CORN GLUTEN HYDROLYSIS BY ALCALASE: KINETICS OF HYDROLYSIS

In the present study, the reaction kinetics of corn gluten hydrolysis by Alcalase, a bacterial protease produced by Bacillus licheniformis, was investigated. The reactions were carried out for 10 min in 0.1 L of aqueous solutions containing 10, 20, 30, 40, and 50 g protein L^?1 corn gluten at various temperature and pH values. The amount of enzyme added to the reaction solution was 0.25% (v/v). Also, to determine decay and product inhibition effects for Alcalase, a series of inhibition experiments were conducted with the addition of various amounts of hydrolysate. For each experimental run, both the amount of hydrolysis (meqv L^?1) and the soluble protein amount (g L^?1) were investigated with respect to time, and the initial reaction rates were determined from the slopes of the linear models that fitted to these experimental data. The kinetic parameters, K_m and V_max were estimated as 53.77 g L^?1 and 5.94 meqv L^?1min^?1. The type of inhibition for Alcalase was determined as uncompetitive, and the inhibition constant, K_i, was estimated as 44.68% (hydrolysate/substrate mixture).     

Enhancing storage stability of emulsions by binding detergents with soy protein isolate and its hydrolysates

Sodium dodecyl sulfate (SDS) is a frequently used anionic detergent, and proteins are among the widely used minor ingredients in cosmetic products. Proteins may enhance the detergent functions and protect human skin from irritation caused by detergents. Soy protein hydrolysates (SPH) were prepared by modifying soy protein (SP) with papain. Varying concentrations of SP or SPH were mixed with various concentrations of SDS at different pH values to determine: (i) molecular characteristics, degree of hydrolysis (DH), and surface hydrophobicity (S ₀) of SP and SPH; (ii) the effect of SDS concentrations on the S ₀ of SP; (iii) the storage stabilities of oil-in-water emulsions formed by SDS, SP, SPH, SP-SDS, and SPH-SDS; and (iv) the effect of protein concentration (0.01 to 1.5%), DH (1.2 to 12.5%), and pH (3.0, 5.0, 7.0, and 9.0) on storage stabilities of emulsions formed by SP-SDS or SPH-SDS. An increase in emulsion stability (ES) with increasing protein concentration was observed. The ES values of emulsions formed by SPH-SDS complexes were significantly higher than those formed by SP-SDS at pH 7.0 and 9.0. The ES of emulsions formed by the complexes were low at pH 5.0 and increased with increasing pH. At pH 3.0 the emulsions formed by SP-SDS at 1 to 1.5% protein concentration and by SPH-SDS at 1.5% protein concentration were very stable. The results indicate that at least one-half of SDS content can be replaced by SP or SPH while maintaining emulsion stability.     

β‐Aminopeptidase‐Catalyzed Biotransformations of β2‐Dipeptides: Kinetic Resolution and Enzymatic Coupling

We have previously shown that the β‐aminopeptidases BapA from Sphingosinicella xenopeptidilytica and DmpA from Ochrobactrum anthropi can catalyze reactions with non‐natural β³‐peptides and β³‐amino acid amides. Here we report that these exceptional enzymes are also able to utilize synthetic dipeptides with N‐terminal β²‐amino acid residues as substrates under aqueous conditions. The suitability of a β²‐peptide as a substrate for BapA or DmpA was strongly dependent on the size of the C_α substituent of the N‐terminal β²‐amino acid. BapA was shown to convert a diastereomeric mixture of the β²‐peptide H‐β²hPhe‐β²hAla‐OH, but did not act on diastereomerically pure β²,β³‐dipeptides containing an N‐terminal β²‐homoalanine. In contrast, DmpA was only active with the latter dipeptides as substrates. BapA‐catalyzed transformation of the diastereomeric mixture of H‐β²hPhe‐β²hAla‐OH proceeded along two highly S‐enantioselective reaction routes, one leading to substrate hydrolysis and the other to the synthesis of coupling products. The synthetic route predominated even at neutral pH. A rise in pH of three log units shifted the synthesis‐to‐hydrolysis ratio (v_S/v_H) further towards peptide formation. Because the equilibrium of the reaction lies on the side of hydrolysis, prolonged incubation resulted in the cleavage of all peptides that carried an N‐terminal β‐amino acid of S configuration. After completion of the enzymatic reaction, only the S enantiomer of β²‐homophenylalanine was detected (ee>99 % for H‐(S)‐β²‐hPhe‐OH, E>500); this confirmed the high enantioselectivity of the reaction. Our findings suggest interesting new applications of the enzymes BapA and DmpA for the production of enantiopure β²‐amino acids and the enantioselective coupling of N‐terminal β²‐amino acids to peptides.     

Optimization of the Aqueous Enzymatic Extraction of Rapeseed Oil and Protein Hydrolysates

An aqueous enzymatic extraction method was developed to obtain free oil and protein hydrolysates from dehulled rapeseeds. The rapeseed slurry was treated by the chosen combination of pectinase, cellulase, and β-glucanase (4:1:1, v/v/v) at concentration of 2.5% (v/w) for 4 h. This was followed by sequential treatments consisting of alkaline extraction and an alkaline protease (Alcalase 2.4L) hydrolysis to both produce a protein hydrolysate product and demulsify the oil. Response surface methodology (RSM) was used to study and optimize the effects of the pH of the alkaline extraction (9.0, 10.0 and 11.0), the concentration of the Alcalase 2.4L (0.5, 1.0 and 1.5%, v/w), and the duration of the hydrolysis (60, 120, and 180 min). Increasing the concentration of Alcalase 2.4L and the duration of the hydrolysis time significantly increased the yields of free oil and protein hydrolysates and the degree of protein hydrolysis (DH), while the alkaline extraction pH had a significant effect only on the yield of the protein hydrolysates. Following an alkaline extraction at pH 10 for 30 min, we defined a practical optimum protocol consisting of a concentration of 1.25–1.5% Alcalase 2.4L and a hydrolysis time between 150 and 180 min. Under these conditions, the yields of free oil and protein hydrolysates were 73–76% and 80–83%, respectively. The hydrolysates consisted of approximately 96% of peptides with a MW less than 1500, of which about 81% had a MW less than 600 Da.     

Hydrophobicity, solubility, and emulsifying properties of soy protein peptides prepared by papain modification and ultrafiltration

Peptide size control is important for obtaining desirable functional properties so that these peptides can be better utilized. Proteolytic enzymatic modification of soy protein isolates (SPI), followed by ultrafiltration, is an effective way to fractionate these proteins into peptides with controlled molecular size. SPI was predenatured by mild alkali at pH 10 and heated at 50°C for 1 h prior to partial hydrolysis by papain at pH 7.0 and 38°C for 10, 30, and 60 min (PMSPI10, PMSPI30, and PMSPI60). The hydrolysate PMSPI60 was further fractionated by ultrafiltration with a stirred cell and disc membranes (100-, 50-, and 20-kDa molecular weight cut-off) into one retentate (R100) and three permeates (P100, P50, and P20). Molecular weight distribution, surface hydrophobicity (S ₀), protein solubility (PS), emulsifying activity index (EAI), and emulsion stability index (ESI) of the control SPI (without added papain), hydrolysates, and ultrafiltrates were investigated. Significant increases (P<0.001) in S ₀, PS, EAI, and ESI were observed in the hydrolysates. Peptides in the permeates had higher PS and EAI but lower S ₀ than the peptides in the retentate and hydrolysate. Soy protein peptides that were prepared from SPI by papain modification and ultrafiltration had lower molecular weight, higher solubility, and higher emulsifying properties. They could find use in products that require these properties, especially in the cosmetic and health food industries.     

Production of fermentable sugars from corn fiber using soaking in aqueous ammonia (SAA) pretreatment and fermentation to succinic acid using <Emphasis Type="Italic">Escherichia coli</Emphasis> AFP184

Conversion of corn fiber (CF), a by-product from the corn-to-ethanol conversion process, into fermentable sugar and succinic acid was investigated using soaking in aqueous ammonia (SAA) pretreatment followed by biological conversions, including enzymatic hydrolysis and fermentation using genetically engineered E. coli (AFP184). The SAA pretreatment (using a 15% w/w NH₄OH solution at a solid-to-liquid ratio of 1: 10 at 60 °C for 24 h) removed 20-38% of lignin and significantly improved the digestibility of the treated solid (85-99% of glucan digestibility). Following the enzymatic hydrolysis, the sugar-rich hydrolysate was subjected to dilute sulfuric acid treatment (1 wt% sulfuric acid and 120 °C for 1 h), which hydrolyzed the oligosaccharides in the hydrolysate into fermentable monomeric sugars. The mixed sugar hydrolysates containing hexose and pentose obtained from the two-step hydrolysis and SAA pretreatment were fermented to succinic acid using a genetically engineered microorganism, Escherichia coli AFP184, for evaluating the fermentability. Engineered E. coli AFP184 effectively converted soluble sugars in the hydrolysate to succinic acid (20.7 g/L), and the production rate and yield were further enhanced with additional nutrients; the highest concentration of succinic acid was 26.3 g/L for 48 h of fermentation.     

Ethanol production from wood via concentrated acid hydrolysis,chromatographic separation,and fermentation

BACKGROUND: Production of bioethanol from wood using concentrated acid hydrolysis has received less attention than the dilute acid hydrolysis route. The feasibility of producing lignocellulosic bioethanol from spruce and birch via concentrated acid hydrolysis was studied experimentally. Hydrolysis with sulfuric acid, chromatographic purification of the hydrolysate, and fermentation of the monosaccharides were investigated. RESULTS: Monosaccharide yields of 70% were obtained in the hydrolysis of spruce and birch. Only low amounts of by‐products were formed. With chromatographic purification of the hydrolysate, over 90% of the hydrolysis acid was recovered for recycling, and furfural and HMF were removed completely. Most of the acetic acid was recovered in a separate fraction. The monosaccharide yield in a single pass separation was approximately 70%. In the fermentation, S. cerevisiae produced higher amounts of ethanol and more efficiently than P. stipitis. Chromatographically purified hydrolysates gave higher ethanol productivities and yields than Ca(OH)₂ neutralized hydrolysates. CONCLUSIONS: Chromatographic purification of concentrated acid lignocellulosic hydrolysates has advantages when compared with neutralization with Ca(OH)₂. With chromatography, most of the inhibitory compounds can be removed from the hydrolysates. In addition, due to the recycling of the hydrolysis acid, the economy of the bioethanol manufacturing process is increased considerably. Copyright © 2011 Society of Chemical Industry     

A Continuous‐Flow Cascade Reactor System for Subtilisin A‐ Catalyzed Dynamic Kinetic Resolution of N‐tert‐Butyloxycarbonylphenylalanine Ethyl Thioester with Benzylamine

Alcalase® (Subtilisin A) was immobilized by simple hydrophobic adsorption onto various surface‐grafted macroporous silica gels resulting in easy‐to‐prepare and stable biocatalysts enabling the efficient kinetic resolution (KR) and dynamic kinetic resolution (DKR) of racemic N‐Boc‐phenylalanine ethyl thioester with benzylamine. The immobilized Alcalase biocatalysts, which retained their activity and selectivity when stored at 4 °C for more than a year, were tested in enzymatic aminolysis in batch and continuous‐flow KRs resulting in (S)‐N‐Boc‐phenylalanine benzylamide in high enantiomeric purity. In KR of the racemic thioester by Alcalase‐catalyzed aminolysis in a continuous‐flow reactor, the productivity (specific reaction rate, r_flow) and enantiomeric ratio (E) were studied in the 0–100 °C range. The effect of the temperature on base‐catalyzed racemization of the non‐transformed (R)‐thioester in a continuous‐flow reactor was also investigated in the 0–150 °C range. The continuous‐mode DKR of the racemic thioester in a serial cascade system of six biocatalyst‐filled columns at 50 °C for KR and five grafted silica gel‐filled columns at 150 °C for racemization resulted in the formation of the (S)‐benzylamide in 79% conversion, 8.17 g L ⁻¹ h⁻¹ volumetric productivity and 98% ee. This is the first example of a dynamic kinetic resolution of an amino acid derivative in continuous‐flow mode using an alternating cascade of packed‐bed enzyme reactors and racemization reactors kept at different temperatures.

     

Optimization of Enzymatic Process Condition for Protein Enrichment,Sugar Recovery and Digestibility Improvement of Soy Flour

Soy protein is a valuable nutritional supplement for food and animal feed. While protein constitutes ~50 % of defatted soy flour (SF), it coexists with complex carbohydrates (30–35 %) which may have anti‐nutritional effects. An enzymatic process can remove the carbohydrate and produce protein‐enriched soy products. The hydrolysate with monomerized carbohydrates is valuable fermentation feedstock. In this study, Aspergillus niger and Trichoderma reesei enzymes were compared for use in the process. Effects of pH (3.2–6.4), temperature (40–60 °C), enzyme‐to‐SF ratio (0–2 ml/g) and SF loading (150–350 g/l) were evaluated for the enzymatic conversion of SF carbohydrate to reducing sugar (Y_RS) and total soluble carbohydrate (Y_TC) in the hydrolysate. Effects of these single factors and interactions between factors were investigated. Optimal pH and temperature were similar for both enzymes: pH 4.8 and 50–51 °C for Y_TC, and pH 5.1–5.2 and 48–51 °C for Y_RS. The two enzymes also gave similar protein contents in resultant soy protein concentrates, i.e., 74–75 % with 2 ml/g enzyme broth and 150 g/l SF, which were higher than the 64–68 % protein in commercial concentrates. A. niger enzyme was significantly more effective in carbohydrate conversion, achieving Y_RS = 75 % and Y_TC = 78 % with 2 ml/g enzyme and 150 g/l SF, higher than the Y_RS (30 %) and Y_TC (64 %) obtained with T. reesei enzyme. Monomerization was essentially complete in hydrolysate produced with A. niger enzyme.     

Production and characterization of an extensive rapeseed protein hydrolysate

Rapeseed protein isolate has been used as starting material for the generation of an extensive protein hydrolysate. Protein hydrolysis was produced by using sequentially an endopeptidase (Alcalase) and an exopeptidase (Flavourzyme). The final hydrolysate has a 60% degree of hydrolysis and was completely soluble between pH values 2.5 and 7. Molecular weight profile of the protein hydrolysate was characterized by gel filtration chromatography. A reduction in protein size was observed during the hydrolysis process with accumulation of small peptides and free amino acids after Flavourzyme digestion. Amino acid composition of fractions with different molecular weights of the final hydrolysate was analyzed. Some of these fractions, enriched or poor in certain amino acids, could be used for supplementation or treatment of determined clinical syndromes.