Conventional Enzymatic Hydrolysis Research Articles

Ultrasound-assisted dilute acid hydrolysis of tea processing waste for production of fermentable sugar.

Lignocellulosic materials that are the most abundant plant biomass in the world have the potential to become sustainable sources of the produced value added products. Tea processing waste (TPW) is a good lignocellulosic source to produce the value added products from fermentable sugars (FSs). Therefore, the present study is undertaken to produce FSs by using ultrasound-assisted dilute acid (UADA) and dilute acid (DA) hydrolysis of TPW followed by enzymatic hydrolysis. UADA hydrolysis of TPW was optimized by response surface methodology (RSM) at maximum power (900 W) for 2 h. The optimum conditions were determined as 50°C, 1:6 (w/v) solid:liquid ratio, and 1% (w/v) DA concentration, which yielded 20.34 g/L FS concentration. Furthermore, its DA hydrolysis was also optimized by using RSM for comparison and the optimized conditions were found as 120°C, 1:8 solid:liquid ratio, and 1% acid concentration, which produced 25.3 g/L FS yield. Even though the produced sugars with UADA hydrolysis are slightly less, but it can provide significant cost saving due to the lower temperature requirement and less liquid consumption. Besides, enzymatic hydrolysis applied after pretreatments of TPW were very more economic than the conventional enzymatic hydrolysis in the literature due to shorter time requiring. In conclusion, ultrasound-assisted is a promising technology that can be successfully applied for hydrolysis of biomass and can be an alternative to the other hydrolysis procedures and also TPW can be considered as suitable carbon source for the production of value-added products like biofuels, organic acids, and polysaccharides. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:393-403, 2016.

Intensification of enzymatic hydrolysis of waste newspaper using ultrasound for fermentable sugar production

An effective conversion of lignocellulose into fermentable sugars is a key step in producing bioethanol in an eco-friendly and cost effective manner. In this study, the effect of ultrasound on enzymatic hydrolysis of newspaper, a potential feedstock for bioethanol production due to its high cellulosic content, was investigated. The effect of substrate loading, enzyme loading, temperature, ultrasonic power and duty cycle on the hydrolysis has been studied. Optimum conditions for conventional enzymatic hydrolysis were substrate loading of 5% (w/v), enzyme loading of 0.14% (w/v), temperature of 323K, and under these conditions and 72h of hydrolysis, reducing sugar yield of 11.569g/L was obtained. In case of ultrasound-assisted enzymatic hydrolysis approach, optimum conditions obtained were substrate loading of 3% (w/v), enzyme loading of 0.8% (w/v), sonication power of 60W, duty cycle of 70%, hydrolysis time of 6.5h and the reducing sugar yield obtained under these conditions was 27.6g/L. Approximately 2.4 times increase in the release of reducing sugar concentration was obtained by the ultrasound-assisted enzymatic hydrolysis approach. Results indicate that there is a synergistic effect obtained from the combination of ultrasound and enzymes which lowers the diffusion-limiting barrier to enzyme/substrate binding and results in an increase in reaction rate. The experimental data were also fitted in a simple three parameter kinetic model.

Optimization of Enzymatic Hydrolysis of Sacha Inchi Oil using Conventional and Supercritical Carbon Dioxide Processes

AbstractSacha inchi (Plukenetia volubilis) oil has high polyunsaturated fatty acids content. The hydrolysis of this oil is an efficient way to obtain desirable free fatty acids (FFA). The optimization of parameters was carried out according to the maximum production of FFA using two enzymatic hydrolysis processes. The effect of enzyme concentration (5–40 % based on weight of oil), temperature (40–60 °C), and oil:water molar ratio (1:5–1:70) were studied for the conventional enzymatic hydrolysis process, while pressure (10–30 MPa) and oil:water molar ratio (1:5–1:30) were studied for the enzymatic hydrolysis in supercritical carbon dioxide (SC‐CO2) media. The hydrolysis in SC‐CO2 media resulted in higher production of FFA (77.98 % w/w) at 30 MPa and an oil:water molar ratio equal to 1:5 compared to the conventional process (68.40 ± 0.98 % w/w) at 60 °C, oil:water molar ratio equal to 1:70, and 26.17 % w/w, enzyme/oil. The only significant parameter on the production of FFA for conventional enzymatic hydrolysis was enzyme concentration, while for the hydrolysis in SC‐CO2 media both pressure and the molar ratio of oil:water were significant. Lipid class analyses showed that with both methods, FFA, monoglycerides, and diglycerides content in the final product increased compared to pure oil, while triglycerides content decreased. Fatty acid composition analysis showed that the content of fatty acids in the FFA form were similar to their triglyceride form.

Matrix solid-phase dispersion on column clean-up/pre-concentration as a novel approach for fast isolation of abuse drugs from human hair

A simple and fast sample pre-treatment method based on matrix solid-phase dispersion (MSPD) for isolating cocaine, benzoylecgonine (BZE), codeine, morphine and 6-monoacethylmorphine (6-MAM) from human hair has been developed. The MSPD approach consisted of using alumina (1.80 g) as a dispersing agent and 0.6 M hydrochloric acid (4 mL) as an extracting solvent. For a fixed hair sample mass of 0.050 g, the alumina mass to sample mass ratio obtained was 36. A previously conditioned Oasis HLB cartridge (2 mL methanol, plus 2 mL ultrapure water, plus 1 mL of 0.2 M/0.2 M sodium hydroxide/boric acid buffer solution at pH 9.2) was attached to the end of the MSPD syringe for on column clean-up of the hydrochloric acid extract and for transferring the target compounds to a suitable solvent for gas chromatography (GC) analysis. Therefore, the adsorbed analytes were directly eluted from the Oasis HLB cartridges with 2 mL of 2% acetic acid in methanol before concentration by N 2 stream evaporation and dry extract derivatization with N-methyl-tert-butylsilyltrifluoroacetamide (BSTFA) and chlorotrimethylsilane (TMCS). The optimization/evaluation of all the factors affecting the MSPD and on column clean-up procedures has led to a fast sample treatment, and analytes extraction and pre-concentration can be finished in approximately 30 min. The developed method has been applied to eight hair samples from poli-drug abusers and measured analyte concentrations have been found to be statistically similar (95% confidence interval) to those obtained after a conventional enzymatic hydrolysis method (Pronase E).

Alternative Solid Sample Pretreatment Methods in Green Analytical Atomic Spectrometry

ABSTRACT A review of recent literature utilizing alternative sample pretreatment methods for solid environmental and biological materials analysis to assess total metals content by atomic spectrometry is provided. All the selected treatments coincide with all or some of the fundamentals of Green Chemistry policy such as prevention of waste generation; safer and less toxic solvents and reagents; and designs for energy efficiency. The review covers the classic slurry sampling technique, mainly, its current trends, as well as developments based on the use of diluted acids, chelating agents solutions, or even water for leaching (extraction) methods assisted by ultrasounds, microwave energy, or pressurization. In addition, the use of conventional or assisted enzymatic hydrolysis procedures is also discussed.

Acceleration of enzymatic hydrolysis of protein-bound selenium by focused microwave energy

This paper demonstrates for the first time that enzymatic hydrolysis of protein-bound selenium can be significantly accelerated by microwave energy. Extraction of selenomethionine from selenised yeast, used as the model sample, was performed using two-step enzymatic hydrolysis with protease/lipase/driselase in a focused microwave reactor. HPLC-ICP-MS with species-specific isotope dilution analysis (IDA) was used for species quantitation. To investigate enzymatic hydrolysis assisted by microwave energy the effect of parameters such as microwave power and extraction time on the extraction efficiency of SeMet from the Se-yeast CRM SELM-1 was investigated. The main chemical variables affecting enzymatic extraction of SeMet, such as pH, enzyme type, sample to enzyme mass ratio and sample to solvent mass ratio were set at the conditions used for conventional enzymatic hydrolysis (in a hybridisation oven). Using a power of 60 W and two consecutive extraction cycles, each of 15 min (total extraction time of 30 min) at 37 °C, the recovery of SeMet obtained by the proposed methodology and calculated based on the certified value of 3389 ± 173 mg kg−1SeMet was 99.6 ± 1.9% (n = 3). The major advantage of the newly proposed method is the dramatic reduction of extraction time in comparison with that of conventional enzymatic hydrolysis, which requires a two-step extraction, each cycle of approximately 20 h. This was achieved without compromising extraction efficiency. Application of the proposed method to the accurate quantification of SeMet in pharmaceutical yeast tablets with a total Se concentration of approximately 300 mg kg−1 was also addressed. The recovery of SeMet, which was calculated based on the mean of results [561.5 mg kg−1 (n = 11, with a standard deviation of 44.3 mg kg−1)] obtained for the yeast tablets in the international intercomparison CCQM-P86,1 was found to be 100.1 ± 3.3%.

Efficient Cleavage of Conjugates of Drugs or Poisons by Immobilized β-Glucuronidase and Arylsulfatase in Columns

Cleavage of conjugates is an important step in toxicological analysis, especially of urine samples. The aim of this study was to combine the advantages and to reduce the disadvantages of acid hydrolysis and conventional enzymatic hydrolysis procedures. beta-Glucuronidase (GRD; EC 3.2.1.31) and arylsulfatase (ARS; EC 3.1.6.1) were purified and coimmobilized on an agarose gel matrix and packed into columns. In columns packed with GRD and ARS, the test conjugates 4-nitrophenyl glucuronide and 4-nitrophenyl sulfate added into urine could be completely cleaved within 25 min. Even the relatively stable morphine conjugates could be completely hydrolyzed within 60 min in authentic urine samples. Therefore, an incubation time of 1 h is recommended. Enzyme inhibition by matrix or by rather high concentrations of acetaminophen conjugates was tested and found to be up to 50%. However, a large excess of GRD and ARS was used. The immobilizate columns could be reused for at least 70 incubations and had a storage stability of at least 12 weeks. Carryover of analytes in reused columns could be avoided by rinsing with 200 mL/L methanol in acetate buffer. Thus, five drugs known to be contaminants added in very high concentrations into urine could be completely removed from the columns. A study on the applicability in systematic toxicological analysis showed that 120 different drugs and/or their metabolites could be detected in 35 different authentic urine samples. Use of immobilized and column-packed GRD and ARS is an efficient alternative for the cleavage of urinary conjugates in clinical toxicology.