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β-glucan derived from the black yeast Aureobasidium pullulans (A. pullulans) is one of the natural products attracting attention due to its high potential for application as a functional food and pharmaceutical. Our team of researchers obtained a highly soluble, low-molecular-weight β-glucan from the fermentation culture medium of A. pullulans via mechanochemical ball milling method, that is, the low-molecular-weight A. pullulans-fermented β-D-glucan (LMW-AP-FBG). We investigated the anti-inflammatory effect of LMW-AP-FBG using lipopolysaccharide (LPS)-stimulated murine macrophages (RAW264.7 cells) in the current study. LMW-AP-FBG altered LPS-stimulated inflammatory responses by reducing the release of inflammatory mediators such as nitric oxide (NO), interleukin (IL)-1β, IL-6 and tumor necrosis factor-α. As well, the mitogen-activated protein kinases (MAPKs) and nuclear factor-κB (NF-κB) signaling pathways were downregulated by LMW-AP-FBG. Furthermore, LMW-AP-FBG markedly reduced LPS-induced expression of cell surface molecules, CD14, CD86, and MHC class II, which mediate macrophage activation. These findings suggest that LMW-AP-FBG can be used as an effective immune modulator to attenuate the progression of inflammatory disease.

Xylooligosaccharides (XOS) are non-digestible and fermentable oligomers that stand out for their efficient production by enzymatic hydrolysis and beneficial effects on human health. This study aimed to investigate the influence of the main reaction parameters of the beechwood xylan hydrolysis using crude xylanase from Aureobasidium pullulans CCT 1261, thus achieving the maximum XOS production. The effects of temperature (40 to 50 °C), reaction time (12 to 48 h), type of agitation, substrate concentration (1 to 6%, w/v), xylanase loading (100 to 300 U/g xylan), and pH (4.0 to 6.0) on the XOS production were fully evaluated. The most suitable conditions for XOS production included orbital shaking of 180 rpm, 40 °C, and 24 h of reaction. High contents of total XOS (10.1 mg/mL) and XOS with degree of polymerization (DP) of 2-3 (9.7 mg/mL), besides to a high percentage of XOS (99.1%), were obtained at 6% (w/v) of beechwood xylan, xylanase loading of 260 U/g xylan, and pH 6.0. The establishment of the best hydrolysis conditions allowed increasing both the content of total XOS 1.5-fold and the percentage of XOS by 9.4%, when compared to the initial production (6.7 mg/mL and 89.7%, respectively). Thus, this study established an efficient enzymatic hydrolysis process that results in a hydrolysate containing XOS with potential prebiotic character (i.e., rich in XOS with DP 2-3) and low xylose amounts.

In this study, cost-effective substrates such as cassava starch, corn steep liquor (CSL) and soybean meal hydrolysate (SMH) were used for pullulan production by Aureobasidium pullulans CCTCC M 2012259. The medium was optimized using response surface methodology (RSM) and artificial neural network (ANN) approaches, and analysis of variance indicated that the ANN model achieved higher prediction accuracy. The optimal medium predicted by ANN was used to produce high molecular weight pullulan in high yield. SMH substrates increased both biomass and pullulan titer, while CSL substrates maintained higher pullulan molecular weight. Results of kinetic parameters, key enzyme activities and intracellular uridine diphosphate glucose contents revealed the physiological mechanism of changes in pullulan titer and molecular weight using different substrates. Economic analysis of batch pullulan production using different substrates was performed, and the cost of nutrimental materials for CSL and SMH substrates was decreased by 46.1% and 49.9%, respectively, compared to the control using glucose and yeast extract as substrates, which could improve the competitiveness of pullulan against other polysaccharides in industrial applications.

To improve β-1,3-1,6-D-glucan (β-glucan) production by Aureobasidium pullulans, an Agrobacterium tumefaciens-mediated transformation method was developed to screen a mutant A. pullulans CGMCC 19650. Based on thermal asymmetric-interlaced PCR detection, DNA sequencing, BLAST analysis, and quantitative real-time PCR assay, the T-DNA was identified to be inserted in the coding region of mal31 gene, which encodes a sugar transporter involved in pullulan biosynthesis in the mutant. The maximal biomass and β-glucan production under batch fermentation were significantly increased by 47.6% and 78.6%, respectively, while pullulan production was decreased by 41.7% in the mutant, as compared to the parental strain A. pullulans CCTCC M 2012259. Analysis of the physiological mechanism of these changes revealed that mal31 gene disruption increased the transcriptional levels of pgm2, ugp, fks1, and kre6 genes; increased the amounts of key enzymes associated with UDPG and β-glucan biosynthesis; and improved intracellular UDPG contents and energy supply, all of which favored β-glucan production. However, the T-DNA insertion decreased the transcriptional levels of ags2 genes, and reduced the biosynthetic capability to form pullulan, resulting in the decrease in pullulan production. This study not only provides an effective approach for improved β-glucan production by A. pullulans, but also presents an accurate and useful gene for metabolic engineering of the producer for efficient polysaccharide production. KEY POINTS: • A mutant A. pullulans CGMCC 19650 was screened by using the ATMT method. • The mal31 gene encoding a sugar transporter was disrupted in the mutant. • β-Glucan produced by the mutant was significantly improved.

Cv. Arbequina fruits were inoculated during one day with suspensions of Aureobasidium pullulans, Epicoccum nigrum or both species (104 spores/g olives), isolated from olive leaves, aiming to increase the phenolic content of the olive oils. Oils were extracted from non-inoculated (control) and inoculated olives, being evaluated for their oxidative stability, and phenolic composition. Compared to the control oils, those extracted from inoculated olives showed higher oxidative stability (+1629%), phenolic acids contents (up to+37%), phenolic alcohols (up to+101%), flavonoids (up to+154%) and secoiridoid derivatives (up to + 12%). These positive effects could be attributed to the hydrolytic activity of enzymes secreted by the studied fungi on the fruit cellular membranes and extracted phenolics, increasing the extractability and, consequently, altering the phenolic concentration. Considering the two fungi species, E. nigrum inoculation had a more significant positive impact on the contents of tyrosol, oleuropein, and ligstroside derivatives, showing an inhibition effect regarding the oleacein formation. Contrary, A. pullulans inoculation mainly resulted into an accentuated increase of luteolin and oleacein contents (3 and 12-times higher compared with control oils, respectively). Overall, no enhanced positive synergetic effect could be attributed to the inoculation with a suspension containing both fungi. The significant changes observed on the phenolic contents, due to the fungal inoculation, allowed the successful discrimination of the four types of extracted oils using an electronic tongue. This device acts as an artificial taste device allowing anticipating an impact at the oils basic taste sensations due to the fungi inoculation., The authors are grateful to the Foundation for Science and Technology (FCT, Portugal) for fnancial support by national funds FCT/MCTES (PIDDAC) to CIMO (UIDB/00690/2020 and UIDP/00690/2020), CEB (UIDB/04469/2020), REQUIMTE-LAQV (UIDB/50006/2020) units and SusTEC (LA/P/0007/2020); to Project “GreenHealth—Digital strategies in biological assets to improve well being and promote green health” (Norte-01–0145-FEDER-000042) and to Project AgriFood XXI (NORTE-01–0145-FEDER-000041), funded by the European Regional Development Fund under the scope of Norte2020 ‐ Programa Operacional Regional do Norte. Ítala M.G. Marx acknowledges the Ph.D. grant (SFRH/BD/137283/2018) pro vided by FCT. Nuno Rodrigues thanks to National funding by FCT-Foundation for Science and Technology, P.I., through the institutional scientifc employment program-contract, info:eu-repo/semantics/publishedVersion

In this study, crude and partially purified xylanase from Aureobasidium pullulans CCT 1261 were characterized and applied in beechwood xylan hydrolysis to obtain xylooligosaccharides (XOS). Fractional precipitation (0–30 %/30–60 %) with ammonium sulfate allowed partial purification of xylanases in 6.8-fold and recovery of 69.4 %. Both extracts exhibited optimum activity at 50 °C and pH 4.5, and greater stability at 40 °C within the studied range. The thermostability parameters (kd, t1/2, ΔH*, ΔG*, ΔS*) of the extracts indicated the enzyme denaturation by the increment in temperature from 40 °C to 60 °C; its process was endothermic and led to protein unfolding in a more random structure. Crude xylanase showed a lower value of the Michaelis-Menten constant (25 mg/mL) than the partially purified enzyme (50 mg/mL). In hydrolysis reactions, total XOS contents (7.7 mg/mL and 7.9 mg/mL) and XOS yields (25.7 % and 26.5 %) were equal statistically using the crude or partially purified extracts, differing only in the amount of xylose released. The hydrolysates were mainly composed of potential prebiotics, i.e., xylobiose (50.9 % and 53.8 %) and xylotriose (30.9 % and 32.8 %). Based on these results, the use of crude and partially purified xylanase from A. pullulans showed a promising perspective for xylan hydrolysis to produce XOS.

Cucumber mosaic virus (CMV), the most prevalent virus in chili pepper (Capsicum annuum L.), negatively affects chili pepper production in South Korea. In this study, foliar spraying with β-glucans obtained from the mycelial walls of the yeast-like fungus Aureobasidium pullulans inhibited CMV infection of chili pepper if applied before virus inoculation. At three concentrations, β-glucans from A. pullulans significantly ameliorated CMV symptoms in treated chili pepper; the effect was greater in plants treated with 0.01% β-glucans than 0.005% or 0.001% β-glucans. Double antibody sandwich enzyme-linked immunosorbent assay showed that these β-glucans treatments resulted in 1.7- to 10-fold reductions in CMV accumulation in the treated chili pepper. The glucans did not act directly on the virus and did not interfere with virus disassembly or replication. Foliar spraying with 0.01% β-glucans from A. pullulans at 24 hr intervals for 3 days significantly increased plant height, the total number of fruit, and the fresh weight of chili pepper fruit. However, the stem diameter of chili pepper treated with β-glucans did not increase significantly. These results indicate that foliar spraying with β-glucans from A. pullulans acts an antiviral agent against CMV infection and stimulates chili pepper growth.

Seed sprouts are nutrient-rich components, and their use in human and animal diets have increased in recent times. Microbial fermentation of seed sprouts not only improves the nutrient digestibility but also elevates the nutritional characteristics by lowering the level of antinutritional factors (ANFs). This study evaluates the effect of sprouting followed by fungal fermentation on the nutritional characteristics of canola seed sprouts. Canola seeds were soaked overnight and sprouted over a 6-day period. Dried and ground seed sprouts were subjected to submerged fermentation for 5 days using three fungal strains (Aureobasidium pullulans, Trichoderma reesei, Neurospora crassa). Results showed an improvement in proteins and reduction in ANFs of sprouts when compared to seeds. Fermentation with T. reesei resulted in higher protein titers, while N. crassa fermented sprouts exhibited slight reduction in crude fiber. Sprouting prior to fermentation helped in enhancing the protein solubility of canola seed and reducing the overall fermentation duration. Based on these findings it could be concluded that sprouting prior to fermentation can potentially lead to the production of nutritionally enriched canola based high protein ingredient. However, further research needs to be conducted to develop a mechanism for minimizing the oil loss during seed sprouting process.

Pullulan is an imperative microbial exo-polymer commercially produced by yeast like fungus Aureobasidium pullulans. Its structure contains maltosyl repeating units which comprises two α-(1 → 4) linked glucopyranose rings attached to one glucopyranose ring through α-(1 → 6) glycosidic bond. The co-existence of α-(1 → 6) and α-(1 → 4) glycosidic linkages endows distinctive physico-chemical properties to pullulan. It is highly biocompatible, non-toxic and non-carcinogenic in nature. It is extremely resistant to any mutagenicity or immunogenicity. The unique properties of pullulan make it a potent candidate for biomedical applications viz. drug delivery, gene delivery, tissue engineering, molecular chaperon, plasma expander, vaccination, etc. This review highlights the potential of pullulan in biomedical research and development.

Extracellular rhodanese obtained from Aureobasidium pullulans was employed in both free and immobilized forms for the biodegradation of cyanide present in cassava processing mill effluent (CPME). Crosslinking with glutaraldehyde (at an optimum concentration of 5% v/v) before entrapment in alginate beads resulted in the highest immobilization yield of 94.5% and reduced enzyme leakage of 1.8%. Rhodanese immobilized by cross-linking before entrapment (cbe) retained about 46% of its initial activity after eight cycles of catalysis compared to the entrapment in alginate alone (eaa) which lost more than 79% after the fifth catalytic cycle. A cross-examination of thermodynamic ( Δ G d * , Δ S d * , Δ H d * ) kinetic ( k d , t 1 / 2 , D and z - values ) parameters at 30-70 °C showed that cbe displayed a higher resistance to thermal inactivation when compared to the free enzyme (fe) and (eaa). The efficiency of cyanide biodegradation from the CPME by the fe, eaa and cbe were 55, 62, and 74% respectively after 6 h. Biodegradation of cyanide using the cbe was monitored using FTIR spectroscopy. Rhodanese immobilized via cbe had a higher resistance to thermal denaturation over other enzyme forms. Hence, this makes cbe adaptable for large-scale detoxification of cyanide from CPME.

β-poly(L-malic acid) (PMLA) has attracted industrial interest for its potential applications in medicine and other industries. For a sustainable PMLA production, it requires replacing/reducing the CaCO3 usage, since the residual CaCO3 impeded the cells’ utilization, and a large amount of commercially useless gypsum was accumulated. In this study, it was found that more glucose was converted into CO2 using soluble alkalis compared with CaCO3 usage. Moreover, since the high ion strength and respiration effect of soluble alkalis also inhibited PMLA production, they could not effectively replace CaCO3. Furthermore, comparing the fermentations with different neutralizers (soluble alkali vs. CaCO3), it was found that the differential genes are mainly involved in the pathway of starch and sucrose metabolism, pentose and glucuronate interconversions, histidine metabolism, ascorbate and aldarate metabolism, and phagosome. In detail, in the case with CaCO3, 562 genes were downregulated and 262 genes were upregulated, and especially, those genes involved in energy production and conversion were downregulated by 26.7%. Therefore, the irreplaceability of CaCO3 was caused by its effect on the PMLA metabolic pathway rather than its usage as neutralizer. Finally, a combined pH shift control strategy with CaCO3 addition was developed. After the fermentation, 64.8 g/L PMLA and 38.9 g/L biomass were obtained with undetectable CaCO3 and less CO2 emission. • The effect of CaCO3 on PMLA metabolic pathway resulted in its irreplaceability. • A pH shift control strategy with CaCO 3 addition was developed. • Undetectable CaCO 3 and less CO 2 emission were detected with the new strategy.

Aureobasidium pullulans LB83 was evaluated for cellulase production under submerged fermentation conditions. Different process variables such as carbon sources (corn cob, sugarcane bagasse, and sug...

Epiphytic yeasts were isolated from different cultivars of apples and lemons and identified by a combination of PCR-RFLP of 5.8S rRNA region and sequencing of D1/D2 domain of the 26S rRNA gene. Among 69 isolates, Aureobasidium pullulans GE17 and Meyerozyma guilliermondii KL3 strains showed the greatest antagonistic activity against two significant apple and lemon postharvest pathogens, Penicillium expansum DSM62841 (blue mold) and Penicillium digitatum DSM2750 (green mold), after preliminary screening. Yeasts were applied as single and mixed cultures with two different cell concentrations of 106 and 108 cells/ml in the present study. It was determined that antagonistic activity of two yeast strains studied emerged with a combination of several mechanisms of action including competition for space and nutrients, production of volatile organic compounds (VOCs), secretion of extracellular lytic enzymes and inhibition of fungal spore germination. The highest inhibition of mycelial growth on P. expansum DSM62841 and P. digitatum DSM2750 (83.4% and 74.7%, respectively) was achieved by utilization of single culture of A. pullulans GE17. Otherwise, the application of mixed culture at the ratio of 108 cells/ml inhibited spore germination of both pathogens from 86% to 95%. Results of this study suggest that an increase in yeast cell concentrations positively affected their biocontrol activity against blue and green molds. According to the results, employing single culture of M. guilliermondii KL3 did not exhibit effective antagonistic activity against blue and green molds. However, utilization of A. pullulans GE17 alone and mixed culture showed succesfull controlling against both P. expansum DSM62841 and P. digitatum DSM2750.

Fungal strain PA-2 was isolated from infected poplar leaves from the Ping'an District, Haidong City of Qinghai Province, China. Based on the culture characteristics and the internal transcribed spacer (ITS) sequence of its 16S rDNA, the strain was identified as Aureobasidium pullulans. The culture and metabolites of strain PA-2 showed high herbicidal potential to five tested weeds Galium aparine var. tenerum, Chenopodium album, Malva crispa, Polygonum lapathifolium and Avena fatua. For the in vitro test, 5 days after the detached leaves were inoculated with PA-2 culture, all leaves infected by the hyphae and became black and rotten. For the in vivo test, a metabolite filtrate of PA-2 culture was sprayed over the living weed plants, and five days after inoculation, the weed plants became withered and necrotic. Seven days after inoculation, the fresh weight reductions of G. aparine var. tenerum, C. album, M. crispa, P. lapathifolium and A. fatua were 87.25, 78.46, 82.25, 62.11, and 80.27%, respectively. Galium aparine var. tenerum and M. crispa exhibited significant reductions in fresh weight. The bio-safety test on the five crops showed no significant plant height reductions, which was also observed for wheat (Triticum aestivum), faba bean (Vicia faba), and barley (Hordeum vulgare). By contrast, oilseed rape (Brassica napus) and pea (Pisum sativum) exhibited light spots but no significant reductions in plant height. These results indicated that A. pullulans could be a potential microbial herbicide for the control of the target weeds in crops. Optimization of the carbon and nitrogen sources for cultural media and substances for solid-state fermentation indicated that PA-2 had better colony growth and spore production with the optimal carbon source of glucose (C4), nitrogen source of soybean flour (N2), and the optimal substance was wheat bran. The results in this study provide useful information for the development of A. pulluans PA-2 as an herbicide for bio-control of the weed.

Polymalic acid (PMA) is a novel biopolymer produced by the polyextremotolerant fungus Aureobasidium pullulans. In this study, a GATA-family transcriptional factor, Gat1, which regulates nitrogen uptake and PMA biosynthesis, was investigated. PMA production increased to 11.2% in the mutant overexpressing gat1 but decreased to 49.1% of the PMA titre when gat1 was knocked out from the genome of A. pullulans. Comparative transcriptome analysis of wild-type and mutant strains (∆gat1 and OE::gat1) revealed that 23 common differentially expressed genes were related to oxidative phosphorylation, ribosome biogenesis, and nitrogen metabolism. Under nitrogen-limited stress, regardless of the preferred nitrogen (glutamine, Gln) or non-preferred nitrogen (proline, Pro), 70% of Gat1 in the cells was located in the nucleus-cytoplasm, which resulted in an increase in nitrogen uptake and PMA biosynthesis regulation. Quantitative RT-PCR revealed that glucosekinase (GLK) in the glycolytic pathway and malate synthase (MLS) in the glyoxylate shunt pathway may be cross-regulated by Gat1 and nitrogen concentration (Gln or Pro), Therefore, glk was overexpressed in mutant strain (OE::gat1), which resulted in an increased PMA titre and yield of 12.6% and 13.0% respectively. These findings indicate that Gat1 may play an important role in the dual regulation of the nitrogen and carbon metabolisms in PMA biosynthesis.

A low molecular weight intracellular and extracellular fructosyltransferase was purified from the wild (Wt) and improved mutant-type (Mt) Aureobasidium pullulans and its characteristics and thermod...

A colour-variant strain of Aureobasidium pullulans FB-1 was used for the production of pullulan in a stirred tank reactor in a medium composed of sucrose, ammonium sulphate, yeast extract, K2HPO4, NaCl and MgSO4. Maximum pullulan production (4.8%, w/v) was obtained at an agitation rate of 300 rpm and aeration of 0.75 vvm in a stirred tank reactor (1 L) with a fermentation time of 6 days. Fourier transformed infrared and NMR spectrum of crude pullulan from A. pullulans FB-1 revealed solely α-(1→6) linked maltosyl units, in accord with the generally accepted structure of pullulan. Hydrolysis of crude pullulan, i.e. digestion with pullulanase also confirmed the identity as well as homogeneity of the produced pullulan. XRD analysis of crude pullulan verified its amorphous nature. Thermal analysis showed melting range of crude pullulan from 245.99–295.71 °C.

Bacterial cellulose (BC11 Uncommon abbreviation used: BC – Bacterial Cellulose) is a biomaterial produced by various strains of microorganisms. BC has improved strength and unique structural properties as compared to plant cellulose, thus has many usages in the food and pharmaceutical industries. In our previous study, a novel co-culture agitated fermentation of Komagataeibacter hansenii, a BC producer, with Aureobasidium pullulans, a producer of pullulan polysaccharide, had been demonstrated where the BC produced exhibited improved mechanical properties. Therefore, this study is undertaken to analyze BC production under different medium composition using response surface methodology (RSM) in shake-flasks and benchtop bioreactors. A verified local high point provided 22.4% higher BC production and 4.5- to 6- folds higher elastic moduli in shake-flasks and bioreactors compared to the baseline media. Overall, the study had revealed the potential of the co-culturing method to enhance BC production while maintaining the desired mechanical properties of BC produced in shake-flasks and larger scale bioreactors.

More than 30,000 tons of menthol are produced every year as a flavor and fragrance compound or as a medical component. So far, only extraction from plant material and chemical synthesis are possible. An alternative approach for menthol production could be a biotechnological-chemical process with ideally only two conversion steps, starting from (+)-limonene, which is a side product of the citrus processing industry. The first step requires a limonene-3-hydroxylase (L3H) activity that specifically catalyzes hydroxylation of limonene at carbon atom 3. Several protein engineering strategies have already attempted to create limonene-3-hydroxylases from bacterial cytochrome P450 monooxygenases (CYPs, or P450s), which can be efficiently expressed in bacterial hosts. However, their regiospecificity is rather low compared to that of the highly selective L3H enzymes from the biosynthetic pathway for menthol in Mentha species. The only naturally occurring limonene-3-hydroxylase activity identified in microorganisms so far was reported for a strain of the black yeast-like fungus Hormonema sp. in South Africa. We have discovered additional fungi that can catalyze the intended reaction and identified potential CYP-encoding genes within the genome sequence of one of the strains. Using heterologous gene expression and biotransformation experiments in yeasts, we were able to identify limonene-3-hydroxylases from Aureobasidium pullulans and Hormonema carpetanum. Further characterization of the A. pullulans enzyme demonstrated its high stereospecificity and regioselectivity, its potential for limonene-based menthol production, and its additional ability to convert α- and β-pinene to verbenol and pinocarveol, respectively. IMPORTANCE (−)-Menthol is an important flavor and fragrance compound and furthermore has medicinal uses. To realize a two-step synthesis starting from renewable (+)-limonene, a regioselective limonene-3-hydroxylase enzyme is necessary. We identified enzymes from two different fungi which catalyze this hydroxylation reaction and represent an important module for the development of a biotechnological process for (−)-menthol production from renewable (+)-limonene.

The production of cell-wall degrading enzymes (CWDE) such as cellulase and pectinase by Aureobasidium pullulans NAC8 through induction using orange peels was investigated for the potential ...

The Kururu no β-glu® (KBG) is a commercial hydrothermal-treated Aureobasidium pullulans β-glucan produced by a unique hydrothermal process that results in high solubility of the β-glucan. In this study, we examined the biological activities of this reagent. RAW264.7 cells do not express Dictin-1 on the cell surface, but cells still respond to various pathogen molecular patterns. Lipopolysaccharide (LPS) induced nitrogen oxide (NO) synthesis and TNF−α production in RAW264.7 cells, and those were suppressed by KBG in a dose-dependent manner. The major signaling cell surface receptor respond to LPS is the TLR4/MD-2 complex. The UT12 antibody against to the TLR4/MD-2 complex mimics LPS function and induces cell responses. NO generation and TNF-α production were similarly induced in cells by stimulation with the antibody, but those were not suppressed by KBG. Cell responses induced by other TLR ligands, such as CPG (TLR9 ligand) and Pam3CSK4 (TLR1/TLR2 ligand), were also suppressed by KBG. Therefore, the target molecule for KBG is different from TLR receptors and Dictin-1. Although we also examined the suppressive activities of several other β-glucan products, comparable activities were not detected with other reagents. A unique hydrothermal process may produce the active reagent. Reprocessing KBG increased low molecular weight fractions, and suppressive activities were markedly enhanced. Therefore, low molecular weight fractions obtained by hydrothermal processing of KBG may result in potential reagents that control inflammation induced by various pathogens.

Pullulan is a biopolymer used in food industry produced by Aureobasidium pullulans from starch. In the present work, sugarcane bagasse (SCB) hydrolysate was evaluated as an alternative substrate in fermentation process assisted by blue LED lights. The best fermentation conditions in Erlenmeyer flasks were 25.3 °C, stirring speed of 232 rpm and yeast extract concentration of 1.88 g/L, yielding 25.19 g/L of pullulan, that corresponded to yield of 0.48 g/g and 0.28 g/(L·h) of volumetric productivity. By using a column bubble photobioreactor, similar yield values were obtained. Thermal properties of the produced pullulan as glass transition (Tg) and melting (Tm) temperatures were 38 °C and 160 °C, respectively, which were similar to the corresponding values of commercial food grade pullulan. Therefore, SCB hydrolysate is a promising substrate to produce good quality pullulan (86% of purity) with application in food industry, besides to represent a new alternative for biorefineries.

Advanced drug delivery systems are now designed with biodegradable, biocompatible and non-toxic materials such as polysaccharides. Pullulan is a polysaccharide consisting of maltotriose units. Pullulan is produced from starch by the fungus Aureobasidium pullulans. Pullulan and derivatives are able to conjugate or complex with hydrophobic drugs. Scientists are optimizing the physical interactions and hydrophilic–hydrophobic balance to create systems with controlled loading and targeted delivery of the drugs to cancer cells or liver cell receptors. This article reviews pullulan-based systems of various therapeutic properties. Pullulan structure can be easily modified to transfer various drugs. Pullulan can form microparticles, nanoparticles, micelles, films and hydrogels. Therapeutic properties include antibacterial, antifungal, antitumor, anti-inflammatory, immunomodulatory, antioxidant, antiglycemic, antilipidemic, bone regenerator and systemic protective role. The medical suitability of pullulan-based drug carriers is confirmed by profiles of drug release and cytotoxicity tests carried out in vivo or in vitro.

β-poly(L-malic acid) (PMLA) is a highly water-soluble biopolymer used in medicine and other industries. PMLA is usually biosynthesized by Aureobasidium pullulans, but in low concentrations, which has restricted its further large-scale production. Here effects of surfactants on PMLA production were investigated. Of various surfactants examined, the addition of Tween 80 at the start of cell growth increased mycelial growth, PMLA production, PMLA productivity and the ratio of PMLA to cell biomass concentration (Yp/x) of A. pullulans. Transmission electron microscopy and flow cytometry showed that cell membrane permeability was increased, improving the secretion of PMLA and uptake of nutrients and oxygen. Proteomic analysis showed that key pathways of PMLA synthesis, along with cellular energy metabolism, were enhanced. Moreover, glutathione (GSH) synthesis and anti-oxidation capacity were also improved. These results indicated that the addition of Tween 80 is an efficient approach for improving the production of PMLA.

Gluconic acid (GA) has many applications such as in the food and pharmaceutical industry. Aureobasidium pullulans P25 strain is able to produce high levels of Ca2+-GA. The genome length, GC content and the gene number of this yeast were found to be 30.97 Mb, 50.28% and 10,922, respectively. The pathways for gluconic acid biosynthesis were annotated. Glucose oxidase (Gox) sequences from different strains of A. pullulans were highly similar but were distinct from those of other fungi. The glucose oxidase had two FAD binding sites and a signal sequence. Deletion of the GOX gene resulted in a strain that showed no Gox activity and that was unable to produce Ca2+-GA. Overexpression of the GOX gene in strain P25 generated strain GA-6 that produced 200.2 ± 2.3 Ca2+-GA g/l and 2480 U/mg of Gox activity. The productivity of Ca2+-GA was 2.78 g/l/h and the yield was 1.1 g/g.

Made available in DSpace on 2021-06-25T11:46:57Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-12-29 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The objectives of the present study were to purify and assess the killer toxin effect produced by Aureobasidium pullulans under casual agents of green mold (Penicillum digitatum) and sour rot (Geotrichum citri-aurantii). Initially, different methods of protein precipitation were tested. The proteolytic activity and the presence of proteins acting on cell wall receptors, beta-1,3-glucanase and chitinase were determined, and toxin purification was conducted by Sephadex G-75 gel exclusion chromatography and cellulose chromatography (medium fibers). Subsequently, purification was confirmed by polyacrylamide gel electrophoresis, and the detection of killer activity was performed in solid YEPD-methylene blue buffered with citrate-phosphate (0.1 M, pH 4.6). Toxin identification was performed by liquid chromatography-mass spectrometry. The results showed that the best protein precipitation method was 2:1 ethanol (vol/vol ethanol/supernatant). It was possible to observe the presence of enzymes with proteolytic activity, including beta-1,3-glucanase and chitinase. During the purification process, it was verified that the killer toxin produced by the yeast has a low-molecular-weight protein belonging to the ubiquitin family, which presents killer activity against P. digitatum and G. citri-aurantii. Julio de Mesquita Filho Paulista State Univ, Grad Agr Microbiol Program, Agr & Veterinarian Fac, Sao Paulo, Brazil Univ Ctr Herminio Ometto Fdn FHO, Grad Program Biomed Sci, Sao Paulo, Brazil Sylvio Moreira Citriculture Ctr IAC, Rodovia Anhanguera,Km 158,CP 04, BR-13470970 Cordeiropolis, SP, Brazil Julio de Mesquita Filho Paulista State Univ, Grad Agr Microbiol Program, Agr & Veterinarian Fac, Sao Paulo, Brazil FAPESP: 2014/25067-3

More than 30,000 tons of menthol are produced every year as a flavor and fragrance compound or as medical component. So far, only extraction from plant material or chemical synthesis is possible. A sustainable alternative approach for menthol production could be a biotechnological-chemical two-step conversion, starting from (+)-limonene, which is a side product of the citrus processing industry. The first step requires a limonene-3-hydroxylase (L3H) activity that specifically catalyzes hydroxylation of limonene at carbon atom 3. Several protein engineering strategies already attempted to create limonene-3-hydroxylases from bacterial cytochrome P450 monooxygenases (CYPs or P450s), which can be efficiently expressed in bacterial hosts. However, their regiospecificity is rather low, if compared to the highly selective L3H enzymes from the biosynthetic pathway towards menthol in Mentha species. The only naturally occurring limonene-3-hydroxylase activity identified in microorganisms so far, was reported for a strain of the black yeast-like fungus Hormonema sp. in South Africa.We have discovered further fungi that can catalyze the intended reaction and identified potential CYP-encoding genes within the genome sequence of one of the strains. Using heterologous gene expression and biotransformation experiments in yeasts, we were able to identify limonene-3-hydroxylases from Aureobasidium pullulans and Hormonema carpetanum. Further characterization of the A. pullulans enzyme demonstrated its high stereospecificity and regioselectivity, its potential for limonene-based menthol production and its additional ability to convert α-and β-pinene to verbenol and pinocarveol, respectively.Importance(−)-Menthol is an important flavor and fragrance compound and furthermore has medicinal uses. To realize a two-step synthesis starting from renewable (+)-limonene, a regioselective limonene-3-hydroxylase enzyme is necessary. We identified enzymes from two different fungi, which catalyze this hydroxylation reaction and represent an important module for the development of a biotechnological process for (−)-menthol production from renewable (+)-limonene.

Coenzyme Q (CoQ, ubiquinone) is an essential component of the electron transport system in aerobic organisms. Human type CoQ10, which has 10 units of isoprene in its quinone structure, is especially valuable as a food supplement. Therefore, studying the biosynthesis of CoQ10is important not only for increasing metabolic knowledge, but also for improving biotechnological production. Herein, we show thatSchizosaccharomyces pombeutilizesp-aminobenzoate (PABA) in addition top-hydroxybenzoate (PHB) as a precursor for CoQ10synthesis. We explored compounds that affect the synthesis of CoQ10and found benzoic acid (Bz) at >5 μg/mL inhibited CoQ biosynthesis without accumulation of apparent CoQ intermediates. This inhibition was counteracted by incubation with a 10-fold lower amount of PABA or PHB. Overexpression of PHB-polyprenyl transferase encoded byppt1(coq2) also overcame the inhibition of CoQ biosynthesis by Bz. Inhibition by Bz was efficient inS.pombeandSchizosaccharomyces japonicus, but less so inSaccharomyces cerevisiae,Aureobasidium pullulans, andEscherichia coli. Bz also inhibited aS.pombe ppt1(coq2) deletion strain expressing humanCOQ2, and this strain also utilized PABA as a precursor of CoQ10. Thus, Bz is likely to inhibit prenylation reactions involving PHB or PABA catalyzed by Coq2.

Many yeasts have demonstrated intrinsic insensitivity to certain antifungal agents. Unlike the fungicide resistance of medically relevant yeasts, which is highly undesirable, intrinsic insensitivity to fungicides in antagonistic yeasts intended for use as biocontrol agents may be of great value. Understanding how frequently tolerance exists in naturally occurring yeasts and their underlying molecular mechanisms is important for exploring the potential of biocontrol yeasts and fungicide combinations for plant protection. Here, yeasts were isolated from various environmental samples in the presence of different fungicides (or without fungicide as a control) and identified by sequencing the internal transcribed spacer (ITS) region or through matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Among 376 isolates, 47 taxa were identified, and Aureobasidium pullulans was the most frequently isolated yeast. The baseline sensitivity of this yeast was established for 30 isolates from different environmental samples in vitro to captan, cyprodinil, and difenoconazole. For these isolates, the baseline minimum inhibitory concentration (MIC50) values for all the fungicides were higher than the concentrations used for the control of plant pathogenic fungi. For some isolates, there was no growth inhibition at concentrations as high as 300 µg/mL for captan and 128 µg/mL for cyprodinil. This information provides insight into the presence of resistance among naturally occurring yeasts and allows the choice of strains for further mechanistic analyses and the assessment of A. pullulans for novel applications in combination with chemical agents and as part of integrated plant-protection strategies., Antibiotics, 9 (9), ISSN:2079-6382

Aureobasidium pullulans is a yeast-like fungus that produces volatile organic compounds (VOCs) with antifungal properties. VOCs have the potential to trigger the production of intracellular reactive oxygen species (ROS), lipid peroxidation and electrolyte loss in microorganisms. The relationship among A. pullulans VOCs, induced ROS accumulation and electrolyte leakage was investigated in Botrytis cinerea and Alternaria alternata in vitro. Exposure to a mixture of A. pullulans VOCs: ethanol, 2-methyl-1-propanol, 3-methyl-1-butanol and 2-phenylethanol, resulted in electrolyte leakage in both B. cinerea and A. alternata. Fluorescence microscopy using 2′,7′-dichlorofluorescein diacetate indicated triggered ROS accumulation in exposed fungal mycelia and the presence of the superoxide radical was evident by intense red fluorescence with dihydroethidium. Partial inhibition of enzymes of the mitochondrial respiratory chain complex I of B. cinerea and A. alternata by pre-treatment with rotenone reduced ROS accumulation in hypha exposed to A. pullulans VOCs and reversed the VOCs inhibition of fungal growth. Scanning electron micrographs revealed that B. cinerea and A. alternata hypha exposed to A. pullulans VOCs had altered cell wall structures. Our findings give insights into the potential mechanisms involved in the antifungal properties of A. pullulans in the suppression of B. cinerea and A. alternata growth in vitro.

The content of pullulan and melanin in 500 mutants of Aureobasidum pullulans obtained by ultraviolet mutagenesis were examined and statistically analyzed, and a strong positive correlation was found between them. The result was further confirmed by culturing wild type strain As3.3984 in different media. Then we constructed melanin-deletion mutant As-Δalb1 and pullulan-deletion mutant As-Δpul. As-Δalb1 was a melanin-free strain with the yield of pullulan decreased by 41.01%. The supplementation of melanin in the culture of As-Δalb1 increased the production of pullulan. As-Δpul synthesized neither pullulan nor melanin and recovered melanin synthesis by adding pullulan to the medium. The results suggested that high concentration- of pullulan induced morphological transformation and synthesis of melanin, and melanin promoted the synthesis of pullulan. The pullulan biosynthetic genes, upt, pgm, ugp, and pul, were down-regulated, while the negative regulatory gene of pullulan synthesis, creA, was up-regulated by melanin deficiency.

In this study, a new strain suspected to be Aureobasidium pullulans was isolated from trees leaves. The molecular characterization and the resulting phylogenetic tree showed that the isolated strain was A. pullulans. Also, the results of methylation analysis, monosaccharide composition, FTIR, NMR and XRD confirmed that the obtained exo-polysaccharide from the mentioned strain was pullulan. The pullulan production optimization by central composite design (CCD) indicated that the maximum yield obtained under optimum conditions (pH of 6.5, sucrose concentration of 5.5% (w/v) and yeast extract concentration of 0.1% (w/v)) was 51.4 ± 0.50 g/L. The produced pullulan had an average molecular weight (Mw) of 2.07 × 105 g.mol−1 based on gel permeation chromatography results. The decomposition temperature (Td) of the produced pullulan was ~300 °C and also, the resulting pullulan had a Newtonian flow behavior in a wide range of concentrations.

Pullulan production from Aureobasidium pullulans was explored to increase yield. Non-linear hybrid mathematical tools for optimization of process variables as well as the pullulan yield were analyzed. The one variable at a time (OVAT) approach was used to optimize the maximum pullulan yield of 35.16 &plusmn, 0.29 g/L. The tools predicted maximum pullulan yields of 39.4918 g/L (genetic algorithm coupled with artificial neural network (GA&ndash, ANN)) and 36.0788 g/L (GA coupled with adaptive network based fuzzy inference system (GA&ndash, ANFIS)). The best regression value (0.94799) of the Levenberg&ndash, Marquardt (LM) algorithm for ANN and the epoch error (6.1055 &times, 10&minus, 5) for GA&ndash, ANFIS point towards prediction precision and potentiality of data training models. The process parameters provided by both the tools corresponding to their predicted yield were revalidated by experiments. Among the two of them GA&ndash, ANFIS results were replicated with 98.82% accuracy. Thus GA&ndash, ANFIS predicted an optimum pullulan yield of 36.0788 g/L with a substrate concentration of 49.94 g/L, incubation period of 182.39 h, temperature of 27.41 &deg, C, pH of 6.99, and agitation speed of 190.08 rpm.

Liamocins are a class of biosurfactants with growing interest. However, methods for identification and quantification of liamocins on the molecular level are lagging behind. Therefore, we developed a chromatographic separation based on supercritical fluid chromatography (SFC) for liamocins and structurally related exophilins. The different congeners could be separated on a charge modulated hydroxyethyl amide functionalized silica-based column. Coupling to high-resolution mass spectrometry (MS) revealed four exophilin species and four liamocin species with mannitol and arabitol as polyol head group in a sample of the yeast-like fungus Aureobasidium pullulans (A. pullulans). In contrast to a recently published reversed phase high-performance liquid chromatography (HPLC) method, the different subclasses (exophilins, mannitol liamocins and arabitol liamocins) were additionally separated by means of SFC. The structures were confirmed by their accurate masses and tandem mass spectrometry (MS/MS). A complementary quantification method was developed using SFC coupled to charged-aerosol detection (CAD) to overcome the disadvantages of quantification by means of MS without authentic standards. A flow compensation by varying the make-up flow was used to obtain a constant composition of the mobile phase during detection and to ensure a stable detector response. The concentrations of the individual liamocin species were determined using an external calibration with n-octyl-β-d-glycopyranoside. The total amount of these concentrations agrees with the dry weight of an aliquot of the heavy oil. The developed SFC-based method has the advantage of shorter analysis time and superior selectivity compared to the previously published LC separation. In brief, the here presented SFC hyphenations enable comprehensive analysis of liamocin biosurfactants providing identification and absolute quantification of individual congeners.

Pullulan, a water-soluble polysaccharide consisting of maltotriose units used in the preparation of edible films and drug delivery, is generally produced from starch by Aureobasidium pullulans (de Bary & Löwenthal) G.Arnaud fungus. In this article, the characterisation of an alternative pullulan source − the stromata of Cyttaria hariotii E.Fisch. fungus − by elemental analysis, infrared spectroscopy and thermal analysis techniques is reported. With a view to a possible valorisation of this pullulan and its derivatives as bioactive formulations in agriculture, low-molecular-weight pullulan (

Polymalic acid (PMA) is a linear anionic polyester composed of l-malic acid monomers, which have potential applications as drug carriers, surgical suture, and biodegradable plastics. In this study, a novel strain of Aureobasidium pullulans var. melanogenum GXZ-6 was isolated and identified according to the morphological observation and deoxyribonucleic acid internal-transcribed spacer sequence analysis, and the product of PMA was characterized by FT-IR, 13C-NMR, and 1H-NMR spectra. The PMA titer of GXZ-6 reached 62.56 ± 1.18 g L−1 with productivity of 0.35 g L−1 h−1 using optimized medium with addition of metabolic intermediates (citrate and malate) and inhibitor (malonate) by batch fermentation in a 10-L fermentor. Besides that the malate for PMA synthesis in GXZ-6 might mainly come from the glyoxylate cycle, based on results, citrate, malate, malonate, and maleate increased while succinate and fumarate inhibited the production of PMA, which was different from that of other A. pullulans. This study provided a potential strain and a simple metabolic control strategy for high-titer production of PMA and shared novel information on the biosynthesis pathway of PMA in A. pullulans.

Antagonistic yeasts suppress plant pathogenic fungi by various mechanisms, but their biocontrol efficacy also depends on the ability to compete and persist in the environment. The goal of the work presented here was to quantify the composition of synthetic yeast communities in order to determine the competitiveness of different species and identify promising candidates for plant protection. For this purpose, colony counting of distinct species and matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF MS; MALDI biotyping) were used to distinguish different yeast species and to quantify the composition of a synthetic community of six yeasts (Aureobasidium pullulans, Candida subhashii, Cyberlindnera sargentensis, Hanseniaspora sp., Metschnikowia pulcherrima and Pichia kluyveri) over time, on apples and in soil, and in different growth media. These studies revealed important characteristics that predispose the different species for particular applications. For example, the competitiveness and antagonistic activity of C. subhashii was strongly increased in the presence of N‐acetylglucosamin as the sole carbon source, M. pulcherrima and A. pullulans were the strongest competitors on apple, and C. sargentensis competed the best in soil microcosms. Based on these laboratory studies, M. pulcherrima and A. pullulans are promising candidates for biocontrol applications against fungal phyllosphere diseases, while C. sargentensis may hold potential for use against soilborne fungal pathogens. These results document the potential of MALDI‐TOF MS for the quantitative analysis of synthetic yeast communities and highlight the value of studying microorganisms with relevant functions in moderately complex, synthetic communities and natural substrates rather than as individual isolates.

A panel of microorganisms was screened for selective reduction ability towards a racemic mixture of prochiral 2-amino-3-methyl-4-ketopentanoate (rac-AMKP). Several of the microorganisms tested produced greater than 0.5mM 4-hydroxyisoleucine (HIL) from rac-AMKP, and the stereoselectivity of HIL formation was found to depend on the taxonomic category to which the microorganism belonged. The enzymes responsible for the AMKP-reducing activity, ApAR and FsAR, were identified from two of these microorganisms, Aureobasidium pullulans NBRC 4466 and Fusarium solani TG-2, respectively. Three AMKP reducing enzymes, ApAR, FsAR, and the previously reported BtHILDH, were reacted with rac-AMKP, and each enzyme selectively produced a specific composition of HIL stereoisomers. The enzymes appeared to have different characteristics in recognition of the stereostructure of the substrate AMKP and in control of the 4-hydroxyl group configuration in the HIL product.

Killer toxin resistant 6 (Kre6) and its paralog, suppressor of Kre null 1 (Skn1), are thought to be involved in the biosynthesis of cell wall β-(1 → 6)-D-glucan in baker's yeast, Saccharomyces cerevisiae. The Δkre6Δskn1 mutant of S. cerevisiae and other fungi shows severe growth defects due to the failure to synthesize normal cell walls. In this study, two homologs of Kre6, namely, K6LP1 (Kre6-like protein 1) and K6LP2 (Kre6-like protein 2), were identified in Aureobasidium pullulans M-2 by draft genome analysis. The Δk6lp1, Δk6lp2, and Δk6lp1Δk6lp2 mutants were generated in order to confirm the functions of the Kre6-like proteins in A. pullulans M-2. The cell morphologies of Δk6lp1 and Δk6lp1Δk6lp2 appeared to be different from those of wild type and Δk6lp2 in both their yeast and hyphal forms. The productivity of the extracellular polysaccharides, mainly composed of β-(1 → 3),(1 → 6)-D-glucan (β-glucan), of the mutants was 5.1-17.3% less than that of wild type, and the degree of branching in the extracellular β-glucan of mutants was 14.5-16.8% lower than that of wild type. This study showed that the gene disruption of Kre6-like proteins affected the cell morphology, the productivity of extracellular polysaccharides, and the structure of extracellular β-glucan, but it did not have a definite effect on the cell viability even in Δk6lp1Δk6lp2, unlike in the Δkre6Δskn1 of S. cerevisiae.

Intracellular glycerol accumulation is one of the main fungal adaptations to osmotic and also cold stress. We investigated the management of glycerol metabolism in polyextremotolerant black yeasts Aureobasidium pullulans and Aureobasidium subglaciale. We show that increased salinity (5 % and 10 %; w/v), but not cold (10 °C) trigger intracellular glycerol accumulation. The transcriptional response of the genes involved in glycerol synthesis, degradation and import, to increased salinity, low temperature or a combination of both was analysed with real-time PCR. Each of the two species contains an NAD+-dependent glycerol-3-phosphate dehydrogenase, a glycerol-3-phosphate phosphatase, a mitochondrial glycerol-3-phosphate dehydrogenase, two copies of a glycerol kinase, and more than ten copies of major facilitator superfamily transporters similar to glycerol proton symporters. Similarly to glycerol accumulation itself, transcriptional response to hypersaline stress was larger compared to low temperature stress and was more consistent in A. pullulans compared to A. subglaciale, reflecting the different stress tolerance and ecological strategy of each species.

This article contains two-dimensional (2D) NMR experimental data, obtained by the Bruker BioSpin 500 MHz NMR spectrometer (Germany) which can used for the determination of primary structures of schizophyllan from Schizophyllum commune (SPG) and a water-soluble β-(1â3, 1â6)-glucan from Aureobasidium pullulans. Data include analyzed the 2D NMR spectra of these β-glucans, which are related to the subject of an article in Carbohydrate Polymers, entitled âNMR spectroscopic structural characterization of a water-soluble β-(1â3, 1â6)-glucan from A. pullulansâ (Kono et al., 2017) [1]. Data can help to assign the 1H and 13C chemical shifts of the structurally complex polysaccharides. Keywords: NMR, β-(1â3, 1â6)-glucan, Aureobasidium pullulans, Schizophyllan, Spectral data

Here, we report an efficient endoglucanase from Aureobasidium pullulans (termed ApCel5A) was expressed in Pichia pastoris. ApCel5A shows two different enzyme activities of endoglucanase (1270 U/mg) and mannanase (31.2 U/mg). Through engineering the signal peptide and fed-batch fermentation, the enzyme activity of endoglucanase was improved to 6.63-folds, totally. Its efficient synergism with Celluclast 1.5 L, excellent tolerance to low pH (2.5), cholate and protease suggests potential application in bioresources, food and feed industries. Site-directed mutagenesis experiments present that ApCel5A residues Glu245 and Glu358 are key catalytic sites, while Asp118, Asp122, Asp198 and Asp314 play an auxiliary role. More importantly, ApCel5A display high degradation efficiency of glucan and glucomannan substrates by using tetrasaccharide contained reducing end of glucose residue as an intermediate. This study elucidated the effective methods to improve an endoglucanase expression and detailed catalytic mechanism for degradation of various substrates, which provides a new insight for endoglucanase application.

The amylase from Aureobasidium pullulans Cau 19 was purified by ammonium sulfate precipitation and Sephadex G-100 chromatography with a 9.25-fold increase in specific activity as compared to crude enzyme. Km and turn over values of the enzyme were 6.25 mg/mL and 5.0 × 102/min, respectively. Effect of different metal ions on the purified enzyme was investigated; 1 mM calcium (Ca) and cobalt (Co) enhanced enzyme activity by twofold; copper (Cu) had no effect on the activity of the enzyme. Mercury (Hg) 1 mM caused 90% inactivation whereas iron (Fe) and manganese (Mn) caused 10 to 16% inhibition. Amylase from A. pullulans Cau 19 was bioconjugated to gold nanoparticles synthesized using the biomass of A. pullulans Cau 19. Fourier transform infrared spectroscopy confirmed the conjugation of the enzyme to the gold nanoparticles. Though, only 20% of the added enzyme was adsorbed/conjugated on gold nanoparticles, 80% of the adsorbed activity could be estimated in the assay. The conjugated enzyme exhibited better tolerance to a broad pH range of 3.0–9.0 and higher temperatures compared with native enzyme.