Raw Starch-degrading Enzyme Research Articles

A RsrC-RsrA-RsrB transcriptional circuit positively regulates polysaccharide-degrading enzyme biosynthesis and development in Penicillium oxalicum

Filamentous fungi produce polysaccharide-degrading enzymes, which is controlled by poorly understood transcriptional circuits. Here we show that a circuit comprising RsrC-RsrA-RsrB (Rsr: production of raw-starch-degrading enzyme regulator) that positively regulates production of raw starch-degrading enzymes in Penicillium oxalicum. Transcription factor (TF) RsrA is essential for biosynthesis of raw starch-degrading enzymes. RsrB and RsrC containing Zn2Cys6- and C2H2-zinc finger domains, act downstream and upstream of RsrA, respectively. RsrA activates rsrB transcription, and three nucleotides (G-286, G-287 and G-292) of rsrB promoter region are required for RsrA, in terms of TF, for binding. RsrB165−271 binds to DNA sequence 5’-TCGATCAGGCACGCC-3’ in the promoter region of the gene encoding key raw-starch-degrading enzyme PoxGA15A. RsrC specifically binds rsrA promoter, but not amylase genes, to positively regulate the expression of rsrA and the production of raw starch-degrading enzymes. These findings expand complex regulatory network of fungal raw starch-degrading enzyme biosynthesis.

Raw starch degrading alkaline α-amylase from Geobacillus kaustophilus TSCCA02: Production, characterization, and its potential for application as a detergent additive.

Geobacillus kaustophilus TSCCA02, a newly isolated strain from cassava (Manihot esculenta L.) rhizosphere soil in Thailand, showed maximum raw starch degrading enzyme (RSDE) activity at 252.3 ± 9.32 U/mL with cassava starch and peptone at 5.0 and 3.0 g/L, respectively. 16 S ribosomal RNA (rRNA) sequencing and phylogenetic tree analyses indicated that the TSCCA02 strain was closely related to G. kaustophilus. The crude RSDE had optimal activity at 60°C and pH 9.0. This enzyme degraded various kinds of starch including potato starch, cassava starch, rice flour, corn starch, glutinous rice flour, and wheat flour to produce sugar syrup at 60°C, as confirmed by scanning electron microscopy (SEM), thin-layer chromatography (TLC), and Fourier-transform infrared spectroscopy (FTIR). The major end products of starch hydrolysis were maltose and maltotriose with a small amount of glucose, confirming this enzyme as an α-amylase. The enzyme improved the washing efficiency of cotton fabric with commercial detergent. Results indicated the potential of alkaline α-amylase produced from a new isolate of G. kaustophilus TSCCA02 for application as a detergent additive on an industrial scale.

Regulation of fungal raw-starch-degrading enzyme production depends on transcription factor phosphorylation and recruitment of the Mediator complex

Filamentous fungus can produce raw-starch-degrading enzyme (RSDE) that efficiently degrades raw starch below starch gelatinization temperature. Employment of RSDE in starch processing can save energy. A key putative transcription factor PoxRsrA (production of raw-starch-degrading enzyme regulation in Penicilliumoxalicum) was identified to regulate RSDE production in P. oxalicum; however, its regulatory mechanism remains unclear. Here we show that PoxRsrA1434–1730 was the transcriptional activation domain, with essential residues, D1508, W1509 and M1510. SANT (SWI3, ADA2, N-CoR and TFIIIB)-like domain 1 (SANT1) bound to DNA at the sequence 5′-RHCDDGGD-3′ in the promoter regions of genes encoding major amylases, with an essential residue, R866. SANT2 interacted with a putative 3-hydroxyisobutyryl-CoA hydrolase, which suppressed phosphorylation at tyrosines Y1127 and Y1170 of PoxRsrA901–1360, thereby inhibiting RSDE biosynthesis. PoxRsrA1135–1439 regulated mycelial sporulation by interacting with Mediator subunit Med6, whereas PoxRsrA1440–1794 regulated RSDE biosynthesis by binding to Med31. Overexpression of PoxRsrA increased sporulation and RSDE production. These findings provide insights into the regulatory mechanisms of fungal RSDE biosynthesis.

Kinase POGSK-3β modulates fungal plant polysaccharide-degrading enzyme production and development

The filamentous fungus Penicillium oxalicum secretes integrative plant polysaccharide-degrading enzymes (PPDEs) applicable to biotechnology. Glycogen synthase kinase-3β (GSK-3β) mediates various cellular processes in eukaryotic cells, but the regulatory mechanisms of PPDE biosynthesis in filamentous fungi remain poorly understood. In this study, POGSK-3β (POX_c04478), a homolog of GSK-3β in P. oxalicum, was characterised using biochemical, microbiological and omics approaches. Knockdown of POGSK-3β in P. oxalicum using a copper-responsive promoter replacement system led to 53.5 - 63.6%, 79.0 - 92.8% and 76.8 - 94.7% decreases in the production of filter paper cellulase, soluble starch-degrading enzyme and raw starch-degrading enzyme, respectively, compared with the parental strain ΔKu70. POGSK-3β promoted mycelial growth and conidiation. Transcriptomic profiling and real-time quantitative reverse transcription PCR analyses revealed that POGSK-3β dynamically regulated the expression of genes encoding major PPDEs, as well as fungal development-associated genes. The results broadened our understanding of the regulatory functions of GKS-3β and provided a promising target for genetic engineering to improve PPDE production in filamentous fungi. KEY POINTS: • The roles of glycogen synthase kinase-3β were investigated in P. oxalicum. • POGSK-3β regulated PPDE production, mycelial growth and conidiation. • POGSK-3β controlled the expression of major PPDE genes and regulatory genes.

Promoter-proximal introns impact recombinant amylase expression in Saccharomyces cerevisiae.

Consolidated bioprocessing (CBP) of starch requires recombinant Saccharomyces cerevisiae strains that produce raw starch-degrading enzymes and ferment the resultant sugars to ethanol in a single step. In this study, the native S. cerevisiae COX4 and RPS25A promoter-proximal introns were evaluated for enhanced expression of amylase genes (ateA, temA or temG_Opt) under the control of an S. cerevisiae promoter (ENO1P, TEF1P, TDH3P, or HXT7P). The results showed that different promoters and promoter-intron combinations differentially affected recombinant amylase production: ENO1P-COX4i and TDH3P-RPS25Ai were the best promoters for AteA, followed closely by HXT7P. The latter was also the best promoter for TemA and TemG production, followed closely by TDH3P-RPS25Ai for both these enzymes. Introducing promoter-proximal introns increased amylase activity up to 62% in Y294[ENO-COX-AteA] and Y294[TDH3-RPS-TemA], a significant improvement relative to the intron-less promoters. Strains co-expressing both an α-amylase and glucoamylase genes yielded up to 56g/L ethanol from 20%w/v raw starch, with a higher carbon conversion observed with strains co-expressing TDH3P-RPS25Ai-temG_Opt than HXT7P-temG_Opt. The study showed that promoter-proximal introns can enhance amylase activity in S. cerevisiae and suggest that these alternative cassettes may also be considered for expression in more efficient ethanol-producing industrial yeast strains for raw starch CBP.

Functional Characterization of Recombinant Raw Starch Degrading α-Amylase from Roseateles terrae HL11 and Its Application on Cassava Pulp Saccharification

Exploring new raw starch-hydrolyzing α-amylases and understanding their biochemical characteristics are important for the utilization of starch-rich materials in bio-industry. In this work, the biochemical characteristics of a novel raw starch-degrading α-amylase (HL11 Amy) from Roseateles terrae HL11 was firstly reported. Evolutionary analysis revealed that HL11Amy was classified into glycoside hydrolase family 13 subfamily 32 (GH13_32). It contains four protein domains consisting of domain A, domain B, domain C and carbohydrate-binding module 20 (CMB20). The enzyme optimally worked at 50 °C, pH 4.0 with a specific activity of 6270 U/mg protein and 1030 raw starch-degrading (RSD) U/mg protein against soluble starch. Remarkably, HL11Amy exhibited activity toward both raw and gelatinized forms of various substrates, with the highest catalytic efficiency (kcat/Km) on starch from rice, followed by potato and cassava, respectively. HL11Amy effectively hydrolyzed cassava pulp (CP) hydrolysis, with a reducing sugar yield of 736 and 183 mg/g starch from gelatinized and raw CP, equivalent to 72% and 18% conversion based on starch content in the substrate, respectively. These demonstrated that HL11Amy represents a promising raw starch-degrading enzyme with potential applications in starch modification and cassava pulp saccharification.

Application of raw starch degrading enzyme from Laceyella sacchari LP175 for development of bacterial cellulose fermentation using colored rice as substrate.

The online version contains supplementary material available at 10.1007/s13205-021-02673-3.

ARTP/EMS-combined multiple mutagenesis efficiently improved production of raw starch-degrading enzymes in Penicillium oxalicum and characterization of the enzyme-hyperproducing mutant

BackgroundApplication of raw starch-degrading enzymes (RSDEs) in starch processing for biofuel production can effectively reduce energy consumption and processing costs. RSDEs are generally produced by filamentous fungi, such as Penicillium oxalicum, but with very low yields, which seriously hampers industrialization of raw starch processing. Breeding assisted by random mutagenesis is an efficient way to improve fungal enzyme production.ResultsA total of 3532 P. oxalicum colonies were generated after multiple rounds of mutagenesis, by atmospheric and room-temperature plasma (ARTP) and/or ethyl methanesulfonate (EMS). Of these, one mutant A2-13 had the highest RSDE activity of 162.7 U/mL, using raw cassava flour as substrate, a yield increase of 61.1%, compared with that of the starting strain, OXPoxGA15A. RSDE activity of A2-13 further increased to 191.0 U/mL, through optimization of culture conditions. Increased expression of major amylase genes, including the raw starch-degrading glucoamylase gene, PoxGA15A, and its regulatory gene, PoxAmyR, as well as several single-nucleotide polymorphisms in the A2-13 genome, were detected by real-time reverse transcription quantitative PCR and genomic re-sequencing, respectively. In addition, crude RSDEs produced by A2-13, combined with commercial α-amylase, could efficiently digest raw corn flour and cassava flour at 40 °C.ConclusionsOverall, ARTP/EMS-combined mutagenesis effectively improved fungal RSDE yield. An RSDE-hyperproducing mutant, A2-13, was obtained, and its RSDEs could efficiently hydrolyze raw starch, in combination with commercial α-amylase at low temperature, which provides a useful RSDE resource for future starch processing.

Production and development of vinegar fermentation from broken Riceberry rice using raw starch-degrading enzyme hydrolysis.

Broken Riceberry rice was used as a substrate for sugar syrup production by the hydrolysis of raw starch-degrading enzyme as a low-temperature amylase (iKnowZyme® LTAA, Thailand). Response surface methodology (RSM) with a central composite design (CCD) showed that an optimized substrate concentration of 250g/L yielded 13°Brix of total soluble solid (TSS) content when incubated at 50°C for 12h. The major product from the broken Riceberry rice hydrolysis was glucose with lesser amounts of maltose and maltotriose. Maximum alcohol content (16% w/v) for broken Riceberry rice wine was obtained after fermentation with two mixed strains of Saccharomyces cerevisiae for 10days. Scanning electron micrographs showed that yeast strains could grow on the solid residue of broken Riceberry rice that supported yeast cell survival under stress conditions. Broken Riceberry rice wine was used as the substrate for vinegar fermentation by Acetobacter aceti TISTR 354. Maximum acetic acid concentration was achieved at 5.4% when incubated at room temperature for 6days, containing 10.92mg/L and 965.53 ± 7.74mL sample/g DPPH of anthocyanin content and antioxidant assay, respectively. Our finding revealed the feasibility of broken Riceberry rice substrate for sugar syrup, wine and vinegar production by raw starch-degrading enzyme hydrolysis which increased the value of low-cost agricultural crops through biotechnological processes.

Very high Gravity (VHG) Bioethanol Production Using Modified Simultaneous Saccharification and Fermentation of Raw Cassava Chips with Molasses by Kluyveromyces marxianus DMKU-KS07

Purpose: The aim of this study was to investigate the enhancement of very high gravity (VHG) bioethanol production using co-substrates of cassava chip hydrolysates and molasses by Kluyveromyces marxianus DMKU-KS07. Methods: The factors effecting cassava chip hydrolysis by raw starch degrading enzyme (RSDE) from the thermophilic bacterium Laceyella sacchari LP175 and commercial glucoamylase were investigated. The obtained raw cassava chip hydrolysate was co-fermented with molasses by the thermotolerant K. marxianus DMKU-KS07 at 42°C under nonsterile system. Results: Factors affecting sugar syrup production from raw cassava chips by synergistic hydrolysis and enzyme saccharification were optimized at temperature, substrate concentration and agitation rate of 50 °C, 250 g/L and 200 rpm, respectively. A yield of 98.6 g/L was obtained at 6 h of incubation, equivalent to 45.9% saccharification and 51.4% hydrolysis of starch, respectively. High bioethanol concentration at 118 g/L, with highest productivity of 2.19 g/L/h and ethanol yield (YP/S) at 0.44 g EtOH/g total sugar, equivalent to 86.3% theoretical yield, was obtained by modified simultaneous saccharification and fermentation (Modified SSF) with co-fermentation of substrates from the enzymatic hydrolysates of raw cassava chips at 42 °C for 12 h. Subsequent addition of molasses increased the final concentration to 100 g total sugar/L at 36 h. Conclusions: Co-fermentation of raw cassava chip hydrolysates with molasses enhanced production of bioethanol at VHG condition, and showed potential for application in ethanol production by enhancing the fermentation process and reducing energy consumption.

Identification of a Novel Transcription Factor TP05746 Involved in Regulating the Production of Plant-Biomass-Degrading Enzymes in Talaromyces pinophilus.

Limited information on transcription factor (TF)-mediated regulation exists for most filamentous fungi, specifically for regulation of the production of plant-biomass-degrading enzymes (PBDEs). The filamentous fungus, Talaromyces pinophilus, can secrete integrative cellulolytic and amylolytic enzymes, suggesting a promising application in biotechnology. In the present study, the regulatory roles of a Zn2Cys6 protein, TP05746, were investigated in T. pinophilus through the use of biochemical, microbiological and omics techniques. Deletion of the gene TP05746 in T. pinophilus led to a 149.6% increase in soluble-starch-degrading enzyme (SSDE) production at day one of soluble starch induction but an approximately 30% decrease at days 2 to 4 compared with the parental strain ΔTpKu70. In contrast, the T. pinophilus mutant ΔTP05746 exhibited a 136.8–240.0% increase in raw-starch-degrading enzyme (RSDE) production, as well as a 90.3 to 519.1% increase in cellulase and xylanase production following induction by culturing on wheat bran plus Avicel, relative to that exhibited by ΔTpKu70. Additionally, the mutant ΔTP05746 exhibited accelerated mycelial growth at the early stage of cultivation and decreased conidiation. Transcriptomic profiling and real-time quantitative reverse transcription-PCR (RT-qPCR) analyses revealed that TP05746 dynamically regulated the expression of genes encoding major PBDEs and their regulatory genes, as well as fungal development-regulated genes. Furthermore, in vitro binding experiments confirmed that TP05746 bound to the promoter regions of the genes described above. These results will contribute to our understanding of the regulatory mechanism of PBDE genes and provide a promising target for genetic engineering for improved PBDE production in filamentous fungi.

Secretory overproduction of a raw starch-degrading glucoamylase in Penicillium oxalicum using strong promoter and signal peptide.

Raw starch-degrading enzymes (RSDEs) are capable of directly degrading raw starch granules below the gelatinization temperature of starch, which may significantly reduce the cost of starch-based biorefining. However, low yields of natural RSDEs from filamentous fungi limit their industrial application. In this study, transcriptomic and secretomic profiling was employed to screen strongest promoters and signal peptides for use in overexpression of a RSDE gene in Penicillium oxalicum. Top five strong promoters and three signal peptides were detected. Using a green fluorescent protein (GFP) as the reporter, the inducible promoter pPoxEgCel5B of an endoglucanase gene PoxEgCel5B and the signal peptide spPoxGA15A of a raw starch-degrading glucoamylase PoxGA15A were respectively identified as driving the highest GFP production in P. oxalicum. PoxGA15A-overexpressed P. oxalicum strain OXPoxGA15A, which was constructed based on both pPoxEgCel5B and spPoxGA15A, produced significantly higher amounts of recombinant PoxGA15A than the parental strain ∆PoxKu70. Furthermore, crude enzyme from the OXPoxGA15A strain exhibited high activities towards raw starch from cassava, potato, and uncooked soluble starch. Specifically, raw cassava starch-degrading enzyme activity reached 241.6U/mL in the OXPoxGA15A, which was 3.4-fold higher than that of the ∆PoxKu70. This work provides a feasible method for hyperproduction of RSDEs in P. oxalicum.

Production of raw starch-degrading enzyme by Aspergillus sp. and its use in conversion of inedible wild cassava flour to bioethanol

The major bottlenecks in achieving competitive bioethanol fuel are the high cost of feedstock, energy and enzymes employed in pretreatment prior to fermentation. Lignocellulosic biomass has been proposed as an alternative feedstock, but because of its complexity, economic viability is yet to be realized. Therefore, research around non-conventional feedstocks and deployment of bioconversion approaches that downsize the cost of energy and enzymes is justified. In this study, a non-conventional feedstock, inedible wild cassava was used for bioethanol production. Bioconversion of raw starch from the wild cassava to bioethanol at low temperature was investigated using both a co-culture of Aspergillus sp. and Saccharomyces cerevisiae, and a monoculture of the later with enzyme preparation from the former. A newly isolated strain of Aspergillus sp. MZA-3 produced raw starch-degrading enzyme which displayed highest activity of 3.3 U/mL towards raw starch from wild cassava at 50°C, pH 5.5. A co-culture of MZA-3 and S. cerevisiae; and a monoculture of S. cerevisiae and MZA-3 enzyme (both supplemented with glucoamylase) resulted into bioethanol yield (percentage of the theoretical yield) of 91 and 95 at efficiency (percentage) of 84 and 96, respectively. Direct bioconversion of raw starch to bioethanol was achieved at 30°C through the co-culture approach. This could be attractive since it may significantly downsize energy expenses.

Co-production of poly(l-lactide)-degrading enzyme and raw starch-degrading enzyme by Laceyella sacchari LP175 using agricultural products as substrate, and their efficiency on biodegradation of poly(l-lactide)/thermoplastic starch blend film

The co-production of poly-(l-lactide)-degrading enzyme and raw starch-degrading enzyme by the thermophilic filamentous bacterium Laceyella sacchari LP175 in liquid medium using low-cost agricultural crops as substrates was investigated. Statistical mixture design experiments indicated that 5 g of raw material – consisting of 2.35 g L−1 cassava chips and 2.65 g L−1 soybean meal in a suspension of 2.0 g L−1 K2HPO4 and 1.0 g L−1 KH2PO4 – gave the highest production of both enzymes when the culture was grown at 50 °C for 24 h cultivation. Addition of 1.0 g L−1 of poly-(l-lactide) powder and 1.0 g L−1 cassava starch to the medium increased poly-(l-lactide)-degrading enzyme and raw starch-degrading enzyme, respectively. Response surface methodology by central composite design found that the optimized concentration of 0.52 g L−1 poly(l-lactide) powder and 3.34 g L−1 cassava starch increased poly-(l-lactide)-degrading enzyme and raw starch-degrading enzyme activities up to 68.8 U mL−1 and 86.1 U mL−1, respectively. The 2% poly-(l-lactide)/thermoplastic starch (PLLA/TPS) blend (50:50) film was degraded up to 99.7% of weight loss by the crude enzyme at an initial pH of 9.0 for 4 h. The high efficiency on biodegradation of poly-(l-lactide)/thermoplastic starch blend polymer by the obtained mixed enzymes from cheap and abundant agricultural products could be applied to reduce global environment from non-biodegradable materials.

Production and characterization of raw starch degrading enzyme from a newly isolated thermophilic filamentous bacterium, Laceyella sacchari LP175

Medium optimization for production of raw starch degrading enzyme (RSDE) from a newly isolated thermophilic filamentous bacterium, Laceyella sacchari LP175, is described. Yeast extract and raw cassava starch were found to be the best nitrogen and carbon sources, respectively, for enzyme production. Response surface methodology with Plackett–Burman design and central composite design showed that an optimized concentration of 4.93 g/L raw cassava starch and 2.8 g/L yeast extract in basal medium yielded 181.1 U/mL RSDE activity in a shaking flask at 50°C and pH 7.0 after 48 h. Maximum RDSE activity in crude enzyme was achieved at pH 6.5 and 50°C. The major end product of raw cassava starch hydrolysis was maltose (88%). The Km and Ki values of raw cassava starch and maltose, respectively, were found to be 2.85 mg/mL and 1.04 mg/mL. Raw cassava starch at 100 g/L was hydrolyzed with 200 U/mL RSDE for 12 h, yielding 36.8% hydrolysis. The addition of commercial glucoamylase (1.6 U/mL) synergistically enhanced the hydrolysis reaction, yielding 70% hydrolysis (equivalent to 78.0 g/L glucose). The high efficiency of RSDE produced from L. sacchari LP175 could be useful for replacing conventional enzymes currently used in various raw starch processing industries.

Statistical Optimization for Simultaneous Production of PLA Degrading and Raw Starch Degrading Enzymes by Thermophilic Filamentous Bacterium, Laceyella sacchari LP175 Using Agricultural Crops as Substrates

Statistical Optimization for Simultaneous Production of PLA Degrading and Raw Starch Degrading Enzymes by Thermophilic Filamentous Bacterium, Laceyella sacchari LP175 Using Agricultural Crops as Substrates

Preferential and rapid degradation of raw rice starch by an α-amylase of glycoside hydrolase subfamily GH13_37

The α-amylase (AmyP) from a marine metagenomic library belongs to the recently classified glycoside hydrolase subfamily GH13_37. The degradation abilities of AmyP on a broad range of raw starch granules were examined at 40 °C and pH 7.5. It was found that AmyP is a raw starch-degrading enzyme, exhibiting a unique and remarkable ability to preferentially and very rapidly digest raw rice starch. The specific activity of raw rice starch was reached 118.5 ± 0.6 Umg(-1), which was much higher than that of other raw starches. The final hydrolysis degrees were obtained in 4 h for 1 % raw rice starch and 1 h for 8 % concentration, indicating a very rapid speed of hydrolysis. The presence of a starch residue resistant was the main limiting factor for complete hydrolysis, although end product inhibition also existed, especially at high starch concentrations. AmyP randomly attacks unique or susceptible sites on raw rice starch granules, and releases glucose, maltose, and maltotriose as end products. This is the first biochemical characterization of the raw starch-degrading ability of an α-amylase of family GH13_37. The specific ability towards raw rice starch has never been described before, and this makes AmyP a promising candidate for use as a novel enzyme in rice starch processing.

Production of raw cassava starch-degrading enzyme by Penicillium and its use in conversion of raw cassava flour to ethanol

A newly isolated strain Penicillium sp. GXU20 produced a raw starch-degrading enzyme which showed optimum activity towards raw cassava starch at pH 4.5 and 50 °C. Maximum raw cassava starch-degrading enzyme (RCSDE) activity of 20 U/ml was achieved when GXU20 was cultivated under optimized conditions using wheat bran (3.0% w/v) and soybean meal (2.5% w/v) as carbon and nitrogen sources at pH 5.0 and 28 °C. This represented about a sixfold increment as compared with the activity obtained under basal conditions. Starch hydrolysis degree of 95% of raw cassava flour (150 g/l) was achieved after 72 h of digestion by crude RCSDE (30 U/g flour). Ethanol yield reached 53.3 g/l with fermentation efficiency of 92% after 48 h of simultaneous saccharification and fermentation of raw cassava flour at 150 g/l using the RCSDE (30 U/g flour), carried out at pH 4.0 and 40 °C. This strain and its RCSDE have potential applications in processing of raw cassava starch to ethanol.

Recent Advances in Microbial Raw Starch Degrading Enzymes

Raw starch degrading enzymes (RSDE) refer to enzymes that can directly degrade raw starch granules below the gelatinization temperature of starch. These promising enzymes can significantly reduce energy and simplify the process in starch industry. RSDE are ubiquitous and produced by plants, animals, and microorganisms. However, microbial sources are the most preferred one for large-scale production. During the past few decades, RSDE have been studied extensively. This paper reviews the recent development in the production, purification, properties, and application of microbial RSDE. This is the first review on microbial RSDE to date.

Improved Production of Raw Starch Degrading Enzyme by Aspergillus oryzae F-30 Using Methyl Glucoside Sesqui-Stearate

The effect of methyl glucoside sesqui-stearate (MGS) on the production of raw starch degrading enzyme (RSDE) by Aspergillus oryzae F-30 was studied in this paper. The activity of RSDE formed by Aspergillus oryzae F-30 was enhanced dramatically by the addition of MGS to the medium. As a result, with the addition of 1.5 g MGS in 1 L basal medium, RSDE activity and productivity were 107 U mL(-1) and 1.49 U mL(-1) h(-1), 4.3-fold and 7.1-fold greater than the values obtained in the basal medium, respectively. The effect of MGS on the synthesis of RSDE by Aspergillus oryzae F-30 was also studied on a molecular level. It was observed that the regulation of RSDE synthesis in Aspergillus oryzae F-30 occurs at both transcriptional and translational level and the enzyme synthesis was provoked by the addition of MGS at transcriptional level.