• Journal of Microbiology and Biotechnology
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• v.20 no.1
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• pp.132-137
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• 2010

The skc gene encoding streptokinase (SK) with a molecular mass of approximately 47.4 kDa was cloned from Streptococcus equisimilis ATCC 9542 and heterologously overexpressed in Streptomyces lividans TK24 and E. coli using various strong promoters. When the promoter for sprT [Streptomyces griseus trypsin (SGT)] was used in the host S. lividans TK24, a 47.4-kDa protein was detected along with a smaller hydrolyzed protein (44 kDa), suggesting that posttranslational hydrolysis had occurred as has been reported in other expression systems. The casein/plasminogen plate assay revealed that the plasmid construct containing the SGT signal peptide was superior to that containing the SK signal peptide in terms of SK production. Maximal production of SK was calculated to be about 0.25 unit/ml of culture broth, a value that was five times higher than that obtained with other expression systems using ermE and tipA promoters in the same host. When the skc gene was expressed in E. coli BL21(${\Delta}DE3$)pLys under the control of the T7 promoter, a relatively large amount of SK was expressed in soluble form without hydrolysis. SK activity in E. coli/pET28a-$T7_pSK_m$ was more than 2 units/ml of culture broth, even though about half of the expressed protein formed an inactive inclusion body.

• Journal of Animal Science and Technology
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• v.44 no.5
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• pp.549-560
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• 2002

This study, including two in vitro experiments and an in vivo experiment were conducted to evaluate effects of Passtein$^{(R)}$ on crude protein degradability, ruminal fermentation characteristics and nutrient digestibility. In in vitro experiment protein degradability was examined using borate-phosphate buffer and neutral detergent, and using protease from Stroptomyces griseus at 39$^{\circ}C$ for 0, 2, 4, 8, 12, and 48 h. In addition, an in vivo experiment was conducted in a switch back design and ruminal fermentation and nutrient digestibility were determined. Four ruminal-fistulated Holstein cows weighing 300kg in mean body weight randomly allotted to 2 treatments (control and Passtein$^{(R)}$ supplementation). Although there was no significant difference on protein fraction between treatments, it appears that Passtein$^{(R)}$ supplementation decreased buffer soluble protein fraction compared to control. Protein degradability was not affected by Passtein$^{(R)}$ from 0 h to 4 h, but decreased at 12 h and 48 h compared to control. Degradation of immediately degradable fraction was higher in Passtein$^{(R)}$ treatment, but degradation of fermentable fraction was lower in Passtein$^{(R)}$ treatment compared to control. The pH and $NH_3$-N concentration tended to increase in Passtein$^{(R)}$ treatment, but VFA production, microbial counts and enzyme activity tended to decrease in Passtein$^{(R)}$ treatment compared to control. In addition, nutrient digestibility in the total tract tended to increase in Passtein$^{(R)}$ treatment compared to control.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.3
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• pp.364-370
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• 2008

Chemical analysis and in vitro studies were conducted to investigate the nutritive value for ruminants of cell mass from lysine production (CMLP) which is a by-product of the lysine manufacturing process. Proximate analysis, protein fractionation, and in vitro protein degradation using protease from Streptomyces griseus and strained ruminal fluid were carried out to estimate ruminal protein degradability of CMLP with two reference feedstuffs-soybean meal (SBM) and fish meal (FM). Amino acid composition and pepsin-HCl degradability were also determined to evaluate postruminal availability. CMLP contained 67.8% crude protein with a major portion being soluble form (45.4% CP) which was composed of mainly ammonium nitrogen (81.8% soluble CP). The amount of nucleic acids was low (1.15% DM). The total amount of amino acids contained in CMLP was 40.60% DM, which was lower than SBM (47.69% DM) or FM (54.08% DM). CMLP was composed of mainly fraction A and fraction B2, while the protein fraction in SBM was mostly B2 and FM contained high proportions of B2 and B3 fractions. The proportion of B3 fraction, slowly degradable protein, in CP was the highest in fish meal (23.34%), followed by CMLP (7.68%) and SBM (1.46%). CMLP was degraded up to 51.40% at 18 h of incubation with Streptomyces protease, which was low compared to FM (55.23%) and SBM (83.01%). This may be due to the insoluble portion of CMLP protein being hardly degradable by the protease. The in vitro fermentation by strained ruminal fluid showed that the amount of soluble fraction was larger in CMLP (40.6%) than in SBM (17.8%). However, because the degradation rate constant of the potentially degradable fraction of CMLP (2.0%/h) was lower than that of SBM (5.8%/h), the effective ruminal protein degradability of CMLP (46.95%) was slightly lower than SBM (53.77%). Unavailable fraction in the rumen was higher in CMLP (34.0%) compared to SBM (8.8%). In vitro CP degradability of CMLP by pepsin was 80.37%, which was lower than SBM (94.42%) and FM (89.04%). The evaluation of protein degradability using different approaches indicated that soluble protein in CMLP may supply a large amount of ammonia in the rumen while insoluble protein can be by-passed from microbial attacks due to its low degradability. The results from this study suggest that CMLP can be used as a protein supplement to ruminants for supplying both non-protein nitrogen to rumen microbes and rumen undegradable protein to the host animal.

• Asian-Australasian Journal of Animal Sciences
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• v.13 no.10
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• pp.1377-1387
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• 2000

Two laboratory techniques: (1) an in vitro method with two procedures for measuring protein degradabilities and (2) an in vitro method with three procedures for measuring protein solubility, were investigated to determine which laboratory techniques could most accurately predict the quantity of rumen protein degradation kinetics of legume seeds after dry roasting under various conditions, in terms of (1) rumen protein disappearance ($D_j$, where j=0, 2, 4, 8, 12, 24 and 48 h incubation), (2) rumen protein effective degradability (EDCP), (3) the parameters describing rumen degradation characteristics (the soluble fraction: S, the potentially degradable fraction: D, undegradable fraction: U, lag time: T0 and the degradation rate: Kd) and (4) rumen bypass protein (BCP), which were determined by the method accepted internationally at present, in sacco nylon bag technique using the standardized Dutch method. Feeds evaluated were the raw and dry roasted whole faba (Vicia faba) beans (WFB) and whole lupin (Lupinus albus) seeds (WLS), each was dry roasted under various conditions (at 110, 130 or $150^{\circ}C$ for 15, 30 or 45 min). In vitro protein degradability ($D_1$_Auf and $D_{24}$_Auf) were determined using the modified Aufr re method by enzymatic hydrolysis for 1 h and 24 h using a protease extracted from Streptomyces griseus in a borate-phosphate buffer. In vitro protein solubility ($bf_1$_S, $bf_2$_S, $bf_3$_S) was measured in a borate-phosphate buffer with three different procedures. Results from laboratory techniques (in vitro) were correlated and linearly regressed with in sacco results. Of the three procedures of in vitro protein solubility evaluated, none of them could predict in sacco results with good precision. The highest Pearson correlation coefficient ($R^2$) was less than 0.50. Of two procedures of in vitro protein degradability studied, the $D_1$_Auf values were closely correlated with in sacco parameters: Kd, EDCP and %BCP with high R' values: 0.82, 0.85 and 0.85, respectively, and closely correlated with in sacco $D_j$ at 2, 4, 8 and 12 h rumen incubation with high $R^2$ values: 0.83, 0.91, 0.93 and 0.83, respectively. The $D_{24}$_Auf values could not predict in sacco results. The highest $R^2$ value was less then 0.40. These results indicated that in vitro protein solubility measured in borate-phosphate failed to identify differences in the rate and extent of protein degradation of legume seeds after dry roasting under various conditions and thus should not be used to predict rumen degradation, particularly for heat processed feedstuffs. But in vitro protein degradability using the modified Aufr re method by enzymatic hydrolysis for 1 h or possibly an intermediate time (>1 h and <24 h) is a promising laboratory procedure to detect effectiveness of dry roasting legume seeds on rumen protein degradation characteristics and could be used as a simple laboratory method to predict the rate and extent of protein degradation in the rumen in sacco with high accuracy. The equations to predict EDCP, Kd and BCP of dry roasted legume seeds (WLS and WFB) under various conditions are as follow: For both: EDCP (%)=-1.37+1.06*$D_1$_Auf ($R^2=0.85$, p<0.01). For both: Kd (%/h)=-21.81+0.49*$D_1$_Auf ($R^2=0.82$, p<0.01). For both: %BCP=103.37-1.07*$D_1$_Auf ($R^2=0.85$, p<0.01).

• Microbiology and Biotechnology Letters
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• v.1 no.1
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• pp.51-57
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• 1973

2,2'-Methylene bis (3,4,6-trichloroacetoxy benzene) had been synthesized from Hexachlorophene. The eleven new derivatives -(2,2'Methylene bis [3,4,6-trichloro o-(${\beta}$-anilinopropionoxy) benzene]: m. p 173∼4$^{\circ}C$, C$\_$31/H$\_$24/N$_2$O$_4$Cl$\_$6/, 2,2' Methylene bis [3,4,6-trichloro (${\beta}$-Cyclohexylaminopropionoxy) benzene]: M. P, 187∼8$^{\circ}C$, C$\_$31/H$\_$36/N$_2$O$_4$Cl$\_$6/, 2,2'-Methylene bis [3,4,6-trichloro (${\beta}$-phenyl hydrazinopropionoxy) benzene]; M. P. 151∼3$^{\circ}C$, C$\_$33/H$\_$28/N$_2$O$_4$Cl$\_$6/, 2,2'-Methylene bis [3,4,6-trichloro (${\beta}$-o-toluidinopropionoxy) benzene]: M. P. 172∼3$^{\circ}C$, C$\_$33/H$\_$28/N$_2$O$_4$Cl$\_$6/, 2,2'-Methylene bis [3,4,6-trichloro (${\beta}$-p-todudinopropionoxy) benzene]: 153∼4$^{\circ}C$, C$\_$33/H$\_$28/N$_2$O$_4$Cl$\_$6/, 2,2'-Methylene bis [3,4,6-trichloro (${\beta}$-o-chloro anilinopropionoxy) benzene]: 170∼2$^{\circ}C$, C$\_$31/H$\_$27/N$_2$O$_4$Cl$\_$8/, 2,2'-Methylene bis [3,4,6-trichloro (${\beta}$-p-sulfamilinopropionoxy) bengene]: M. P. 310-5$^{\circ}C$, C$\_$31/H$\_$24/N$_2$S$_2$O$\_$10/Cl$\_$8/, 2,2'-Methylene bis [3,4,6-trichloro (${\beta}$-piperidinopropionoxy) benzene]: M. P. 168∼2$^{\circ}C$, C$\_$29/H$\_$32/N$_2$O$_4$Cl$\_$6/, 2,2'-Methylene bis (3,4,6-trichloro (${\beta}$-morpholinopropionoxy) benzene]: M. P. 226∼8$^{\circ}C$, C$\_$27/H$\_$28/N$_2$O$\_$6/Cl$\_$6/, 2,2'-Methylene bis (3,4,6-trichloro (${\beta}$-2-amino pyridino propionoxy) benzene]; M. P. 145∼6$^{\circ}C$, C$\_$29/H$\_$22/N$_4$O$_4$Cl$\_$5/-were synthesized by Mannichs reaction as potential antimicrobial agents and their antimicrobial activity were tested against Bacillus subtilis, Pseudomonas ovalis, Escherichia coli, Staphylococcus aureus, aerogenes, Bacillus Acerobacter Polymyxa, Bacillus brevis, Streptomyces griseus, Candida troficalis, Rhodotorula glutinis, Candida utilis, Hansenula anamola, Saccharomyces cerevisiae in vitro. Among them, compounds of benzylamine and p-toludine showed an effective antimibrobial activity againt Bacillus subtilis and Pseudomonas ovalis.

• Korean Journal of Organic Agriculture
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• v.15 no.2
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• pp.195-205
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• 2007

This study was conducted to determine effects of probiotics on performance, nutrient digestibility and nitrogen retention in Korean black goats fed two diets differing in forage to concentrate (F:C) ratio (30:70 and 70:30), for the establishment of their more efficient feeding management system. The probiotics employed in this trial were mixtures of different microbial species, which consisted of Lactobacillus casei, Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus oryzae and Streptomyces griseus. Additional levels of probiotics to each F:C ratio (70:30 or 30:70) were 0 and 0.2%, respectively. Thus, twelve Korean black male goats were allotted to treatments in four groups of three goats per treatment and then they were housed in individual metabolism cages with a randomized complete block design for 21 days. Dry matter (DM) intakes were not affected by dietary F:C ratio and probiotics. Digestible DM amounts were significantly (p<0.05) decreased with increasing levels of dietary forage, but they were not affected by probiotics addition. Dry matter intakes per metabolic body weight and their ratio per body weight had a similar trend to DM intakes with no significant difference by F:C ratio and probiotics addition. The nutrient digestibility was significantly (p<0.05) increased with decreasing levels of forage in the diet, but it was not affected by probiotics addition. Urinary nitrogen loss was significantly (p<0.05) decreased with decreasing levels of dietary forage, but there was no significant difference between probiotics-supplemented and unsupplemented groups within the same F:C ratio. On the contrary, nitrogen retention was decreased with increasing levels of dietary forage, and probiotics supplementation to two diets differing in F:C ratio showed slightly increasing trends in the nitrogen retention. From the above results, probiotics supplementation to two diets differing in F:C ratio did not have the significant influence on feed intakes, nutrient digestibility and nitrogen retention. Consequently, these parameters of Korean black goats were dominated rather by F:C ratio than by dietary probiotics.