• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.197-208
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• 2014

A novel protease gene from Bacillus gibsonii, aprBG, was cloned, expressed in B. subtilis, and characterized. High-level expression of aprBG was achieved in the recombinant strain when a junction was present between the promoter and the target gene. The purified recombinant enzyme exhibited similar N-terminal sequences and catalytic properties to the native enzyme, including high affinity and hydrolytic efficiency toward various substrates and a superior performance when exposed to various metal ions, surfactants, oxidants, and commercial detergents. AprBG was remarkably stable in 50% organic solvents and retained 100% activity and stability in 0-4 M NaCl, which is better than the characteristics of previously reported proteases. AprBG was most closely related to the high-alkaline proteases of the subtilisin family with a 57-68% identity. The secretion and maturation mechanism of AprBG was dependent on the enzyme activity, as analyzed by site-directed mutagenesis. Thus, when taken together, the results revealed that the halo-solvent-tolerant protease AprBG displays significant activity and stability under various extreme conditions, indicating its potential for use in many biotechnology applications.

• Applied Biological Chemistry
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• v.41 no.1
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• pp.73-77
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• 1998

The effects of soymilk residues solubilization by cellulase, protease, koji and yeast were investigated on dry matter and protein yields, amino acid and organic acid contents. Co-treatment of soymilk residues by cellulase and protease gave high dry matter yield and protein yield. Koji treatment followed by yeast fermentation was effective for increasing organic acid content and producing soy sauce-like taste and odor. Organic acid content of fermented hydrolysates was improved by cellulase treatment. Protease treatment rather than koji treatment gave high amino acid content, and cellulase treatment seemed to have little effect on increasing free amino acid content. In sensory evaluation, koji-treated hydrolysate showed higher overall acceptance than other hydrolysates, however it showed lower overall acceptance than commercial fermented soy sauce.

• Journal of Animal Science and Technology
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• v.62 no.3
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• pp.313-320
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• 2020

This research was performed to investigate the hypothesis that dietary mono-component protease (PRO) might improve growth performance, nutrient digestibility, and carcass characteristics of growing-finishing pigs. A total of eighty-four pigs [Duroc × (Landrace × Yorkshire), 25.3 ± 2.16 kg initial body weight] were randomly assigned to three dietary treatments (7 replicates/treatment; 2 barrows and 2 gilts/replicate) in a randomized complete block design (block = sex). The dietary treatments were prepared as follows; (1) a positive control (PC) as a typical growing-finishing diet based on corn and soybean meal, (2) PC added with 0.015% of PRO (PCPRO), and (3) a negative control (NC) added with 0.015% of PRO (NCPRO). The NC had a lower concentration of crude protein (CP) compared with PC. The PRO was a commercial product that contained 75,000 protease units/g and derived from Nocardiopsis prasina produced in Bacillus licheniformis. Dietary treatments were offered to pigs during growing and finishing periods. Measurements were growth performance, apparent total tract digestibility (ATTD) of nutrients, and carcass characteristics. The PCPRO and/or NCPRO increased average daily gain (ADG) and gain to feed ratio (G:F) during growing (p < 0.10), finishing (p < 0.05), and growing-finishing periods (p < 0.10) compared with PC. Furthermore, pigs fed PCPRO and NCPRO had higher (p < 0.05) ATTD of CP and energy during growing and/or finishing periods than those fed PC. In conclusion, the supplementation of PRO in diets improved growth performance and protein digestibility of growing-finishing pigs.

• Applied Biological Chemistry
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• v.41 no.4
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• pp.270-272
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• 1998

Angiotensin converting enzyme (ACE) inhibitor was isolated from beef bone extract hydrolysate. After hydrolysis of beef bone extract with a commercial protease, ACE inhibitory peptide was purified by using ultrafiltration, gel permeation chromatography, and reverse-phase high pressure liquid chromatography. The purified ACE inhibitor was a pentapeptide, Gly-Pro-X-Gly-Pro.

• Journal of Animal Science and Technology
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• v.59 no.10
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• pp.23.1-23.11
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• 2017

Background: Although soybean meal (SBM) is excellent source of protein in diets for poultry, it is sometimes inaccessible, costly and fluctuates in supply. The SBM can partially be replaced by full-fat SBM, but the meals prepared from raw full-fat soybean contain antinutritional factors. To avoid the risk of antinutritional factors, heat treatment is always advisable, but either excessive or under heating the soybean could negatively affect the quality. However, the potential for further improvement of SBM by supplementing with microbial enzymes has been suggested by many researchers. The objective of this study was to evaluate the performance and ileal nutrient digestibility of birds fed on diets containing raw soybeans and supplemented with microbial protease. Methods: A $3{\times}2$ factorial, involving 3 levels of raw full-fat soybean (RFFS; 0, 45 or 75 g/kg of diet) and 2 levels of protease (0 or 15,000 PROT/kg) was used. The birds were raised in a climate-controlled room. A nitrogen-free diet was also offered to a reference group from day 19 to 24 to determine protein and amino acid flow at the terminal ileum and calculate the standardized ileal digestibility of nutrients. On days 10, 24 and 35, body weight and feed leftover were recorded to calculate the body weight gain (BWG), feed intake (FI) and feed conversion ratio (FCR). On day 24, samples of ileal digesta were collected at least from two birds per replicate. Results: When RFFS was increased from 0 to 75 g/kg of diet, the content of trypsin inhibitors was increased from 1747 to 10,193 trypsin inhibitors unit (TIU)/g of diets, and feed consumption of birds was also reduced (P < 0.05). Increasing RFFS level reduced the BWG from hatch 0 to 10 d (P < 0.01) and hatch to 24 d (P < 0.05). The BWG of birds from hatch to 35 was not significantly (P = 0.07) affected. Feed intake was also reduced (P < 0.05) during 0 to 35 d. However, protease supplementation improved (P < 0.05) the BWG and FCR during 0 to 24 d. Rising levels of RFFS increased the weight of pancreas (P < 0.001) and small intestine (P < 0.001) at day 24. Except for methionine, apparent and the corresponding standardized ileal digestibility of CP and AA were reduced (P < 0.01) by increasing levels of RFFS in diets. Conclusion: This study showed that some commercial SBM could be replaced by RFFS in broiler diets, without markedly compromising productivity. The AID and SID of CP and lysine were slightly improved by dietary supplementation of microbial protease.

• KSBB Journal
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• v.27 no.6
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• pp.352-360
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• 2012

Bacillus clausii I-52 which produced SDS- and $H_2O_2$-tolerant extracellular alkaline protease (BCAP) was isolated from heavily polluted tidal mud flat of West Sea in Incheon, Korea and stable strain (transformant C5) of B. clausii I-52 harboring another copy of BCAP gene in the chromosome was developed using the chromosome integration vector, pHPS9-fuBCAP. When investigated the production of BCAP using B. clausii transformant C5 through pilot-scale submerged fermentation (500 L) at $37^{\circ}C$ for 30 h with an aeration rate of 1 vvm and agitation rate of 250 rpm, protease yield of approximately 105,700 U/mL was achieved using an optimized medium (soybean meal 2%, wheat flour 1%, sodium citrate 0.5%, $K_2HPO_4$ 0.4%, $Na_2HPO_4$ 0.1%, NaCl 0.4%, $MgSO_4{\cdot}7H_2O$ 0.01%, $FeSO_4{\cdot}7H_2O$ 0.05%, liquid maltose 2.5%, $Na_2CO_3$ 0.6%). The enzyme stability of BCAP was increased by addition of polyols (10%, v/v) and also, the stabilities of BCAP towards not only the thermal-induced inactivation at $50^{\circ}C$ but also the SDS and $H_2O_2$-induced inactivation at $50^{\circ}C$ were enhanced. Among the polyols examined, the best result was obtained with propylene glycol (10%, v/v). The BCAP supplemented with propylene glycol exhibited extreme stability against not only the detergent components such as ${\alpha}$-orephin sulfonate (AOS) and zeolite but also the commercial detergent preparations. The granulized enzyme of BCAP was prepared with approximately 1,310,000 U/g of granule. Wash performance analysis using EMPA test fabrics revealed that BCAP granule exhibited high efficiency for removal of protein stains in the presence of anionic surfactants as well as bleaching agents. When compared to Savinase 6T$^{(R)}$ and Everlase 6T$^{(R)}$ manufactured by Novozymes, BCAP under this study probably showed similar or higher efficiency for the removal of protein stains. These results suggest that the alkaline protease produced from B. clausii transformant C5 showing high stability against detergents and high wash performance has significant potential and a promising candidate for use as a detergent additive.

• 한국생물공학회:학술대회논문집
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• 2000.04a
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• pp.554-557
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• 2000

Strain JV3 was isolated from commercial Swiss cheese products and identified as a bacteriocin producer, which has bactericidal activity against Leuconostoc mesenteroides KCCM 11324. Strain JW3 was identified tentatively as Lactococcus lactis by the API test. The activity of lacticin JW3, named tentatively as the bacteriocin produced by Lactococcus lactis JW3, was detected during the mid-log growth phase, and reached a maximum during the early stationary phase, and decreased after the late stationary phase. Its antimicrobial activity on sensitive indicator cells was completely disappeared by protease IV. The inhibitory activities of lacticin JW3 were detected during treatments of up to $121\'^{circ}C$ for 15 min. Lacticin JW3 was very stable over a pH range of 2.0 to 9.0 The apparent molecular mass of lacticin JW3 was estimated to be in the region of 3-3.5kDa, which was determined by the direct detection of bactericidal activity after SDS-PAGE.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.32 no.4
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• pp.458-465
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• 1999

Distribution of the proteolytic activities of crude pretense extracted from the viscera of ten kinds of fish was examined. Their proteolytic activities on proteinous substrates (azocasein, hemoglobin, and casein) from the viscera of anchovy, bastard flatfish, mackerel and red sea bream were higher than those of other fishes, and the crude pretenses were further fractoinated with acetone or ammonium sulfate. Optimum concentrations for pretenses fractionation were $0\~55\%$ for acetone and $30\~70\%$ for ammonium sulfate. The fractionated viscera pretense of mackerel showed the highest proteolytic activity among four kinds of fishes. Activities of cathepsin D- and pepsin-like enzymes at pH 3.0, cathepsin L-, B-, H- and G-like enzyme at pH 6,0, and Hypsin- and chymotrypsin- like enzymes (pH 8.0) were detected in the fractionated viscera pretense, whereas activities of cathepsin L- and chymoeypsin-like enzyme were observed in commercial pretenses. Proteolytic activities of Alcalase, Protamex, and Aroase AP-10 for azocasein were slightly higher than the fractionated viscera pretenses, but their amidolytic activities at pH 6.0 and 8.0 toward synthetic substrates were lower than counterpart. The fractionated pretenses from fish viscera would be utilized as commercial pretenses.

• The Korean Journal of Food And Nutrition
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• v.18 no.1
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• pp.11-18
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• 2005

Angiotensin Ⅰ converting enzyme(ACE) inhibitory activities of laver(Porphyra tenera) protein hydrolysates were investigated by enzymes used for hydrolysis, molecular fractions and drying methods. For the enzymatic hydrolysis, crude laver protein, separated by filtration of water extract of dried laver extracted with 20 times(w/v) water for 3 hours at boiling temperature, were hydrolyzed with three commercial protease, Pepsin, alcalase and maxazyme NNP at optimal conditions. The yield of hydrolysis and ACE inhibitory activities of which were high in order of pepsin, alcalase and maxazyme NNP. ACE inhibitory activities of laver hydrolysates by molecular levels were high in order of 3 kDa > 10 kDa > 3∼10 kDa, and the IC/sub 50/ ACE inhibitory activities by molecular lebels were 4 mg/mL(3 kDa), 5 mg/mL(total hydrolysate), and 20 mg/mL(10 kDa), respectively. The storage stability of dried laver hydrolysates at 20℃ were strongly affected by drying methods, hot air dried of which were much stabler than freeze-dried one.

• Journal of the Korean Society of Tobacco Science
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• v.13 no.2
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• pp.27-33
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• 1991

Current studies indicated that emphysema in smokers might be due, in part, to the local suppression of G, -protease inhibitor(u, -Pl) in lung by reactive oxygen species in cigarette smoke or smoke-activated lung neutrophiles. In the present works, we examined the possibility that a measure which inactivated $\alpha$l-Pl by cigarette smoke could be an alternative method to evaluate the cigarette quality, In order to determine the inactivation of $\alpha$1, -Pl, trypsin inhibitory capacity(TIC) was assayed. A rapid loss of $\alpha$1, -Pl activity occurred when $\alpha$1-Pl solutions was exposed the gas phase or total particulate matter(TPM) obtained from various brands. The inactivation of $\alpha$1-Pl by gas phase was dependent upon the number of puffs and the age of the smoke. However, that by TPM was rather decreased since 2 puffs and also showed no more change over 24hrs after exposing. Inactivation of $\alpha$1-Pl determined by our suggested method(5 puffs, 24hours of aging after exposing) using various commercial cigarettes exhibited that high tar brands has inactivated it more strongly than low tar cigarettes. But the ability of some brands to inactivate $\alpha$1-Pl does not correlate with the content of tar or nicotine. These results so여esc that the degree of $\alpha$1-Pl inactivation by cigarette smoke may be a useful index for the evaluation of cigarette quality and that it should be also contribute to the manufacture of less hazardous cigarettes.