Expression of a bifunctional cellulase with exoglucanase and endoglucanase activities to enhance the hydrolysis ability of cellulase from a marine Aspergillus niger

Low exoglucanase and endoglucanase activities of marine Aspergillus niger cellulase decreased the hydrolyzing ability of cellulase. To increase the activity of halostable cellulase obtained from a marine A. niger, a cellulase with endoglucanase and exoglucanase activity was efficiently expressed by constructing a vector with promoter glaA. Exoglucanase and endoglucanase activities increased from 0.21 and 4.51 U/ml of the original strain to 0.89 U/ml and 15.12 U/ml of the transformant, respectively. Filter paper activity (FPA) increased by 7.1 folds from 0.63 to 4.47 U/ml. The release of glucose by hydrolysis of wheat straw with cellulase from the transformant was 1.37 folds higher than that with cellulase from the original strain under high salinity condition. Cellulase with endoglucanase and exoglucanase activities could be well expressed in marine A. niger. The cellulase from the transformant not only showed higher activity, but also retained halostability. An appreciate proportion of β-glucosidase, exoglucanase, endgolucanasein cellulase was important for hydrolyzing cellulose.     

Cloning and Expression of the Exo-β-D-1,3-glucanase Gene (exgS) from Aspergillus saitoi

A gene of exo-1,3-β-D-glucanase (exgS) was cloned from a koji mold, Aspergillus saitoi, genomic DNA using PCR. The exgS has an ORF comprising 2832 bp, which contains one intron of 45 bp, and encodes 945 amino acids. The deduced amino acid sequences showed that the ExgS has a non-homologous linker region consisting of 180 amino acids, which encompassed highly conserved regions observed in Exg homologues from filamentous fungi. A recombinant protein (ExgS) has been recovered from the cultural filtrate of an Aspergillus oryzae strain that carried an expression vector containing full length of the exgS. The N-terminal amino acid sequences of the recombinant exo-1,3-β-D-glucanase (ExgS) were identical to that of native ExgS from A. saitoi.     

Process engineering of cellulosic n-butanol production from corn-based biomass using Clostridium cellulovorans

The cellulolytic Clostridium cellulovorans has been engineered to produce n-butanol from low-value lignocellulosic biomass by consolidated bioprocessing (CBP). The objective of this study was to establish a robust cellulosic biobutanol production process using a metabolically engineered C. cellulovorans. First, various methods for the pretreatment of four different corn-based residues, including corn cob, corn husk, corn fiber, and corn bran, were investigated. The results showed that better cell growth and a higher concentration of n-butanol were produced from corn cob that was pretreated with sodium hydroxide. Second, the effects of different carbon sources (glucose, cellulose and corn cob), basal media and culture pH values on butanol production were evaluated in the fermentations performed in 2-L bioreactors to identify the optimal CBP conditions. Finally, the engineered C. cellulovorans produced butanol with final concentration >3 g/L, yield >0.14 g/g, and selectivity >3 g/g from pretreated corn cob at pH 6.5 in CBP. This study showed that the fermentation process engineering of C. cellulovorans enabled a high butanol production directly from agricultural residues.     

Cellulosome and noncellulosomal cellulases of Clostridium cellulovorans

This paper reviews the properties of the cellulosome and noncellulosome cellulases produced by Clostridium cellulovorans, an anaerobic, mesophilic, spore-forming microorganism that produces copious amounts of cellulase. The three major subunits of the cellulosome, CbpA, exoglucanase S (ExgS), and P100, are described, as well as the properties of the functional domains of CbpA. The properties of two noncellulosomal endoglucanases, EngD and EngF, are compared. The functions of the cellulose-binding domain (CBD) of CbpA indicate its potential uses in biotechnology. Received: November 18, 1997 / Accepted: November 26, 1997     

Carbonic anhydrase activators: Activation of the β-carbonic anhydrase from the pathogenic yeast Candida glabrata with amines and amino acids

The protein encoded by the NCE103 gene of Candida glabrata, a β-carbonic anhydrase (CA, EC 4.2.1.1) designated as CgCA, was investigated for its activation with amines and amino acids. CgCA was weakly activated by amino acids such as l-/d-His, l-Phe, l-DOPA, and l-Trp and by histamine or dopamine (K_As of 21.2–37 μM) but more effectively activated by d-Phe, d-DOPA, d-Trp as well as serotonin, pyridyl-alkylamines, aminoethyl-piperazine/morpholine (K_As of 10.1–16.7 μM). The best activators were l-/d-Tyr, with activation constants of 7.1–9.5 μM. This study may bring a better understanding of the catalytic/activation mechanisms of β-CAs from pathogenic fungi.     

Carbonic anhydrase activators: Activation of the β-carbonic anhydrase Nce103 from the yeast Saccharomyces cerevisiae with amines and amino acids

The protein encoded by the Nce103 gene of Saccharomyces cerevisiae, a β-carbonic anhydrase (CA, EC 4.2.1.1) designated as scCA, was investigated for its activation with amines and amino acids. scCA was poorly activated by amino acids such as l-/d-His, Phe, DOPA, Trp (K_As of 82–90 μM) and more effectively activated by amines such as histamine, dopamine, serotonin, pyridyl-alkylamines, aminoethyl-piperazine/morpholine (K_As of 10.2–21.3 μM). The best activator was l-adrenaline, with an activation constant of 0.95 μM. This study may help to better understand the catalytic/activation mechanisms of the β-CAs and eventually to design modulators of CA activity for similar enzymes present in pathogenic fungi, such as Candida albicans and Cryptococcus neoformans.     

Physical and genetic characterization of an outer-membrane protein (OmpM1) containing an N-terminal S-layer-like homology domain from the phylogenetically Gram-positive gut anaerobe Mitsuokella multacida

Thin sectioning and freeze-fracture-etch of the ovine ruminal isolate Mitsuokella multacida strain 46/5(2) revealed a Gram-negative envelope ultra-structure consisting of a peptidoglycan wall overlaid by an outer membrane. Sodium-dodecyl-sulfate-polyacrylamide gel electrophoretic (SDS-PAGE) analysis of whole cells, cell envelopes and Triton X-100 extracted envelopes in combination with thin-section and N-terminal sequence analyses demonstrated that the outer membrane contained two major proteins (45 and 43 kDa) sharing identical N-termini (A-A-N-P-F-S-D-V-P-A-D-H-W-A-Y-D). A gene encoding a protein with a predicted N-terminus identical to those of the 43 and 45 kDa outer-membrane proteins was cloned. The 1290 bp open reading frame encoded a 430 amino acid polypeptide with a predicted molecular mass of 47,492 Da. Cleavage of a predicted 23 amino acid leader sequence would yield a protein with a molecular mass of 45,232 Da. Mass spectroscopic analysis confirmed that the cloned gene (ompM1) encoded the 45 kDa outer-membrane protein. The N-terminus of the mature OmpM1 protein (residues 24–70) shared homology with surface-layer homology (SLH) domains found in a wide variety of regularly structured surface-layers (S-layers). However, the outer-membrane locale, resistance to denaturation by SDS and high temperatures and the finding that the C-terminal residue was a phenylalanine suggested that ompM1 encoded a porin. Threading analysis in combination with the identification of membrane spanning domains indicated that the C-terminal region of OmpM1 (residues 250–430) likely forms a 16-strand β-barrel and appears to be related to the unusual N-terminal SLH-domain-containing β-barrel-porins previously described in the cyanobacterium Synechococcus PCC6301.     

Cloning and characterization of a recombinant family 5 endoglucanase from Bacillus licheniformis strain B-41361

The gene encoding a family 5 endoglucanase, cel5A, was cloned from the moderate thermophile Bacillus licheniformis strain B-41361. The primary structure of the translated cel5A gene predicts a 49 amino acid putative secretion signal and a 485 residue endoglucanase consisting of an N-terminal family 5 catalytic domain and C-terminal family 3 cellulose binding domain. The endoglucanase portion of the gene was expressed in Escherichia coli, but soluble activity in cell lysates was due to a truncated enzyme with an apparent mass of 42 kDa, the equivalent of the predicted catalytic domain. Insoluble protein renatured from inclusion bodies was protected against truncation, yielding an active holoenzyme (rCel5A) with apparent mass of 62 kDa. The recombinant rCel5A was optimally active at 65 °C and pH 6.0, but retained only 10% activity after 1 h incubation at this temperature. At 55 °C, rCel5A had a broad pH range for activity and stability, with greater than 75% relative activity from pH 4.5–7.0, and retaining greater than 80% relativity activity across the range pH 4.5–8.0 following 1 h incubation at 55 °C. It readily hydrolyzed pNPC, carboxymethylcellulose, barley β-glucan, and lichenan, but despite binding to cellulose, had only weak activity against avicel. Hydrolysis products from soluble polysaccharides included glucose, cellobiose, cellotriose, and cellotetraose. The catalytic properties, broad pH range and thermostability of the recombinant B. licheniformis endoglucanase may prove suitable for industrial applications.     

Sequence analysis of a bacteriocinogenic plasmid of Clostridium butyricum and expression of the bacteriocin gene in Escherichia coli

A small cryptic plasmid, namely, pCBM588, was obtained from Clostridium butyricum MIYAIRI 588 (CBM588) — a bacterium used in probiotics. The complete sequence of pCBM588 was determined. The size of pCBM588 was 8060 bp and the G + C content was 24.3%. Nine open reading frames (ORFs) were predicted, and ORF3 showed significant homologies with a structural bacteriocin gene of Clostridium tyrobutyricum. The putative bacteriocin gene was inserted into the pET21d expression vector in frame; it was expressed as a His-tagged recombinant protein in Escherichia coli BL21 (DE3). A total of 10240 AU of the recombinant bacteriocin were purified from 100 ml of E. coli culture. The bacteriocin was cleaved into 2 portions, and the small C-terminal polypeptide consisting of 83 amino acids possessed bactericidal activity. These results demonstrated that the ORF3 of pCBM588 encoded a bacteriocin, which is identical or very similar to the previously reported butyricin 7423.     

Biochemical and molecular characterization of a novel laccase from selective lignin-degrading white-rot fungus Echinodontium taxodii 2538

A novel laccase was isolated and characterized from a new selective lignin-degrading white-rot fungus Echinodontium taxodii 2538, in which a high yield of laccase was obtained. No laccase isoenzyme was detected in the synthetic liquid media. The purified laccase (designated as EtL2538) had an apparent molecular mass of 56 kDa, pI value of 3.1, and N-terminal amino acid sequence of GIGPVTDLHIVNAAV. EtL2538 showed optimum pH at 3.0 and optimum temperature at 60 °C using ABTS as the substrate. EtL2538 revealed superior thermostability, and retained over 80% of its original activity after incubation for 2 h at 50 °C. The laccase gene, etl2538, was also cloned and sequenced. This gene encoded a mature laccase protein containing 499 amino acids (aa) preceded by a signal peptide of 21 aa, and the deduced protein sequence contained four copper-binding conserved domains of typical laccase protein. EtL2538 was further used in lignin oxidation and dye decolorization. Even without the existence of redox mediators, EtL2538 could cleave the methoxyl groups and β-O-4 ether linkages in lignin from bamboo, and significantly decolorize malachite green and RBBR. These novel properties of EtL2538 may render it as a potential biocatalyst for biotechnological and environmental applications.     

Synergistic effects on crystalline cellulose degradation between cellulosomal cellulases from Clostridium cellulovorans

Clostridium cellulovorans produces a multienzyme cellulose-degrading complex called the cellulosome. In this study, we determined the synergistic effects on crystalline cellulose degradation by three different recombinant cellulosomes containing either endoglucanase EngE, endoglucanase EngH, or exoglucanase ExgS bound to mini-CbpA, a part of scaffolding protein CbpA. EngE, EngH, and ExgS are classified into the glycosyl hydrolase families 5, 9, and 48, respectively. The assembly of ExgS and EngH with mini-CbpA increased the activity against insoluble cellulose 1.5- to 3-fold, although no effects on activity against soluble cellulose were observed. These results indicated that mini-CbpA could help cellulase components degrade insoluble cellulose but not soluble cellulose. The mixture of the cellulosomes containing ExgS and EngH showed higher activity and synergy degrees than the other cellulosome mixtures, indicating the synergistic effect between EngH and ExgS was the most dominant effect among the three mixtures for crystalline cellulose degradation. Reactions were also performed by adding different cellulosomes in a sequential manner. When ExgS was used for the initial reaction followed by EngE and EngH, almost no synergistic effect was observed. On the other hand, when EngE or EngH was used for the first reaction followed by ExgS, synergistic effects were observed. These results indicated that the initial reactions by EngH and/or EngE promoted cellulose degradation by ExgS.     

Association of TP53 codon 72 and CDH1 genetic polymorphisms with colorectal cancer risk in Bangladeshi population

Till now no pharmacogenetic study of TP53 codon 72 (Arg72Pro) and CDH1 rs16260 (-160C<A) genes has been reported on Bangladeshi population relating those with colorectal cancer. So the aim of the study is to determine whether there is an elevated risk of colorectal cancer development with TP53 codon 72 and CDH1 rs16260 genetic polymorphism in Bangladeshi population for the first time. To investigate the association of these two SNPs, we conducted a case-control study with 288 colorectal cancer patients and 295 healthy volunteers by using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) method. We found an increased risk of association between Arg/Pro heterozygosity (adjusted OR = 2.58, 95% CI = 1.77–3.77, p < 0.05) and Pro/Pro mutant homozygosity (adjusted OR = 2.92, 95% CI = 1.78–4.78, p < 0.05) along with the combined genotype (Arg/Pro + Pro/Pro) (adjusted OR = 2.70, 95% CI = 1.90–3.82, p < 0.05) and colorectal cancer predisposition. In case of CDH1 rs16260 polymorphism, C/A heterozygous and A/A mutant homozygous are significantly (p < 0.05) found to be associated with colorectal cancer risk with adjusted OR of 1.94 and 2.63, respectively. The combined genotype of C/A and A/A was also found to be strongly associated with colorectal cancer risk compared to C/C genotype (adjusted OR = 2.02, 95% CI = 1.42–2.87, p < 0.05). In conclusion, heterozygosity and mutant homozygosity as well as the combination of both TP53 Arg72Pro and CDH1 rs16260 polymorphisms are responsible to increase the risk of colorectal cancer development in Bangladeshi population.     

Simultaneous saccharification and fermentation of cellulose by Fusarium oxysporum F3—growth characteristics and metabolite profiling

The simultaneous saccharification and fermentation (SSF) of cellulose by Fusarium oxysporum was investigated in the present study. It was found that F. oxysporum grow with a maximum specific growth rate of 0.023 h⁻¹ on cellulose at aerobic conditions and that it can produce ethanol with a volumetric productivity of 0.044 g/L/h and a yield of 0.35 g/g cellulose under anaerobic conditions. The cellulase system in F. oxysporum is well balanced as no cellobiose accumulated. The profile of the phosphorylated intermediates from Pentose Phosphate Pathway (PPP), Embden–Meyerhof–Parnas Pathway (EMP) and the key intermediates of the glycolytic pathway as well as extracellular organic and amino acids were determined during the fermentation in order to investigate the potential metabolic bottlenecks of the process. The high levels of intracellular glucose-1,6-DP a metabolic downstream of phoshoglucomutase also indicates limiting activities of this enzyme and difficulty of glucose to be channelled into biosynthetic and glycolytic pathways. The presence of high levels of γ-aminobutyrate (GABA) under anaerobic conditions suggests a functional GABA bypass and possible block in the Krebs cycle.     

Molecular cloning and characterization of the partial major ampullate silk protein gene from the spider Araneus ventricosus

Spider silks have great potential as biomaterials with extraordinary properties. Here, we report the cloning and characterization of the major ampullate silk protein gene from the spider Araneus ventricosus. A cDNA encoding the partial major ampullate silk protein (AvMaSp) was cloned from A. ventricosus. An analysis of the cDNA sequence shows that AvMaSp consists of a 240 amino acid repetitive region and a 99 amino acid C-terminal non-repetitive domain. The peptide motifs that were found in the spider major ampullate silk proteins, (A)n, (GA)n, and (GGX)n, were conserved in the repetitive region of AvMaSp. Phylogenetic analysis further confirmed that AvMaSp belongs to the spider major ampullate spidroin family of proteins. The AvMaSp-R cDNA, which encodes the 240 amino acid repetitive domain, was expressed as a soluble 22 kDa polypeptide in baculovirus-infected insect cells. Recombinant AvMaSp-R was degraded abruptly by trypsin. However, AvMaSp-R was stable at 100 °C for at least 30 min. Additionally, the AvMaSp-R was stable at pH values from 2 to 12 for at least 1 h. Taken together, our findings describe the molecular structure and biochemical properties of the A. ventricosus major ampullate silk protein and demonstrate its potential as a biomaterial.     

Cloning and molecular analysis of a cDNA and the Cs-mnp1 gene encoding a manganese peroxidase isoenzyme from the lignin-degrading basidiomycete Ceriporiopsis subvermispora

A cDNA (MnP13-1) and the Cs-mnp1 gene encoding for an isoenzyme of manganese peroxidase (MnP) from C. subvermispora were isolated separately and sequenced. The cDNA, identified in a library constructed in the vector Lambda ZIPLOX, contains 1285 nucleotides, excluding the poly(A) tail, and has a 63% G+C content. The deduced protein sequence shows a high degree of identity with MnPs from other fungi. The mature protein contains 364 amino acids, which are preceded by a 24-amino-acid leader sequence. Consistent with the peroxidase mechanism of MnP, the proximal histidine, the distal histidine and the distal arginine are conserved, although the aromatic binding site (L/V/I–P–X–P) is less hydrophilic than those of other peroxidases. A gene coding for the same protein (Cs-mnp1) was isolated from a genomic library constructed in Lambda GEM-11 vector using the cDNA MnP13-1 as a probe. A subcloned SacI fragment of 2.5 kb contained the complete sequence of the Cs-mnp1 gene, including 162 bp and 770 bp of the upstream and downstream regions, respectively. The Cs-mnp1 gene possesses seven short intervening sequences. The intron splice junction sequences as well as the putative internal lariat formation sites adhere to the GT–AG and CTRAY rules, respectively. To examine the structure of the regulatory region of the Cs-mnp1 gene further, a fragment of 1.9 kb was amplified using inverse PCR. A putative TATAA element was identified 5′ of the translational start codon. Also, an inverted CCAAT element, SP-1 and AP-2 sites and several putative heat-shock and metal response elements were identified.     

Cell biology of adhesive setae in gecko lizards

Adhesive devices of digital pads of gecko lizards are formed by microscopic hair-like structures termed setae that derive from the interaction between the oberhautchen and the clear layer of the epidermis. The two layers form the shedding complex and permit skin shedding in lizards. Setae consist of a resistant but flexible corneous material largely made of keratin-associated beta-proteins (KAβPs, formerly called beta-keratins) of 8–22 kDa and of alpha-keratins of 45–60 kDa. In Gekko gecko, 19 sauropsid keratin-associated beta-proteins (sKAβPs) and at least two larger alpha-keratins are expressed in the setae. Some sKAβPs are rich in cysteine (111–114 amino acids), while others are rich in glycine (169–219 amino acids). In the entire genome of Anolis carolinensis 40 KaβPs are present and participate in the formation of all types of scales, pad lamellae and claws. Nineteen sKAβPs comprise cysteine-rich 9.2–14.4 kDa proteins of 89–142 amino acids, and 19 are glycine-rich 16.5–22.0 kDa proteins containing 162–225 amino acids, and only two types of sKAβPs are cysteine- and glycine-poor proteins. Genes coding for these proteins contain an intron in the 5′-non-coding region, a typical characteristic of most sauropsid KaβPs. Gecko KAβPs show a central amino acid region of high homology and a beta-pleated conformation that is likely responsible for the polymerization of KaβPs into long and resistant filaments. The association of numerous filaments, probably over a framework of alpha-keratins, permits the formation of bundles of corneous material for the elongation of setae, which may be over 100 μm long. The terminals branching off each seta may derive from the organization of the cytoskeleton and from the mechanical separation of keratin bundles located at the terminal apex of setae.     

BSF1 fibrinolytic enzyme from a marine bacterium Bacillus subtilis A26: Purification,biochemical and molecular characterization

A novel fibrinolytic enzyme, subtilisin BSF1, from a newly isolated Bacillus subtilis A26 was purified, characterized and the gene was isolated and sequenced. The subtilisin BSF1 was purified to homogeneity by five-step procedure with a 4.97-fold increase in specific activity and 6.28% recovery. The molecular weight of the purified enzyme was estimated to be 28 kDa by SDS-PAGE and gel filtration. The purified enzyme exhibited high fibrinolytic activity on fibrin agar plates.Interestingly, the enzyme was highly active over a wide range of pH from 7.0 to 12.0, with an optimum at pH 9.0. The relative activities at pH 10.0 and 11.0 were 97.8% and 85.2% of that at pH 9.0. The optimum temperature for enzyme activity was 60 °C. The activity of subtilisin BSF1 was totally lost in the presence of PMSF, suggesting that the purified enzyme is a serine protease. The N-terminal amino acid sequence of the first 11 amino acids (aa) of the purified fibrinolytic enzyme was AQSVPYGISQI.The bsf1 gene encoding the subtilisin BSF1 was isolated and its DNA sequence was determined. The bsf1 gene consisted of 1146 bp encoding a pre-pro-protein of 381 amino acids organized into a signal peptide (29 aa), a pro-peptide (77 aa) and a mature domain (275 aa). The deduced amino acids sequence of the mature enzyme (BSF1) differs from those of nattokinase from B. subtilis natto and subtilisin DFE from Bacillus amyloliquefaciens DC-4 by 5 and 39 amino acids, respectively.     

Marinitoga lauensis sp. nov., a novel deep-sea hydrothermal vent thermophilic anaerobic heterotroph with a prophage

A novel moderately thermophilic, heterotrophic anaerobe, designated strain LG1^T, was isolated from the Mariner deep-sea hydrothermal vent field along the Eastern Lau Spreading Center and Valu Fa Ridge. Cells of strain LG1^T were motile rods, occurring singly or in pairs, 0.6 μm in width and 1.2 μm in length. The strain LG1^T grew between 40 and 70 °C (optimum 50–55 °C), at a pH between 5 and 8 (optimum pH 6.5) and with 7.5–50 g L⁻¹ NaCl (optimum 30 g L⁻¹). Sulfur, cystine and thiosulfate were reduced to sulfide, and cell yield was improved in the presence of cystine. Strain LG1^T was an organotroph able to use a variety of organic compounds. Phylogenetic analysis based on 16S rRNA gene sequence comparisons indicated that strain LG1^T was affiliated to the genus Marinitoga within the order Petrotogales. It shared 95.34–96.31% 16S rRNA gene sequence similarity with strains of other Marinitoga species, and is most closely related to Marinitoga okinawensis. Genome analysis revealed the presence of a prophage sharing high sequence homology with the viruses MPV1, MCV1 and MCV2 hosted by Marinitoga strains. Based on the data from the phylogenetic analyses and the physiological properties of the novel isolate, we propose that strain LG1^T is a representative of a novel species, for which the name Marinitoga lauensis sp. nov. is proposed; the type strain is LG1^T (=DSM 106824 = JCM 32613).     

Combined effect of probiotics and specific immunoglobulin Y directed against Escherichia coli on growth performance,diarrhea incidence,and immune system in calves

Enterotoxigenic Escherichia coli (ETEC) K99 is one of the major pathogens associated with calf diarrhea. The induction of passive immunity in animals by immunoglobulin Y and using probiotics are inexpensive alternatives to antibiotics for the prevention and treatment of a number of bacterial infections, including diarrhea. Hence, the aim of this research was to evaluate the impact of dietary probiotics and ETEC K99-specific egg yolk antibody supplements, alone and in combination with each other, on health and growth parameters, diarrhea incidence and immune stimulation in newborn Holstein calves. One hundred and twenty neonatal calves were allocated randomly into 4 dietary groups (n = 30 per group) received colostrum/milk without any additives (control group), or supplemented with egg yolk powder contained E. coli K99-specific antibody (Ab group; 1 g/day), a commercial probiotic, Hypro-calves (Pro group; 3 g/day), and their combination (Ab + Pro group), from day (d) 1 to d28 of age. Analyses of the growth parameters, feed efficiency, fecal score, and microbiota and immune function were carried out on d0, 14, 21, and 28 of the experiment. Calves in Ab or Ab + Pro group had higher (P < 0.05) average daily gain compared to control and Pro groups during 0–14d. Feed efficiency of calves in Ab and Ab + Pro groups was significantly higher than that in control group during the period of 0–14d; however, no significant differences were observed in 0–28d period. Diarrhea prevalence and fecal score in Ab + Pro group were lower than control group (P < 0.05). Calves in Ab + Pro group had the lowest number of fecal E. coli in comparison to other groups on d28 (P < 0.05). Feeding Ab + Pro supplement increased (P < 0.05) concentrations of blood IgA and serum CD4 compared to the control group. Likewise, calves in Pro group had higher CD4 levels as compared to the control calves (P < 0.05). Serum concentration of interferon-gamma in control group was lower than other groups (P < 0.05). Overall, these data suggest that feeding a combination of probiotic and specific antibody against ETEC to neonate Holstein calves enhances feed efficiency, boosts immunity, and reduces diarrhea prevalence.     

Urinary amino acid analysis: A comparison of iTRAQ®–LC–MS/MS,GC–MS,and amino acid analyzer

Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) of propyl chloroformate and iTRAQ^® derivatized amino acids, respectively, to conventional amino acid analysis. The GC–MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC–MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ^® tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC–MS, and iTRAQ^®–LC–MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27 ± 5.22, 21.18 ± 10.94, and 18.34 ± 14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39 ± 5.35, 6.23 ± 3.84, and 35.37 ± 29.42. Both GC–MS and iTRAQ^®–LC–MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.