Characterization of an alcohol acetyltransferase GcAAT responsible for the production of antifungal volatile esters in endophytic Geotrichum candidum PF005.

Alcohol acetyltransferases (AATs) are a group of enzymes that catalyze the formation of esters from different alcohols and acetyl-CoA. However, these enzymes are not well characterized with regard to synthesis of antifungal compounds. The present study aims to investigate the AAT enzyme from Geotrichum candidum PF005, an endophytic yeast-like fungus that emits fruity scented antifungal volatiles, primarily comprising of acetate esters. After PCR-based cloning of the GcAAT gene, the encoded enzyme was characterized structurally through in silico methods and functionally via heterologous expression in Saccharomyces cerevisiae. In native host, the single copy GcAAT gene exhibited induced expression upon supplementation with metabolic precursors, like L-leucine (Leu) or α-ketoisocaproate (α-KIC). Docking studies using the modelled structure of GcAAT revealed differential but favourable binding interactions for three alcohol substrates (i.e., isoamyl alcohol, isobutyl alcohol and 2-phenylethanol) and the co-substrate acetyl-CoA. Binding sites for both substrate and co-substrate are found to be located inside a tunnel identified in the structure, wherein the H208 of the acetyltransferase conserved motif HXXXD was found at a hydrogen bond distance from the substrate. Functional complementation of GcAAT in S. cerevisiae AAT knockout strain caused 32% decrease in dry biomass weight of the test phytopathogenic fungus, Rhizoctonia solani as compared to the control (AAT knockout strain with empty plasmid) after 72 h of incubation due to the emitted volatiles. When the transformed yeast cells were fed with Leu and α-KIC, the relative abundance of the isoamyl acetate ester increased by 21% and 48%, respectively as compared to the control (without precursor). Further analysis documented that volatiles from α-KIC fed GcAAT transformant exhibited 58% higher antifungal activity against the test fungus R. solani than the control, engendered by increased oxidative stress that led to distorted mycelial morphology and increased hyphal branching. Together, the augmented antifungal effect displayed by the GcAAT expressing S. cerevisiae AAT knockout strain is clearly attributable to the acetate esters, especially isoamyl acetate, which are inherently produced in endophytic G. candidum PF005 as antifungal volatiles.

Supporting role of lignin in immobilization of yeast on sugarcane bagasse for continuous pectinase production.

BACKGROUND: Lignocellulosic wastes are pretreated prior to their utilization in fermentation processes. Such pretreatment also alters the topological features of the substrates, and therefore the suitability of pretreated waste as immobilization matrix for microbial cells needs investigation. RESULTS: In this study, the effect of chemical pretreatment of sugarcane bagasse (SB) for its subsequent utilization as a matrix to immobilize a pectinolytic yeast, Geotrichum candidum AA15, was evaluated using cell retention, concentration of immobilized cells, immobilization efficiency, scanning electron microscopy and Fourier transform infrared spectroscopy of the substrate and pectinase titers obtained after recycling. The results revealed that untreated SB is more efficient for immobilization with higher values of cell retention and pectinase productivity (99.78%) retained for up to six production cycles. It was deduced that removal of lignin by pretreatment negatively influenced the ability of SB to support cell adhesion, as lignin acts as a sealing agent that provides strength to the substrate. CONCLUSIONS: The strategy of utilizing SB as immobilization matrix was found effective at the laboratory scale as it improved pectinase production and may be investigated further for large-scale and cost-effective production. © 2020 Society of Chemical Industry.

Elucidation of fungal dye-decolourizing peroxidase (DyP) and ligninolytic enzyme activities in decolourization and mineralization of azo dyes.

AIM: The aim of the study is to investigate the efficiency of Geotrichum candidum in the decolourization and mineralization of synthetic azo dyes. METHODS AND RESULTS: It includes screening of enzymes from G. candidum and its optimization, followed by decolourization and mineralization studies. Decolourization was observed to be maximum in methyl orange (94·6%) followed by Congo red (85%), trypan blue (70·4%) and Eriochrome Black T (55·6%) in 48 h, suggesting the plausible degradation of the azo dyes by G. candidum. The enzyme activity study showed that DyP-type peroxidase has highest activity of 900 mU ml-1 compared to that of laccase (405 mU ml-1 ) and lignin peroxidase (LiP) (324 mU ml-1 ) at optimized pH (6) and temperature (35°C). Moreover, the rate of decolourization was found to be directly proportional to the production of laccase and LiP, unlike DyP-type peroxidase. Furthermore, mineralization study demonstrated reduction in aromatic amines, showing 20% mineralization of methyl orange. CONCLUSION: Geotrichum candidum with its enzyme system is able to efficiently decolourize and mineralize the experimental azo dyes. SIGNIFICANCE AND IMPACT OF THE STUDY: The efficient decolourization and mineralization of azo dyes makes G. candidum a promising alternative in the treatment of textile effluent contaminated with azo dyes.

Determination of allosteric and active sites responsible for catalytic activity of delta 12 fatty acid desaturase from Geotrichum candidum and Mortierella alpina by domain swapping.

Cheese lacks essential fatty acids (EFAs). Delta 12 fatty acid desaturase (FADS12) is a critical enzyme required for EFA biosynthesis in fermentation of the predominant strains of cheese. Previously, we identified the FADS12 gene and characterized its function for the first time in Geotrichum candidum, a dominant strain used to manufacture soft cheese with white rind. In this study, we analyzed the molecular mechanism of FADS12 function by swapping domains from Mortierella alpina and G. candidum that had, respectively, high and low oleic acid conversion rates. The results revealed three regions that are essential to this process, including regions from the end of the second transmembrane domain to the beginning of the third transmembrane domain, from the end of the third transmembrane domain to the beginning of the fourth transmembrane domain, and from the 30-amino acid from the end of the sixth transmembrane domain to the C-terminal end region. Based on our domain swapping analyses, nine pairs of amino acids including H112, S118, H156, Q161, K301, R306, E307, A309 and S323 in MaFADS12 (K123, A129, N167, M172, T302, D307, I308, E310 and D324 in GcFADS12) were identified as having a significantly effect on FADS12 catalytic efficiency, and linoleic acid and its analogues (12,13-cyclopropenoid fatty acid) were found to inhibit the catalytic activity of FADS12 and related recombinant enzymes. Furthermore, the molecular mechanism of FADS12 inhibition was analyzed. The results revealed two allosteric domains, including one domain from the N-terminal region to the beginning of the first transmembrane domain and another from the 31st amino acid from the end of the sixth transmembrane domain to the C terminus. Y4 and F398 amino acid residues from MaFADS12 and eight pairs of amino acids including G56, L60, L344, G10, Q13, S24, K326 and L344 in MaFADS12 (while Y66, F70, F345, F20, Y23, Y34, F327 and F345 in GcFADS12) played a pivotal role in FADS12 inhibition. Finally, we found that both allosteric and active sites were responsible for the catalytic activity of FADS12 at various temperatures, pH, and times. This study offers a solid theoretical basis to develop preconditioning methods to increase the rate at which GcFADS12 converts oleic and linoleic acids to produce higher levels of EFAs in cheese.

Co-culturing corncob-immobilized yeasts on orange peels for the production of pectinase.

OBJECTIVE: Pectinase is an industrially important enzyme which is employed in an array of commercial processes; cost of production, however, impedes its application. The main objective of this study was to design a two-layered strategy for the reduction of production cost, firstly by using a yeast co-culture in an immobilized form on an agricultural waste matrix, corncob (CB), secondly by utilizing orange peels (OP) as substrate. RESULTS: Two yeast strains, Saccaromyces cerevisiae MK-157 and Geotrichum candidum AA15 were cultivated as mono-, as well as, co-culture after immobilization on CB and pectinase production was monitored. Initial experiments revealed that co-culture is beneficial to get sustainable product in subsequent 2nd and 3rd production cycles. The factors affecting pectinase production in consecutive three production cycles were studied by employing Plackett-Burman design and the significant factors were optimized through Box-Behnken design. Under optimized conditions, 17.89 IU mL-1 of pectinase was obtained. Scanning electron micrographs presented damaged immobilized yeast cells on CB after the 3rd production cycle. CONCLUSION: The pectinase production was improved substantially by using immobilized co-culture and hence the strategy was found effective at lab scale. Since, pectinase is applied in orange juice clarification, therefore, the study can be extended to move forward towards circular economy.

Ethanol Production and Other Bioproducts by Galactomyces geotrichum from Sugarcane Bagasse Hydrolysate.

This study investigated the enzymatic saccharification of alkaline-pretreated sugarcane bagasse (PSB) and the bioconversion of simple sugars from hydrolysates to ethanol and other bioproducts by the yeast Galactomyces geotrichum. The effects of percentage of dry substrate (3 and 10% w/v) and time of hydrolysis (24 and 72 h) in the content of released sugars were evaluated. The concentrations of monosaccharides and total reducing sugars (TRS) were calculated by high-performance liquid chromatography (HPLC) and by 3.5-dinitrosalicylic acid (DNS) method, respectively. The highest concentrations of TRS, glucose and xylose (73.96, 31.78 and 10.85 g/L, respectively) were obtained after the saccharification of 10% of PSB with Cellic CTec3 multi-enzyme cocktail (10 FPU/g cellulose) during 72 h (hydrolysate IV). G. geotrichum UFVJM-R150 fermented both glucose and xylose from the hydrolysates. The most efficient ethanol production was obtained after the fermentation of hydrolysate IV (9.99 g/L of ethanol, volumetric productivity-QP of 0.42 g/L.h and yield of ethanol as a function of the substrate-YP/S of 0.27 gethanol/gsugar). Besides ethanol, G. geotrichum was also able to produce other high-value chemicals such as isoamyl alcohol and galacturonic acid. This is the first report of the potential of the yeast G. geotrichum to fermentate sugarcane bagasse hydrolysates with the production of important bioproducts to further use by biorefineries.

Reversible control of enantioselectivity by the length of ketone substituent in biocatalytic reduction.

Enzyme engineering has been widely employed to tailor the substrate specificity and enantioselectivity of enzymes. In this study, we mutated Trp288, an unconserved residue in the small binding pocket of an acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD). Trp288 mutants showed substrate specificity expansion towards bulky-bulky ketones and enantioselectivity alteration which was highly dependent on the substrate substituent length. In aliphatic ketone reduction, enantioselectivity inverted from (S) to (R) when one of the substituents to the carbonyl carbon was elongated from propyl to butyl or pentyl. The best (R)-selective mutant, Trp288Val, achieved the reduction of 3-heptanone to its corresponding (R)-alcohol with 97% ee. Our docking simulation suggested that when enantioselectivity inverted to (R), only pro-R binding poses were productive. Gly94 played an important role to stabilize the butyl or pentyl group for their productive pro-R poses. Interestingly, when the substituent was further elongated, the enantioselectivity inverted back to the (S) form.

Structural basis for a highly (S)-enantioselective reductase towards aliphatic ketones with only one carbon difference between side chain.

Aliphatic ketones, such as 2-butanone and 3-hexanone, with only one carbon difference among side chains adjacent to the carbonyl carbon are difficult to be reduced enantioselectively. In this study, we utilized an acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD) to reduce challenging aliphatic ketones such as 2-butanone (methyl ethyl ketone) and 3-hexanone (ethyl propyl ketone) to their corresponding (S)-alcohols with 94% ee and > 99% ee, respectively. Through crystallographic structure determination, it was suggested that residue Trp288 limit the size of the small binding pocket. Docking simulations imply that Trp288 plays an important role to form a C-H⋯π interaction for proper orientation of ketones in the pro-S binding pose in order to produce (S)-alcohols. The excellent (S)-enantioselectivity is due to a non-productive pro-R binding pose, consistent with the observation that the (R)-alcohol acts as an inhibitor of (S)-alcohol oxidation.

Optimization of free fatty acid production by enzymatic hydrolysis of vegetable oils using a non-commercial lipase from Geotrichum candidum.

This study aimed to optimize free fatty acid production by enzymatic hydrolysis of cottonseed, olive and palm kernel oils in stirred-tank reactors using a lipase from Geotrichum candidum (GCL-I). The effect of pH, temperature and substrate concentration on the hydrolytic activity of GCL-I using these vegetable oils was investigated. Thermal stability tests and thermodynamic studies were also performed. A complete hydrolysis of cottonseed oil was obtained after 120 min of reaction, while for olive and palm kernel maximum hydrolysis percentage was 96.4% and 60.1%, respectively. GCL-I exhibited the highest activity in the hydrolysis of vegetable oils that are rich in unsaturated-fatty acids (cottonseed and olive oils). Under optimal conditions (46.8% m/m of oil, 6.6 U/g of the reaction mixture at 40 °C), complete cottonseed oil hydrolysis was observed at 60 min of reaction performed in an emulsifier-free system with no buffer.

Δ12 fatty acid desaturase gene from Geotrichum candidum in cheese: molecular cloning and functional characterization.

Soft cheese with white rind lacks essential fatty acids (EFAs), and as a result its long-term consumption may lead to various kinds of cardiovascular and cerebrovascular diseases, such as hyperlipidemia, hypertension, and atherosclerosis. Geotrichum candidum is a dimorphic yeast that plays an important role in the ripening of mold cheese. A gene coding for Δ12 fatty acid desaturase, a critical bifunctional enzyme desaturating oleic acid (OA) and linoleic acid (LA) to produce LA and α-linolenic acid (ALA), respectively, was isolated from G. candidum, and then cloned and heterologously expressed in Saccharomyces cerevisiae. This gene, named GcFADS12, had an open reading frame of 1257 bp and codes for a protein of 419 amino acids with a predicted molecular mass of 47.5 kDa. Characterization showed that GcFADS12 had the ability to convert OA to LA and LA to ALA, and the conversion rates for OA and LA were 20.40 ± 0.66% and 6.40 ± 0.57%, respectively. We also found that the protein product of GcFADS12 catalyzes the conversion of the intermediate product (LA) to ALA by addition of OA as the sole substrate. The catalytic activity of GcFADS12 on OA and LA was unaffected by fatty acid concentrations. Kinetic analysis revealed that GcFADS12 had stronger affinity for the OA than for the LA substrate. This study offers a solid basis for improving the production of EFAs by G. candidum in cheese.

Immobilized enzymes from Geotrichum spp. improve wine quality.

Higher alcohols are the byproducts of yeasts in alcohol fermentation and are harmful to human health at high concentrations. In this study, immobilized crude enzymes extracted from Geotrichum spp. strains S12 and S13 were separately employed to treat red wine, then GC and GC-MS analyses were used to determine the profiles of volatile compounds in untreated and treated wine samples. Immobilized enzymes from S13 (SA-S13E) were more active in decreasing higher alcohols than enzymes from S12. Conditions for preparing SA-S13E were optimized, and best results were obtained at a sodium alginate concentration of 35 g/L, calcium chloride of 20 g/L, and crude enzyme dosage of 3 mL. Treatment with SA-S13E significantly increased the ester content and sensory quality of wine. After being reused three times, SA-S13E still exhibited approximately 80% activity towards 1-propanol, isobutanol, and hexanol and had certain activity even after 3 months storage at -20 °C, indicating high stability in application and storage and thus showing potential in wine processing.

Purification and characterization of a melanin biodegradation enzyme from Geotrichum sp.

OBJECTIVE: Melanin is a black or brown phenolic polymer present mainly in skin and hair. Although melanin can be degraded by some microbial species, the melanin degradation capacity of Geotrichum sp. is unknown. The aim of this study was to characterize a melanin biodegradation enzyme from Geotrichum sp. METHODS: In this study, we assessed the melanin degradation activity of Geotrichum sp. in comparison with the major melanin-degrading enzymes, manganese-dependent peroxidase (MnP), manganese-independent peroxidase, lignin peroxidase and laccase. Furthermore, the effect of several carbohydrates on melanin degradation by Geotrichum sp. was determined. The MnP enzyme was purified using ammonium sulphate precipitation and Sephadex G-200 column chromatography, and then the conditions for optimal enzymatic activity were determined by adjusting the pH, temperature and Tween-80 concentration. RESULTS: Compared with extracellular ligninolytic enzymes of Geotrichum sp., MnP had the highest ligninolytic enzyme activity; and the highest enzymatic activity was observed in the presence of glucose. The final purified MnP enzyme exhibited 6 U mL-1 activity and had a molecular weight of 54.2 kDa. The enzymatic activity was highest at pH 4.5 and 25-35°C in the absence of Tween-80. CONCLUSION: These results indicate the potential of MnP purified from Geotrichum sp. as a skin-lightening agent in the cosmetic industry.

Optimization of the production and characterization of lipase from Candida rugosa and Geotrichum candidum in soybean molasses by submerged fermentation.

This present work describes the production and biochemical characterization of lipase by Candida rugosa and Geotrichum candidum in a culture supplemented with soybean molasses. After optimizing the fermentation times for both microorganisms, the effects of changing the soybean molasses concentration, the fermentative medium pH and the fermentation temperature were evaluated using the Central Composite Planning. When soybean molasses was used at a concentration of 200 g/L at 27 ± 1 °C and pH 3.5, the lipolytic activity measured in the broth was 12.3 U/mL after 12 h for C. rugosa and 11.48 U/mL after 24 h for G. candidum. The molecular masses were 38.3 kDa to G. candidum lipase and 59.7 kDa to C. rugosa lipase, determined by SDS-PAGE. The lipase from both microorganisms exhibited maximal hydrolytic activity at a temperature of 40 °C and were inhibited at pH 10.0. Using different concentration of p-nitrophenylbutyrate (p-NPB), the kinetic parameters were calculated, as follows: the Km of lipase from G. candidum was 465.44 µM and the Vmax 0.384 µmol/min; the Km and Vmax of lipase from C. rugosa were 129.21 µM and 0.034 µmol/min, respectively. Lipases activity were increased by metallic ions Mg(2+) and Na(+) and inhibited by metallic ion Cu(3+).

Sequencing, biochemical characterization, crystal structure and molecular dynamics of cellobiohydrolase Cel7A from Geotrichum candidum 3C.

The ascomycete Geotrichum candidum is a versatile and efficient decay fungus that is involved, for example, in biodeterioration of compact discs; notably, the 3C strain was previously shown to degrade filter paper and cotton more efficiently than several industrial enzyme preparations. Glycoside hydrolase (GH) family 7 cellobiohydrolases (CBHs) are the primary constituents of industrial cellulase cocktails employed in biomass conversion, and feature tunnel-enclosed active sites that enable processive hydrolytic cleavage of cellulose chains. Understanding the structure-function relationships defining the activity and stability of GH7 CBHs is thus of keen interest. Accordingly, we report the comprehensive characterization of the GH7 CBH secreted by G. candidum (GcaCel7A). The bimodular cellulase consists of a family 1 cellulose-binding module (CBM) and linker connected to a GH7 catalytic domain that shares 64% sequence identity with the archetypal industrial GH7 CBH of Hypocrea jecorina (HjeCel7A). GcaCel7A shows activity on Avicel cellulose similar to HjeCel7A, with less product inhibition, but has a lower temperature optimum (50 °C versus 60-65 °C, respectively). Five crystal structures, with and without bound thio-oligosaccharides, show conformational diversity of tunnel-enclosing loops, including a form with partial tunnel collapse at subsite -4 not reported previously in GH7. Also, the first O-glycosylation site in a GH7 crystal structure is reported--on a loop where the glycan probably influences loop contacts across the active site and interactions with the cellulose surface. The GcaCel7A structures indicate higher loop flexibility than HjeCel7A, in accordance with sequence modifications. However, GcaCel7A retains small fluctuations in molecular simulations, suggesting high processivity and low endo-initiation probability, similar to HjeCel7A. DATABASE: Structural data are available in the Protein Data Bank under the accession numbers 5AMP, 4ZZV, 4ZZW, 4ZZT, and 4ZZU. The Geotrichum candidum GH family 7 cellobiohydrolase nucleotide sequence is available in GenBank under accession number KJ958925. ENZYMES: Glycoside hydrolase family 7 reducing end acting cellobiohydrolase.

Crystallization and preliminary crystallographic analysis of acetophenone reductase from Geotrichum candidum NBRC 4597.

Acetophenone reductase (APRD) from Geotrichum candidium NBRC 4597 was crystallized by the hanging-drop vapour-diffusion method using PEG 3350 as a precipitant. The crystal belonged to space group P6522, with unit-cell parameters a = b = 104.5, c = 273.7 Å, and diffracted to 2.6 Šresolution. Phasing using the single-wavelength anomalous diffraction method was successful. Model building and crystallographic refinement are in progress.

Mycelium-bound lipase from a locally isolated strain of Geotrichum candidum.

Mycelium-bound lipase (MBL), from a locally isolated Geotrichum candidum strain, was produced and characterized as a natural immobilized lipase. A time course study of its lipolytic activity in 1 L liquid broth revealed the maximum MBL activity at 4 h for mycelium cells harvested after 54 h. The yield and specific activity of MBL were 3.87 g/L dry weight and 508.33 U/g protein, respectively, while less than 0.2 U/mL lipase activity was detected in the culture supernatant. Prolonged incubation caused release of the bound lipase into the growth medium. The growth pattern of G. candidum, and production and properties of MBL were not affected by the scale. The stability of mycelia harboring lipase (MBL), harvested and lyophilized after 54 h, studied at 4 °C depicted a loss of 4.3% and 30% in MBL activity after 1 and 8 months, while the activity of free lipase was totally lost after 14 days of storage. The MBL from G. candidum displayed high substrate selectivity for unsaturated fatty acids containing a cis-9 double bond, even in crude form. This unique specificity of MBL could be a direct, simple and inexpensive way in the fats and oil industry for the selective hydrolysis or transesterification of cis-9 fatty acid residues in natural triacylglycerols.

Malting of barley with combinations of Lactobacillus plantarum, Aspergillus niger, Trichoderma reesei, Rhizopus oligosporus and Geotrichum candidum to enhance malt quality.

Good quality malt is characterised by the presence of high levels of fermentable sugars, amino acids and vitamins. To reach the starch-rich endosperm of the kernel, ß-glucan- and arabinoxylan-rich cell walls have to be degraded. ß-Glucanase is synthesized in vast quantities by the aleurone layer and scutellum during germination. Secretion of hydrolytic enzymes is often stimulated by addition of the plant hormone gibberellic acid (GA3) during germination. We have shown an enhanced ß-glucanase and α-amylase activity in malt when germinating barley was inoculated with a combination of Lactobacillus plantarum B.S1.6 and spores of Aspergillus niger MH1, Rhizopus oligosporus MH2 or Trichoderma reesei MH3, and L. plantarum B.S1.6 combined with cell-free culture supernatants from each of these fungi. Highest malt ß-glucanase activity (414 Units/kg malt) was recorded with a combination of L. plantarum B.S1.6 and spores of A. niger MH1. Highest α-amylase activities were recorded with a combination of L. plantarum B.S1.6 and spores of R. oligosporus MH2 (373 Ceralpha Units/g malt). Highest FAN levels were recorded when L. plantarum was inoculated in combination with spores of either R. oligosporus MH2 or T. reesei MH3 (259 and 260 ppm, respectively). This is the first study showing that cell-free culture supernatants of Aspergillus, Rhizopus and Trichoderma have a stimulating effect on ß-glucanase and α-amylase production during malting. A combination of L. plantarum B.S1.6, and spores of A. niger MH1 and R. oligosporus MH2 may be used as starter cultures to enhance malt quality.

Structure of Acidothermus cellulolyticus family 74 glycoside hydrolase at 1.82 Å resolution.

Here, a 1.82 Šresolution X-ray structure of a glycoside hydrolase family 74 (GH74) enzyme from Acidothermus cellulolyticus is reported. The resulting structure was refined to an R factor of 0.150 and an Rfree of 0.196. Structural analysis shows that five related structures have been reported with a secondary-structure similarity of between 75 and 89%. The five similar structures were all either Clostridium thermocellum or Geotrichum sp. M128 GH74 xyloglucanases. Structural analysis indicates that the A. cellulolyticus GH74 enzyme is an endoxyloglucanase, as it lacks a characteristic loop that blocks one end of the active site in exoxyloglucanases. Superimposition with the C. thermocellum GH74 shows that Asp451 and Asp38 are the catalytic residues.

Acetophenone reductase with extreme stability against a high concentration of organic compounds or an elevated temperature.

The gene encoding acetophenone reductase (APRD), a useful biocatalyst for producing optically pure alcohols, was cloned from the cDNA of Geotrichum candidum NBRC 4597. The gene contained an open reading frame that consisted of 1,029 nucleotides corresponding to 342 amino acid residues. The subunit molecular weight was calculated to be 36.7 kDa. The predicted amino acid sequence did not have significant similarity to those of the acetophenone reductase reported previously. The gene was inserted into the pET-21b(+) expression vector and expressed in Escherichia coli Rosetta™(DE3)pLysS by induction with 1 mM of isopropyl-ß-D-thiogalactopyranoside. E. coli cell-free extract gave 21.9 U/mg APRD activity, which was 81 times that of the G. candidum cell-free extract. The enzyme was purified with a HisTrap FF crude column. The enzyme exhibited the highest activity at 60 °C, and optimum reducing and oxidizing activity were observed in a pH range around 7.0-8.0 and 8.5, respectively. The enzyme was most stable at 60 °C and pH 6.5-7.5. The Vmax and the apparent Km value of the reductase were 67.6 µmol/min per milligram of protein and 0.146 mM for acetophenone, respectively. From 4 % (v/v) 4-phenyl-2-butanone, (S)-4-phenyl-2-butanol was obtained with a yield >80 % and an enantiomeric excess >99 % in a 20 h reaction recycling NADH with 15 % (v/v) 2-propanol.

Inulinase production by Geotrichum candidum OC-7 using migratory locusts as a new substrate and optimization process with Taguchi DOE.

Utilization of migratory locusts (Locusta migratoria) as a main substrate due to its high protein content for inulinase (2,1-ß-d-fructan fructanohydrolase) production by Geotrichum candidum OC-7 was investigated in this study. To optimize fermentation conditions, four influential factors (locust powder (LP) concentration, sucrose concentration, pH and fermentation time) at three levels were investigated using Taguchi orthogonal array (OA) design of experiment (DOE). Inulinase yield obtained from the designed experiments with regard to Taguchi L9 OA was processed with Minitab 15 software at 'larger is better' as quality character. The results showed that optimal fermentation conditions determined as LP 30 g/l, sucrose 20 g/l, pH 6.0 and time 48 h. Maximum inulinase activity was recorded as 30.12 U/ml, which was closer to the predicted value (30.56 U/ml). To verify the results, analysis of variance test was employed. LP had the greatest contribution (71.96%) among the other factors. Sucrose had lower contribution (13.96%) than LP. This result demonstrated that LP had a strong effect on inulinase activity and can be used for enzyme production. Taguchi DOE application enhanced enzyme activity to about 3.05-fold versus unoptimized condition and 2.34-fold versus control medium. Consequently, higher inulinase production can be achieved by the utilization of an edible insect material as an alternative substrate and Taguchi DOE presents suitable optimization method for biotechnological process.