@article{langeveld_balkema-buschmann_becher_thomzig_nonno_andreoletti_davidse_di bari_pirisinu_agrimi_et al._2021, title={Stability of BSE infectivity towards heat treatment even after proteolytic removal of prion protein}, volume={52}, ISSN={["1297-9716"]}, DOI={10.1186/s13567-021-00928-8}, abstractNote={AbstractThe unconventional infectious agents of transmissible spongiform encephalopathies (TSEs) are prions. Their infectivity co-appears with PrPSc, aberrant depositions of the host’s cellular prion protein (PrPC). Successive heat treatment in the presence of detergent and proteolysis by a keratinase from Bacillus licheniformis PWD-1 was shown before to destroy PrPSc from bovine TSE (BSE) and sheep scrapie diseased brain, however data regarding expected reduction of infectivity were still lacking. Therefore, transgenic Tgbov XV mice which are highly BSE susceptible were used to quantify infectivity before and after the bovine brain treatment procedure. Also four immunochemical analyses were applied to compare the levels of PrPSc. After heating at 115 °C with or without subsequent proteolysis, the original BSE infectivity of 106.2–6.4 ID50 g−1 was reduced to a remaining infectivity of 104.6–5.7 ID50 g−1 while strain characteristics were unaltered, even after precipitation with methanol. Surprisingly, PrPSc depletion was 5–800 times higher than the loss of infectivity. Similar treatment was applied on other prion strains, which were CWD1 in bank voles, 263 K scrapie in hamsters and sheep PG127 scrapie in tg338 ovinized mice. In these strains however, infectivity was already destroyed by heat only. These findings show the unusual heat resistance of BSE and support a role for an additional factor in prion formation as suggested elsewhere when producing prions from PrPC. Leftover material in the remaining PrPSc depleted BSE preparation offers a unique substrate for searching additional elements for prion infectivity and improving our concept about the nature of prions.}, number={1}, journal={VETERINARY RESEARCH}, author={Langeveld, Jan P. M. and Balkema-Buschmann, Anne and Becher, Dieter and Thomzig, Achim and Nonno, Romolo and Andreoletti, Olivier and Davidse, Aart and Di Bari, Michele A. and Pirisinu, Laura and Agrimi, Umberto and et al.}, year={2021}, month={Apr} }
 @article{zang_shih_cheng_liu_liu_lu_2020, title={Thermophilic solid state anaerobic digestion of switchgrass for liquid digestate reuse and organic fertilizer production}, volume={35}, ISSN={["1742-1713"]}, DOI={10.1017/S1742170519000139}, abstractNote={AbstractTwo solid state anaerobic digesters (SSADs), 15 L each, were set up for co-digestion of switchgrass with primary digestate of a liquid anaerobic digester (LAD) and the recirculating leachate. Both the LAD and two SSADs were operated at 50°C. The results showed that the bioreactors were not started up stably until day 16 and day 47 for reactors A and B, respectively. The supplement of LAD digestate or injection of sodium hydroxide (NaOH) into the recirculating leachate readily reinitiated the biogas production to normal daily high rates of the two individual SSADs, one on day 16 and the other on day 47. In contrast to reactor A, there was a longer lag phase for bioreactor B, however, it showed 46.2% [77.9 vs 53.3 L kg−1 volatile solid (VS)] more cumulative biogas yields, and higher reduction rate of total solid, VS, cellulose and hemicellulose of 29.5, 31, 40.6 and 15%, respectively, which was likely due to optimized pH and NaOH pretreated switchgrass during start-up period. Methane contents of biogas increased gradually and stabilized at 50% for both reactors, indicating a normal operation of anaerobic digestion lasted for at least 100 days. The determined parameters of digested residues met China organic fertilizer standard (NY 525-2012) except for high moisture and low total nutrient contents. Therefore, the product of SSAD has the potential value of organic fertilizer. It is concluded that the LAD digestate can be reused as inoculums by co-digestion with agricultural residues for biogas and organic fertilizer production in SSAD.}, number={5}, journal={RENEWABLE AGRICULTURE AND FOOD SYSTEMS}, author={Zang, Jianjun and Shih, Jason C. H. and Cheng, Jay J. and Liu, Zhimin and Liu, Ying and Lu, Wenqing}, year={2020}, month={Oct}, pages={503–512} }
 @article{shih_2012, title={From biogas energy, biotechnology to new agriculture}, volume={68}, ISSN={["1743-4777"]}, DOI={10.1017/s0043933912000529}, abstractNote={Agriculture in the 21st Century is facing new challenges and transformation according to five general trends: commercialisation, internationalisation, science and technology, environment and energy production. In this laboratory, a series of research, development and commercialisation of biogas and enzyme technologies reflect these trends. Anaerobic digestion is a microbial process that converts organic waste into biogas, containing 60–70% methane. A simple thermophilic anaerobic digestion (TAnD) system for poultry manure was developed from the lab to the farm. TAnD at 50 to 60°C produces biogas at high rates and, as a result, the process is efficient and the digester is a compact unit. A pilot plug-flow TAnD was first constructed and operated at NCSU research farm. Multiple benefits of the system were demonstrated. Supported by UNDP, a TAnD to process five tons of manure daily (from 50,000 hens) was constructed in China and has been operating for 20 years. Large scale digesters processing hundreds of tons of manure daily are increasingly popular in China and Europe. Biogas from large digesters can be a significant source of energy to generate electricity or to fuel transportation. Beyond energy production, the digester is a rich source of biomaterials. A feather-degrading bacterium was discovered and isolated from TAnD, and its keratinase enzyme and the gene encoding for the enzyme were subsequently isolated. Genetic manipulation for over-expression and up-scale fermentation have now made the industrial production of the enzyme possible. As a heat-stable protease, the keratinase was found to be useful in processing feather meal, in enhancing feed digestibility and was able to degrade prion proteins (implicated as the disease vector in bovine spongiform encephalopathy).}, number={3}, journal={WORLDS POULTRY SCIENCE JOURNAL}, author={Shih, J. C. H.}, year={2012}, month={Sep}, pages={409–416} }
 @article{stark_spencer_shih_chewning_wang_2009, title={Evaluation of keratinase stability in pelleted broiler diets}, volume={18}, ISSN={["1537-0437"]}, DOI={10.3382/japr.2008-00038}, abstractNote={SUMMARY Versazyme (VZ) is an approved keratinase-based feed additive produced naturally as a fermentation product of Bacillus licheniformis PWD-1. The experiment was designed to test the recovery and efficacy of VZ using typical feed industry pelleting parameters. The pelleted diets were conditioned to 85°C for 30 s and pelleted using a 4 × 32 mm die, cooled, and crumbled. A total of 192 male broiler chicks were fed a corn-soybean meal starter diet formulated at 18.5% CP to 21 d of age. The 4 treatments used in the feeding study were as follows: VZ 0.0%-mash, VZ 0.0%-pellet, VZ 0.1%-mash, and VZ 0.1%-pellet. Body weight and feed consumption were determined at 21 d of age. The recovery of VZ in feed was 93% in the mash diet and 66% in the pelleted diet. There was a 55-g increase in BW gain and 0.17 improvement of FCR when birds were supplemented with 0.1% VZ. Pelleting improved BW gain (P < 0.05) but did not significantly improve the FCR. Growth performance results indicate that pelleting did not affect the efficacy of VZ.}, number={1}, journal={JOURNAL OF APPLIED POULTRY RESEARCH}, author={Stark, C. R. and Spencer, B. E. and Shih, J. C. H. and Chewning, C. G. and Wang, J. J.}, year={2009}, pages={30–33} }
 @article{wang_guo_shih_2008, title={Effects of dietary supplementation of keratinase on growth performance, nitrogen retention and intestinal morphology of broiler chickens fed diets with soybean and cottonseed meals}, volume={140}, ISSN={["1873-2216"]}, DOI={10.1016/j.anifeedsci.2007.04.003}, abstractNote={A preparation of keratinase (KE, 400,000 enzyme units/g) was evaluated as a feed additive for its effects on growth performance, nutrient digestibility and intestinal development in broiler chickens. Four experimental diets were arranged as a 2 × 2 factorial with two sources of protein, either soybean meal (SBM) alone or a combination of SBM and cottonseed meal (1:1 on crude protein basis) and with or without 1.0 g KE/kg diet. Dietary supplementation of KE increased body weight gain from 1 to 21 days of age and improved feed conversion ratio from 1 to 42 days of age. Also, KE supplementation enhanced nitrogen retention (P<0.001) and starch digestibility (P<0.001), reflecting the high level of KE and some amylase activities in the enzyme preparation. Microscopic examination of the small intestine on day 21 indicated that KE increased the ratio of villus height to crypt depth (P<0.05). The results suggest that KE has positive effects on growth performance, dietary component digestibility and intestinal development of broilers.}, number={3-4}, journal={ANIMAL FEED SCIENCE AND TECHNOLOGY}, author={Wang, Haiying and Guo, Yuming and Shih, Jason C. H.}, year={2008}, month={Jan}, pages={376–384} }
 @article{wang_garlich_shih_2006, title={Beneficial effects of versazyme, a keratinase feed additive, on body weight, feed conversion, and breast yield of broiler chickens}, volume={15}, ISSN={["1537-0437"]}, DOI={10.1093/japr/15.4.544}, abstractNote={Abstract Versazyme (VZ) is a keratinase-based feed additive. Broiler diets were formulated with low (95%), medium (100%), or high (105%) CP and amino acid levels relative to US Poultry Industry Reporting Service averages (2003). In each diet, amino acid minimums were maintained relative to the percentage of CP. The protein levels were as follows: starter—21, 22, 23%; grower—19, 20, 21%; finisher—17, 18, 19%; and withdrawal—16, 17, 18%. Mixed sex broilers 0 to 48 d of age were fed the 3 protein levels with and without VZ (0.10%) in a 3 × 2 factorial experiment. Statistical main effects indicated 48-d BW was greater on the high protein diets (2.42 kg) compared with low protein (2.36 kg), with the medium protein diet being intermediate (2.40 kg). Overall, VZ significantly (P}, number={4}, journal={JOURNAL OF APPLIED POULTRY RESEARCH}, author={Wang, J. J. and Garlich, J. D. and Shih, J. C. H.}, year={2006}, pages={544–550} }
 @article{shih_wang_2006, title={Keratinase technology: from feather degradation and feed additive,to prion destruction}, volume={1}, ISBN={1749-8848}, DOI={10.1079/pavsnnr20061042}, abstractNote={Abstract
          
            A bio-process called thermophilic anaerobic digestion that converts poultry waste into biogas energy was developed in our laboratory. During operation, feathers in the waste were completely degraded. This observation prompted a series of studies that have led to new technology and commercial developments. A feather-degrading bacterium was discovered and isolated. An enzyme called keratinase capable of hydrolysing the feather protein keratin was purified. The gene encoding this enzyme was isolated and sequenced. Genetic modification enabled the over expression of the gene in the parent strain as well as in other
            Bacillus
            strains. Scale-up fermentation, first in 150 l and then in 50 kl fermentors, made the mass production of the enzyme possible. Subsequently, in application research, keratinase was shown to partially hydrolyse feathers and convert them into a digestible feed protein. When supplemented as a feed additive on standard diet, the enzyme promoted chicken growth by improving dietary protein digestibility. Ifcommercially developed, keratinase can lower feed cost, increase poultry meat yield, and upgrade the utilization of less digestible protein feedstuffs. From an environmental point of view, keratinase has the potential to reduce waste nitrogen excretion by farm animals. More recently, keratinase was found to be able to degrade prion protein that is believed to cause transmissible spongiform encephalopathies (TSE) including ‘mad cow disease’, sheep scrapie, deer chronic wasting disease and human Creutzfeldt–Jakob disease. This discovery indicated the potential use of keratinase in a new enzymatic process to destroy prion protein and possibly prevent TSE, in a cost-effective and environmentally friendly manner. To test and to develop the enzymatic process, a prion-like surrogate protein (PSP) has been developed as a safe bio-marker. The combination of keratinase and PSP can provide a useful toolbox for TSE prevention. Based on the keratinase platform technology and its applications, BioResource International (BRI), a biotechnology company, was founded to serve the poultry and animal industries.
          }, number={042}, journal={CAB Reviews: Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources}, author={Shih, J. C. H. and Wang, J. J.}, year={2006}, pages={6} }
 @article{wang_borwornpinyo_shih_2007, title={Sup35NM-His6 aggregates: A prion-like protein useful in prion degradation studies}, volume={40}, ISSN={["0141-0229"]}, DOI={10.1016/j.enzmictec.2006.09.019}, abstractNote={A prion-like protein, Sup35NM-His6, which is safe, easy to produce and able to aggregate into stable amyloid, was developed. This study was to test the feasibility of using Sup35NM-His6 amyloid as a marker for standard sterilization methods known to be effective in inactivating infectious prions. Also Sup35NM-His6 aggregates were spiked into cow brain tissue homogenates to mimic prions in tissues to determine if the aggregate protein could be specifically detected by Western blot at the nanogram level. Like mammalian prions, the proteinase K (PK) resistant fractions of Sup35NM-His6 remain intact after autoclaving. Treatments with 0.1N NaOH or 1.0% NaOCl also resulted in PK undigestible residues. Exposure to the strong denaturant guanidine thiocyanate (>3 M) destabilized and made the protein PK-digestible. Under strong alkaline conditions of 1.0N NaOH, 2.5% NaOCl, or a combination of NaOH (0.1 and 1.0N) with autoclaving, Sup35NM-His6 was completely hydrolyzed such that it was no longer detectable by Western blot. Overall, these tests suggest that Sup35NM-His6 could be a useful tool to assess the effectiveness of prion degradation for the prevention of TSE.}, number={4}, journal={ENZYME AND MICROBIAL TECHNOLOGY}, author={Wang, Jeng-Jie and Borwornpinyo, Rattana and Shih, Jason C. H.}, year={2007}, month={Mar}, pages={976–981} }
 @article{chen_rojanatavorn_clark_shih_2005, title={Characterization and enzymatic degradation of Sup35NM, a yeast prion-like protein}, volume={14}, ISSN={["1469-896X"]}, DOI={10.1110/ps.041234405}, abstractNote={AbstractTransmissible spongiform encephalopathies (TSEs) are believed to be caused by an unconventional infectious agent, the prion protein. The pathogenic and infectious form of prion protein, PrPSc, is able to aggregate and form amyloid fibrils, very stable and resistant to most disinfecting processes and common proteases. Under specific conditions, PrPSc in bovine spongiform encephalopathy (BSE) brain tissue was found degradable by a bacterial keratinase and some other proteases. Since this disease‐causing prion is infectious and dangerous to work with, a model or surrogate protein that is safe is needed for the in vitro degradation study. Here a nonpathogenic yeast prion‐like protein, Sup35NM, cloned and overexpressed in E. coli, was purified and characterized for this purpose. Aggregation and deaggregation of Sup35NM were examined by electron microscopy, gel electrophoresis, Congo red binding, fluorescence, and Western blotting. The degradation of Sup35NM aggregates by keratinase and proteinase K under various conditions was studied and compared. These results will be of value in understanding the mechanism and optimization of the degradation process.}, number={9}, journal={PROTEIN SCIENCE}, author={Chen, CY and Rojanatavorn, K and Clark, AC and Shih, JCH}, year={2005}, month={Sep}, pages={2228–2235} }
 @article{wang_greenhut_shih_2005, title={Development of an asporogenic Bacillus licheniformis for the production of keratinase}, volume={98}, ISSN={["1364-5072"]}, DOI={10.1111/j.1365-2672.2004.02515.x}, abstractNote={Aims:  Bacillus licheniformis PWD‐1 is a keratin‐degrading, spore‐forming bacterium isolated from a poultry waste digester. A sporulation‐deficient mutant of B. licheniformis PWD‐1, named B. licheniformis WBG, was developed and characterized.}, number={3}, journal={JOURNAL OF APPLIED MICROBIOLOGY}, author={Wang, JJ and Greenhut, WB and Shih, JCH}, year={2005}, pages={761–767} }
 @article{wang_borwornpinyo_odetallah_shih_2005, title={Enzymatic degradation of a prion-like protein, Sup35NM-His6}, volume={36}, ISSN={["1879-0909"]}, DOI={10.1016/j.enzmictec.2004.12.023}, abstractNote={Recent studies indicate that enzymatic treatment of the infectious PrPSc prion under defined conditions could be an effective method to inactivate infectious prions. However, field studies on prion inactivation are hampered by restricted access to the dangerous and expensive infectious prion material. Hence, a surrogate marker for infectious prions would facilitate more practical prion inactivation research. Protein Sup35p, a non-pathogenic prion-like protein produced in yeast, has physical and chemical properties very similar to the BSE prion. Sup35NM-His6, a derivative of Sup35p, was produced from Escherichia coli by gene cloning, protein expression and purification. Monomeric Sup35NM-His6 is soluble. When aggregated, it forms prion-like amyloid, insoluble and resistant to proteases. Similar to BSE prion, a pre-heating step renders this protein digestible by proteinase K, subtilisin and keratinase but not collagenase and elastase. These results indicated that Sup35NM-His6, being simple and inexpensive to produce and non-pathogenic, can be a potential ideal candidate of prion surrogate protein in the study of prion inactivation and prevention of prion diseases.}, number={5-6}, journal={ENZYME AND MICROBIAL TECHNOLOGY}, author={Wang, JH and Borwornpinyo, R and Odetallah, N and Shih, JCH}, year={2005}, month={Apr}, pages={758–765} }
 @article{odetallah_wang_garlich_shih_2005, title={Versazyme supplementation of broiler diets improves market growth performance}, volume={84}, ISSN={["1525-3171"]}, DOI={10.1093/ps/84.6.858}, abstractNote={Day-old male broiler chicks were randomly assigned to 32 floor pens in a completely randomized block design and grown to 6 wk of age. Birds in experiment 1 were fed 1 of 2 basal diets supplemented with or without a protease containing feed additive, Versazyme (VZ). The 4 treatments were 1) control (C), a corn-soybean meal diet that contained 95% of amino acids recommended by NRC except for threonine and isoleucine; 2) C + 0.1% VZ (wt/wt) (C+) in the starter diet only; 3) high (HP) amino acid diet, a corn-soybean meal diet with 100 to 105% of amino acid recommended by NRC except for threonine and isoleucine; and 4) HP + 0.1% VZ (wt/wt) (HP+) in starter diet only. Supplementing both diets with VZ improved BW and feed conversion ratio (FCR) at 21 d of age and BW at 42 d of age. Cumulative 42-d FCR was only improved in birds fed the HP+ diet. Birds in experiment 2 received the following treatments: 1) HP, 2) HP + 0.1% VZ batch A (wt/wt) (A) in starter diet only, and 3) HP + 0.1% VZ batch B (wt/wt) (B) in starter diet only. Enzyme supplementation improved 22-d BW and FCR. There was no significant difference in BW at 43 d of age. Both A and B improved overall FCR (1.758 and 1.751 vs. 1.79 for A and B vs. HP, respectively). In conclusion, supplementation of starter broiler diets with VZ resulted in improved market growth performance.}, number={6}, journal={POULTRY SCIENCE}, author={Odetallah, NH and Wang, JJ and Garlich, JD and Shih, JCH}, year={2005}, month={Jun}, pages={858–864} }
 @article{kang_wang_shih_lanier_2004, title={Extracellular production of a functional soy cystatin by Bacillus subtilis}, volume={52}, ISSN={["1520-5118"]}, DOI={10.1021/jf049711x}, abstractNote={A recombinant Bacillus subtilis producing soy cystatin was developed by subcloning with a soy cystatin gene cloned in Escherichia coli. An active form of cystatin against the cysteine protease from Pacific whiting fillets contaminated with Myxosporidia parasite was constitutively expressed and secreted extracelluarly into the medium. Two gene fragments of signal peptides from kerA and sacB were introduced and compared for secretion efficiency of cystatin. The secretion level of active cystatin improved with the signal peptide of kerA when compared to that of sacB. Inhibitor activity was reduced rapidly after peak expression of the target protein at 36 h of fermentation. The addition of 1% glucose, a suppressor of protease, into the medium sustained the increase of the cystatin activity during fermentation. This study introduced a potential new method for fermentation production of cystatin.}, number={16}, journal={JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY}, author={Kang, IS and Wang, JJ and Shih, JCH and Lanier, TC}, year={2004}, month={Aug}, pages={5052–5056} }
 @article{wang_rojanatavorn_shih_2004, title={Increased production of Bacillus keratinase by chromosomal integration of multiple copies of the kerA gene}, volume={87}, ISSN={["0006-3592"]}, DOI={10.1002/bit.20145}, abstractNote={AbstractTo increase the production of keratinase, stable strains of Bacillus licheniformis carrying multiple keratinase gene copies in the chromosome were developed. Integrative vectors carrying kerA with or without P43‐promoter were constructed and subcloned into B. licheniformis T399D and Bacillus subtilis DB104. In T399D, multiple copies of kerA integration into the chromosome were identified and determined by Southern blot. The optimal integration of kerA was found in the range of 3–5 copies. Higher integration of gene copies (>5) caused reduced processing and secretion of the extracellular keratinase. In DB104, kerA was cloned in the plasmid, not integrated into the chromosome. The strong constitutive promoter P43 not only increased the keratinase production in plasmid‐based expression in DB104 but also improved the enzyme yield of the integrants of T399D. New strains were able to enhance cell growth and enzyme yield at higher concentrations of medium substrate. When they were grown in either soy or feather medium, the keratinase activity was stable and improved by about 4–6 times. © 2004 Wiley Periodicals, Inc.}, number={4}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Wang, JJ and Rojanatavorn, K and Shih, JCH}, year={2004}, month={Aug}, pages={459–464} }
 @article{langeveld_wang_wiel_shih_garssen_bossers_shih_2003, title={Enzymatic degradation of prion protein in brain stem from infected cattle and sheep}, volume={188}, ISSN={["0022-1899"]}, DOI={10.1086/379664}, abstractNote={Prions-infectious agents involved in transmissible spongiform encephalopathies-normally survive proteolytic and mild protein-destructive processes. Using bacterial keratinase produced by Bacillus licheniformis strain PWD-1, we tested conditions to accomplish the full degradation of prion protein (PrP) in brain-stem tissue from animals with bovine spongiform encephalopathy and scrapie. The detection of PrPSc, the disease-associated isoform of PrP, in homogenates was done by Western blotting and various antibodies. The results indicated that only in the presence of detergents did heat pretreatment at >100 degrees C allow the extensive enzymatic breakdown of PrPSc to a state where it is immunochemically undetectable. Proteinase K and 2 other subtilisin proteases, but not trypsin and pepsin, were also effective. This enzymatic process could lead to the development of a method for the decontamination of medical and laboratory equipment. The ultimate effectiveness of this method of prion inactivation has to be tested in mouse bioassays.}, number={11}, journal={JOURNAL OF INFECTIOUS DISEASES}, author={Langeveld, JPM and Wang, JJ and Wiel, DFM and Shih, GC and Garssen, GJ and Bossers, A and Shih, JCH}, year={2003}, month={Dec}, pages={1782–1789} }
 @article{odetallah_wang_garlich_shih_2003, title={Keratinase in starter diets improves growth of broiler chicks}, volume={82}, ISSN={["1525-3171"]}, DOI={10.1093/ps/82.4.664}, abstractNote={The objective of this study was to determine the efficacy of a broad-spectrum protease enzyme, PWD-1 keratinase, upon supplementation to corn-soy starter diets on growth performance of broiler chickens. Three experiments were conducted. In each experiment, 1-d-old broiler chicks were randomly assigned to 24 cage pens of eight birds per pen in a completely randomized design of five experimental treatments and grown to 21 or 26 d of age. Treatments in experiments 1 and 2 were control (C, 21.39% CP), low protein (LP, 18% CP), and LP supplemented with 0.05, 0.1, or 0.15% enzyme preparation (wt/wt). Treatments in experiment 3 were control (C), C+ 0.1% enzyme preparation (C+E) fed starting at either 1 or 5 d of age, LP and LP+ 0.1% enzyme preparation (LP+E). Feeding the LP+E diet produced numerically higher BW at 21 d of age (experiments 1 and 3) and a significantly higher BW at 26 d of age (experiment 2; 1,025 and 1,032 g vs. 965 g for 0.1 and 0.15% vs. LP, respectively, P < 0.05). Feed conversion ratio (FCR) was also improved when chicks were fed the LP+E diet both at 21 (experiment 3) and 26 d of age (experiment 2). In experiment 3, supplementing the C diets with 0.10% enzyme resulted in improvements (P < 0.05) in BW whether the enzyme was supplemented starting at 1 d (767 vs. 695 g for C+E vs. C, respectively) or 5 d of age (764 vs. 695 g for C+E vs. C, respectively). FCR was numerically improved. Furthermore, diets supplemented with the enzyme at any level resulted in reduction of jejunal viscosity at 22 and 27 d of age (P < 0.05). Results of these experiments indicate that the growth of broiler chickens can be significantly improved by dietary supplementation with PWD-1 keratinase.}, number={4}, journal={POULTRY SCIENCE}, author={Odetallah, NH and Wang, JJ and Garlich, JD and Shih, JCH}, year={2003}, month={Apr}, pages={664–670} }
 @article{wang_swaisgood_shih_2003, title={Production and characterization of bio-immobilized keratinase in proteolysis and keratinolysis}, volume={32}, ISSN={["0141-0229"]}, DOI={10.1016/S0141-0229(03)00060-7}, abstractNote={Extracellular production of keratinase–streptavidin fusion protein (KER–STP) was accomplished by the cloning of Bacillus subtilis with a transforming plasmid carrying the kerA-stp fusion gene. The fusion protein was readily immobilized onto a biotinylated solid matrix by mixing in the culture medium. The properties and reaction kinetics of free and immobilized keratinase (KE) were characterized and compared. Heat stability and pH tolerance were greatly improved by immobilization, but the catalytic efficiency (kcat/Km) was reduced by eightfold. The yield of bio-immobilization using bioselective adsorption of the fusion protein was approximately 20%, as estimated from the activity of free KE. HPLC analysis of reaction products demonstrated the hydrolysis of feather keratin, casein, and bovine serum albumin (BSA) by the immobilized KE. The rates of reactions are lower than those of the free enzyme. On the other hand, the stability of the enzyme was greatly improved.}, number={7}, journal={ENZYME AND MICROBIAL TECHNOLOGY}, author={Wang, JJ and Swaisgood, HE and Shih, JCH}, year={2003}, month={Jun}, pages={812–819} }
 @article{wang_swaisgood_shih_2003, title={Bioimmobilization of keratinase using Bacillus subtilis and Escherichia coli systems}, volume={81}, ISSN={["0006-3592"]}, DOI={10.1002/bit.10485}, abstractNote={AbstractImmobilized keratinase can improve stability while retaining its proteolytic and keratinolytic properties. Conventional purification followed by chemical immobilization is a laborious and costly process. A new genetic construct was developed to produce the keratinase–streptavidin fusion protein. Consequently, the purification and immobilization of the fusion protein onto a biotinylated matrix can be accomplished in a single step. The method was tested in both the Bacillus subtilis and Escherichia coli systems. In B. subtilis, the fusion protein was produced extracellularly and readily immobilized from the medium. In E. coli, the fusion protein was produced intracellularly in inclusion bodies; additional separation and renaturation processes were required prior to immobilization from the cell extract. The overall efficiencies were approximately the same, 24–28%, using both systems.  © 2003 Wiley Periodicals, Inc. Biotechnol Bioeng 81: 421–429, 2003.}, number={4}, journal={BIOTECHNOLOGY AND BIOENGINEERING}, author={Wang, JJ and Swaisgood, HE and Shih, JCH}, year={2003}, month={Feb}, pages={421–429} }
 @article{shih_2001, title={Dr Milton L. Scott: A remembrance}, volume={57}, number={4}, journal={World's Poultry Science Journal}, author={Shih, J. C. H.}, year={2001}, pages={465–466} }
 @article{wang_shih_1999, title={Fermentation production of keratinase from Bacillus licheniformis PWD-1 and a recombinant B-subtilis FDB-29}, volume={22}, ISSN={["1367-5435"]}, DOI={10.1038/sj.jim.2900667}, abstractNote={Fermentation scale-up was studied for the production of keratinase by Bacillus licheniformis PWD-1, the parent strain, and B. subtilis FDB-29, a recombinant strain. In both strains, keratinase was induced by proteinaceous media, and repressed by carbohydrates. A seed culture of B. licheniformis PWD-1 at early age, 6-10 h, is crucial to keratinase production during fermentation, but B. subtilis FDB-29 is insensitive to the seed culture age. During the batch fermentation by both strains, the pH changed from 7.0 to 8.5 while the keratinase activity and productivity stayed at high levels. Control of pH, therefore, is not necessary. The temperature for maximum keratinase production is 37 degrees C for both strains, though B. licheniformis is thermophilic and grows best at 50 degrees C. Optimal levels of dissolved oxygen are 10% and 20% for B. licheniformis and B. subtilis respectively. A scale-up procedure using constant temperature at 37 degrees C was adopted for B. subtilis. On the other hand, a temperature-shift procedure by which an 8-h fermentation at 50 degrees C for growth followed by a shift to 37 degrees C for enzyme production was used for B. licheniformis to shorten the fermentation time and increase enzyme productivity. Production of keratinase by B. licheniformis increased by ten-fold following this new procedure. After respective optimization of fermentation conditions, keratinase production by B. licheniformis PWD-1 is approximately 40% higher than that by B. subtilis FDB-29.}, number={6}, journal={JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY}, author={Wang, JJ and Shih, JCH}, year={1999}, month={Jun}, pages={608–616} }
 @misc{shih_lin_miller_1998, title={DNA encoding Bacillus lichenformis PWD-1 keratinase}, volume={5,712,147}, number={1998 Jan. 27}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Shih, J. C. H. and Lin, X. and Miller, E. S.}, year={1998} }
 @article{lin_wong_miller_shih_1997, title={Expression of the Bacillus licheniformis PWD-1 keratinase gene in B-subtilis}, volume={19}, ISSN={["0169-4146"]}, DOI={10.1038/sj.jim.2900440}, abstractNote={The kerA gene which encodes the enzyme keratinase was isolated from the feather-degrading bacterium Bacillus licheniformis PWD-1. The entire gene, including pre-, pro- and mature protein regions, was cloned with Pker, its own promoter, P43, the vegetative growth promoter, or the combination of P43-Pker into plasmid pUB18. Transformation of the protease-deficient strain B. subtilis DB104 with these plasmids generated transformant strains FDB-3, FDB-108 and FDB-29 respectively. All transformants expressed active keratinase in both feather and LB media, in contrast to PWD-1, in which kerA was repressed when grown in LB medium. With P43-Pker upstream of kerA, FDB-29 displayed the highest activity in feather medium. Production of keratinase in PWD-1 and transformants was further characterized when glucose or casamino acids were supplemented into the feather medium. These studies help understand the regulation of kerA expression and, in the long run, can help strain development and medium conditioning for the production of this industrially important keratinase.}, number={2}, journal={JOURNAL OF INDUSTRIAL MICROBIOLOGY & BIOTECHNOLOGY}, author={Lin, X and Wong, SL and Miller, ES and Shih, JCH}, year={1997}, month={Aug}, pages={134–138} }
 @misc{shih_1996, title={Process and apparatus for anaerobic digestion}, volume={5525229}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Shih, J. C. H.}, year={1996} }
 @misc{shih_lee_1993, title={Method and composition for maintaining animals on a keratin-containing diet}, volume={5186961}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Shih, J. C. H. and Lee, C.-G.}, year={1993} }
 @misc{shih_williams_1992, title={Purified Bacillus licheniformis PWD-1 keratinase}, volume={5171682}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Shih, J. C. H. and Williams, C. M.}, year={1992} }
 @misc{shih_williams_1991, title={Method of degrading keratinaceous material and bacteria useful therefor}, volume={5063161}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Shih, J. C. H. and Williams, C. M.}, year={1991} }
 @misc{shih_williams_1990, title={Feather-lysate, a hydrolyzed feather feed ingredient and animal feeds containing the same}, volume={4908220}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Shih, J. C. H. and Williams, C. M.}, year={1990} }
 @misc{shih_williams_1990, title={Method of degrading keratinaceous material and bacteria useful therefore}, volume={4959311}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Shih, J. C. H. and Williams, C. M.}, year={1990} }
 @article{shih_pullman_kao_1983, title={GENETIC SELECTION, GENERAL CHARACTERIZATION, AND HISTOLOGY OF ATHEROSCLEROSIS-SUSCEPTIBLE AND ATHEROSCLEROSIS-RESISTANT JAPANESE QUAIL}, volume={49}, ISSN={["0021-9150"]}, DOI={10.1016/0021-9150(83)90006-0}, abstractNote={A new animal model of atherosclerosis has been developed through genetic selection of Japanese quail (Coturnix coturnix japonica) into susceptible (SUS) and resistant (RES) lines. Characterization of the selected quail has shown that the RES birds were resistant to the disease and developed little atherosclerosis on a diet containing 1% cholesterol. The SUS birds were sensitive and developed severe atherosclerosis in 8-9 wks on a diet containing only 0.5% cholesterol. The histology of the progression of atherosclerosis in the SUS quail was studied. It bore a morphological similarity to that of human arterial atherosclerosis. The atherosclerotic plaque was characterized by intimal thickening, the presence of foam cells, the proliferation of smooth muscle cells and/or fibroblasts, and the formation of scar with collagen deposition. We believe that these two lines of quail may serve as a valid animal model for the study of the genetic and biochemical basis of cholesterol-induced atherosclerosis.}, number={1}, journal={ATHEROSCLEROSIS}, author={SHIH, JCH and PULLMAN, EP and KAO, KJ}, year={1983}, pages={41–53} }