Enzymatic Preparation of Crystalline Laminaribiose from Curdlan

The enzymatic preparation of laminaribiose was investigated in this research. When curdlan was hydrolyzed with the β-1,3-glucanase system of Streptomyces sp. K27-4, the hydrolyzate mainly consisted of glucose and laminaribiose in an approximate ratio of 1:1 by weight. Yeast strains selected in this study, effectively and selectively metabolized all of the glucose in the hydrolyzate, without any degradation of laminaribiose.

By successive treatment of curdlan with the glucanase system and glucose-metabolizable yeast, 31 g of crystalline laminaribiose was obtained from l00g of curdlan.     

Ethanol production from AFEX pretreated corn fiber by recombinant bacteria

Summary Fermentation of an enzymatic hydrolyzate of ammonia fiber explosion (AFEX) pretreated corn fiber (containing a mixture of different sugars including glucose, xylose, arabinose, and galactose) by genetically-engineered Escherichia coli strain SL40 and KO11 and Klebsiella oxytoca strain P2 was investigated under pH-controlled conditions. Both E. coli strains (SL40 and KO11) efficiently utilized most of the sugars contained in the hydrolyzate and produced a maximum of 26.6 and 27.1 g/l ethanol, respectively, equivalent to 90 and 92% of the theoretical yield. Very little difference was observed in cell growth and ethanol production between fermentations of the enzymatic hydrolyzate and mixtures of pure sugars, simulating the hydrolyzate. These results confirm the fermentability of the AFEX-treated corn fiber hydrolyzate by ethanologenic E. coli. K.oxytoca strain P2, on the other hand, showed comparatively poor growth and ethanol production (maximum 20 g/l) from both enzymatic hydrolyzate and simulated sugar mixtures under the same fermentation conditions.     

Fed-batch cultivation of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> on lignocellulosic hydrolyzate

Saccharomyces cerevisiae grows very poorly in dilute acid lignocellulosic hydrolyzate during the anaerobic fermentation for fuel ethanol production. However, yeast cells grown aerobically on the hydrolyzate have increased tolerance for the hydrolyzate. Cultivation of yeast on part of the hydrolyzate has therefore the potential of enabling increased ethanol productivity in the fermentation of the hydrolyzate. To evaluate the ability of the yeast to grow in the hydrolyzate, fed-batch cultivations were run using the ethanol concentration as input variable to control the feed-rate. The yeast then grew in an undetoxified hydrolyzate with a specific growth rate of 0.19 h⁻¹ by controlling the ethanol concentration at a low level during the cultivation. However, the biomass yield was lower for the cultivation on hydrolyzate compared to synthetic media: with an ethanol set-point of 0.25 g/l the yield was 0.46 g/g on the hydrolyzate, compared to 0.52 g/g for synthetic media. The main reason for the difference was not the ethanol production per se, but a significant production of glycerol at a high specific growth rate. The glycerol production may be attributed to an insufficient respiratory capacity.     

Applying Proteolytic Enzymes on Soybean

The soybean cold-insoluble protein was hydrolyzed with pepsin and the hydrolyzate was dialyzed. The diffusate was submitted to gel permeation chromatography of Sephadex G-10 and thin layer chromatography of silica gel G. A ninhydrin-negative bitter peptide was detected by chlorine-starch-iodine test and was purified by paper electrophoresis and by rechromatography on the silica gel. Azeotropic HCl hydrolysis of the peptide gave equal molar ratio of Ala, Glu, Gly, Ile, Leu, Phe, Ser and Val. N-Terminal residue was composed of pyrrolidone carboxylic acid, which was tentatively identified in comparison with the authentic sample by paper electrophoresis and thin layer chromatography. Hydrazinolysis of the peptide, followed by the 2,4-dinitrophenylation, produced α,α,γ-tri-DNP-glutamic acid-α,γ-dihydrazide and DNP-pyrrolidone carboxylic acid hydrazide, also supporting the N-terminal structure. Hydrolysis of the peptide by carboxypeptidase A gave Leu, Val, Phe, Ile, Ala, etc. in the order of the liberation rate. As the peptide fragments remaining in this hydrolyzate were detected pyrrolidone carboxyl-Gly·OH, pyrrolidone carboxyl-Gly-Ser·OH, pyrrolidone carboxyl-Gly-Ser-Ala·OH, etc. In conclusion, the structure of this peptide was proposed as: pyrrolidone carboxyl-Gly-Ser-Ala-Ile-Phe-Val-Leu·OH. Quantitative information about the total amount of pyrrolidone carboxyl residue contained in the peptic hydrolyzate of the soybean protein and sensory study on the bitterness of the peptide as referred by standard solution of phenylthiourea, were demonstrated.     

Production of ethanol from corn stover hemicellulose hydrolyzate using <Emphasis Type="Italic">Pichia stipitis</Emphasis>

Hemicellulose liquid hydrolyzate from dilute acid pretreated corn stover was fermented to ethanol using Pichia stipitis CBS 6054. The fermentation rate increased with aeration but the pH also increased due to consumption of acetic acid by Pichia stipitis. Hemicellulose hydrolyzate containing 34 g/L xylose, 8 g/L glucose, 8 g/L Acetic acid, 0.73 g/L furfural, and 1 g/L hydroxymethyl furfural was fermented to 15 g/L ethanol in 72 h. The yield in all the hemicellulose hydrolyzates was 0.37–0.44 g ethanol/g (glucose + xylose). Nondetoxified hemicellulose hydrolyzate from dilute acid pretreated corn stover was fermented to ethanol with high yields, and this has the potential to improve the economics of the biomass to ethanol process.     

Comparison of Pseudomonas fragi and Gluconobacter oxydans for production of xylonic acid from hemicellulose hydrolyzates

Summary Xylonic acid was produced efficiently from pure xylose by Pseudomonas fragi ATCC 4973 and Gluconobacter oxydans subsp. suboxydans ATCC 621. The yield from 10% xylose was in both cases over 95% of the theoretical. However, the sensitivities of the strains towards the major inhibitors found in hemicellulose hydrolyzates, ie. acetic acid, furfural and two lignin-derived compounds, varied. G. oxydans tolerated all these inhibitors better than P. fragi. In tests using steamed hemicellulose hydrolyzate, G. oxydans was able to utilize the substrate only at dilute xylose concentrations. After ether extraction or mixed bed resin pretreatment, the fermentability of the hydrolyzate was increased significantly.     

Preparation and characterization of novel bioactive peptides responsible for angiotensin I‐converting enzyme inhibition from wheat germ

Reported is the preparation of wheat germ (WG) hydrolyzate with potent angiotensin I‐converting enzyme (ACE) inhibitory activity, and the characterization of peptides responsible for ACE inhibition. Successful hydrolyzate with the most potent ACE inhibitory activity was obtained by 0.5 wt.%–8 h Bacillus licheniformis alkaline protease hydrolysis after 3.0 wt.%–3 h α‐amylase treatment of defatted WG (IC₅₀; 0.37 mg protein ml⁻¹). The activity of WG hydrolyzate was markedly increased by ODS and subsequent AG50W purifications (IC₅₀; 0.018 mg protein ml⁻¹). As a result of isolations by high performance liquid chromatographies, 16 peptides with the IC₅₀ value of less than 20 μm , composed of 2–7 amino acid residues were identified from the WG hydrolyzate. Judging from the high content (260 mg in 100 g of AG50W fraction) and powerful ACE inhibitory activity (IC₅₀; 0.48 μm ), Ile‐Val‐Tyr was identified as a main contributor to the ACE inhibition of the hydrolyzate. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Fed-batch Cultivation of Mucor indicus in Dilute-acid Lignocellulosic Hydrolyzate for Ethanol Production

Mucor indicus fermented dilute-acid lignocellulosic hydrolyzates to ethanol in fed-batch cultivation with complete hexose utilization and partial uptake of xylose. The fungus was tolerant to the inhibitors present in the hydrolyzates. It grew in media containing furfural (1 g/l), hydroxymethylfurfural (1 g/l), vanillin (1 g/l), or acetic acid (7 g/l), but did not germinate directly in the hydrolyzate. However, with fed-batch methodology, after initial growth of M. indicus in 500 ml enzymatic wheat hydrolyzate, lignocellulosic hydrolyzate was fermented with feeding rates 55 and 100 ml/h. The fungus consumed more than 46% of the initial xylose, while less than half of this xylose was excreted in the form of xylitol. The ethanol yield was 0.43 g/g total consumed sugar, and reached the maximum concentration of 19.6 g ethanol/l at the end of feeding phase. Filamentous growth, which is regarded as the main obstacle to large-scale cultivation of M. indicus, was avoided in the fed-batch experiments.     

Environmental parameters affecting xylitol production from sugar cane bagasse hemicellulosic hydrolyzate by Candida guilliermondii

The bioconversion of xylose to xylitol by Candida guilliermondii FTI 20037 cultivated in sugar cane bagasse hemicellulosic hydrolyzate was influenced by cell inoculum level, age of inoculum and hydrolyzate concentration. The maximum xylitol productivity (0.75 g L⁻¹ h⁻¹) occurred in tests carried out with hydrolyzate containing 54.5 g L⁻¹ of xylose, using 3.0 g L⁻¹ of a 24-h-old inoculum. Xylitol productivity and cell concentration decreased with hydrolyzate containing 74.2 g L⁻¹ of xylose. Received 02 February 1996/ Accepted in revised form 15 November 1996     

Batch and repeated batch production of l(+)-lactic acid by Enterococcus faecalis RKY1 using wood hydrolyzate and corn steep liquor

Lactic acid production was investigated for batch and repeated batch cultures of Enterococcus faecalis RKY1, using wood hydrolyzate and corn steep liquor. When wood hydrolyzate (equivalent to 50 g l⁻¹ glucose) supplemented with 15–60 g l⁻¹ corn steep liquor was used as a raw material for fermentation, up to 48.6 g l⁻¹ of lactic acid was produced with, volumetric productivities ranging between 0.8 and 1.4 g l⁻¹ h⁻¹. When a medium containing wood hydrolyzate and 15 g l⁻¹ corn steep liquor was supplemented with 1.5 g l⁻¹ yeast extract, we observed 1.9-fold and 1.6-fold increases in lactic acid productivity and cell growth, respectively. In this case, the nitrogen source cost for producing 1 kg lactic acid can be reduced to 23% of that for fermentation from wood hydrolyzate using 15 g l⁻¹ yeast extract as a single nitrogen source. In addition, lactic acid productivity could be maximized by conducting a cell-recycle repeated batch culture of E. faecalis RKY1. The maximum productivity for this process was determined to be 4.0 g l⁻¹ h⁻¹.     

Enzymatic Modification of Proteins in Foodstuffs

Peptic hydrolyzate of soy protein was submitted to the plastein reaction with α-chymotrypsin under the following condition: substrate concentration, 20%; enzyme-substrate ratio by weight, 1/100; reaction pH, 7.0; and reaction temperature, 37°C. The plastein yield resulting from the plastein reaction for 24 hr was found to depend on the degree of hydrolysis of the substrate (per cent ratio between nitrogen amount in 10% trichloroacetic acid soluble nitrogen and that in whole hydrolyzate); the optimum degree of hydrolysis for the highest plastein yield seemed to lie around 80%. A turbidity appeared in the process of the plastein reaction, whose intensity was correlative to the plastein yield. The peptic hydrolyzate of soy protein per se had bitterness and its magnitude decreased with increasing plastein yield.

As a result of the plastein reaction applied for 24 hr to the hydrolyzate whose degree of hydrolysis was 80%, the average molecular weight estimated by the change in amino nitrogen content increased by approximately three times. The molecular weight distribution pattern obtained by gel filtration supported the above result. The total amount of amino acids liberated from the plastein reaction product by its treatment with either leucine aminopeptidase or carboxypeptidase A was significantly less than that liberated from the original hydrolyzate by its similar treatment. This result also supports the formation of higher-molecular protein-like substances by the plastein reaction. Deuteration study followed by IR spectrometry showed the occurrence of peptide bond formation, i.e. decrease in ionized carboxyl group at 1575 cm^?1 and increase in deuterated amide at 1450 cm^?1, even at the earlier stages of the plastein reaction.     

Optimization of enzymatic hydrolysis of pretreated rice straw and ethanol production

Cellulase, Tween 80, and β-glucosidase loading were studied and optimized by response surface methodology to improve saccharification. Microwave alkali-pretreated rice straw used as substrate for onsite enzyme production by Aspergillus heteromorphus and Trichoderma reesei. The highest enzymatic hydrolysis (84%) was obtained from rice straw at crude enzyme loading of 10 FPU/gds of cellulase, 0.15% Tween 80, and 100 international unit/g dry solids of β-glucosidase activities. Enzymatic hydrolyzate of pretreated rice straw was used for ethanol production by Saccharomyces cerevisiae, Scheffersomyces stipitis, and by co-culture of both. The yield of ethanol was 0.50, 0.47, and 0.48 g_p/g_s by S. cerevisiae, S. stipitis, and by co-culture, respectively, using pretreated rice straw hydrolyzate. The co-culture of S. cerevisiae and S. stipitis produced 25% more ethanol than S. cerevisiae alone and 31% more ethanol than S. stipitis alone. During anaerobic fermentation 65.08, 36.45, and 50.31 μmol/ml CO₂ released by S. cerevisiae, S. stipitis, and by co-culture, respectively. The data indicated that saccharification efficiency using optimized crude enzyme cocktail was good, and enzymatic hydrolyzate could be fermented to produce ethanol.     

Study of the Protective Activity of Chitosan Hydrolyzate Against Septoria Leaf Blotch of Wheat and Brown Spot of Tobacco

Chitosan hydrolyzate containing low-molecular-weight chitosan (≤24 kDa) and its oligomers (≤1.2 kDa) has been obtained via chemical depolimerization of high-molecular-weight chitosan by nitric acid. The fractions of the obtained chitosan hydrolyzate have been characterized by high performance gel permeation chromatography and proton magnetic resonance. The test performed on detached leaves of wheat has shown that the hydrolyzate completely inhibits the development of Stagonospora nodorum, a casual agent of the septoria leaf blotch, at a concentration of 200 μg/mL. A similar test with detached tobacco leaves has shown that the hydrolyzate at a concentration of 100 μg/mL also inhibits the development of Alternaria longipes, which causes brown spot of tobacco, by 75%.     

Debittering effect of Monascus carboxypeptidase during the hydrolysis of soybean protein

The actions of pepsin and the admixture of pepsin and Monascus pilosus carboxypeptidase 1 (MpiCP-1) on the hydrolysis of soybean protein were studied. The results showed that the pepsin hydrolyzate of soybean protein was much more bitter and contained relatively smaller amounts of total free amino acids than the hydrolyzate obtained with the admixture of pepsin and MpiCP-1. In addition, hydrophilic and hydrophobic amino acids were present in almost equal proportions in the pepsin hydrolyzate, while mainly hydrophobic amino acids made up the hydrolyzate obtained with the admixture of pepsin and MpiCP-1. These results suggest that MpiCP-1 suppresses and reverses the development of the bitterness taste that results from the pepsin hydrolysis of soybean protein by releasing mainly hydrophobic amino acids from the C-termini of the bitter components.     

Constitutional Amino Acids of the Antibiotic YA–56

Acid hydrolysis of the antibiotic YA-56 X (Zorbamycin) and Y belonging to the phleomycin-bleomycin group was carried out and the following constitutional amino acids were isolated from the hydrolyzate of YA–56 X: β-Amino-β-(4-amino-6-carboxy-5-methylpyrimidine-2-yl)- propionic acid, β-aminoalanine, L-erythro-β-hydroxyhistidine and 3 unidentified amino acids. Though the former 3 amino acids were known to be constituents of phleomycins and bleomycins, the latter three were not found in phleomycins and bleomycins. YA–56 Y gave one more unidentified amino acid.

Furthermore, isolation of β-alanine and 2-acetylthiazole-4-carboxylic acid from the hydrolyzate indicated the presence of 2-(2-(2-aminoethyl)-Δ²-thiazoline-4-yl)-thiazole-4-carboxylic acid in YA–56 X and Y as in phleomycins.     

Bioconversion of agro-wastes into acetone butanol

Summary Agricultural residues such as bagasse and rice straw have been hydrolyzed by mixed culture filtrates of T. reesei and A. wentii to obtain fermentable sugars. After the hydrolyzate had been treated to remove undesired impurities, Cl. saccharoper butylacetonicum was used to produce butanol to the extent of 16 g/l.     

Production of Actinomycin D in Complex Media

The production of actinomycin D was examined using Streptomyces antibioticus growing on a complex medium based on glucose and casein hydrolyzate. Purification of the medium to remove calcium increased actinomycin production by about one-third and prevented production of a characteristic brown pigment. The sensitivity to magnesium concentrations was less than was reported by Katz et al.,¹¹⁾ while similar requirements were found for zinc and iron.     

High solid fed‐batch butanol fermentation with simultaneous product recovery: Part II—process integration

In these studies, liquid hot water (LHW) pretreated and enzymatically hydrolyzed Sweet Sorghum Bagasse (SSB) hydrolyzates were fermented in a fed‐batch reactor. As reported in the preceding paper, the culture was not able to ferment the hydrolyzate I in a batch process due to presence of high level of toxic chemicals, in particular acetic acid released from SSB during the hydrolytic process. To be able to ferment the hydrolyzate I obtained from 250 g L^?1 SSB hydrolysis, a fed‐batch reactor with in situ butanol recovery was devised. The process was started with the hydrolyzate II and when good cell growth and vigorous fermentation were observed, the hydrolyzate I was slowly fed to the reactor. In this manner the culture was able to ferment all the sugars present in both the hydrolyzates to acetone butanol ethanol (ABE). In a control batch reactor in which ABE was produced from glucose, ABE productivity and yield of 0.42 g L^?1 h^?1 and 0.36 were obtained, respectively. In the fed‐batch reactor fed with SSB hydrolyzates, these productivity and yield values were 0.44 g L^?1 h^?1 and 0.45, respectively. ABE yield in the integrated system was high due to utilization of acetic acid to convert to ABE. In summary we were able to utilize both the hydrolyzates obtained from LHW pretreated and enzymatically hydrolyzed SSB (250 g L^?1) and convert them to ABE. Complete fermentation was possible due to simultaneous recovery of ABE by vacuum. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:967–972, 2018     

Studies on the Proteolytic Enzymes of Black Aspergilli

In order to determine the specificity of Aspergillus Saitoi protease, the hydrolyzate of B-chain of insulin oxidized by this enzyme was investigated on paperchromatography according to the 2,4-dinitrofluorobenzene technique. Specificity was compared with pepsin and other proteolytic enzymes.     

Poly(L-histidyl-L-alanyl-α-L-glutamic acid). I. Synthesis

Poly(L -histidyl-L -alanyl-α-L -glutamic acid) has been prepared in order to test the acid–base catalytic ability of a carboxyl-imidazole hydrogen-bonded system. Two different blocked histidyl-alanyl-glutamic acid monomers were used in the polymerization step. The imidazole ring was blocked with either a dinitrophenyl or a t-butyloxycarbonyl group. The γ-carboxyl of glutamic acid was protected as the benzyl ester. Both the coupling reactions and the polymerization step were via the N-hydroxysuccinimide active ester method. Thiolysis removed the dinitrophenyl group, while hydrogen bromide removed the t-butyloxycarbonyl and the benzyl groups. The water-soluble unblocked polymers obtained were fractionated on Sephadex G-50 or Bio-Gel P30. Fractions within a range of average molecular weights of 2,000 to 25,000 were isolated. Enzymatic oxidation of the acid hydrolyzate of the polymers revealed that no detectable racemization had occurred.