently derived a ß-N-acetylhexosaminidase, BbhI, from Bifidobacterium bifidum JCM 1254, which could regioselectively synthesize GlcNAcß1-3Galß1-4Glc with a yield of 44.9%. Here, directed evolution of BbhI by domain-targeted mutagenesis was carried out. Firstly, the GH20 domain was selected for random mutagenesis using MEGAWHOP method and a small library of 1300 clones was created. A total of 734 colonies with reduced hydrolytic activity were isolated, and three mutants with elevated transglycosylation yields, GlcNAcß1-3Galß1-4Glc yields of 68.5%, 74.7%, and 81.1%, respectively, were obtained. Subsequently, nineteen independent mutants were constructed according to all the mutation sites in these three mutants. After transglycosylation analysis, Asp714 and Trp773 were identified as key residues for improvement in transglycosylation ability and were chosen for the second round of directed evolution by site-saturation mutagenesis. Two most efficient mutants D714T and W773R that acted as trans-ß-N-acetylhexosaminidase were finally achieved. D714T with the substitution at the putative nucleophile assistant residue Asp714 by threonine showed high yield of 84.7% with unobserved hydrolysis towards transglycosylation product. W773R with arginine substitution at Trp773 residue locating at the entrance of catalytic cavity led to the yield up to 81.8%. The kcat/Km values of D714T and W773R for hydrolysis of pNP-ß-GlcNAc displayed drastic decreases. NMR investigation of protein-substrate interaction revealed an invariable mode of the catalytic cavity of D714T, W773R, and WT BbhI. The collective motions of protein model showed the mutations Thr714 and Arg773 exerted little effect on the dynamics of the inside but a large effect on the dynamics of the outside of catalytic cavity.

Synthesis of Pyrrolidine Monocyclic Analogues of Pochonicine and Its Stereoisomers: Pursuit ofSimplified Structures and Potent ß-N-Acetylhexosaminidase Inhibition.

Ten pairs of pyrrolidine analogues of pochonicine and its stereoisomers have been synthesized from four enantiomeric pairs of polyhydroxylated cyclic nitrones. Among the ten N-acetylamino pyrrolidine analogues, only compounds with 2,5-dideoxy-2,5-imino-d-mannitol (DMDP) and pochonicine (1) configurations showed potent inhibition of ß-N-acetylhexosaminidases (ß-HexNAcases); while 1-amino analogues lost almost all their inhibitions towards the tested enzymes. The assay results reveal the importance of the N-acetylamino group and the possible right configurations of pyrrolidine ring required for this type of inhibitors.

Biochemical characteristics and crystallographic evidence for substrate-assisted catalysis of a ß-N-acetylhexosaminidase in Akkermansia muciniphila.

In this paper, we characterized Am2136 as a ß-N-acetylhexosaminidase from Akkermansia muciniphila to perform the biochemical characteristics and the crystal structure of selenomethionine-labeled Am2136 with GlcNAc complex. Crystallographic evidence suggests that an oxazolinium ion was formed intermediately by the 2-acetamido group during the substrate-assisted catalytic procedure. Structural and kinetic analysis of native Am2136 and D412A, E413A mutants were investigated and the results revealed substantial difference. The Kcat/Km value of D412A was decreased 4297-fold compared to native Am2136 revealed that mutation of Asp-412 results in preventing the 2-acetamido substituent from providing anchimeric assistance and thus reducing the catalytic efficiency. Moreover, Am2136 has a wide dependence on pH and temperature, while sensitive to divalent metal ions such as Ca2+ and Mn2+. These biochemical and crystallographic results provide evidences that Asp-412 residue assists to orient the 2-acetamido group for catalysis. Based on crystallographic evidence and sequence alignment with other GH family 20 enzymes, Asp-412 residue is possibly fundamental for Am2136 during substrate-assisted catalysis.

ß-N-Acetylhexosaminidases-the wizards of glycosylation.

ß-N-Acetylhexosaminidases (EC 3.2.1.52) are a unique family of glycoside hydrolases with dual substrate specificity and a particular reaction mechanism. Though hydrolytic enzymes per se, their good stability, easy recombinant production, absolute stereoselectivity, and a broad substrate specificity predestine these enzymes for challenging applications in carbohydrate synthesis. This mini-review aims to demonstrate the catalytic potential of ß-N-acetylhexosaminidases in a range of unusual reactions, processing of unnatural substrates, formation of unexpected products, and demanding reaction designs. The use of unconventional media can considerably alter the progress of transglycosylation reactions. By means of site-directed mutagenesis, novel catalytic machineries can be constructed. Glycosylation of difficult substrates such as sugar nucleotides was accomplished, and the range of afforded glycosidic bonds comprises unique non-reducing sugars. Specific functional groups may be tolerated in the substrate molecule, which makes ß-N-acetylhexosaminidases invaluable allies in difficult synthetic problems.

Selective ß-N-acetylhexosaminidase from Aspergillus versicolor-a tool for producing bioactive carbohydrates.

ß-N-Acetylhexosaminidases (EC 3.2.1.52) are typical of their dual activity encompassing both N-acetylglucosamine and N-acetylgalactosamine substrates. Here we present the isolation and characterization of a selective ß-N-acetylhexosaminidase from the fungal strain of Aspergillus versicolor. The enzyme was recombinantly expressed in Pichia pastoris KM71H in a high yield and purified in a single step using anion-exchange chromatography. Homologous molecular modeling of this enzyme identified crucial differences in the enzyme active site that may be responsible for its high selectivity for N-acetylglucosamine substrates compared to fungal ß-N-acetylhexosaminidases from other sources. The enzyme was used in a sequential reaction together with a mutant ß-N-acetylhexosaminidase from Talaromyces flavus with an enhanced synthetic capability, affording a bioactive disaccharide bearing an azido functional group. The azido function enabled an elegant multivalent presentation of this disaccharide on an aromatic carrier. The resulting model glycoconjugate is applicable as a selective ligand of galectin-3 - a biomedically attractive human lectin. These results highlight the importance of a general availability of robust and well-defined carbohydrate-active enzymes with tailored catalytic properties for biotechnological and biomedical applications.

A chemo-enzymatic approach for the synthesis of human milk oligosaccharide backbone structures.

The ability of an engineered ß-N-acetylhexosaminidase to utilize a reactive oxazoline as donor molecule for transglycosylation reaction to synthesize human milk oligosaccharide backbone structures was studied. The human milk oligosaccharide precursor lacto-N-triose II and three regioisomers could be synthesized using the oxazoline, which was either in situ-generated resulting in a chemo-enzymatic sequential cascade or was used as a purified compound. The highest observed concentration of overall transglycosylation products in a cascade reaction was 13.7 mM after 18.5 h, whereas the use of purified oxazoline resulted in 25.0 mM of transglycosylation products after 6.5 h. Remarkably, the in situ-generated oxazoline could be used without any further purification and it was shown that the used enzyme tolerated significant amounts of reagents such as triethylamine, which is reported for the first time for an enzyme from the glycoside hydrolase family 20.

Synthesis of Human Milk Oligosaccharides: Protein Engineering Strategies for Improved Enzymatic Transglycosylation.

Human milk oligosaccharides (HMOs) signify a unique group of oligosaccharides in breast milk, which is of major importance for infant health and development. The functional benefits of HMOs create an enormous impetus for biosynthetic production of HMOs for use as additives in infant formula and other products. HMO molecules can be synthesized chemically, via fermentation, and by enzymatic synthesis. This treatise discusses these different techniques, with particular focus on harnessing enzymes for controlled enzymatic synthesis of HMO molecules. In order to foster precise and high-yield enzymatic synthesis, several novel protein engineering approaches have been reported, mainly concerning changing glycoside hydrolases to catalyze relevant transglycosylations. The protein engineering strategies for these enzymes range from rationally modifying specific catalytic residues, over targeted subsite -1 mutations, to unique and novel transplantations of designed peptide sequences near the active site, so-called loop engineering. These strategies have proven useful to foster enhanced transglycosylation to promote different types of HMO synthesis reactions. The rationale of subsite -1 modification, acceptor binding site matching, and loop engineering, including changes that may alter the spatial arrangement of water in the enzyme active site region, may prove useful for novel enzyme-catalyzed carbohydrate design in general.

The ß-N-Acetylhexosaminidase in the Synthesis of Bioactive Glycans: Protein and Reaction Engineering.

N-Acetylhexosamine oligosaccharides terminated with GalNAc act as selective ligands of galectin-3, a biomedically important human lectin. Their synthesis can be accomplished by ß-N-acetylhexosaminidases (EC 3.2.1.52). Advantageously, these enzymes tolerate the presence of functional groups in the substrate molecule, such as the thiourea linker useful for covalent conjugation of glycans to a multivalent carrier, affording glyconjugates. ß-N-Acetylhexosaminidases exhibit activity towards both N-acetylglucosamine (GlcNAc) and N-acetylgalactosamine (GalNAc) moieties. A point mutation of active-site amino acid Tyr into other amino acid residues, especially Phe, His, and Asn, has previously been shown to strongly suppress the hydrolytic activity of ß-N-acetylhexosaminidases, creating enzymatic synthetic engines. In the present work, we demonstrate that Tyr470 is an important mutation hotspot for altering the ratio of GlcNAcase/GalNAcase activity, resulting in mutant enzymes with varying affinity to GlcNAc/GalNAc substrates. The enzyme selectivity may additionally be manipulated by altering the reaction medium upon changing pH or adding selected organic co-solvents. As a result, we are able to fine-tune the ß-N-acetylhexosaminidase affinity and selectivity, resulting in a high-yield production of the functionalized GalNAcß4GlcNAc disaccharide, a selective ligand of galectin-3.

N-glycan maturation mutants in Lotus japonicus for basic and applied glycoprotein research.

Studies of protein N-glycosylation are important for answering fundamental questions on the diverse functions of glycoproteins in plant growth and development. Here we generated and characterised a comprehensive collection of Lotus japonicusLORE1 insertion mutants, each lacking the activity of one of the 12 enzymes required for normal N-glycan maturation in the glycosylation machinery. The inactivation of the individual genes resulted in altered N-glycan patterns as documented using mass spectrometry and glycan-recognising antibodies, indicating successful identification of null mutations in the target glyco-genes. For example, both mass spectrometry and immunoblotting experiments suggest that proteins derived from the α1,3-fucosyltransferase (Lj3fuct) mutant completely lacked α1,3-core fucosylation. Mass spectrometry also suggested that the Lotus japonicus convicilin 2 was one of the main glycoproteins undergoing differential expression/N-glycosylation in the mutants. Demonstrating the functional importance of glycosylation, reduced growth and seed production phenotypes were observed for the mutant plants lacking functional mannosidase I, N-acetylglucosaminyltransferase I, and α1,3-fucosyltransferase, even though the relative protein composition and abundance appeared unaffected. The strength of our N-glycosylation mutant platform is the broad spectrum of resulting glycoprotein profiles and altered physiological phenotypes that can be produced from single, double, triple and quadruple mutants. This platform will serve as a valuable tool for elucidating the functional role of protein N-glycosylation in plants. Furthermore, this technology can be used to generate stable plant mutant lines for biopharmaceutical production of glycoproteins displaying relative homogeneous and mammalian-like N-glycosylation features.

Selective inhibition of ß-N-acetylhexosaminidases by thioglycosyl-naphthalimide hybrid molecules.

To develop selective inhibitors for ß-N-acetylhexosaminidases which are involved in a myriad of physiological processes, a series of novel thioglycosyl-naphthalimide hybrid inhibitors were designed, synthesized and evaluated for inhibition activity against glycosyl hydrolase family 20 and 84 (GH20 and GH84) ß-N-acetylhexosaminidases. These compounds which incorporate groups with varied sizes and lengths at the linker region between thioglycosyl moiety and naphthalimide moiety are designed to improve the selectivity and stacking interactions. The GH84 human O-GlcNAcase (hOGA) was sensitive to the subtle changes in the linker region and the optimal choice is a small size linker with six atoms length. And the GH20 insect ß-N-acetylhexosaminidase OfHex1 could tolerate compounds with a hydrophobic bulky linker. Especially, the compound 5c (hOGA, Ki = 3.46 µM; OfHex1, Ki > 200 µM) and the compound 6f (hOGA, Ki > 200 µM; OfHex1, Ki = 21.81 µM) displayed high selectivity. The molecular docking results indicated that the inhibition mechanism was different between the two families due to their different structural characteristics beyond the active sites. These results provide some promising clues to improve selectivity of potent molecules against ß-N-acetylhexosaminidases.

Design and synthesis of naphthalimide group-bearing thioglycosides as novel ß-N-acetylhexosaminidases inhibitors.

GH20 human ß-N-acetylhexosaminidases (hsHex) and GH84 human O-GlcNAcase (hOGA) are involved in numerous pathological processes and emerged as promising targets for drug discovery. Based on the catalytic mechanism and structure of the catalytic domains of these ß-N-acetylhexosaminidases, a series of novel naphthalimide moiety-bearing thioglycosides with different flexible linkers were designed, and their inhibitory potency against hsHexB and hOGA was evaluated. The strongest potency was found for compound 15j (Ki = 0.91 µM against hsHexB; Ki > 100 µM against hOGA) and compound 15b (Ki = 3.76 µM against hOGA; Ki = 30.42 µM against hsHexB), which also exhibited significant selectivity between these two enzymes. Besides, inhibitors 15j and 15b exhibited an inverse binding patterns in docking studies. The determined structure-activity relationship as well as the established binding models provide the direction for further structure optimizations and the development of specific ß-N-acetylhexosaminidase inhibitors.

Engineered N-acetylhexosamine-active enzymes in glycoscience.

BACKGROUND: In recent years, enzymes modifying N-acetylhexosamine substrates have emerged in numerous theoretical studies as well as practical applications from biology, biomedicine, and biotechnology. Advanced enzyme engineering techniques converted them into potent synthetic instruments affording a variety of valuable glycosides. SCOPE OF REVIEW: This review presents the diversity of engineered enzymes active with N-acetylhexosamine carbohydrates: from popular glycoside hydrolases and glycosyltransferases to less known oxidases, epimerases, kinases, sulfotransferases, and acetylases. Though hydrolases in natura, engineered chitinases, ß-N-acetylhexosaminidases, and endo-ß-N-acetylglucosaminidases were successfully employed in the synthesis of defined natural and derivatized chitooligomers and in the remodeling of N-glycosylation patterns of therapeutic antibodies. The genes of various N-acetylhexosaminyltransferases were cloned into metabolically engineered microorganisms for producing human milk oligosaccharides, Lewis X structures, and human-like glycoproteins. Moreover, mutant N-acetylhexosamine-active glycosyltransferases were applied, e.g., in the construction of glycomimetics and complex glycostructures, industrial production of low-lactose milk, and metabolic labeling of glycans. In the synthesis of biotechnologically important compounds, several innovative glycoengineered systems are presented for an efficient bioproduction of GlcNAc, UDP-GlcNAc, N-acetylneuraminic acid, and of defined glycosaminoglycans. MAJOR CONCLUSIONS: The above examples demonstrate that engineering of N-acetylhexosamine-active enzymes was able to solve complex issues such as synthesis of tailored human-like glycoproteins or industrial-scale production of desired oligosaccharides. Due to the specific catalytic mechanism, mutagenesis of these catalysts was often realized through rational solutions. GENERAL SIGNIFICANCE: Specific N-acetylhexosamine glycosylation is crucial in biological, biomedical and biotechnological applications and a good understanding of its details opens new possibilities in this fast developing area of glycoscience.

The ß-N-acetylhexosaminidase gene family in the brown planthopper, Nilaparvata lugens.

ß-N-Acetylhexosaminidases (HEXs) are enzymes that can degrade the chitin oligosaccharides that are produced by the activity of chitinases on chitin in insects. Using bioinformatic methods based on genome and transcriptome databases, 11 ß-N-acetylhexosaminidase genes (NlHexs) in Nilaparvata lugens were identified and characterized. Phylogenetic analysis revealed a six-grouped tree topology. The O-Linked N-acetylglucosaminidase (O-GlcNAcase) group includes NlHex11, which harbours a catalytic domain that differs from that of the other 10 NlHexs. Observations of the expression of NlHexs during different developmental stages revealed that NlHex4 is expressed with periodicity during moulting. Although the tissue-specific expression patterns of most NlHexs were nonspecific, NlHex4 was found to be expressed mainly in the female reproductive system as well as in the integument. RNA interference (RNAi) demonstrated failure to shed the old cuticle only in the nymphs treated with double-stranded RNA (dsRNA) targeting NlHex4, and these nymphs eventually died; no observable morphological abnormalities were found in insects treated with dsRNAs targeting the other 10 NlHexs. Based on this study and our previous analyses, a '5 + 1 + 3' pattern of chitinolytic enzymes is proposed, in which five chitinases, one NlHEX and three chitin deacetylases are required for moulting in N. lugens. A better understanding of chitin metabolism in the hemimetabolous insect, N. lugens, would be achieved by considering three chitinolytic enzyme families: chitinase, chitin deacetylase and ß-N-acetylhexosaminidase.

Susceptibility of green and conventional building materials to microbial growth.

Green building materials are becoming more popular. However, little is known about their ability to support or limit microbial growth. The growth of fungi was evaluated on five building materials. Two green, two conventional building materials and wood as a positive control were selected. The materials were inoculated with Aspergillus versicolor, Cladosporium cladosporioides and Penicillium brevicompactum, in the absence and presence of house dust. Microbial growth was assessed at four different time points by cultivation and determining fungal biomass using the N-acetylhexosaminidase (NAHA) enzyme assay. No clear differences were seen between green and conventional building materials in their susceptibility to support microbial growth. The presence of dust, an external source of nutrients, promoted growth of all the fungal species similarly on green and conventional materials. The results also showed a correlation coefficient ranging from 0.81 to 0.88 between NAHA activity and culturable counts. The results suggest that the growth of microbes on a material surface depends on the availability of organic matter rather than the classification of the material as green or conventional. NAHA activity and culturability correlated well indicating that the two methods used in the experiments gave similar trends for the growth of fungi on material surfaces.