L-蛋氨酸酰化酶基因工程菌培养条件的研究

为提高L-氨基酰化酶基因工程菌1016的生物量及酶产量,优化了发酵工艺,确定了较佳的培养基成分,并初步考察了该酶的底物特异性。优化后工程菌摇瓶产酶稳定在980U/mL发酵液,发酵周期17 h,生物量A_(420)穗定在0.7。产酶量为原来的2倍以上。乙酰化的脂肪族氨基酸Met是该酶的最适底物。     

Enhanced production of cellulase from a novel strain Trichoderma gamsii M501 through response surface methodology and its application in biomass saccharification

Cellulolytic enzymes produced by Trichoderma sp. have attracted interest in converting the biomass to simple sugars in the production of cellulosic ethanol. In this work, a novel cellulolytic strain M501 was isolated and identified as T. gamsii by sequencing the ITS rDNA region. The production of cellulase (CMCase) by T. gamsii M501 was enhanced by employing statistical methods. The strain grown in the optimized production medium composed of mineral salts, microcrystalline cellulose (13.7 g/l), tryptone (4.8 g/l) and trace elements (2 mL/l) at pH 5.5 and 28 °C for 72 h produced a maximum CMCase of 61.3 U/mL. The optimized production medium also showed the other enzyme activity of FPU (2.6 U/mL), β-glucosidase (2.1 U/mL), xylanase (681 U/mL) and β- xylosidase (0.6 U/mL). The crude cellulase cocktail produced by T. gamsii M501 efficiently hydrolyzed alkali pretreated sugarcane bagasse with glucose and xylose yield of 78 % and 74 % respectively at 10 % solid loading. This study is the first of its kind research on biomass saccharification using T. gamsii cellulase cocktail. Therefore, the novel strain T. gamsii M501 would be useful for further development of an enzyme cocktail for cellulosic ethanol production.     

Application of factorial designs for optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae pneumoniae AS-22.

Production of cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae pneumoniae AS-22 was optimized in shake flasks using a statistical experimental design approach. Effect of various components in the basal medium, like carbon, nitrogen, phosphorus, and mineral sources as well as initial pH and temperature, were tested on enzyme production. The optimum concentrations of the selected media components were determined using statistical experimental designs. Two level fractional factorial designs in five variables, namely, dextrin, peptone, yeast extract, ammonium dihydrogen orthophosphate, and magnesium sulphate concentrations were constructed. The optimum medium composition thus found consisted of 49.3 g/L dextrin, 20.6 g/L peptone, 18.3 g/L yeast extract, 6.7 g/L ammonium dihydrogen orthophosphate, and 0.5 g/L magnesium sulphate. The maximum CGTase activity obtained was 21.4 U/mL in 28 h of incubation. The cell growth and CGTase production profiles were studied with the optimized medium in shake flasks and in 1-L fermenters. It was observed that the enzyme production was growth associated both in shake flask and in fermenter, although it was slower in shake flask. The maximum CGTase activity obtained in the fermenter was 32.5 U/mL in 16 h. The optimized medium resulted in about 9-fold increase in the enzyme activity as compared to that obtained in the basal medium in shake flask as well as in fermenter.     

Statistical optimization of culture media and conditions for production of mannan by Saccharomyces cerevisiae

In view of the increase in Saccharomyces cerevisiae mannan content, the culture medium and condition for S. cerevisiae were optimized in this study. The influence of culture medium ingredients such as carbon and nitrogen sources, inorganic ion, and enzyme activator on mannan production were evaluated using factional design. The mathematical model was established by the quadratic rotary combination design through response surface analysis. The optimized concentrations of culture medium were determined as follows: 4.98 g/100 mL, sucrose; 4.39 g/100 mL, soybean peptone; 3.10 g/100 mL, yeast extract; and 2.21 g/100 mL, glycerol. The optimized culture medium increased mannan production from 82.7 ± 3.4 mg/100 mL to 162.53 ± 3.47 mg/100 mL. The influence of original pH, inoculum size, temperature, and media volume on mannan production was evaluated and confirmed by orthogonale experimental design, with the order of effect as follows: media volume > temperature > initial pH > inoculation size. The optimized culture condition was pH, 5; inoculum size, 5 ml; temperature, 32°C; and media volume, 40 mL. The maximum mannan production increased to 258.5 ± 9.1 mg/100 mL at the optimum culture condition. It was evident that the mannan production was affected significantly by culture medium and condition optimization (p < 0.01).     

Optimization of culture media for enhanced chitinase production from a novel strain of Stenotrophomonas maltophilia using response surface methodology

Chitinase is one of the most important mycolytic enzymes with industrial significance. This enzyme is produced by a number of organisms including bacteria. In this study we describe optimization of media components with increased production of chitinase for selected bacteria Stenotrophomonas maltophilia isolated from the soil. Different components of the defined media responsible for influencing chitinase secretion by the bacterial isolate were screened using Plackett-Burman experimental design and were further optimized by Box-Behnken factorial design of response surface methodology (RSM) in liquid culture. Maximum chitinase production was predicted in medium containing chitin 4.94 g/l, maltose 5.56 g/l, yeast extract 0.62 g/l, KH2PO4 1.33 g/l and MgSO4.7H2O 0.65 g/l using Response surface plots and point prediction tool of DESIGN EXPERT 7.1.6 (Statease, USA) software.     

Enhanced cellulase production through random mutagenesis of Talaromyces pinophilus OPC4-1 and fermentation optimization

Reducing cellulase cost remains a major challenge for lignocellulose to fuel and chemical industries. In this study, mutants of a novel wild-type cellulolytic fungal strain Talaromyces pinophilus OPC4-1 were developed by consecutive UV irradiation, N-methyl-N`-nitro-N-nitrosoguanidine (NTG) and ethylmethane sulfonate (EMS) treatment. A potential mutant EMM was obtained and displayed enhanced cellulase production. Using Solka Floc cellulose as the substrate, through fed-batch fermentation, mutant strain T. pinophilus EMM generated crude enzymes with an FPase activity of 27.0 IU/mL and yield of 900 IU/g substrate. When corncob powder was used, strain EMM produced crude enzymes with an FPase activity of 7.3 IU/mL and yield of 243.3 IU/g substrate. In addition, EMM crude enzymes contained 29.2 and 16.3 IU/mL β-glucosidase on Solka Floc cellulose and corncob power, respectively. The crude enzymes consequently displayed strong biomass hydrolysis performance. For corncob hydrolysis, without supplement of any commercial enzymes, glucose yields of 591.7 and 548.6 mg/g biomass were obtained using enzymes produced from Solka Floc cellulose and corncob powder, respectively. It was 553.9 mg/g biomass using the commercial enzyme mixture of Celluclast 1.5 L and Novozyme 188. Strain T. pinophilus EMM was therefore a potential fungus for on-site enzyme production in biorefinery processes.     

Optimization of culture medium for novel cell-associated tannase production from Bacillus massiliensis using response surface methodology

Naturally immobilized tannase (tannin acyl hydrolase, E.C. 3.1.1.20) has many advantages, as it avoids the expensive and laborious operation of isolation, purification, and immobilization, plus it is highly stable in adverse pH and temperature. However, in the case of cell-associated enzymes, since the enzyme is associated with the biomass, separation of the pure biomass is necessary. However, tannic acid, a known inducer of tannase, forms insoluble complexes with media proteins, making it difficult to separate pure biomass. Therefore, this study optimizes the production of cell-associated tannase using a "protein-tannin complex" free media. An exploratory study was first conducted in shake-flasks to select the inducer, carbon source, and nitrogen sources. As a result it was found that gallic acid induces tannase synthesis, a tryptose broth gives higher biomass, and lactose supplementation is beneficial. The medium was then optimized using response surface methodology based on the full factorial central composite design in a 3 l bioreactor. A 2(3) factorial design augmented by 7 axial points (alpha = 1.682) and 2 replicates at the center point was implemented in 17 experiments. A mathematical model was also developed to show the effect of each medium component and their interactions on the production of cell-associated tannase. The validity of the proposed model was verified, and the optimized medium was shown to produce maximum cell-associated tannase activity of 9.65 U/l, which is 93.8% higher than the activity in the basal medium, after 12 h at pH 5.0, 30 degrees C. The optimum medium consists of 38 g/l lactose, 50 g/l tryptose, and 2.8 g/l gallic acid.     

Engineered Penicillium funiculosum produces potent lignocellulolytic enzymes for saccharification of various pretreated biomasses

PfMig1⁸⁸, a catabolically derepressed engineered strain of the hyper-cellulolytic fungus Penicillium funiculosum NCIM1228, was investigated for the efficacy of its secretome for biomass saccharification. An inexpensive version of media containing microcrystalline cellulose, wheat bran and soya protein was optimized for producing a high-quality secretome from the PfMig1⁸⁸ strain in both shake flasks and in a 20-L bioreactor. The activities of four classes of core cellulolytic enzymes required for saccharification in the PfMig1⁸⁸ secretome, namely, cellobiohydrolase (Avicelase activity), endoglucanase (CMCase activity), β-glucosidase (PNPGase activity) and xylanase (xylanase activity), were found to be 2.29 U/mL, 28.24 U/mL, 150 U/mL and 76 U/mL, respectively. The saccharification potential of the PfMig1⁸⁸ secretome was evaluated on rice straw and sugarcane bagasse pretreated with nitric acid and/or ammonium hydroxide. Saccharification performed using a 15 % (w/v) biomass load and a 3% (w/w) enzyme load released >100 g/L sugar in the hydrolysate, irrespective of the type of biomass and pre-treatment, with >80 % hydrolysis. Furthermore, the presence of lignin in nitric acid-pretreated biomass only marginally affected saccharification. Overall, the results demonstrated that the PfMig1⁸⁸ secretome, having relatively broad substrate specificity, is a viable and efficient substitute for T. reesei-based secretomes for diverse biomass saccharification.     

Design of experiments and artificial neural network linked genetic algorithm for modeling and optimization of L-asparaginase production by <Emphasis Type="Italic">Aspergillus terreus</Emphasis> MTCC 1782

The sequential optimization strategy for design of an experimental and artificial neural network (ANN) linked genetic algorithm (GA) were applied to evaluate and optimize media component for L-asparaginase production by Aspergillus terreus MTCC 1782 in submerged fermentation. The significant media components identified by Plackett-Burman design (PBD) were fitted into a second order polynomial model (R² = 0.910) and optimized for maximum L-asparaginase production using a five-level central composite design (CCD). A nonlinear model describing the effect of variables on L-asparaginase production was developed (R² = 0.995) and optimized by a back propagation NN linked GA. Ground nut oil cake (GNOC) flour 3.99% (w/v), sodium nitrate (NaNO₃) 1.04%, L-asparagine 1.84%, and sucrose 0.64% were found to be the optimum concentration with a maximum predicted L-asparaginase activity of 36.64 IU/mL using a back propagation NN linked GA. The experimental activity of 36.97 IU/mL obtained using the optimum concentration of media components is close to the predicted L-asparaginase activity of the ANN linked GA.     

Nutritional requirements and media development for Lactococcus lactis IL1403

Lactic acid bacteria are extensively used in food technology and for the production of various compounds, but they are fastidious in nutrient requirements. In order to elucidate the role of each component precisely, defined multicomponent media are required. This study focuses on determining nutrient auxotrophies and minimizing media components (amino acids, vitamins, metal ions, buffers and additional compounds) for the cultivation of Lactococcus lactis subsp. lactis IL1403, using microtitre plates and test tubes. It was shown that glutamine and asparagine were the most important media components for achieving higher biomass yields while the branched-chain amino acids were necessary to increase specific growth rate. The amino acid and glucose ratio was reduced to achieve minimal residual concentration of amino acids in the medium after the growth of cells, whereas the specific growth rate and biomass yield of cells were not considerably affected. As the percentage of each consumed amino acid compared to initial amount is larger than measurement error, these optimized media are important for achieving more precise data about amino acid utilization and metabolism.     

Optimization of novel and greener approach for the coproduction of uricase and alkaline protease in Bacillus licheniformis by Box–Behnken model

This study explores a novel concept of coproduction of uricase and alkaline protease by Bacillus licheniformis using single substrate in single step. Seven local bacterial strains were screened for uricase production, amongst which B. licheniformis is found to produce highest uricase along with alkaline protease. Optimization of various factors influencing maximum enzyme coproduction by B. licheniformis is performed. Maximum enzyme productivity of 0.386?U/mL uricase and 0.507?U/mL alkaline protease is obtained at 8?hr of incubation period, 1% (v/v) inoculum, and at 0.2% (w/v) uric acid when the organism is cultivated at 25°C, 180?rpm, in a media containing xylose as a carbon source, urea as a nitrogen source, and initial pH of 9.5. The statistical experimental design method of Box–Behnken was further applied to obtain optimal concentration of significant parameters such as pH (9.5), uric acid concentration (0.1%), and urea concentration (0.05%). The maximum uricase and alkaline protease production by B. licheniformis using Box–Behnken design was 0.616 and 0.582?U/mL, respectively, with 1.6- and 1.13-fold increase as compared to one factor at a time optimized media. This study will be useful to develop an economic, commercially viable, and scalable process for simultaneous production of uricase and protease enzymes.     

Enhancement of <Emphasis Type="Italic">Penicillium echinulatum</Emphasis> glycoside hydrolase enzyme complex

The enhancement of enzyme complex produced by Penicillium echinulatum grown in several culture media components (bagasse sugarcane pretreated by various methods, soybean meal, wheat bran, sucrose, and yeast extract) was studied to increment FPase, xylanase, pectinase, and β-glucosidase enzyme activities. The present results indicated that culture media composed with 10 g/L of the various bagasse pretreatment methods did not have any substantial influence with respect to the FPase, xylanase, and β-glucosidase attained maximum values of, respectively, 2.68 FPU/mL, 2.04, and 115.4 IU/mL. On the other hand, proposed culture media to enhance β-glucosidase production composed of 10 g/L steam-exploded bagasse supplemented with soybean flour 5.0 g/L, yeast extract 1.0 g/L, and sucrose 10.0 g/L attained, respectively, 3.19 FPU/mL and 3.06 IU/mL while xylanase was maintained at the same level. The proteomes obtained from the optimized culture media for enhanced FPase, xylanase, pectinase, and β-glucosidase production were analyzed using mass spectrometry and a panel of GH enzyme activities against 16 different substrates. Culture medium designed to enhance β-glucosidase activity achieved higher enzymatic activities values (13 measured activities), compared to the culture media for FPase/pectinase (9 measured activities) and xylanase (7 measured activities), when tested against the 16 substrates. Mass spectrometry analyses of secretome showed a consistent result and the greatest number of spectral counts of Cazy family enzymes was found in designed β-glucosidase culture medium, followed by FPase/pectinase and xylanase. Most of the Cazy identified protein was cellobiohydrolase (GH6 and GH7), endoglucanase (GH5), and endo-1,4-β-xylanase (GH10). Enzymatic hydrolysis of hydrothermally pretreated sugarcane bagasse performed with β-glucosidase enhanced cocktail achieved 51.4 % glucose yield with 10 % w/v insoluble solids at enzyme load of 15 FPU/g material. Collectively the results demonstrated that it was possible to rationally modulate the GH activity of the enzymatic complex secreted by P. echinulatum using adjustment of the culture medium composition. The proposed strategy may contribute to increase enzymatic hydrolysis of lignocellulosic materials.     

An optimized microplate assay system for quantitative evaluation of plant cell wall-degrading enzyme activity of fungal culture extracts

Developing enzyme cocktails for cellulosic biomass hydrolysis complementary to current cellulase systems is a critical step needed for economically viable biofuels production. Recent genomic analysis indicates that some plant pathogenic fungi are likely a largely untapped resource in which to prospect for novel hydrolytic enzymes for biomass conversion. In order to develop high throughput screening assays for enzyme bioprospecting, a standardized microplate assay was developed for rapid analysis of polysaccharide hydrolysis by fungal extracts, incorporating biomass substrates. Fungi were grown for 10 days on cellulose- or switchgrass-containing media to produce enzyme extracts for analysis. Reducing sugar released from filter paper, Avicel, corn stalk, switchgrass, carboxymethylcellulose, and arabinoxylan was quantified using a miniaturized colorimetric assay based on 3,5-dinitrosalicylic acid. Significant interactions were identified among fungal species, growth media composition, assay substrate, and temperature. Within a small sampling of plant pathogenic fungi, some extracts had crude activities comparable to or greater than T. reesei, particularly when assayed at lower temperatures and on biomass substrates. This microplate assay system should prove useful for high-throughput bioprospecting for new sources of novel enzymes for biofuel production.     

Enhanced α-amylase production by a marine protist,Ulkenia sp. using response surface methodology and genetic algorithm

Amylases are a group of enzymes with a wide variety of industrial applications. Enhancement of α-amylase production from the marine protists, thraustochytrids has been attempted for the first time by applying statistical-based experimental designs using response surface methodology (RSM) and genetic algorithm (GA) for optimization of the most influencing process variables. A full factorial central composite experimental design was used to study the cumulative interactive effect of nutritional components viz., glucose, corn starch, and yeast extract. RSM was performed on two objectives, that is, growth of Ulkenia sp. AH-2 (ATCC® PRA­296) and α-amylase activity. When GA was conducted for maximization of the enzyme activity, the optimal α-amylase activity was found to be 71.20?U/mL which was close to that obtained by RSM (71.93?U/mL), both of which were in agreement with the predicted value of 72.37 U/mL. Optimal growth at the optimized process variables was found to be 1.89A_660nm. The optimized medium increased α-amylase production by 1.2-fold.     

Production and extraction of extracellular lipase from the entomopathogenic fungus Isaria fumosoroseus (Cordycipitaceae; Hypocreales)

Lipases are important cuticle degrading enzymes involved in the infection process of entomopathogens by hydrolysing the ester bonds of lipoproteins, fats and waxes present in the insect integument. Production of extracellular lipase by Isaria fumosoroseus (Cordycipitaceae; Hypocreales) isolate IF28.2 was investigated using different combinations of basal medium components. The effect of different vegetable oils added to a basal medium at different concentrations to improve enzyme production was evaluated. Maximum lipase activity (125.33±2.96 U/mL) as well as maximum biomass production (22.36±0.99 mg/mL) was observed for olive oil when used at a concentration of 2% (v/v) of the basal medium. In the presence of surfactants, the highest lipase activity occurred when SDS and Tween 80 were added at the time of fungal inoculation. SDS proved to be the best surfactant having 110.66±3.52 U/mL lipase activity. The effects of the divalent metal ions (iron and magnesium) on lipase activity were also studied. Iron inhibited, whereas magnesium slightly increased lipase activity. The optimum pH for lipase production was 5.7 while 32°C proved to be the best temperature for lipase production.     

Synergistic effect of Aspergillus niger and Trichoderma reesei enzyme sets on the saccharification of wheat straw and sugarcane bagasse

Plant‐degrading enzymes can be produced by fungi on abundantly available low‐cost plant biomass. However, enzymes sets after growth on complex substrates need to be better understood, especially with emphasis on differences between fungal species and the influence of inhibitory compounds in plant substrates, such as monosaccharides. In this study, Aspergillus niger and Trichoderma reesei were evaluated for the production of enzyme sets after growth on two “second generation” substrates: wheat straw (WS) and sugarcane bagasse (SCB). A. niger and T. reesei produced different sets of (hemi‐)cellulolytic enzymes after growth on WS and SCB. This was reflected in an overall strong synergistic effect in releasing sugars during saccharification using A. niger and T. reesei enzyme sets. T. reesei produced less hydrolytic enzymes after growth on non‐washed SCB. The sensitivity to non‐washed plant substrates was not reduced by using CreA/Cre1 mutants of T. reesei and A. niger with a defective carbon catabolite repression. The importance of removing monosaccharides for producing enzymes was further underlined by the decrease in hydrolytic activities with increased glucose concentrations in WS media. This study showed the importance of removing monosaccharides from the enzyme production media and combining T. reesei and A. niger enzyme sets to improve plant biomass saccharification.     

Comprehensive studies on optimization of cellulase and xylanase production by a local indigenous fungus strain via solid state fermentation using oil palm frond as substrate

The high cost of cellulases remains the most significant barrier to the economical production of bio-ethanol from lignocellulosic biomass. The goal of this study was to optimize cellulases and xylanase production by a local indigenous fungus strain (Aspergillus niger DWA8) using agricultural waste (oil palm frond [OPF]) as substrate. The enzyme production profile before optimization indicated that the highest carboxymethyl cellulose (CMCase), filter paper (FPase), and xylanase activities of 1.06 U/g, 2.55 U/g, and 2.93 U/g were obtained on day 5, day 4, and day 5 of fermentation, respectively. Response surface methodology was used to study the effects of several key process parameters in order to optimize cellulase production. Of the five physical and two chemical factors tested, only moisture content of 75% (w/w) and substrate amount of 2.5 g had statistically significant effect on enzymes production. Under optimized conditions of 2.5 g of substrate, 75% (w/w) moisture content, initial medium of pH 4.5, 1 × 10⁶ spores/mL of inoculum, and incubation at ambient temperature (±30°C) without additional carbon and nitrogen, the highest CMCase, FPase, and xylanase activities obtained were 2.38 U/g, 2.47 U/g, and 5.23 U/g, respectively. Thus, the optimization process increased CMCase and xylanase production by 124.5 and 78.5%, respectively. Moreover, A. niger DWA8 produced reasonably good cellulase and xylanase titers using OPF as the substrate when compared with previous researcher finding. The enzymes produced by this process could be further use to hydrolyze biomass to generate reducing sugars, which are the feedstock for bioethanol production.     

An indirect optimization method for biochemical systems: description of method and application to the maximization of the rate of ethanol, glycerol, and carbohydrate production in Saccharomyces cerevisiae

Three metabolic models for the production of ethanol, glycerol, and carbohydrates in yeast are optimized with respect to different production rates. While originally nonlinear, all three optimization problems are reduced in such a way that methods of linear programming can be used. The optimizations lead to profiles of enzyme activities that are compatible with the physiology of the cells, which guarantees their viability and fitness, and yield higher rates of the desired final end products than the original systems. In order to increase ethanol rate production at least three times, six enzymes must be modulated. By contrast, when the production of glycerol or carbohydrates is optimized, modulation of just one enzyme (in the case of glycerol) or two enzymes (in the case of carbohydrates) is necessary to yield significant increases in product flux rate. Comparisons of our results with those obtained from other methods show great similarities and demonstrate that both are valid methods. The choice of one or the other method depends on the question of interest. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 758-772, 1997.     

Biodetoxification of toxins generated from lignocellulose pretreatment using a newly isolated fungus, Amorphotheca resinae ZN1, and the consequent ethanol fermentation

Background

Enzymes for plant cell wall deconstruction are a major cost in the production of ethanol from lignocellulosic biomass. The goal of this research was to develop optimized synthetic mixtures of enzymes for multiple pretreatment/substrate combinations using our high-throughput biomass digestion platform, GENPLAT, which combines robotic liquid handling, statistical experimental design and automated Glc and Xyl assays. Proportions of six core fungal enzymes (CBH1, CBH2, EG1, β-glucosidase, a GH10 endo-β1,4-xylanase, and β-xylosidase) were optimized at a fixed enzyme loading of 15 mg/g glucan for release of Glc and Xyl from all combinations of five biomass feedstocks (corn stover, switchgrass, Miscanthus, dried distillers' grains plus solubles [DDGS] and poplar) subjected to three alkaline pretreatments (AFEX, dilute base [0.25% NaOH] and alkaline peroxide [AP]). A 16-component mixture comprising the core set plus 10 accessory enzymes was optimized for three pretreatment/substrate combinations. Results were compared to the performance of two commercial enzymes (Accellerase 1000 and Spezyme CP) at the same protein loadings.

Results

When analyzed with GENPLAT, corn stover gave the highest yields of Glc with commercial enzymes and with the core set with all pretreatments, whereas corn stover, switchgrass and Miscanthus gave comparable Xyl yields. With commercial enzymes and with the core set, yields of Glc and Xyl were highest for grass stovers pretreated by AP compared to AFEX or dilute base. Corn stover, switchgrass and DDGS pretreated with AFEX and digested with the core set required a higher proportion of endo-β1,4-xylanase (EX3) and a lower proportion of endo-β1,4-glucanase (EG1) compared to the same materials pretreated with dilute base or AP. An optimized enzyme mixture containing 16 components (by addition of α-glucuronidase, a GH11 endoxylanase [EX2], Cel5A, Cel61A, Cip1, Cip2, β-mannanase, amyloglucosidase, α-arabinosidase, and Cel12A to the core set) was determined for AFEX-pretreated corn stover, DDGS, and AP-pretreated corn stover. The optimized mixture for AP-corn stover contained more exo-β1,4-glucanase (i.e., the sum of CBH1 + CBH2) and less endo-β1,4-glucanase (EG1 + Cel5A) than the optimal mixture for AFEX-corn stover. Amyloglucosidase and β-mannanase were the two most important enzymes for release of Glc from DDGS but were not required (i.e., 0% optimum) for corn stover subjected to AP or AFEX. As a function of enzyme loading over the range 0 to 30 mg/g glucan, Glc release from AP-corn stover reached a plateau of 60-70% Glc yield at a lower enzyme loading (5-10 mg/g glucan) than AFEX-corn stover. Accellerase 1000 was superior to Spezyme CP, the core set or the 16-component mixture for Glc yield at 12 h, but the 16-component set was as effective as the commercial enzyme mixtures at 48 h.

Conclusion

The results in this paper demonstrate that GENPLAT can be used to rapidly produce enzyme cocktails for specific pretreatment/biomass combinations. Pretreatment conditions and feedstock source both influence the Glc and Xyl yields as well as optimal enzyme proportions. It is predicted that it will be possible to improve synthetic enzyme mixtures further by the addition of additional accessory enzymes.     

Improvement of carbonyl reductase production of Geotrichum candidum for the transformation of 1-acetonaphthone to S(-)-1-(1'-napthyl) ethanol

The aim of this work was to study the influence of media components and physico-chemical parameters on the growth and carbonyl reductase production by Geotrichum candidum. Under optimized conditions, the conversion of the substrate increased to >93%, while the specific growth rate and enzyme activity were increased by 200% and 29%, respectively. The rate of conversion of the substrate was also very high in the cells grown in optimized medium. The volumetric productivity of the biotransformation process was much higher (0.27g/lh) with the cells grown in the optimized medium compared to that of grown in un-optimized medium (0.16g/lh). The cells were also highly stable in the operational condition, indicating the feasibility of their use in multiple batches of reaction.