Fungal lysozyme leverages the gut microbiota to curb DSS-induced colitis.

Colitis is characterized by colonic inflammation and impaired gut health. Both features aggravate obesity and insulin resistance. Host defense peptides (HDPs) are key regulators of gut homeostasis and generally malfunctioning in above-mentioned conditions. We aimed here to improve bowel function in diet-induced obesity and chemically induced colitis through daily oral administration of lysozyme, a well-characterized HDP, derived from Acremonium alcalophilum.C57BL6/J mice were fed either low-fat reference diet or HFD ± daily gavage of lysozyme for 12 weeks, followed by metabolic assessment and evaluation of colonic microbiota encroachment. To further evaluate the efficacy of intestinal inflammation, we next supplemented chow-fed BALB/c mice with lysozyme during Dextran Sulfate Sodium (DSS)-induced colitis in either conventional or microbiota-depleted mice. We assessed longitudinal microbiome alterations by 16S amplicon sequencing in both models.Lysozyme dose-dependently alleviated intestinal inflammation in DSS-challenged mice and further protected against HFD-induced microbiota encroachment and fasting hyperinsulinemia. Observed improvements of intestinal health relied on a complex gut flora, with the observation that microbiota depletion abrogated lysozyme's capacity to mitigate DSS-induced colitis.Akkermansia muciniphila associated with impaired gut health in both models, a trajectory that was mitigated by lysozyme administration. In agreement with this notion, PICRUSt2 analysis revealed specific pathways consistently affected by lysozyme administration, independent of vivarium, disease model and mouse strain.Taking together, lysozyme leveraged the gut microbiota to curb DSS-induced inflammation, alleviated HFD-induced gastrointestinal disturbances and lowered fasting insulin levels in obese mice. Collectively, these data present A. alcalophilum-derived lysozyme as a promising candidate to enhance gut health.

A novel GH30 xylobiohydrolase from Acremonium alcalophilum releasing xylobiose from the non-reducing end.

Xylanases of the GH30 family are grouped to subfamilies GH30-7 and GH30-8. The GH30-8 members are of bacterial origin and well characterized, while the GH30-7 members are from fungal sources and their properties are quite diverse. Here, a heterologous expression and characterization of the GH30-7 xylanase AaXyn30A from a cellulolytic fungus Acremonium alcalophilum is reported. From various polymeric and oligomeric substrates AaXyn30A generates xylobiose as the main product. It was proven that xylobiose is released from the non-reducing end of all tested substrates, thus the enzyme behaves as a typical non-reducing-end acting xylobiohydrolase. AaXyn30A is active on different types of xylan, exhibiting the highest activity on rhodymenan (linear ß-1,3-ß-1,4-xylan) from which also an isomeric xylotriose Xyl-ß-1,3-Xyl-ß-1,4-Xyl is formed. Production of xylobiose from glucuronoxylan is at later stage accompanied by a release of aldouronic acids differing from those liberated by the bacterial GH30-8 glucuronoxylanases.

Application of Genetic Engineering Approaches to Improve Bacterial Metabolite Production.

Genetic engineering is a powerful method to improve the fermentation yield of bacterial metabolites. Since many biosynthetic mechanisms of bacterial metabolites have been unveiled, genetic engineering approaches have been applied to various issues of biosynthetic pathways, such as transcription, translation, post-translational modification, enzymes, transporters, etc. In this article, natamycin, avermectins, gentamicins, piperidamycins, and ß-valienamine have been chosen as examples to review recent progress in improving their production by genetic engineering approaches. In these cases, not only yields of target products have been increased, but also yields of by-products have been decreased, and new products have been created.

The flavonoid degrading fungus Acremonium sp. DSM 24697 produces two diglycosidases with different specificities.

AbstractDiglycosidases hydrolyze the heterosidic linkage of diglycoconjugates, releasing the disaccharide and the aglycone. Usually, these enzymes do not hydrolyze or present only low activities towards monoglycosylated compounds. The flavonoid degrading fungus Acremonium sp. DSM 24697 produced two diglycosidases, which were termed 6-O-α-rhamnosyl-ß-glucosidase I and II (αRßG I and II) because of their function of releasing the disaccharide rutinose (6-O-α-L-rhamnosyl-ß-D-glucose) from the diglycoconjugates hesperidin or rutin. In this work, the genome of Acremonium sp. DSM 24697 was sequenced and assembled with a size of ~ 27 Mb. The genes encoding αRßG I and II were expressed in Pichia pastoris KM71 and the protein products were purified with apparent molecular masses of 42 and 82 kDa, respectively. A phylogenetic analysis showed that αRßG I grouped in glycoside hydrolase family 5, subfamily 23 (GH5), together with other fungal diglycosidases whose substrate specificities had been reported to be different from αRßG I. On the other hand, αRßG II grouped in glycoside hydrolase family 3 (GH3) and thus is the first GH3 member that hydrolyzes the heterosidic linkage of rutinosylated compounds. The substrate scopes of the enzymes were different: αRßG I showed exclusive specificity toward 7-O-ß-rutinosyl flavonoids, whereas αRßG II hydrolyzed both 7-O-ß-rutinosyl- and 3-O-ß-rutinosyl- flavonoids. None of the enzymes displayed activity toward 7-O-ß-neohesperidosyl- flavonoids. The recombinant enzymes also exhibited transglycosylation activities, transferring rutinose from hesperidin or rutin onto various alcoholic acceptors. The different substrate scopes of αRßG I and II may be part of an optimized strategy of the original microorganism to utilize different carbon sources.

Complete biosynthetic pathways of ascofuranone and ascochlorin in Acremonium egyptiacum.

Ascofuranone (AF) and ascochlorin (AC) are meroterpenoids produced by various filamentous fungi, including Acremonium egyptiacum (synonym: Acremonium sclerotigenum), and exhibit diverse physiological activities. In particular, AF is a promising drug candidate against African trypanosomiasis and a potential anticancer lead compound. These compounds are supposedly biosynthesized through farnesylation of orsellinic acid, but the details have not been established. In this study, we present all of the reactions and responsible genes for AF and AC biosyntheses in A. egyptiacum, identified by heterologous expression, in vitro reconstruction, and gene deletion experiments with the aid of a genome-wide differential expression analysis. Both pathways share the common precursor, ilicicolin A epoxide, which is processed by the membrane-bound terpene cyclase (TPC) AscF in AC biosynthesis. AF biosynthesis branches from the precursor by hydroxylation at C-16 by the P450 monooxygenase AscH, followed by cyclization by a membrane-bound TPC AscI. All genes required for AC biosynthesis (ascABCDEFG) and a transcriptional factor (ascR) form a functional gene cluster, whereas those involved in the late steps of AF biosynthesis (ascHIJ) are present in another distantly located cluster. AF is therefore a rare example of fungal secondary metabolites requiring multilocus biosynthetic clusters, which are likely to be controlled by the single regulator, AscR. Finally, we achieved the selective production of AF in A. egyptiacum by genetically blocking the AC biosynthetic pathway; further manipulation of the strain will lead to the cost-effective mass production required for the clinical use of AF.

Enzyme-mediated transglycosylation of rutinose (6-O-α-l-rhamnosyl-d-glucose) to phenolic compounds by a diglycosidase from Acremonium sp. DSM 24697.

The structure of the carbohydrate moiety of a natural phenolic glycoside can have a significant effect on the molecular interactions and physicochemical and pharmacokinetic properties of the entire compound, which may include anti-inflammatory and anticancer activities. The enzyme 6-O-α-rhamnosyl-ß-glucosidase (EC 3.2.1.168) has the capacity to transfer the rutinosyl moiety (6-O-α-l-rhamnopyranosyl-ß-d-glucopyranose) from 7-O-rutinosylated flavonoids to hydroxylated organic compounds. This transglycosylation reaction was optimized using hydroquinone (HQ) and hesperidin as rutinose acceptor and donor, respectively. Since HQ undergoes oxidation in a neutral to alkaline aqueous environment, the transglycosylation process was carried out at pH values ≤6.0. The structure of 4-hydroxyphenyl-ß-rutinoside was confirmed by NMR, that is, a single glycosylated product with a free hydroxyl group was formed. The highest yield of 4-hydroxyphenyl-ß-rutinoside (38%, regarding hesperidin) was achieved in a 2-h process at pH 5.0 and 30 °C, with 36 mM OH-acceptor and 5% (v/v) cosolvent. Under the same conditions, the enzyme synthesized glycoconjugates of various phenolic compounds (phloroglucinol, resorcinol, pyrogallol, catechol), with yields between 12% and 28% and an apparent direct linear relationship between the yield and the pKa value of the aglycon. This work is a contribution to the development of convenient and sustainable processes for the glycosylation of small phenolic compounds.

Expression of the 5-enoylpyruvylshikimate-3-phosphate synthase domain from the Acremonium sp. aroM complex enhances resistance to glyphosate.

OBJECTIVE: To discover and isolate a glyphosate-resistant gene from a microorganism through gene mining. RESULTS: The full aroM gene from Acremonium sp. (named aroMA.sp.) was cloned using rapid amplification of cDNA ends. The transcriptional expression level of each domain increased significantly after glyphosate treatment in the aroMA.sp. complex and reached its maximum at 48 h. The aroA domain of the aroMA.sp. (named aroA A.sp.) was expressed in Escherichia coli BL21 (DE3) and the product was purified through Ni-NTA affinity chromatography. Furthermore, 45 KDa was indicated by SDS-PAGE and its enzyme activity was optimal at 30 °C and PH 7.0. The Ki/Km value of aroAA.sp. was 0.106, and the E. coli BL21 harboring aroAA.sp. could grow in the M9 minimal medium with 100 mM glyphosate. CONCLUSION: The aroAA.sp. from the aroMA.sp. complex had high enzyme activity and glyphosate resistance. Therefore, this research offers a new strategy for improving glyphosate resistance using the aroA domain of the aroM complex in the fungi.

Expression, Purification, and Activity of ActhiS, a Thiazole Biosynthesis Enzyme from Acremonium chrysogenum.

Thiamine pyrophosphate is an essential coenzyme in all organisms. Its biosynthesis involves independent syntheses of the precursors, pyrimidine and thiazole, which are then coupled. In our previous study with overexpressed and silent mutants of ActhiS (thiazole biosynthesis enzyme from Acremonium chrysogenum), we found that the enzyme level correlated with intracellular thiamine content in A. chrysogenum. However, the exact structure and function of ActhiS remain unclear. In this study, the enzyme-bound ligand was characterized as the ADP adduct of 5-(2-hydroxyethyl)-4-methylthiazole-2-carboxylic acid (ADT) using HPLC and 1H NMR. The ligand-free ActhiS expressed in M9 minimal medium catalyzed conversion of NAD+ and glycine to ADT in the presence of iron. Furthermore, the C217 residue was identified as the sulfur donor for the thiazole moiety. These observations confirm that ActhiS is a thiazole biosynthesis enzyme in A. chrysogenum, and it serves as a sulfur source for the thiazole moiety.

Characterisation of three fungal glucuronoyl esterases on glucuronic acid ester model compounds.

The glucuronoyl esterases (GEs) that have been identified so far belong to family 15 of the carbohydrate esterases in the CAZy classification system and are presumed to target ester bonds between lignin alcohols and (4-O-methyl-)D-glucuronic acid residues of xylan. Few GEs have been cloned, expressed and characterised to date. Characterisation has been done on a variety of synthetic substrates; however, the number of commercially available substrates is very limited. We identified novel putative GEs from a wide taxonomic range of fungi and expressed the enzymes originating from Acremonium alcalophilum and Wolfiporia cocos as well as the previously described PcGE1 from Phanerochaete chrysosporium. All three fungal GEs were active on the commercially available compounds benzyl glucuronic acid (BnGlcA), allyl glucuronic acid (allylGlcA) and to a lower degree on methyl glucuronic acid (MeGlcA). The enzymes showed pH stability over a wide pH range and tolerated 6-h incubations of up to 50 °C. Kinetic parameters were determined for BnGlcA. This study shows the suitability of the commercially available model compounds BnGlcA, MeGlcA and allylGlcA in GE activity screening and characterisation experiments. We enriched the spectrum of characterised GEs with two new members of a relatively young enzyme family. Due to its biotechnological significance, this family deserves to be more extensively studied. The presented enzymes are promising candidates as auxiliary enzymes to improve saccharification of plant biomass.

Silage fermentation and ruminal degradation of stylo prepared with lactic acid bacteria and cellulase.

In order to improve the silage fermentation of stylo (Stylosanthes guianensis) in tropical areas, stylo silages were prepared with commercial additives Lactobacillus plantarum Chikuso-1 (CH1), L. rhamnasus Snow Lact L (SN), Acremonium cellulase (CE) and their combination as SN+CE or CH1 + CE, and the fermentation quality, chemical composition and ruminal degradation of these silages were studied. Stylo silages treated with lactic acid bacteria (LAB) or cellulase, the pH value and NH3 -N / total-N were significantly (P < 0.05) decreased while the ruminal degradability of dry matter (DM), crude protein (CP), neutral detergent fiber (aNDFom) and acid detergent fiber (ADFom) were significantly (P < 0.05) increased compared to control. Compared to LAB or cellulase-treated silages, the DM, CP contents and relative feed value (RFV), and the ruminal degradability in LAB plus cellulase-treated silages were significantly (P < 0.05) higher, but the aNDFom content was significantly (P < 0.05) lower. CH1 + CE treatment was more effective in silage fermentation and ruminal degradation than SN+CE treatment. The results confirmed that LAB or LAB plus cellulase treatment could improve the fermentation quality, chemical composition and ruminal degradation of stylo silage. Moreover, the combined treatment with LAB and cellulase may have beneficial synergistic effects on ruminal degradation.

Hydrophobic adsorption in ionic medium improves the catalytic properties of lipases applied in the triacylglycerol hydrolysis by synergism.

It is known that lipases may have their catalytic properties improved by the action of some salts or by the adsorption on hydrophobic supports. However, what we present in this work is more than that: we evaluate the combination of these two factors of hyperactivation of lipases from Acremonium-like ROG 2.1.9, a study that has not been done so far. This work proves that a synergistic effect occurs when the lipases are immobilized on hydrophobic supports at the presence of sodium chloride and are applied in triacylglycerol hydrolysis. This assay made it possible to achieve the highest hyperactivation of 500 % with the lipases immobilized on Phenyl-Sepharose and applied with 0.1 M of sodium chloride. Besides this positive effect on enzyme activity, the use of these two factors led to the thermal stability increasing of the immobilized lipases. For this derivative, the recovered activity was approximately 85 % after 6 h incubated at 55 °C and 1.0 M of the sodium chloride against 50 % of the same derivative without this salt. Furthermore, others assays were performed to prove the evidences about the synergistic effect, showing a promising method to improve the catalytic properties of the lipases from Acremonium-like ROG 2.1.9.

A glucuronoyl esterase from Acremonium alcalophilum cleaves native lignin-carbohydrate ester bonds.

The Glucuronoyl esterases (GE) have been proposed to target lignin-carbohydrate (LC) ester bonds between lignin moieties and glucuronic acid side groups of xylan, but to date, no direct observations of enzymatic cleavage on native LC ester bonds have been demonstrated. In the present investigation, LCC fractions from spruce and birch were treated with a recombinantly produced GE originating from Acremonium alcalophilum (AaGE1). A combination of size exclusion chromatography and (31) P NMR analyses of phosphitylated LCC samples, before and after AaGE1 treatment provided the first evidence for cleavage of the LC ester linkages existing in wood.

Soluble inhibitors generated during hydrothermal pretreatment of oil palm mesocarp fiber suppressed the catalytic activity of Acremonium cellulase.

Oil palm mesocarp fiber was subjected to hydrothermal pretreatment under isothermal and non-isothermal conditions. The pretreated slurries were separated by filtration, pretreated liquids and solids were characterized. An enzymatic digestibility study was performed for both pretreated slurries and solids to understand the effect of soluble inhibitors generated during the pretreatment process. The highest glucose yield obtained from pretreated slurries was 70.1%, and gradually decreased with higher pretreatment severities. The highest glucose yield obtained in pretreated solids was 100%, after pretreatment at 210°C for 20min. In order to study the inhibitory effects of compounds generated during pretreatment with cellulase, technical grade solutions that mimic the pretreated liquid were prepared and their effect on Acremonium cellulase activity was monitored using Avicel. Xylo-oligomers and tannic acid were identified as powerful inhibitors of Acremonium cellulase, and the lowest hydrolysis rate of Avicel of 0.18g/g-glucose released/L/h was obtained from tannic acid.

Acthi, a thiazole biosynthesis enzyme, is essential for thiamine biosynthesis and CPC production in Acremonium chrysogenum.

BACKGROUND: The filamentous fungus Acremonium chrysogenum is an important industrial fungus and is used in the production of the ß-lactam antibiotic cephalosporin C. Little is known regarding the molecular and biological mechanisms of how this industrial strain was improved by mutagenesis and molecular breeding. Comparative proteomics is one of the most powerful methods to evaluate the influence of gene expression on metabolite production. RESULTS: In this study, we used comparative proteomics to investigate the molecular mechanisms involved in the biosynthesis of cephalosporin C between a high-producer (HY) strain and a wide-type (WT) strain. We found that the expression levels of thiamine biosynthesis-related enzymes, including the thiazole biosynthesis enzyme (Acthi), pyruvate oxidase, flavin adenine dinucleotide (FAD)-dependent oxidoreductase and sulfur carrier protein-thiS, were up-regulated in the HY strain. An Acthi-silencing mutant of the WT strain grew poorly on chemically defined medium (MMC) in the absence of thiamine, and its growth was recovered on MMC medium supplemented with thiamine. The intracellular thiamine content was changed in the Acthi silencing or over-expression mutants. In addition, we demonstrated that the manipulation of the Acthi gene can affect the hyphal growth of Acremonium chrysogenum, the transcription levels of cephalosporin C biosynthetic genes, the quantification levels of precursor amino acids for cephalosporin C synthesis and the expression levels of thiamine diphosphate-dependent enzymes. Over-expression of Acthi can significantly increase the cephalosporin C yield in both the WT strain and the HY mutant strain. CONCLUSIONS: Using comparative proteomics, four differently expressed proteins were exploited, whose functions may be involved in thiamine diphosphate metabolism. Among these proteins, the thiazole biosynthesis enzyme (ActhiS) may play an important role in cephalosporin C biosynthesis. Our studies suggested that Acthi might be involved in the transcriptional regulation of cephalosporin C biosynthesis. Therefore, the thiamine metabolic pathway could be a potential target for the molecular breeding of this cephalosporin C producer for industrial applications.

Polyethyleneimine coating of magnetic particles increased the stability of an immobilized diglycosidase.

The diglycosidase, α-rhamnosyl-ß-glucosidase, from Acremonium sp. DSM24697 was immobilized by adsorption and cross-linking onto polyaniline-iron (PI) particles. The immobilization yield and the immobilization efficiency were relatively high, 31.2% and 8.9%, respectively. However, the heterogeneous preparation showed lower stability in comparison with the soluble form of the enzyme in operational conditions at 60 °C. One parameter involved in the reduced stability of the heterogeneous preparation was the protein metal-catalyzed oxidation achieved by iron traces supplied from the support. To overcome the harmful effect, iron particles were coated with polyethyleneimine (PEI; 0.84 mg/g) previously for the immobilization of the catalyst. The increased stability of the catalyst was correlated with the amount of iron released from the support. Under operational conditions, the uncoated particles lost between 76% and 52% activity after two cycles of reuse, whereas the PEI-coated preparation reduced 45-28% activity after five cycles of reuse in the range of pH 5.0-10, respectively. Hence, polymer coating of magnetic materials used as enzyme supports might be an interesting approach to improve the performance of biotransformation processes.

Characterization of the xylanase regulator protein gene, xlnR, in Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus).

We cloned a putative Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) xlnR gene and isolated a xlnR disruptant strain. XlnR protein was localized in the nucleus. Xylanase production by the xlnR disruptant was lower than in the control strain at both the enzyme and transcriptional level. These data suggest that the XlnR protein regulates xylanase production in T. cellulolyticus.

Structural insights into rice straw pretreated by hot-compressed water in relation to enzymatic hydrolysis.

Pretreatment-induced structural alteration is critical in influencing the rate and extent of enzymatic saccharification of lignocellulosic biomass. The present work has investigated structural features of rice straw pretreated by hot-compressed water (HCW) from 140 to 240 °C for 10 or 30 min and enzymatic hydrolysis profiles of pretreated rice straw. Compositional profiles of pretreated rice straw were examined to offer the basis for structural changes. The wide-angle X-ray diffraction analysis revealed possible modification in crystalline microstructure of cellulose and the severity-dependent variation of crystallinity. The specific surface area (SSA) of pretreated samples was able to achieve more than 10-fold of that of the raw material and was in linear relationship with the removal of acetyl groups and xylan. The glucose yield by enzymatic hydrolysis of pretreated materials correlated linearly with the SSA increase and the dissolution of acetyl and xylan. A quantitatively intrinsic relationship was suggested to exist between enzymatic hydrolysis and the extraction of hemicellulose components in hydrothermally treated rice straw, and SSA was considered one important structural parameter signaling the efficiency of enzymatic digestibility in HCW-treated materials in which hemicellulose removal and lignin redistribution happened.

Crystal structure of Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) GH family 11 xylanase.

Talaromyces cellulolyticus (formerly known as Acremonium cellulolyticus) is one of the mesophilic fungi that can produce high levels of cellulose-related enzymes and are expected to be used for the degradation of polysaccharide biomass. In silico analysis of the genome sequence of T. cellulolyticus detected seven open reading frames (ORFs) showing homology to xylanases from glycoside hydrolase (GH) family 11. The gene encoding the GH11 xylanase C (TcXylC) with the highest activity was used for overproduction and purification of the recombinant enzyme, permitting solving of the crystal structure to a resolution of 1.98 Å. In the asymmetric unit, two kinds of the crystal structures of the xylanase were identified. The main structure of the protein showed a ß-jelly roll structure. We hypothesize that one of the two structures represents the open form and the other shows the close form. The changing of the flexible region between the two structures is presumed to induce and accelerate the enzyme reaction. The specificity of xylanase toward the branched xylan is discussed in the context of this structural data and by comparison with the other published structures of xylanases.

Ball milling pretreatment of oil palm biomass for enhancing enzymatic hydrolysis.

Oil palm biomass, namely empty fruit bunch and frond fiber, were pretreated using a planetary ball mill. Particle sizes and crystallinity index values of the oil palm biomass were significantly reduced with extended ball mill processing time. The treatment efficiency was evaluated by the generation of glucose, xylose, and total sugar conversion yields from the pretreatment process compared to the amount of sugars from raw materials. Glucose and xylose contents were determined using high-performance liquid chromatography. An increasing trend in glucose and xylose yield as well as total sugar conversion yield was observed with decreasing particle size and crystallinity index. Oil palm frond fiber exhibited the best material yields using ball milling pretreatment with generated glucose, xylose, and total sugar conversion yields of 87.0, 81.6, and 85.4%, respectively. In contrast, oil palm empty fruit bunch afforded glucose and xylose of 70.0 and 82.3%, respectively. The results obtained in this study showed that ball mill-treated oil palm biomass is a suitable pretreatment method for high conversion of glucose and xylose.

Application of endo-ß-1,4,D-mannanase and cellulase for the release of mannooligosaccharides from steam-pretreated spent coffee ground.

Spent coffee ground (SCG), a present waste stream from instant coffee production, represents a potential feedstock for mannooligosaccharides (MOS) production. MOS can be used in nutraceutical products for humans/animals or added to instant coffee, increasing process yield and improving product health properties. The SCG was evaluated for MOS production by steam pretreatment and enzymatic hydrolysis with a recombinant mannanase and a commercial cellulase cocktail (Acremonium, Bioshigen Co. Ltd, Japan). The mannanase was produced using a recombinant strain of Yarrowia lipolytica, used to produce and secrete endo-1,4-ß,D-mannanase from Aspergillus aculeatus in bioreactor cultures. Endo-1,4-ß,D-mannanase was produced with an activity of 183.5 U/mL and 0.23 mg protein/mL. The enzyme had an optimum temperature of 80 °C, and the activity in the supernatant was improved by 150 % by supplementation with 0.2 % sodium benzoate and 35 % sorbitol as a preservative and stabiliser, respectively. The steam pretreatment of SCG improved the enzymatic digestibility of SCG, thus reducing the required enzyme dosage for MOS release. Combined enzymatic hydrolysis of untreated or steam-pretreated (150, 190 and 200 °C for 10 min) SCG with mannanase and cellulase cocktail resulted in 36-57 % (based on mannan content) of MOS production with a degree of polymerization of up to 6. The untreated material required at least 1 % of both mannanase and cellulase loading. The optimum mannanase and cellulase loadings for pretreated SCG hydrolysis were between 0.3 and 1 and 0.4 and 0.8 %, respectively. Statistical analysis suggested additive effect between cellulase cocktail and mannanase on MOS release, with no indication of synergism observed.