D-Amino acid dipeptide production utilizing D-alanine-D-alanine ligases with novel substrate specificity

D-Alanine-D-alanine ligase (Ddl) is an important enzyme in the synthesis of bacterial peptidoglycan. The genes encoding Ddls from Escherichia coli K12 (EcDdlB), Oceanobacillus iheyensis JCM 11309 (OiDdl), Synechocystis sp. PCC 6803 (SsDdl) and Thermotoga maritima ATCC 43589 (TmDdl), the genomic DNA sequences of which have been determined, were cloned and the substrate specificities of these recombinant Ddls were investigated. Although OiDdl had a high substrate specificity for D-alanine; EcDdlB, SsDdl and TmDdl showed broad substrate specificities for D-serine, D-threonine, D-cysteine and glycine, in addition to D-alanine. Four D-amino acid dipeptides were produced using EcDdlB, and D-amino acid homo-dipeptides were successfully produced at high yields except for D-threonyl-D-threonine.     

ATP再生系统及其应用

构建ＡＴＰ再生系统是提高生物合成酶反应经济性的一个有效措施。本文讨论了ＡＴＰ再生系统的两种形式自耦合反应系统与种间耦合反应系统在生产ＧＭＰ、ＡＴＰ、ＩＭＰ、ＧＳＨ和ＣＤＰ胆碱中的应用，提出了构建ＡＴＰ再生系统的必要条件并分析了该系统目前存在的问题，以期为国内同行开展类似研究提供参考。     

A novel ATP regeneration system using polyphosphate-AMP phosphotransferase and polyphosphate kinase

Polyphosphate-AMP phosphotransferase (PAP) and polyphosphate kinase (PPK) were used for designing a novel ATP regeneration system, named the PAP-PPK ATP regeneration system. PAP is an enzyme that catalyzes the phospho-conversion of AMP to ADP, and PPK catalyzes ATP formation from ADP. Both enzymes use inorganic polyphosphate [poly(P)] as a phosphate donor. In the PAP-PPK ATP regeneration system, ATP was continuously synthesized from AMP by the coupling reaction of PAP and PPK using poly(P). Poly(P) is a cheap material compared to acetyl phosphate, phosphoenol pyruvate and creatine phosphate, which are phosphate donors used for conventional ATP regeneration systems. To achieve efficient synthesis of ATP from AMP, an excessive amount of poly(P) should be added to the reaction solution because both PAP and PPK consume poly(P) as a phosphate donor. Using this ATP generation reaction, we constructed the PAP-PPK ATP regeneration system with acetyl-CoA synthase and succeeded in synthesizing acetyl-CoA from CoA, acetate and AMP. Since too much poly(P) may chelate MG2+ and inhibit enzyme activity, the Mg2+ concentration was optimized to 24 mM in the presence of 30 mM poly(P) in the reaction. In this reaction, ATP was regenerated 39.8 times from AMP, and 99.5% of CoA was converted to acetyl-CoA. In addition, since the PAP-PPK ATP regeneration system can regenerate GTP from GMP, it could also be used as a GTP regeneration system.     

Heat inactivation of exogenous proteinases from Pseudomonas fluorescens. I. Possibility of inactivation in milk

The inactivation reaction of the proteinase of a P. fluorescens strain of biotype I in milk was investigated at 130-150 degrees C, also in milk and in buffer with and without added CaCl2 at temperatures below 100 degrees C. The decline in activity corresponded to first order kinetics in the UHT region; Ea = 115 kJ/mol. D values were 290 (130 degrees C), 124 (140 degrees C) and 54 s (150 degrees C); therefore, the usual temperature time combinations of UHT treatment are not sufficient to achieve the required rates of inactivation. At temperatures below 80 degrees C, inactivation corresponded increasingly to second order kinetics with considerably higher reaction rates; at 55 degrees C, an inactivation reaction corresponding to that induced by UHT treatment could be achieved at a thermal stress lower by a factor of 500. This "low temperature inactivation" was observed in a further 20 strains representing the spectrum of P. fluorescens. The average rates of inactivation following heat treatment in milk for 20 min are 47% at 55 degrees C and 44% at 60 degrees C. This can be regarded as the most effective temperature range for the inactivation of the proteinases in milk. Clear connections can be seen between the biotype groups and the optimum temperature for inactivation: biotype group I ca. 55 degrees C, group II (with a few exceptions) less than or equal to 50 degrees C and group III greater than or equal to 60 degrees C. The inactivation reaction is systematically influenced by the proteins and Ca++ ions present in milk.     

Effect of Storage Temperature on Rigor-Mortis and ATP Degradation in Plaice Paralichthys olivaceus Muscle

Live specimens of the plaice Paralichthys olivaceus were spiked at the brain, stored at various temperatures ranging from 0° to 20°C and examined for changes in rigor tension and ATP degradation in the muscle. The ATP degradation rate was clearly slower at 5–15°C than at WC, resulting in retardation of rigor- mortis onset at the former temperatures. Lactic acid accumulation in the muscle correlated well with the decrease of ATP. The muscle showed an ATP concentration of 3 μmol/g and “rigor index” (full rigor = 100%) at 30% when lactic acid increased up to 30 μmol/g at most storage temperatures. The muscle showed full rigor when ATP completely disappeared and lactic acid attained the maximum plateau (40–50 μmol/g).     

A simplified approach for the kinetic characterization of acrylamide formation in fructose-asparagine model system.

The potential of acrylamide formation and degradation was studied in fructose-asparagine reaction system at different temperatures (120-200 degrees C). Kinetic data for concurrent formation and degradation of acrylamide was analysed based on a simplified form of chemical reaction in series in which acrylamide occurred as an intermediate. Experimental results revealed that the reaction proceeds zero order and first order with respect to asparagine and fructose, respectively. The thermal degradation of acrylamide was determined to be first order in fructose-glycine reaction system. The concurrent formation and degradation of acrylamide followed a typical kinetic pattern at the temperatures studied. Thermal degradation was observed within 60 min at T>150 degrees C, while only the accumulation was noted at T<150 degrees C. The mathematical model fitted to experimental data very well within temperature range of 120-200 degrees C. The temperature dependence of both acrylamide formation and degradation were found to obey Arrhenius law, and the activation energies were 52.1 kJ/mol and 72.9 kJ/mol, respectively.     

Ethyl esterification of docosahexaenoic acid in an organic solvent-free system with immobilized Candida antarctica lipase

Ethyl docosahexaenoate (EtDHA) is regarded as a potentially useful pharmaceutical substance on account of its beneficial physiological activities. We attempted the ethyl esterification of docosahexaenoic acid (DHA) in an organic solvent-free system using Candida antarctica lipase, which acts strongly on DHA and ethanol. Esterification of 88% was attained by shaking a mixture of DHA/ethanol (1:1, mol/mol) and 2 wt% immobilized C. antarctica lipase at 30 degrees C for 24 h. However, even in the presence of an excess amount of ethanol, the extent of esterification could not be raised above 90%. To attain a higher level of esterification, a two-step reaction was found to be effective. The first step was performed in a mixture of DHA/ethanol (1:1, mol/mol), and the reaction mixture was then dehydrated. In the second step, the resulting mixture was shaken at 30 degrees C for 24 h with 5 molar equivalents of ethanol against the remaining DHA using 2 wt% immobilized lipase. By means of this two-step procedure, 96% esterification was attained. Repetition of the first and second reactions showed that the immobilized lipase was reusable for at least 50 cycles. In addition, DHA remaining in the second-step reaction mixture was removed by a conventional alkali refining process, giving purified EtDHA with a high yield.     

Open L-lactic acid fermentation of food refuse using thermophilic Bacillus coagulans and fluorescence in situ hybridization analysis of microflora

In the production of commercially useful poly-L-lactic acid plastic from biomass wastes, a feasible fermentation process to produce optically active L-lactic acid would be required. Here, model kitchen refuse (MKR) was inoculated with Bacillus coagulans NBRC12583 under nonsterilized openculture conditions. At temperatures below 45 degrees C, a racemic mixture of D- and L-lactic acids was accumulated, whereas only L-lactic acid was selectively accumulated by incubation at 50-65 degrees C. At 45 degrees C, the results of fermentation could not be consistently reproduced. To analyze microflora in this type of mixed culture system, whole-cell fluorescence in situ hybridization (FISH) using 16S rRNA-targeted oligonucleotide probes for B. coagulans, Bcoa191, and LAC722(L), a group-specific probe for a wide range of mesophilic lactic acid bacteria was applied. The dominancy of mesophilic lactic acid bacteria at lower temperatures, and that of B. coagulans at higher temperatures were confirmed. By using a saccharified liquid of collected kitchen refuse, 86 g/l of L-lactic acid was accumulated under nonsterile conditions by a 5-d incubation at 55 degrees C, pH 6.5, with 53% carbon yield and 97% optical purity. To conclude, high temperature open lactic acid fermentation is a simple and promising method for producing high-grade L-lactic acid from biomass waste, and FISH analysis of such mixed-culture systems is helpful for monitoring the microflora in these cultures.     

Inhibition of foodborne bacteria by the lactoperoxidase system in a beef cube system

Biopreservatives are being developed to inhibit the growth of foodborne pathogens and thus improve food safety. The lactoperoxidase system (LPS) is a naturally occurring system that has potential for use as an antimicrobial agent in foods. Growth of single strains of the pathogens Staphylococcus aureus, Listeria monocytogenes, Escherichia coli O157:H7, Salmonella enterica subsp. enterica serovar Typhimurium, Yersinia enterocolitica, Pseudomonas aeruginosa and beef microflora were assessed on LPS-treated meat surfaces in an experimental system. Beef cubes inoculated with approximately 10(4) cfu cm(-2) of bacteria were treated with the LPS and incubated at 37 degrees C for 24 h, 12 degrees C for 7 days or in a chilling regime: 12 to -1 degrees C over 1 week and held at -1 degrees C for 4 weeks. Treatment with LPS was more effective at storage temperatures non-permissive for rapid bacterial growth with strong inhibition of growth achieved on LPS-treated cubes at 12 degrees C and reduction in pathogen viable counts at chilling temperatures. At chilling temperatures, the LPS inhibited the growth of native pseudomonads but did not prevent the development of native lactic acid bacteria.     

Formation kinetics of hydroxymethylfurfural, lactulose and furosine in milk heated under isothermal and non-isothermal conditions

A detailed kinetic study of hydroxymethylfurfural, lactulose and furosine formation was performed upon heating milk at temperatures between 90 degrees C and 140 degrees C. In case of prolonged heating, formation kinetics could be described by a fractional conversion model. Considering only the first phase of the model, kinetics could be simplified to a pseudo-zero order model. A first assessment of kinetic parameters was made by isothermal experiments. Data were analysed using both a 2-step linear and a 1-step non-linear regression method. Only for furosine, did the global 1-step regression approach seem to give better results than the individual 2-step regression approach. Next, the estimated parameters k(ref) and Ea were re-evaluated under non-isothermal conditions by subjecting milk to a time variable temperature profile. Given the complexity of Maillard reaction, it seemed better to estimate kinetic parameters under non-isothermal conditions when using a simplified model. Formation of hydroxymethylfurfural, lactulose and furosine was characterized by an Ea value of 90.2 kJ/mol (k(110 degrees C) = 1.2 micromol/l, min), 99.1 kJ/mol (k(110 degrees C) = 51.5 mg/l, min) and 88.7 kJ/mol (k(110 degrees C) = 16.3 mg/100 g protein, min) respectively. Additionally, 90% joint confidence regions were constructed in order to obtain an accurate representation of the statistical confidence associated with the simultaneously estimated parameters.     

Fermentation by Lactobacillus fermentum Ogi E1 of different combinations of carbohydrates occurring naturally in cereals: consequences on growth energetics and alpha-amylase production

Glucose, fructose, sucrose and starch are naturally present in cereals. Fermentation of different combinations of these carbohydrates by Lactobacillus fermentum Ogi E1, a sourdough heterofermentative lactobacillus, was investigated to determine effects on fermentation kinetics, growth energetics and alpha-amylase production. Irrespective of the substrate combination, the strain was able to simultaneously produce alpha-amylase and consume starch, glucose, fructose and sucrose. In mixtures of starch with either sucrose or fructose or with both fructose and glucose, yields of alpha-amylase from biomass (Y(amy/x)) were similar to those observed for starch. However, for starch and glucose or starch, glucose, fructose and sucrose mixtures, both Y(amy/x) and the specific rate of alpha-amylase production decreased markedly. In fructose- or sucrose-containing mixtures, mannitol was formed stoichiometrically indicating that fructose served as electron acceptor, and acetate was produced at constant yield from biomass (Y(ac/x)) (1 g acetate g biomass(-1)). Acetate production was expected to confer to the strain a competitive advantage during natural fermentation by improving biomass formation and growth through an increase in the ATP gain. Y(ATP) varied depending on the carbohydrate mixture, indicating different effects of substrate mixtures on the efficiency in ATP coupling to biomass formation. Compared to starch fermentation, the highest value of Y(ATP) (29 g biomass mol ATP(-1)) was estimated for the starch/fructose mixture but no increase in mu(max) was observed. The lowest value (16 g biomass mol ATP(-1)) was obtained for the starch, glucose and fructose mixture, whereas for the mixture of all carbohydrates, Y(ATP) was similar to that obtained with starch alone (20 g biomass mol ATP(-1)) and it was intermediary for the starch and sucrose mixture (17 g biomass mol ATP(-1)). It is concluded that competitiveness of the strain cannot be based on expected energy gain in mixed substrate fermentation involving fructose and sucrose with glucose and starch, but rather on its ability to simultaneously use carbohydrates while producing alpha-amylase and to produce acetic acid. Acetic acid production could enhance the strain capacity to inhibit nonacid-tolerant, competitive microflora at the earlier stage of natural fermentation.     

Characterization of FtsZ homolog from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1

The gene of bacterial type ftsZ homolog in hyperthermophilic archaeon, Pyrococcus kodakaraensis KOD1 (Pk-ftsZ), was identified. The gene product of the Pk-ftsZ gene is composed of 380 amino acids with a molecular mass of 41,354 Da. In the deduced amino acid sequence of the Pk-ftsZ gene, a glycine-rich sequence (Gly-Gly-Gly-Thr-Gly-Ala-Gly) implicated in GTP binding was well conserved. The Pk-ftsZ gene was overexpressed using Escherichia coli as a host and the recombinant protein was purified. The purified Pk-FtsZ protein exhibited GTPase activity with optimum temperatures higher than 80 degrees C. However, the protein showed little GTPase activity at 40 degrees C, indicating that a high reaction temperature is required for the GTPase activity in accordance with the thermophilic nature of P. kodakaraensis KOD1. The GTP-binding ability of Pk-FtsZ protein could also be detected by UV-induced cross-linking of a protein to [alpha-32P] GTP. The Pk-ftsZ gene was expressed in E. coli cells with a temperature-sensitive ftsZ mutation, E. coli ftsZ84 (ts), but its mutant phenotype of elongated cell form at a nonpermissive temperature (42 degrees C) could not be compensated, possibly because of the thermophilic nature of the Pk-FtsZ. Pk-FtsZ could form protofilaments in a GTP-dependent manner at 90 degrees C. Results of phylogenetic analysis suggest that there might be additional factors required for formation of the Z ring in P. kodakaraensis KOD1.     

Factors affecting the oxidative activity of laccase towards biphenyl derivatives in homogeneous aqueous-organic systems

Catalytic oxidation of biphenyl derivatives was investigated using laccase in a homogeneous aqueous-organic system. A thermostable laccase from Trametes sp. showed the highest catalytic activity for the oxidation of 4-hydroxybiphenyl (4-HB) at a reaction temperature of 60 degrees C when dimethylsulfoxide (DMSO) was employed as a co-solvent. Furthermore, the catalytic performance was successfully enhanced by the incorporation of a laccase mediator system (LMS) into the aqueous-DMSO media. The catalytic performance strongly depended on the type of mediator, and the highest activity was observed with 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as mediator, suggesting the importance of the selection of a suitable mediator. It was verified that this mediator system is applicable to the oxidation of several biphenyl derivatives with hydroxyl groups.     

Properties of Rhodotorula gracilis D-amino acid oxidase immobilized on magnetic beads through his-tag

D-amino acid oxidase catalyzes one of the key steps in the production of semisynthetic cephalosporins. We expressed and purified recombinant Rhodotorula gracilis D-amino acid oxidase with C-terminal his-tags. This engineered enzyme was immobilized onto Ni(2+)-chelated nitrilotriacetic acid magnetic beads through the interaction between his-tag and Ni(2+). The kinetic constants, storage properties, and the reusability of the immobilized d-amino acid oxidase were determined. The effects of temperature, pH, and hydrogen peroxide on the activity of immobilized d-amino acid oxidase were also studied. The highest activity recovery was 75%. Thermal stability was improved after immobilization; the relative activity of the immobilized enzyme was 56% whereas the free enzyme was completely inactivated after incubation at 50 degrees C for 1 h. In the presence of 10 mM hydrogen peroxide, the immobilized enzyme did not show a rapid loss of activity during the first 2 h of incubation, which was observed in the case of the free enzyme; the residual activity of the immobilized enzyme after 9 h was 72% compared with 22% of the free form. The long-term storage stability was improved; the residual activity of the immobilized enzyme was 74% compared with 20% of the free enzyme when stored at room temperature for 10 d. The immobilized form retained 37% of its initial activity after 20 consecutive reaction cycles.     

Methanol oxidation using ozone on titania-supported vanadia catalyst

Ozone-enhanced catalytic oxidation of methanol has been conducted at mild temperatures of 100-250 degrees C using a V2O5/ TiO2 catalyst prepared by the sol-gel method. The catalyst was characterized using XRD, surface area measurements, and temperature-programmed desorption of methanol. The oxidation of methanol with ozone in the absence of a catalyst gave about 30% conversion at 100 degrees C. Methanol oxidation over a V2O5/TiO2 catalyst at 100 degrees C gave very little conversion with oxygen, whereas the conversion increased to 80% with ozone. Methanol, having an inlet stream concentration of 15 000 ppmv, can be completely oxidized to CO(x) with an ozone-to-methanol ratio of 1.2, a temperature of 150 degrees C, and a gas hourly space velocity (GHSV) of 60 000 h(-1). The apparent activation energy with ozone was calculated to be ca. 40 kJ/mol, which is much lower than that calculated with oxygen (60 kJ/mol). At low methanol conversion methyl formate was the main product, whereas higher conversions favored oxidation to CO(x). The results imply a consecutive reaction of adsorbed methanol species, favoring selectivity toward methyl formate at lower temperatures and ozone-to-methanol ratios and CO(x) at higher temperatures and ozone-to-methanol ratios. Langmuir-Hinshelwood kinetics was used to model the reaction with and without ozone in the feed. The model parameters were obtained using least-squares fit to a selected set of experimental data, and the model was subsequently compared to all experimental data obtained in this study.     

Isolation and characterization of proline iminopeptidase from Propionibacterium freudenreichii ATCC 9614

A dimeric, 90 kDa subunit intracellular proline iminopeptidase from Propionibacterium freudenreichii ATCC 9614 was purified to homogeneity by chromatography on hydroxyapatite, Sephacryl 200, Phenyl Superose and Mono Q. The enzyme was specific on Pro-p-nitroanilide and Pro-X dipeptides. It hydrolyzed 2 fragments of hormone oligopeptides with an N-terminal proline: bradykinin, f2-7 and substance P, f4-11. A number of oligopeptides containing 5-11 amino acids residues and proline at the penultimate position from N-terminus or other internal position were not hydrolyzed. The enzyme was most active at pH 7-7.5 and at 37-40 degrees C but it retained 9% of maximal activity at pH 5.5 and >12% of maximal activity at 10 or 60 degrees C. The enzyme was inhibited strongly by the serine protease inhibitor 3,4-dichloroisocoumarin, and stimulated markedly by 1 mol/l of NaCl. The results indicate that the enzyme may lead to the accumulation of proline from dipeptides and oligopeptides during the ripening of cheese.     

Theoretical investigation of fructose 1,6-diphosphate production and simultaneous ATP regeneration by conjugated enzymes in an ultrafiltration hollow-fiber reactor

The performance of an ultrafiltration hollow-fiber reactor, in which enzymatic synthesis of fructose 1,6-diphosphate (FDP) from glucose and enzymatic ATP regeneration are performed simultaneously, was analyzed theoretically. The reaction system consists of three-step synthetic reactions catalyzed by glucokinase (GK), phosphoglucose isomerase and phosphofructokinase, and the ATP regeneration reaction catalyzed by acetate kinase. Based on a simple analytical model developed previously in which the liquid flowing in a tube was assumed to be plug flow and the radial concentration gradients in the tubes and shell side space were both neglected, a computer program was developed to calculate the concentration profiles of all the components along the flow direction in the tubes and shell side space of the reactor. From the FDP concentrations at the reactor outlet calculated under various operational conditions, reactor performances such as the FDP yield and the ATP recycle number were determined. The calculation showed the interesting phenomenon that under some conditions the FDP yield was higher when GK concentration was lower.     

Oxidation of benzoic acid by electrochemically generated Ce(IV)

This paper presents a study of the oxidation of benzoic acid in aqueous solutions by Ce(IV), which functions as an electrogenerated mediator. In this reaction, benzoic acid is oxidized to CO2, and the reaction displayed pseudo-first-order kinetics for the conditions 0.15-0.45 mM Ce(SO4)2, 0.5-3.0 mM benzoic acid, and 0.2 M H2SO4 at 25 degrees C. The k1 values were in the range 0.40 x 10(-4)-0.59 x 10(-4) s(-1) and increased with temperature within the range studied, 25-45 degrees C. The activation parameters for the reaction, deltaH++, deltaG++, and deltaS++, were determined to be 10.5 kcal/mol, 23.3 kcal/mol, and -43 cal/(mol K), respectively. The electrolysis of Ce(III) in a stirred solution at 25 degrees C and with 1.5 V constant potential generated a 96% Ce(IV)-4% Ce(III) distribution in 140 min. The k for the generation of Ce(IV) was 0.0003 s(-1). Electrolysis under similar conditions but with a large excess of benzoic acid in solution produced a steady-state conversion of 87% Ce (IV) and the corresponding k was 0.00027 s(-1). This reduction in k is consistent with the relative magnitudes of the oxidation reactions for Ce(III) and benzoic acid. Correspondingly, the electrolysis of benzoic acid (0.594 mM) in a 0.2 M H2SO4 solution containing 0.25 mM Ce2(SO4)3 was accomplished by applying 1.5 V to the stirred solution at 25 degrees C. The benzoic acid concentration was reduced to 0.026 mM, or by 96%, in 456 h. In this experiment the potential was applied for 3 h and then turned off for 48 h, and the sequence was repeated 8 times.     

Enzymes acting on peptides containing D-amino acid

Mainly microorganisms but only a few higher organisms are presently known to express enzymes that hydrolyze peptides containing D-amino acids. These enzymes can be involved in proceedings at the bacterial cell wall, in either assembly or modification, and thus cause resistance to glycopeptide antibiotics, or mediate resistance against beta-lactam antibiotics. In other cases the in vivo function is still unknown. New enzymes screened from nature, such as D-aminopeptidase, D-amino acid amidase, alkaline D-peptidase or D-aminoacylase, offer potential application in the production of D-amino acids, the synthesis of D-amino acid oligomers by promoting the reversed reaction under appropriate conditions, or in the field of semi-synthetic antibiotics.     

Response of forage fiber degradation by ruminal microorganisms to branched-chain volatile fatty acids,amino acids,and dipeptides

This study evaluated the effect of branched-chain volatile fatty acids (VFA; isobutyric acid, isovaleric acid), amino acids (valine, leucine), and dipeptides (valine-valine, leucine-leucine) on neutral detergent fiber (NDF) degradation by rumen microorganisms in vitro. The CP (%) and in situ NDF degradation rate (%/h) for alfalfa, bermudagrass, and pangolagrass hays, and napiergrass silage were 17.2 and 7.5, 4.7 and 3.1, 8.3 and 5.3, and 9.6 and 3.4, respectively. In vitro NDF digestibility was the lowest for bermudagrass; alfalfa and napiergrass were the highest. When the incubation contained more ammonia initially, digestibilities increased, but relative differences among forages were unchanged. Adding branched-chain VFA (2 mM) to incubations increased digestibilities more than controls on 15 out of 16 occasions. The effectiveness varied with isoacids and forages used. Amino acid (2 mM) or dipeptide (1 mM) addition consistently increased digestibility over controls. Amino acids further increased digestibility over corresponding isoacids on 14 occasions. Improvement in digestibility over control by leucine appeared to be greater than that by valine. Digestibilities with dipeptides were always greater than those with isoacids, except for one case. Dipeptide addition further increased digestibility significantly over corresponding amino acids on only six occasions, while percent improvement in digestibility numerically by dipeptides occurred in 10 cases. Valine-valine seemed to exert different effect than leucine-leucine, depending on initial ammonia availability. The results indicate that dipeptides could be more effective than isoacids and amino acids in improving NDF digestion. Forages with high CP content or rapid NDF degradation rate appeared to respond to additives to smaller degrees.