Rhizopus Microsporus Var Research Articles

Purification and Characterization of β-Mannanase Derived from Rhizopus microsporus var. rhizopodiformis Expressed in Komagataella phaffii.

The demand for food-grade β-mannanases, ideal for high-temperature baking, is increasing. Using the Komagataella phaffii (P. pastoris) expression system for β-mannanase production, this study aimed to enhance purification methods. We evaluated better conditions for production and purification of β-mannanase (PpRmMan134A) from recombinant P. pastoris X-33, focusing on a higher purity and reducing the production of endogenous secretory proteins in fermentation. By adjusting carbon and nitrogen sources, culture time, and temperature, we controlled cell growth to reduce the production of endogenous secretory proteins. The better-evaluated conditions involved culturing recombinant P. pastoris in 70 mL buffered glycerol complex medium for 24 h at 30 °C, then in modified buffered methanol-complex medium with 0.91% (w/v) methanol, 0.56% (w/v) sorbitol, and 0.48% (w/v) mannitol for another 24 h, which improved the PpRmMan134A yield and reduced endogenous secretory proteins, shortening the fermentation time by 72 h. An affordable purification method using ultrafiltration and salt-out precipitation was utilized. PpRmMan134A showed thermostability up to 100 °C and effectively degraded locust bean gum into smaller fragments, mainly producing mannotriose. In conclusion, with its enhanced purity due to reduced levels of endogenous secretory proteins, purified PpRmMan134A emerges as a promising enzyme for high-temperature baking applications.

Pilot scale production of high-content mycoprotein using Rhizopus microsporus var. oligosporus by submerged fermentation and agro-industrial by-products

While mycoprotein has gained traction as a human food source, its potential as a nutrient for animals remains largely unexplored. The mycoprotein-producing Rhizopus microsporus var. oligosporus, a fungus traditionally used for human food in Indonesia, is promising. It could revolutionise animal nutrition once it is Generally Recognized as Safe (GRAS) and is a biosafety level 1 (BSL1) organism. To enhance sustainably, we propose using sugar cane molasses (SM) and corn steep liquor (CSL) as nutrient sources. Also, we investigated the growth of R. microsporus var. oligosporus in five 14 L external-loop airlift bioreactors using CSL as the sole nutrient source. After 96 h of fermentation, at 25 °C and 0.5 vvm, the mycelium produced had an average biomass yield of 38.34 g L−1, with 70.18 % (m v−1) crude protein (mycoprotein). This bioprocess, which is scalable and economically viable, produces high amounts of mycoprotein for animal feed using CSL, a cost-effective agro-industrial by-product, providing a practical solution to the growing demand for animal protein.

First report of Pectobacterium aroidearum causing soft rot on leaf mustard in China.

Leaf mustard (Brassica juncea [L.] Czern. et Coss.) is widely planted in China as an important leaf vegetable. In March 2022, water-soaked and mushy rot symptoms were observed on leaf mustard plants in the field of Zhaotong (27.85°N; 105.05°E), Yunnan, China. The incidence of symptomatic leaf mustard was approximately 10%. The isolation of the causal agent followed the method of Peng et al. (2023). Briefly, infected tissues from four diseased plants were mixed and teased apart, and homogenized by vortex shaking. The bacterial suspension was diluted and spread on nutrient agar (NA). About 10 single colonies exhibiting different colony morphologies were picked and purified separately by successive streaking. A pinprick method was used for pathogenicity tests with an inoculum concentration of 108 CFU/ml (Singh et al. 2013). Among 10 isolates, only strain YKX exhibited soft rot symptoms on detached mustard leaves. In addition, ten two-month-old leaf mustard plants grown in the greenhouse were used for in vivo pathogenicity tests. Briefly, sterilized pins were dipped in the bacterial suspension, and then leaf mustard petioles were pricked with these pins. After inoculation, each plant was kept in a plastic bag for 12 hours to maintain high humidity. As expected, strain YKX caused obvious rot symptoms on eight plants at 1-2 days post-inoculation while the control group including two plants treated with sterile water showed no symptoms. The colonies of strain YKX on NA were white, roughly circular, and convex. For a preliminary identification, total DNA was extracted and used as the template in PCR amplification of 16S rDNA with the universal PCR primer pair 27F/1492R (Weisburg et al. 1991). The quality-filtered DNA sequence (871 bp) showed 100% query coverage and 99.47% identity to the 16S rDNA sequences of type strain Pectobacterium aroidearum SCRI 109T (GenBank: NR_159926) found in the NCBI rRNA/ITS database. Whole-genome sequencing of strain YKX was then performed using the Illumina and Nanopore sequencing platforms by Tsingke Biotechnology Co., Ltd. (Beijing, China). A single contig (GenBank: CP129239) with a length of approximately 4.9 Mb was obtained by de novo hybrid assembly using Unicycler v0.5.0 (Wick et al. 2017). The quality of the genomic data was evaluated by BUSCO v5.4.7 (Manni et al. 2021) against the gammaproteobacteria_odb10 dataset. A BUSCO complete score of 99.5% indicated high assembly quality. The genome sequence of strain YKX was uploaded to the Type Strain Genome Server for a genome-based taxonomic analysis (Meier-Kolthoff et al. 2022). The distance-based phylogeny showed that strain YKX and P. aroidearum L6 (GenBank: CP065044) and P. aroidearum PC1 (GenBank: NC_012917) form a clade. When comparing strain YKX with L6 or PC1, the digital DNA-DNA hybridization value (83.5-83.8%) was above the species delineation threshold (70% for DDH), clearly indicating that strain YKX should be classified as P. aroidearum. Additionally, P. aroidearum was reisolated from inoculated leaves and identified based on morphological similarities and 16S rDNA sequencing, thus fulfilling Koch's postulates. It is worth noting that a previous study reported occurrences of soft rot disease on leaf mustard attributed to Rhizopus microsporus var. chinensis (Wang et al. 2020). To our knowledge, this is the first report of P. aroidearum causing soft rot on leaf mustard in China, which expands the known host range of this pathogen and benefits the control of this disease.

β(1,3) β(1,6) glucogalactan from Rhizopus microsporus var. oligosporus: extraction, characterization, antioxidant and α-amylase inhibitory activities.

In this study, the Box-Behnken experimental planning was used to optimize the extraction of polysaccharides from the cell wall of Rhizopus microspore var. oligosporus, with analysis of the quantitative effects of parameters pH, temperature and extraction time for polysaccharide yield. The optimal conditions for extraction were determined by the regression equation and evaluation of the response surface graphs, which indicated: pH 13, temperature of 120ºC and time of 60 min, with maximum yield around 18.5%. Fourier transform infrared spectroscopy analysis indicated typical polysaccharide signals. Nuclear magnetic resonance spectroscopy and monosaccharide analysis indicated a β(1,3) β(1,6) glucogalactan. The polysaccharide exhibited an average molecular weight of 120 kDa and a polymerization degree of 741. Antioxidant assays in vitro revealed the potential of polysaccharide in elimination of ABTS+ radical and hydroxyl radicals. EC50 values for free radical elimination were 7.69 and 17.8 mg/mL, for ABTS+ and hydroxyls, respectively. The polysaccharides showed potential for α-amylase inhibition with an EC50 of 1.66 mg/mL. The results suggest that β(1,3) β(1,6) glucogalactan from Rhizopus microsporus var. oligosporus can be used in biotechnological applications.

Fibrinolytic Protease Production: Impact of Initial pH and Temperature in Solid-State Fermentation by Rhizopus microsporus var. oligosporus FNCC 6010

Background: Fibrinolytic enzyme is one of the cardiovascular disease therapies. Rhizopus microsporus var. oligosporus is microorganism that has been evaluated to produce fibrinolytic protease by fermentation. This study conducted fermentation of helianthi annui semen (sunflower seed) by Rhizopus microsporus var. oligosporus to produce fibrinolytic enzyme. Objective: This study aims to determine the effect of Initial pH and incubation temperature and its optimization in the production of fibrinolytic protease by Rhizopus microsporus var. oligosporus FNCC 6010 in solid-state fermentation on helianthi annui semen (sunflower seed) substrate. Optimum condition was determined by maximum protease and fibrinolytic activity. Method: A crude enzyme of protease fibrinolytic was obtained from the supernatant extract of fermented sunflower seed. Protease activity was measured by the skimmed milk agar (SMA) plate method, and fibrinolytic activity was determined by the fibrin agar plate method. Result: It was found that the starting pH affects both the proteolytic and fibrinolytic activity of enzymes that are produced in fermentation. The starting pH of 5.0 showed higher fibrinolytic and proteolytic activity values compared to the starting pH of 7.0. The incubation temperature 33±1 °C had the higher activity compared to 28±1 °C or 37±1 °C. Conclusion: Initial pH and incubation temperature affect the proteolytic and fibrinolytic activity of crude enzyme extracted from fermented sunflower seed by Rhizopus microsporus var. oligosporus. The optimum condition for producing fibrinolytic protease in the state fermentation method was an initial pH of 5.0 and an incubation temperature of 33±1°C.

Fibrinolytic Protease Activity of Crude Enzyme from Fermented Sunflower (Helianthus annuus) and Common Bean (Phaseolus vulgaris) seeds by Rhizopus microsporus var. oligosporus FNCC 6010 in Solid State Fermentation

In the entire world, cardiovascular diseases (CVDs) are the main cause of death. For the treatment of CVDs, microbial fibrinolytic enzymes are highly regarded as novel therapeutic candidates. This study was purposed to determine the fibrinolytic protease activity produced by fungus source, which is Rhizopus microsporus var. oligosporus FNCC 6010 in fermented sunflower (Helianthus annuus) seed and common bean (Phaseolus vulgaris) seed. Fermentation was carried out by solid-state fermentation method at an initial pH of 5, incubation temperature of 33±1°C, and incubation time of 24 hours. The fermented seed was extracted to obtain supernatant as the crude enzyme. The proteolytic activity assay was done by the skimmed milk agar (SMA) plate method to obtain the proteolytic index, and the fibrinolytic activity assay was conducted by the fibrin-agarose plate method to get the fibrinolytic index. The results show that crude enzymes from fermented H. annuus and P. vulgaris seeds by R. microsporus have fibrinolytic protease activity with proteolytic index 2.64 ± 0.01 and 2.23 ± 0.04, respectively. The fibrinolytic index is 2.40 ± 0.06 and 1.64 ± 0.06, respectively. Therefore, the crude enzyme has the potential to be further researched as a candidate for thrombolytic agents. The purification, characterization, and in-depth research are needed to develop enzymes into preparations for preventing and treating CVDs.

Solid-state fungal fermentation transforms low-quality plant-based foods into products with improved protein quality

To meet global protein demand sustainably in the future, we have to move to alternative, non-animal sources. A problem of many plant-derived foods is their low protein quality compared to animal-based proteins, in particular their deficiency in lysine. To improve the protein quality, plant proteins could be converted to fungal proteins using solid-state fungal fermentation (SSFF). In this project Rhizopus microsporus var. oligosporus and Aspergillus oryzae were used to ferment barley and rice, producing tempeh and koji, respectively. SSFF yielded products with 6–13% (dry weight basis) fungal biomass. Protein quality was defined by the parameters indispensable amino acid index (IAAI) and protein digestibility corrected amino acid score (PDCAAS). SSFF improved both parameters in all samples. Lysine was the limiting amino acid in unfermented barley and rice, with PDCAAS of 0.54 and 0.57, respectively. SSFF increased the PDCAAS of lysine by approximately 30% in barley koji, barley tempeh and rice koji and 10% in rice tempeh. These results demonstrate the use of SSFF to improve the protein quality of staple foods. Thereby, this fermentation method can aid in meeting protein demands sustainably in the future through plant-based diets.

Antioxidant activity of tempe fermented with three different <i>Rhizopus</i> species

Tempe, a traditional Indonesian fermented soy-based food, reportedly has higher antioxidant activity than unfermented soybeans. In 2015, the Codex Alimentarius defined the starters of soybean tempe to be Rhizopus microsporus var. oligosporus, Rhizopus oryzae, and/or Rhizopus stolonifer. Most previous studies have focused only on tempe prepared with R. oligosporus. In this study, we compared the antioxidant activities of tempe fermented with three Rhizopus species and unfermented soybeans using assays for β-carotene bleaching (antioxidant activity), antioxidative potential, and oxidative stabilization. The β-carotene bleaching and antioxidative potential assays indicated that the tempe fermented with R. stolonifer had higher antioxidant activity than the tempe fermented with R. oligosporus and R. oryzae. We speculate that the high antioxidant properties of this product are attributable to the isoflavone aglycones and hydroxylated compounds produced by fermentation.

Purification and characterization of an iron-activated alkaline phosphatase produced by Rhizopus microsporus var. microsporus under submerged fermentation using rye flour

Purification and characterization of an iron-activated alkaline phosphatase produced by Rhizopus microsporus var. microsporus under submerged fermentation using rye flour

Produção e caracterização da amilase obtida de Rhizopus microsporus var. oligosporus

As amilases possuem a capacidade de catalisar a ligação α-1,4 do amido liberando glicose e dextrina, com destaque em diversos campos industriais. A pesquisa buscou caracterizar a enzima amilase obtida do fungo Rhizopus microsporus var. oligosporus, através da metodologia de Doehlert, avaliando o comportamento da enzima frente as variações de pH e temperatura. A produção da amilase ocorreu com a utilização do amido como indutor, e a caracterização foi realizada através da metodologia de superfície de resposta, com a análise do pH em 5 níveis (3,0, 4,0, 5,0, 6,0 e 7,0) e temperatura em 3 níveis (30, 50 e 70 °C). A avaliação da termoestabilidade da amilase ocorreu à 60, 70 e 80ºC. A aplicação do modelo experimental indicou que a amilase obtida de R. microsporus var. oligosporus apresenta melhor desenvolvimento catalítico em temperaturas entre 40ºC e 55ºC e pH entre 2,5 e 3,2. A avaliação da termoestabilidade indicou que o aumento da temperatura influencia negativamente na atividade catalítica da amilase. O modelo experimental conduziu à compreensão das condições favoráveis à produção de amilases de R. microsporus var. oligosporus.

First Report of Rot Disease Caused by Rhizopus microsporus var. chinensis on Leaf Mustard (Brassica juncea) in Guangzhou, China.

First Report of Rot Disease Caused by Rhizopus microsporus var. chinensis on Leaf Mustard (Brassica juncea) in Guangzhou, China.

Ethanol from rice byproduct using amylases secreted by Rhizopus microsporus var. oligosporus. Enzyme partial purification and characterization

A three-stage bioethanol bioprocess was developed. Firstly, amylases were obtained from Rhizopus microsporus var. oligosporus using wheat bran in solid-state fermentation. Secondly, amylases hydrolyzed a rice byproduct to make a glucose-rich solution, and this sugar was finally metabolized by Saccharomyces cerevisiae to produce bioethanol. Besides, the secreted enzymes were also partially purified and characterized. The amylase activity (AA) in the crude extract was 358 U/g substrate, and the partially purified enzyme showed the best activity in the 4.0–5.5 pH range. A wide pH stability range (3.5–8.5) was confirmed. The amylase was thermostable up to 60 °C. The ion Mn+2 (10 mM) improved by 60% the AA. There was a 54.9% yield in the conversion of rice residues into reducing sugars in 10 h. The glucose-rich solution was undergone fermentation by S. cerevisiae and showed high ethanol efficiency, 95.8% of the theoretical value. These results suggested a promising technology for bioethanol production.

Preparation, characterization, and prebiotic activity of manno-oligosaccharides produced from cassia gum by a glycoside hydrolase family 134 β-mannanase

To produce manno-oligosaccharides from cassia gum, a mutated glycoside hydrolase family 134 β-mannanase gene (mRmMan134A) from Rhizopus microsporus var. rhizopodiformis F518 was expressed in Pichia pastoris and a high expression level (3680 U mL−1) was obtained through high cell density fermentation. mRmMan134A exhibited maximum activity at pH 5.5 and 50 °C. It was then subjected to hydrolyze cassia gum with 70.6% of overall yield of manno-oligosaccharides. From the hydrolysate, seven components (F1–F7) were separated and identified as mannose, mannobiose, galactose, mannotriose, mannotetraose, 61-α-d-galactosyl-β-d-mannobiose, and mannopentaose, respectively. According to in vitro fermentation, the manno-oligosaccharides were able to promote the growth of three Bifidobacterium strains and six Lactobaillus strains with 3.0-fold increment in culture absorbance, and these strains preferred manno-oligosaccharides with degree of polymerization (DP) 2–3 rather than those with DP 4–5. Novel manno-oligosaccharides from cassia gum with promising prebiotic activity were provided in the present study.

Fungal strain selection for protease production by solid-state fermentation using agro-industrial waste as substrates

Screening of the fungal strains, Aspergillus niger, Trichoderma harzianum, and Rhizopus microsporus var. chinensis for protease production was realised by monitoring the presence of clear zones on skimmed milk agar plates. Additionally, screening of the radial growth on vanilla waste, cane bagasse, and pineapple crown plates was performed. Radial growth data of the fungal strains on skimmed milk agar and agro-industrial waste plates were adjusted to the Gompertz model. The results indicate that R. microsporus var. chinensis and pineapple crown were adequate for the production of milk-clotting enzymes by solid-state fermentation. Crude enzyme extract was produced via solid-state fermentation using two particle sizes [2–2.6 mm (SSF1) and a mixture of less than 1 mm with 2–2.6 mm, at a ratio of 1:1(SSF2)]. The highest milk-clotting and proteolytic activities were shown within 12 h and 48 h of fermentation for SSF1 and SSF2, respectively. The optimum milk-clotting activity presented by the crude enzyme extract was at pH 6.5, 40 °C, and 0.04 M CaCl2. The ratio of milk-clotting activity to proteolytic activity (9.7) presented by the crude enzyme extract indicates potential for use as a calf rennet substitute.

Purification and characterization of an alkalistable phytase produced by Rhizopus microsporus var. microsporus in submerged fermentation

Abstract The filamentous fungus Rhizopus microsporus var. microsporus produces an alkaline phytase in submerged fermentation. This alkaline phytase was purified 83-fold with 1.9% protein yield. The molecular mass of the glycoprotein was estimated in 12% SDS-PAGE as 55 kDa and 63 kDa as revealed by gel filtration, indicating a monomeric structure for the enzyme. The alkaline phytase was optimally active at 65 °C and pH 9.5, with a half-life (T1/2) of 280 min at 50 °C. At 80 °C, the enzyme maintained 87% of its initial activity for 280 min. The phytase activity was characterized as alkalistable and this activity was stimulated when maintained at pH 8.0 and 9.0. It was inhibited mainly by KH2PO4, β-mercaptoethanol, EDTA, HgCl2, and urea. The alkaline phytase activity was also inhibited by detergents (Triton X-100, Tween 20, and SDS) and solvents (acetone, acetonitrile, ethanol, isopropanol, methanol, and n-butanol). The alkaline phytase showed a preference for sodium phytate as substrate (100%), followed by ATP (27.9%) and p-NPP (11.5%). The Km value for sodium phytate was 0.413 mmol L−1. This study represents the first description of the purification and characterization of a fungal alkaline phytase.

Controlled fermentation of rapeseed presscake by Rhizopus, and its effect on some components with relevance to human nutrition

The use of rapeseed protein could contribute to meeting the increasing demand for plant proteins with high biological value in human nutrition. In order to make rapeseed presscake fit for human consumption, the presscake was fermented by using the tempeh mould, Rhizopus microsporus var. oligosporus. Fermentation was satisfactory at initial levels of added acetic acid of 40–60 mmoles/Kg, aw of 0.97, pasteurization, surface inoculation and incubation at 32 °C and 90–95% relative humidity. It was crucial to stop the fermentation once the mould had grown and metabolized sufficiently but before a major rise in pH and subsequent growth of acid-sensitive sporeforming bacteria occurred. Some degradation of glucosinolates, cell wall polysaccharides and phenolic compounds was found, but there was some evidence that growth and metabolism of the mould also depended on the texture of the presscake, as these factors affect the oxygen supply to the mould. In conclusion, it is possible to ferment rapeseed presscake by using the “Tempeh starter” Rhizopus oligosporus, but in order to use the resulting product to enrich various foods with protein or replace other proteins, the degree of degradation of undesired compounds should be further standardized, especially by the control of the pH, oxygen supply, and fermentation time.

English

This study screened four Rhizopus species as biosurfactant producers using different markers. First of all, Rhizopus spp. UCP 1607 was identified as Rhizopus arrhizus by morphological and molecular methods. The production of biosurfactant/bioemulsifier was investigated by submerged fermentation using soybean post-frying oil (5% v/v) and sodium glutamate (1% w/v) medium. The primary markers’ hemolysis and parafilm M tests showed that R. arrhizus UCP1607 strain exhibited higher hemolytic activity (49 mm of clear zone) on sheep blood agar and a larger drop diameter (12 mm) on parafilm hydrophobic surface. The experimental results showed the most promising biosurfactant production by R. arrhizus UCP 1607 strain led to a reduction of surface tension (31.8 mN/m) and the diameter of the oil-spreading covered an area of 66.4 cm2. The strains Rhizopus microsporus var. chinensis UCP1296, R. microsporus var. microsporus UCP1304, and R. arrhizus UCP1607 were capable of forming stable emulsions corresponding to 91.7, 94.8, and 82.6%, respectively in crude oil. Key words: Tensio-active agent, bioemulsifier, screening of Rhizopus strains, submerged fermentation.

The Active Role of Spray-Dried Phytase Produced by Rhizopus microsporus var. microsporus Biofilm in Feeding Broiler Chickens

Objective: Phytases are enzymes that can catalyze the hydrolysis of phytic acid, and have potential for use in the formulation of broiler chickens’ diets. As such, we evaluated the effects of supplementation of broiler chicken diets with a spraydried phytase derived from the fungal Rhizopus microsporus var. microsporus biofilm on the animal performance. Methods: Cobb 500 male chicks were housed for 42 days under controlled environment conditions and provided with three different dietary regimes. These included a control diet meeting nutritional requirements, a diet with phosphorus reduction of 0.175% over days 1-21 and 0.225% over days 22-42, and diets with spray-dried phytase added at 250 FTU/kg, 500 FTU/kg and 750 FTU/kg. Animal performance was assessed based on their body weight, feed intake, weight gain, feed conversion rate, and viability. Results: At the first phase of development (1-21 days of age), performance was not improved by phytase addition, but at the latter phase (22-42 days of age); performance improved significantly, especially in birds fed phytase at 750 FTU/ kg. In addition, phytase promoted an increase in villous height for animals of both 21- and 42 day-old animals, improving nutrient absorption and performance at the end phase of development. Conclusion: Phytase supplementation positively affected animal development and jejunum morphology, highlighting its potential to be used as an additive for broiler chicken diet formulation.

Backbone and Ile-δ1, Leu, Val methyl 1H, 15N, and 13C, chemical shift assignments for Rhizopus chinensis lipase.

Lipase r27RCL is a 296-residue, 33kDa monomeric enzyme with high ester hydrolysis activity, which has significant applications in the baking, paper and leather industries. The lipase gene proRCL from Rhizopus microsporus var. chinensis (also Rhizopus chinensis) CCTCC M201021 was cloned as a fusion construct C-terminal to a maltose-binding protein (MBP) tag, and expressed as MBP-proRCL in an Escherichia coli BL21 trxB (DE3) expression system with uniform 2H,13C,15N-enrichment and Ile-δ1, Leu, and Val 13CH3 methyl labeling. The fusion protein was hydrolyzed by Kex2 protease at the recognition site Lys-Arg between residues -29 and -28 of the prosequence, producing the enzyme form called r27RCL. Here we report extensive backbone 1H, 15N, and 13C, as well as Ile-δ1, Leu, and Val side chain methyl, NMR resonance assignments for r27RCL.

UV/Vis spectroscopy combined with chemometrics for monitoring solid‐state fermentation with Rhizopus microsporus var. oligosporus

AbstractBACKGROUNDDifficulties in bioprocess monitoring are a drawback of solid‐state fermentation (SSF). Specifically, monitoring of enzyme activities in SSF is not an easy task. This work aimed to calibrate partial least squares (PLS) and artificial neural network (ANN) models for inferring protease and amylase activities, as well as protein concentration, from UV‐Vis spectra of aqueous extracts of samples removed during SSF using Rhizopus microsporus var. oligosporus.RESULTSSSFs were performed using single agro‐industrial wastes (wheat bran, type II wheat flour, sugarcane bagasse and soybean meal) and ternary mixtures of them. Enzyme activities and protein concentrations in the aqueous extracts were quantified biochemically. The corresponding UV‐Vis spectra of diluted extracts were also collected. The prediction quality of the ANN was higher than that of the PLS model. The relative errors considering the range for amylolytic and proteolytic enzymes were 4% (3–442 U g−1) and 6% (0–256 U g−1), respectively, for the best ANN architectures (8 and 6 neurons in hidden layer, respectively).CONCLUSIONThese results, in combination with correlation coefficients (R > 0.94), suggest that this approach is suitable for developing a chemosensor for monitoring SSFs, reducing the analytical work for quantification of enzyme activities. No satisfactory results were obtained for protein concentration. © 2017 Society of Chemical Industry