• Title/Summary/Keyword: Protease

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Predicting In Sacco Rumen Degradation Kinetics of Raw and Dry Roasted Faba Beans (Vicia faba) and Lupin Seeds (Lupinus albus) by Laboratory Techniques

  • Yu, P.;Egan, A.R.;Leury, B.J.
    • 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).

Effect of Natural Tenderizers or Phosphates on Quality Improvement of the Low-grade Seasoned Hanwoo Ribs (천연연화제 및 인산염의 첨가가 저급양념한우갈비의 품질개선에 미치는 효과)

  • Kim, K. J.;Min, J. S.;Lee, S. O.;Jang, A.;Jang, S. H.;Cheon, Y. H.;Lee, M.
    • Journal of Animal Science and Technology
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    • v.45 no.2
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    • pp.309-318
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    • 2003
  • In order to improve the quality of low-grade seasoned ribs, kiwi powder, pear powder and phosphates were studied. To seasoned ribs, 0.1%, 0.3% or 0.5% of kiwi powder was added and their MFI(Myofibrillar Fragmentation Index) values were 341.4, 368.3 and 405.1, respectively. As the amount of kiwi increased, MFI value increased(p<0.001). Also, when 0.5%, 1.0% or 3.0% of pear powder was added to seasoned ribs, their MFI values were increased as the addition levels of pear powder increased. As the amount of kiwi and pear powder were increased, WHC(Water Holding Capacity) decreased(p<0.001). On the other hand, as the amount of phosphates added increased, WHC increased. In drip loss, as the amount of kiwi and pear powder increased, it increased. However, drip loss was decreased as the amount of phosphates increased. For meat color, ‘L’ values of pear treatment groups were similar to those of control and ‘a’ values were higher in the treatment groups of 0.1${\sim}$0.3% kiwi powder, 0.5${\sim}$1.0% pear powder and 0.3${\sim}$0.5% phosphate than those of control. In sensory evaluation, treatment group of 0.3% kiwi powder and 0.5% phosphate showed the highest values in juiciness, tenderness and acceptability among the treatments(p<0.01).

Studies on Offspring Production by Transfer of Bisected Demi-Embryos in Mice (생쥐에 있어서 양분분할구(兩分分割球) 이식(移植)에 의한 새끼생산(生産)에 관한 연구(硏究))

  • Suh, Tae Kwang;Park, Hang Kyun
    • Current Research on Agriculture and Life Sciences
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    • v.6
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    • pp.145-156
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    • 1988
  • This study was conducted to investigate the effect of embryo stage on bisection rates of embryos, on development of separated demi-embryos and on subsequent development to full term following transfer of demi-embryos to recipients. The results obtained in this study are summarized as follows. The 2-, 4-, 8-cell and morula embryos were mostly obtained at 36-42, 48-54, 62-66 and 72-78 hours after injection of hCG, respectively, and the average number of embryos recovered per head was 17.0. The bisection rate of 8-cell embryos was 83.6%, which was significantly higher than that of morula embryos, 65.5%. But the development of morula demi-embryos to blastocyst after in-vitro culture was better than that of 8-cell demi-embryos and the rate was 76.5% and 60.9%, respectively. When the 2, 10 and 15 cultured demi-embryos or intact embryos were transferred to each recipient mouse, respectively. the highest pregnancy rate was obtained when 15 embryos were transferred. And the overall offspring production rate of intact embryos was higher than that of demi-embryos.

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EFFECT OF CHLORHEXIDINE ON MICROTENSILE BOND STRENGTH OF DENTIN BONDING SYSTEMS (Chlorhexidine 처리가 상아질 접착제의 미세인장결합강도에 미치는 영향)

  • Oh, Eun-Hwa;Choi, Kyoung-Kyu;Kim, Jong-Ryul;Park, Sang-Jin
    • Restorative Dentistry and Endodontics
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    • v.33 no.2
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    • pp.148-161
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    • 2008
  • The purpose of this study was to evaluate the effect of chlorhexidine (CHX) on microtensile bond strength (${\mu}TBS$) of dentin bonding systems. Dentin collagenolytic and gelatinolytic activities can be suppressed by protease inhibitors, indicating that MMPs (Matrix metalloproteinases) inhibition could be beneficial in the preservation of hybrid layers. Chlorhexidine (CHX) is known as an inhibitor of MMPs activity in vitro. The experiment was proceeded as follows: At first, flat occlusal surfaces were prepared on mid-coronal dentin of extracted third molars. GI (Glass Ionomer) group was treated with dentin conditioner, and then, applied with 2 % CHX. Both SM (Scotchbond Multipurpose) and SB (Single Bond) group were applied with CHX after acid-etched with 37% phosphoric acid. TS (Clearfil Tri-S) group was applied with CHX, and then, with adhesives. Hybrid composite Z-250 and resin-modified glass ionomer Fuji-II LC was built up on experimental dentin surfaces. Half of them were subjected to 10,000 thermocycle, while the others were tested immediately. With the resulting data, statistically two-way ANOVA was performed to assess the ${\mu}TBS$ before and after thermo cycling and the effect of CHX. All statistical tests were carried out at the 95 % level of confidence. The failure mode of the testing samples was observed under a scanning electron microscopy (SEM). Within limited results, the results of this study were as follows; 1. In all experimental groups applied with 2 % chlorhexidine, the microtensile bond strength increased, and thermo cycling decreased the micro tensile bond strength (P > 0.05). 2. Compared to the thermocycling groups without chlorhexidine, those with both thermocycling and chlorhexidine showed higher microtensile bond strength, and there was significant difference especially in GI and TS groups. 3. SEM analysis of failure mode distribution revealed the adhesive failure at hybrid layer in most of the specimen. and the shift of the failure site from bottom to top of the hybrid layer with chlorhexidine groups. 2 % chlorhexidine application after acid-etching proved to preserve the durability of the hybrid layer and microtensile bond strength of dentin bonding systems.

Characteristics of Intestinal Microflora and Their Extracellular Pretense of Sardine, Sardinops melanosticta (정어리 내장세균의 특성과 균체외 단백분해효소에 관한 연구)

  • CHANG Dong-Suck;CHO Hak-Rae;CHOI Seung-Tae
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.17 no.3
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    • pp.184-190
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    • 1984
  • Sardine, Sardinops melanosticta, has been caught more than fifty thousand metric tons every year in adjacent sea of Korea, but most of them used for uneatable fish meal because of their rapid spoilage. Usually it is known that the main reason of putrefaction of foods is caused by the maicro-organisms included in them. Therefore, this experiment was carried out to identify the micro-organisms isolated from the intestine of fresh sardine and characterize their proteolytic enzymes produced from them. Aerobic cell count ranged from $1.7{\times}10^4\;to\;3.6{\times}10^5/g$, while anaerobic cell count, from $2.9{\times}10^4\;to\;5.5{\times}10^5/g$. Most of the isolated strains were psychrotrophic mesophiles. Among the two hundred and eighty strains isolated from the fresh samples, fifty-six strains ($20.0\%$) were proteolytics, one hundred and seventy-five strains ($62.5\%$) were lipolytics and tenty-nine strains ($10.5\%$) had the ability to produce hydrogn sulfide. The most predominantly isolated microbial groups from the fresh sardine were Moraxella ($31.4\%$) and Pseudomonas sup. ($28.6\%$). Flavobacterium-Cytophaga, Vibrio, Acinetobacter, Micrococcus spp. and Enterobacteriaceae appeared from $7.9\%\;to\;5\%$ out of total tested strains. The average bacterial count in the spoiled samples (stored at about $18^{\circ}C$ for 48 hours) was increased to the level of $2.9{\times}10^8/g$ for aerobes, $1.5{\times}10^8/g$ for anaerobes, then one hundred and ten strains, corresponding to $52\%$, out of two hundred and thirteen strains submitted to the test were proteolytics. The strongest proteolytic bacterium among the two hundred and eighty strains was identified as Pseudomonas 101 which grew best at $25^{\circ}C$. The optimum condition for the activity of the proteolytic enzyme produced by Pseudomonas 101 appeared $35^{\circ}C$ and pH 9.0, but the activity was relatively unchanged between 5.0 and 11.0 of pH and between $30^{\circ}C\;and\;50^{\circ}C$ of temperature.

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Fungal Microflora on Korean Home-made Meju (재래식(在來式) 메주에 분포(分布)하고 있는 진균(眞菌)에 관한 조사(調査) 연구(硏究))

  • Park, Kyoung-Ja;Kim, Young-Mi;Lee, Bae-Ham;Lee, Bok-Kwon
    • The Korean Journal of Mycology
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    • v.5 no.1
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    • pp.7-12
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    • 1977
  • For this study 3 samples of the home-made meju and 3 samples of the improved meju were selected according to their characteristics. A total of 25 strains of true fungi were isolated from the samples of the home-made meju and identified by the Key of Alexopoulos and Raper, using a strain Aspergillus oryzae A-113 as a control. Amylolytic and proteolytic enzyme activities of the isolated strains were investigated ana the results obtained were as follows. 1. The 25 strains from the home-made meju were identified into 2 Aspergillus oryzae, 14 Asp. flavus, 6 Penicillum spp. 1 Candida sp 1 Spicaria sp and 1 Rhizopus sp. 2. The 3 strains from the improved meju were all identified as Aspergillus oryzae. 3. Aspergillus flavus, A-B, from the home-made meju was found to he the strongest strain in ${\alpha}-amylase$ activity and also to be similar to the strains of Aspergillus orzae from improved meju. 4. Aspergillus flavus, A-7, from the home-made meju was found to be the strogest strain in ${\beta}-amylase$ activity and stronger than that from the improved meju. 5. Aspergillus flavus B-3, was found to be the strongest strain in protease activity and stronger than that from the improved meju. 6. Some of the strains from the home-made meju turned out to be harmful strains, such as Penicillium spp. which secrete antibiotics, Asp. flavus which secretes mycotoxin, Candida sp which causes skin diseases, Spicaria sp. which is a insect pathogen. 7. Rhizopus sp was also found but it has not been proved to be harmful.

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Quality and Storage Characteristics of Low Salted Onion and Five Cereals-Doenjang (저염 양파 오곡된장의 품질 및 저장 특성)

  • Shin, A-Ga;Lee, Ye-Kyung;Jung, Yoo-Kyung;Kim, Soon-Dong
    • Food Science and Preservation
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    • v.15 no.2
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    • pp.174-184
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    • 2008
  • The quality and storage characteristics of low salted onion and five cereals-doenjang (DFO) were investigated. At the DFO, soybean koji ($57{\sim}62%$), onion (3%) and salt (8%) were mixed with equal amount of rice, barley, glutinous millet and glutinous indian millet ($10{\sim}30%$), and water ($7{\sim}12%$). The storage of DFO was done by vacuum packing in polypropylene tube, and sterilized at $121^{\circ}C$. The fermentation and storage was conducted for 60 days at $25^{\circ}C$ at each condition. Control doenjang (GD) was the salinity of 16% soybean doenjang that was not sterilized and packed in plastic containers for storage. During the fermentation, pH of DFO was lower than GD. The pH maintained stability during the storage, and revealed to be lowered, as the proportion of mixed cereals was higher. During the fermentation of DFO, the brix degree revealed to be higher than GD and maintained s1ability during the storage. During the fermentation and storage, the amino nitrogen content of DFO was ranged $400{\sim}470mg%$ by showing higher content than GD and maintained stable content during the storage. During the fermentation, the activities of protease and ${\beta}$-amylase were maintained to be high at DFO, but the activities during the storage were high at GD. The color $L^*$ value of DFO during fermentation and storage maintained higher values than GD, but $a^*$ value revealed lower pattern. Total free amino acids of DFO was ranged $1,918{\sim}2,290mg%$ which was higher than GD that recorded 1,291 mg%. When the sensory evaluation was conducted for DFO that was fermented and stored for 60 days, the DFO mixed with $20{\sim}30%$ of cereals resulted to have more savory taste, flavor, and sweeter than GD, and overall acceptability for color and overall taste was high.

A Study on the Cooking in 'The Kosa-sibi Jip' (2) ("고사십이집(攷事十二集)"의 조리가공에 관한 분석적 연구(2))

  • 김성미
    • Journal of the East Asian Society of Dietary Life
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    • v.4 no.3
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    • pp.1-19
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    • 1994
  • In this paper, twenty-five kinds of food presented in Sooljip(戌集) 5 and 6 of Food collections of 'Kosa-sibi Jip(攷事十二集)' have been classified into four : Staple food, subsidiary food, Tuck(rice cake) and Han-gwa(Korean confectionery), and Tang-jng and tea. Cooking processes have been examined and scientifically analyzed in terms of cooking, Fourteen kinds of Jook (thick gruel with cereal) as well as Urak-Jook were presented among the methods of making Jook, one of staple foods. Milk and ground rice were boiled together into Urak-Jook, which was nutritious because of carbohydrate, added to milk. Hong-sa Myun was mode of ground shrimps, ground bean, ground rice and flour which were kneaded together. It was a nutritiously balanced food. Nineteen kinds of Kimchi presented in this book were classified by the recipes. The five of Jook-soon Ja, U-so Ja, Tam-bok Ja and Jo-gang were made by adding red malt and cereals(boiled rice or candies). Jo-gang, Jo-ga and Jo-gwa-chae were made by adding salt and rice wine. With salt and fermenters added, eight were made. Chim-jup-jeo-ga was made by adding Jang(soy-bean sauce) and the inner chaff of wheat instead of salt. The four of Ka-za-san, Hwang-gwa-san, Tong-gwa-san and Jo-gang were made by adding salt and vinegar. Jo-gang was made by adding salt, rice wine, residue of rice wine and candies. The four of Kae-mal-ga, Ku-cho-chim-chae, Un-gu-hwa and Suk-hwa-chim-chim-chae were made by adding salt and spices. San-got-Kimchi was made without salt. San-got-Kimchi and Suk-hwa-chim-chae were made originally in Korea. Suk-hwa-chim-chae, in particular, was first classified as a kind of Kimchi in this book and oysters were added, which is notable. Pork could be preserved longer when smoked oven the weak fire of thatch ten days and nights. Dog meat was sauced and placed on the bones in a pot. A porcelain was put on the top of the pot. Flour paste sealed the gap between the porcelain and the pot. Some water was poured into the porcelain, and the meat was steamed, with two or three thatched sacks burned, which was a distilled dry steaming. This process has been in use up to now. Various cooking methods of chicken were presented from in Umsik-dimi-bang to in Chosun Musang Sinsik Yori Jebup. These methods were ever present regardless of ages. Such measuring units as Guin(斤) and Nyang(兩) were most frequently used in cooking processes of this book, except in case of Jang(soy bean sauce), vinegar and liquor. Twenty eight kinds of kitchenware and cookers were used, of which porcelains wee most used and pans and sieves followed. The scientific eight cooking methods were as follows. First, salt was refined through saturated solution. Next, it was recommended Hong-sa Myun containing shrimps should not be taken along with pork, which is thought to be a proper diet in terms of cholesterol contained by shrimps and pork. Third, meat was coated with thin gruel and quickly roasted and cleared of the dried gruel membrane, which prevented nutrients from exuding and helped to make the meat well-done. Fourth, The fruit of paper mulberry trees has the protease which can soften meat. Therefore when meat was boiled with th fruit of paper mulberry trees, it can be softened easily. Fifth, pork was smoked over the weak fire of thatch. Sixth, in cooking dog meat, distilled dry steaming raised the boiling point and made it possible to preserve meat longer. Seventh, in boiling the sole of a bear, lime was added, which made meat tender by making the pH lower or higher than that of raw meat. Finally, in boiling down rice gluten, a porcelain in the pot prevented boiling over the brim, which is applied to pots in which to boil medical herbs.

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Urokinase Plasminogen Activator Receptor Gene Expression and Clinico-Pathologic Feature in Gastric Cancer Patients (위암 환자의 Urokinase Plasminogen Activator Receptor 유전자의 발현양상)

  • Kim Yong Gil;Lee Kyung Hee;Kim Min Kyung;Lee Jae Lyun;Hyun Myung Sue;Kim Sang Hun;Kim Hee Sun
    • Journal of Gastric Cancer
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    • v.4 no.4
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    • pp.207-212
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    • 2004
  • Purpose: Invasion and metastasis in solid tumors require the action of tumor-associated proteases. The serine protease urokinase-type plasminogen (uPA) and receptor (uPAR) appear to have a major function in these processes. Expression of the uPAR is elevated in breast and colon carcinomas, and this is often associated with invasiveness and poor prognosis. The purpose of this study was to determine whether the expression of the uPAR gene correlates with clinico-pathological parameters in human gastric carcinomas. Materials and Methods: We examined the expression of uPAR mRNA by using northern blot analysis and RT-PCR in 35 gastric carcinomas and the surrounding normal mucosa. Macroscopic and histopathological tumor findings and survival rates were obtained from the patient records and from endoscopic, surgical, and pathological reports. Results: The expression of uPAR and was higher in most neoplasms than in the corresponding normal mucosal tissue. uPAR mRNA expression in tumors correlated well with lymph-node metastasis (P<0.02) and tumor stage (P<0.01). The survival rate of patients with tumors displaying high uPAR expression levels was significantly lower (P<0.04) than that of patients without uPAR expression, but IL-8 showed only the tendency of survival difference. Conclusion: These results suggest that uPAR may be an important prognostic factor in human gastric carcinomas.

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Biochemical Characterizations of Phenylalanine Ammonia-Lyase and its Mutants to Develop an Enzymatic Therapy for Phenylketonuria (페닐케톤뇨증의 효소치료 개발을 위한 phenylalanine ammonia-lyase 및 유전자 변이형의 생화학적 특성)

  • Kim, Woo-Mi
    • Journal of Life Science
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    • v.19 no.9
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    • pp.1226-1231
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    • 2009
  • Enzyme substitution with recombinant phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) is currently being explored for treatment of phenylketonuria (PKU), an autosomal recessive genetic disorder with mutations of the gene encoding phenylalanine-4-hydroxylase (EC 1.14.16.1). However, oral administration of PAL is limited because of proteolytic digestion in the gastrointestinal tract. The aim of this study was to determine the biochemical properties of PAL and delinate the susceptibility of wild-type PAL to pancreatic proteolysis by exploring several mutants, and to develop therapeutic drugs with PAL for PKU. The specific activity of PAL was assayed and its optimal pH, temperature stability, and intestinal protease susceptibility were investigated. Its $V_{max}$ values for phenylalanine and tyrosine were 1.77 and $0.47{\mu}mol$/ min/mg protein, respectively, and its $K_m$ values were $4.77{\times}10^{-4}$ and $4.37{\times}10^{-4}\;M$, respectively. PAL showed an optimal pH at 8.5, corresponding to the average pH range of the small intestine. It showed no loss of activity at $-80^{\circ}C$ for 5 months and possessed 93.4% of its activity under $4^{\circ}C$ for 4 wks. PAL was susceptible to chymotrypsin digestion and, to a lesser extent, to trypsin, elastase, carboxypeptidase A, and B. The trypsin and chymotrypsin cleaving sites were mutated to investigate protection from pancreatic digestion and the specific activities of these mutants were evaluated. The six mutants displayed low specific activities compared to the wild-type, suggesting that the primary trypsin and chymotrypsin cleaving sites may be essential for catalytic reaction. The PAL mutants could therefore be applied as a pretreatment modality without susceptibility to proteolytic attack, however, additional modification for enhancing enzymatic activity is needed to reduce the Phe levels effectively.