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1.
Dan Wu Xiao Wei Yu Tong Chun Wang Rui Wang Yan Xu 《Biotechnology and Bioprocess Engineering》2011,16(2):305-311
Rhizopus lipases have been successfully expressed in Pichia pastors and different fermentation strategies have been investigated. However, there is no sufficient study on the effects of methanol
concentration on the production of Rhizopus lipases in P. pastors. In this study, the lipase from Rhizopus chinensis CCTCC M20102 was expressed under different fed-batch fermentation conditions at methanol concentrations ranging from 0.5
to 3.5 g/L. The lipase activity, stability, and productivities were analyzed. The optimum methanol concentration was 1 g/L,
with the highest lipase activity of 2,130 U/mL, without degradation. Additional information was obtained from the analysis
of methanol consumption and production rates. The results also suggested that the cell concentration at the end of the glycerol
fed-batch phase was very important for cell viability and protease activity. 相似文献
2.
The study was designed to determine whether methionine-enkephalin (M-ENK) was present in the digestive system of the scallop
Chlamys farreri and investigate the effects of M-ENK on the activity of amylase, protease and lipase in the digestive system of C. farreri. The results indicated that M-ENK-like material was present in the epithelium and connective tissue of labial palps, mouth
labia, stomach, intestine, rectum, and hepatopancreas of the scallop C. farreri. Moreover, it was also found that many isolated small cells showing M-ENK-like immunoreactivity were scattered in the epithelial
layer of intestine, and many isolated big epithelial cells showing M-ENK-like immunoreactivity were scattered in tubules of
hepatopancreas of the scallop. The activity levels of amylase and lipase in crystalline style, hepatopancreas and intestine
were enhanced at 1 h after injection of exogenous M-ENK into adductor muscle of the scallops, whereas protease activity levels
were significantly suppressed. Our report constitutes the first characterization of M-ENK in the digestive system of scallop
C. farreri and investigates the effects of M-ENK on the activities of digestive enzymes of mollusk for the first time. The results suggest
an involvement of M-ENK in the functional regulation of the digestive system of C. farreri. 相似文献
3.
Timo Thünken Sebastian A. Baldauf Nicole Bersau Theo C. M. Bakker Harald Kullmann Joachim G. Frommen 《Hydrobiologia》2010,654(1):137-145
Parasites with a complex life cycle are supposed to influence the behaviour of their intermediate host in such a way that
the transmission to the final host is enhanced, but reduced to non-hosts. Here, we examined whether the trophically transmitted
bird parasite Polymorphus minutus increases the antipredator response of its intermediate host, the freshwater amphipod Gammarus pulex to fish cues, i.e. non-host cues (‘increased host abilities hypothesis’). Aggregation behaviour and reduced activity are
assumed to decrease the predation risk of gammarids by fishes. Uninfected G. pulex are known to aggregate in the presence of a fish predator. In the present study, gammarids were allowed to choose either
to join a group of conspecifics or to stay solitary (experiment 1) or between two groups differing in infection status (experiment
2), both in the presence or absence of fish odour. The perception of the groups was limited to mainly olfactory cues. Contrary
to the ‘increased host abilities hypothesis’, in infected gammarids of experiment 1, fish cues induced similar aggregation
behaviour as in their uninfected conspecifics. In experiment 2, uninfected as well as infected gammarids did not significantly
discriminate between infected and uninfected groups. Although only uninfected gammarids reduced their activity in the presence
of predator cues, infected G. pulex were generally less active than uninfected conspecifics. This might suggest that P. minutus manipulates rather the general anti-predator behaviour than the plastic response to predation risk. 相似文献
4.
Yaping Lu Qian Lin Jin Wang Yufan Wu Wuyundalai Bao Fengxia Lv Zhaoxin Lu 《Journal of industrial microbiology & biotechnology》2010,37(9):919-925
A Proteus vulgaris strain named T6 which produced lipase (PVL) with nonpositional specificity had been isolated in our laboratory. To produce
the lipase in large quantities, we cloned its gene, which had an opening reading frame of 864 base pairs and encoded a deduced
287-amino-acid protein. The PVL gene was inserted into the Escherichia coli expression vector pET-DsbA, and active lipase was expressed in E. coli BL21 cells. The secretive expression of PVL gene in Bacillus subtilis was examined. Three vectors, i.e., pMM1525 (xylose-inducible), pMMP43 (constitutive vector, derivative of pMM1525), and pHPQ
(sucrose-inducible, constructed based on pHB201), were used to produce lipase in B. subtilis. Recombinant B. subtilis WB800 cells harboring the pHPQ-PVL plasmid could synthesize and secrete the PVL protein in high yield. The lipase activity
reached 356.8 U/mL after induction with sucrose for 72 h in shake-flask culture, representing a 12-fold increase over the
native lipase activity in P. vulgaris. The characteristics of the heterologously expressed lipase were identical to those of the native one. 相似文献
5.
Rosa María Camacho Juan Carlos Mateos Orfil González-Reynoso Lilia Arely Prado Jesús Córdova 《Journal of industrial microbiology & biotechnology》2009,36(7):901-909
The present study was conducted to investigate the capability of Haloarcula marismortui to synthesize esterases and lipases, and the effect of physicochemical conditions on the growth and the production of esterases
and lipases. Finally, the effect of NaCl concentration and temperature on esterase and lipase activities was studied using
intracellular crude extracts. In order to confirm the genomic prediction about the esterase and lipase synthesis, H. marismortui was cultured on a rich medium and the crude extracts (intra- or extracellular) obtained were assayed for both activities
using p-nitrophenyl esters and triacylglycerides as substrates. Studies on the kinetics of growth and production of esterase and
lipase of H. marismortui were performed, reaching a maximum growth rate of 0.053 h−1 and maximal productions of intracellular esterase and lipase of 2.094 and 0.722 U l−1 using p-nitrophenyl valerate and p-nitrophenyl laurate, respectively. Both enzymes were produced as growth-associated metabolites. The effects of temperature,
pH, and NaCl concentration on the growth rate and production of enzymes were studied by using a Box–Behnken response surface
design. The three response variables were significantly influenced by the physicochemical factors and an interaction effect
between temperature and NaCl concentration was also evidenced. The surface response method estimated the following maximal
values for growth rate and productions of esterase and lipase: 0.086 h−1 (at 42.5°C, pH 7.4, and 3.6 mol l−1 NaCl), 2.3 U l−1 (at 50°C, pH 7.5, and 4.3 mol l−1 NaCl), and 0.58 U l−1 (at 50°C, pH 7.6, and 4.5 mol l−1 NaCl), respectively. Esterases were active at different salt concentrations, showing two optimal activities (at 0.5 and 5 mol l−1 NaCl), which suggested the presence of two different esterases. Interestingly, in the absence of salt, esterase retained
50% residual activity. Esterases and lipase activities were maximal at 45°C and inactive at 75°C. This study represents the
first report evidencing the synthesis of esterase and lipase by H. marismortui. 相似文献
6.
Prathumpai W Flitter SJ McIntyre M Nielsen J 《Applied microbiology and biotechnology》2004,65(6):714-719
Two recombinant strains of Aspergillus niger (NW 297-14 and NW297-24) producing a heterologous lipase from Thermomyces lanuginosus were constructed. The heterologous lipase was expressed using the TAKA amylase promoter from Aspergillus oryzae. The production kinetics of the two strains on different carbon sources in batch and carbon-limited chemostat cultivations were evaluated. In batch cultivations, the highest total product yield coefficient (Yxp total), given as the sum of extracellular and intracellular yields, was obtained during growth on glucose for the transformant strain NW297-24 (5.7±0.65 KU/g DW), whereas the highest total product yield coefficient was obtained during growth on maltose for the transformant strain NW297-14 (6.3±0.02 KU/g DW). Both transformants were evaluated in glucose-limited chemostat cultures. Strain NW297-14 was found to be the best producer and was thus employed for further analysis of the influence of carbon source in chemostat cultures. Here, the highest total specific lipase productivity (rp total, the sum of extracellular and intracellular lipase productivity) was found to be 1.60±0.81 KU/g DW/h in maltose-limited chemostats at a dilution rate of 0.08 h–1, compared with a total specific lipase productivity of 1.10±0.41 KU/g DW/h in glucose-limited chemostats. At the highest specific productivity obtained in this study, the heterologous enzyme accounted for about 1% of all cellular protein being produced by the cells, which shows that it is possible to obtain high productivities of heterologous fungal enzymes in A. niger. However, SDS-PAGE analysis showed that most of the produced lipase was bound to the cell wall. 相似文献
7.
Shockey J Chapital D Gidda S Mason C Davis G Klasson KT Cao H Mullen R Dyer J 《Applied microbiology and biotechnology》2011,92(6):1207-1217
Saccharomyces cerevisiae is frequently used as a bioreactor for conversion of exogenously acquired metabolites into value-added products, but has
not been utilized for bioconversion of low-cost lipids such as triacylglycerols (TAGs) because the cells are typically unable
to acquire these lipid substrates from the growth media. To help circumvent this limitation, the Yarrowia lipolytica lipase 2 (LIP2) gene was cloned into S. cerevisiae expression vectors and used to generate S. cerevisiae strains that secrete active Lip2 lipase (Lip2p) enzyme into the growth media. Specifically, LIP2 expression was driven by the S. cerevisiae PEX11 promoter, which maintains basal transgene expression levels in the presence of sugars in the culture medium but is rapidly
upregulated by fatty acids. Northern blotting, lipase enzyme activity assays, and gas chromatographic measurements of cellular
fatty acid composition after lipid feeding all confirmed that cells transformed with the PEX11 promoter–LIP2 construct were responsive to lipids in the media, i.e., cells expressing LIP2 responded rapidly to either free fatty acids or TAGs and accumulated high levels of the corresponding fatty acids in intracellular
lipids. These data provided evidence of the creation of a self-regulating positive control feedback loop that allows the cells
to upregulate Lip2p production only when lipids are present in the media. Regulated, autonomous production of extracellular
lipase activity is a necessary step towards the generation of yeast strains that can serve as biocatalysts for conversion
of low-value lipids to value-added TAGs and other novel lipid products. 相似文献
8.
In the northern hemisphere, pike (Esox lucius Linnaeus) is one of the most important recreational fisheries resources, and regulatory or voluntary catch-and-release angling
is common. No information is available about the potential sublethal impacts on pike that catch-and-release fishing may cause,
such as behavioural alterations and changes in habitat choice after release. Radio telemetry with N = 20 pike was used to test the hypothesis that fish modify behaviour by reducing movement as a reaction to a catch-and-release
event in a previously unfished, slightly eutrophic lake, having an area of 25 ha and located in northeastern Germany. During
a 7-month tracking period, activity of pike was monitored for consecutive 24 h every week. Minimum displacement per hour (m)
and distance to shore (m) were significantly lower upon the first post release tracking compared to tracking before the capture.
Two tracking events after capture, both movement and distance to shore were similar to those measured during pre-angling.
There were no significant relations between the change in movement and distance to shore and size of pike. In terms of habitat
choice, pike significantly selected for reed and avoided the pelagic area over the whole study period that was not influenced
by catch-and-release angling. The results indicated that catch-and-release induces short-term behavioural alterations in pike,
probably explained by physiological disturbances and facilitated by evolved anti-predation behaviours. Such alterations, however,
seem to be of short duration and reversible suggesting sublethal catch-and-release impacts on pike behaviour are limited.
Guest editors: J. M. Farrell, C. Skov, M. Mingelbier, T. Margenau & J. E. Cooper
International Pike Symposium: Merging Knowledge of Ecology, Biology, and Management for a Circumpolar Species 相似文献
9.
Xiaoting Zheng Yafei Duan Hongbiao Dong Jiasong Zhang 《Probiotics and antimicrobial proteins》2018,10(3):504-510
A 15-day feeding trial was conducted to investigate the effect of dietary Lactobacillus plantarum on growth performance, digestive enzyme activities and gut morphology of juvenile Pacific white shrimp, Litopenaeus vannamei (initial body weight = 7.96 ± 0.59 g). Four microbound diets were formulated to contain fermentation supernatant (FS), live bacteria (LB), dead bacteria (DB), and cell-free extract (CE) of L. plantarum. Results indicated that final weight was significantly higher in FS, DB, and CE group in comparison to the control group (P < 0.05). The maximum weight gain rate (WGR) and specific growth rate (SGR) of the CE diet group were significantly higher than that of other groups (P < 0.05). The FCR of CE diet group was lower than that of the control, LB, DB, and FS diets groups (P < 0.05). The highest digestive enzyme activities (amylase, lipase, and pepsin activity) in the hepatopancreas and gut of shrimp were observed in the CE diet group. Histological study revealed that dietary CE diet could significantly increase the enterocytes height of shrimp. The administration of cell-free extract of L. plantarum could effectively improve the growth performance of L. vannamei via the improvement of digestive enzyme activities and the enterocytes height of shrimp. The results of this study will be essential to promote application of probiotics in shrimp aquaculture. 相似文献
10.
Elena P. Ivanova I. Yu. Bakunina Olga I. Nedashkovskaya Nataliya M. Gorshkova Yulia V. Alexeeva Elena A. Zelepuga T.N. Zvaygintseva Dan V. Nicolau V.V. Mikhailov 《Current microbiology》2003,46(1):0006-0010
The ecophysiological variabilities in the ectohydrolytic enzyme profiles of the three species of Pseudoalteromonas, P. citrea, P. issachenkonii, and P. nigrifaciens, have been investigated. Forty-one bacteria isolated from several invertebrates, macroalgae, sea grass, and the surrounding
water exhibited different patterns of hydrolytic enzyme activities measured as the hydrolysis of either native biopolymers
or fluorogenic substrates. The activities of the following enzymes were assayed: proteinase, tyrosinase, lipase, amylase,
chitinase, agarase, fucoidan hydrolase, laminaranase, alginase, pustulanase, cellulase, β-glucosidase, α- and β-galactosidases,
β-N-acetylglucosaminidase, β-glucosaminidase, β-xylosidase, and α-mannosidase. The occurrence and cell-specific activities
of all enzymes varied over a broad range (from 0 to 44 μmol EU per hour) and depended not only on taxonomic affiliation of
the strain, but also on the source/place of its isolation. This suggests ‘specialization’ of different species for different
types of polymeric substrates as, for example, all strains of P. citrea and P. issachenkonii hydrolyzed alginate and laminaran, while strains of P. nigrifaciens were lacking the ability to hydrolyze most of the algal polysaccharides. The incidence of certain enzymes such as fucoidan
hydrolases, alginate lyases, agarases, and α-galactosidases might be strain specific and reflect its particular ecological
habitat.
Received: 15 February 2002 / Accepted: 27 March 2002 相似文献
11.
The foodborne pathogen Bacillus cereus can form biofilms on various food contact surfaces, leading to contamination of food products. To study the mechanisms of
biofilm formation by B. cereus, a Tn5401 library was generated from strain UW101C. Eight thousand mutants were screened in EPS, a low nutrient medium. One mutant
(M124), with a disruption in codY, developed fourfold less biofilm than the wild-type, and its defective biofilm phenotype was rescued by complementation.
Addition of 0.1% casamino acids to EPS prolonged the duration of biofilms in the wild-type but not codY mutant. When decoyinine, a GTP synthesis inhibitor, was added to EPS, biofilm formation was decreased in the wild-type but
not the mutant. The codY mutant produced three times higher protease activity than the wild-type. Zymogram and SDS-PAGE data showed that production
of the protease (∼130 kDa) was repressed by CodY. Addition of proteinase K to EPS decreased biofilm formation by the wild-type.
Using a dpp-lacZ fusion reporter system, it was shown that that the B. cereus CodY can sense amino acids and GTP levels. These data suggest that by responding to amino acids and intracellular GTP levels
CodY represses production of an unknown protease and is involved in biofilm formation. 相似文献
12.
A 8-week trial was conducted to evaluate the effect of Codonopsis pilosula on immune and digestion in sea cucumber. The results showed that the total coelomocytes counts (TCC), phagocytic activity and superoxide anion production (O2-), superoxide dismutase (SOD), acid phosphatase (ACP), phenoloxidase (PO) activities in coelomocytes of sea cucumbers were improved significantly after 28 or 42 days. Again dietary C. pilosula had a positive impact by activating the RNA expression levels of complement component 3 (C3), lysozyme and protease, amylase and alginase activities in the gut. Finally the resistance of A. japonicus against Vibrio splendidus was enhanced. 相似文献
13.
The extracellular lipase structural gene was isolated from cDNA of Aureobasidium pullulans HN2-3 by using SMARTTM RACE cDNA amplification kit. The gene had an open reading frame of 1245 bp long encoding a lipase. The coding region of the
gene was interrupted by only one intron (55 bp). It encodes 414 amino acid residues of a protein with a putative signal peptide
of 26 amino acids. The protein sequence deduced from the extracellular lipase structural gene contained the lipase consensus
sequence (G-X-S-X-G) and three conserved putative N-glycosylation sites. According to the phylogenetic tree of the lipases,
the lipase from A. pullulans was closely related to that from Aspergillus fumigatus (XP_750543) and Neosartorya fischeri (XP_001257768) and the identities were 50% and 52%, respectively. The mature peptide encoding cDNA was subcloned into pET-24a
(+) expression vector. The recombinant plasmid was expressed in Escherichia coli BL21(DE3). The expressed fusion protein was analyzed by SDS-PAGE and western blotting and a specific band with molecular
mass of about 47 kDa was found. Enzyme activity assay verified the recombinant protein as a lipase. A maximum activity of
0.96 U/mg was obtained from cellular extract of E. coli BL21(DE3) harboring pET-24a(+)LIP1. Optimal pH and temperature of the crude recombinant lipase were 8.0 and 35 °C, respectively and the crude recombinant lipase
had the highest hydrolytic activity towards peanut oil. 相似文献
14.
Vesta Skrodenytė-Arbačiauskienė Nijolė Kazlauskienė Milda Zita Vosylienė Tomas Virbickas 《Central European Journal of Biology》2012,7(5):878-885
Experimental studies of infection transmission via water from infected to healthy fish were conducted. The dark-brown bacterial colonies typical for Aeromonas salmonicida on tryptone soya agar (TSA) have been isolated and counted (from 3.0±0.6×102 to 3.5±0.5×105 c.f.u. g−1) from the internal organs of naturally infected (NI) and experimentally infected (EI) perch and sea trout. No significant differences in dark-brown bacterial counts were detected between EI perch and EI sea trout. The assessment and comparison of the alterations of the biological parameters of EI European perch and sea trout with bacterium Aeromonas salmonicida subsp. salmonicida with naturally infected perch were conducted. No mortality was recorded in groups of EI perch and sea trout. Whereas, the mortality of NI perch (collected from the main sites of outbreak of disease) was observed from the second day of the experiments. Changes in morphophysiological parameters of EI perch and sea trout were similar. Different alterations in blood cell parameters of EI fish were observed, and the most noticeable was the decrease (P≤0.01) in white blood cell count (WBC) of EI perch and sea trout. Based on these results it can be deduced that there is infection transmission of bacterium A. salmonicida from European perch via water to other fish species. 相似文献
15.
Zhenming Chi Fang Wang Zhe Chi Lixi Yue Guanglei Liu Tong Zhang 《Applied microbiology and biotechnology》2009,82(5):793-804
It has been well documented that Aureobasidium pullulans is widely distributed in different environments. Different strains of A. pullulans can produce amylase, proteinase, lipase, cellulase, xylanase, mannanase, transferases, pullulan, siderophore, and single-cell
protein, and the genes encoding proteinase, lipase, cellulase, xylanase, and siderophore have been cloned and characterized.
Therefore, like Aspergillus spp., it is a biotechnologically important yeast that can be used in different fields. So it is very important to sequence
the whole genomic DNA of the yeast cells in order to find new more bioproducts and novel genes from this yeast. 相似文献
16.
Biswendu Chaudhuri Jennifer Rojek M Margaret Vickerman Jason M Tanzer Frank A Scannapieco 《BMC microbiology》2007,7(1):60
Background
Glucosyltransferases (Gtfs), enzymes that produce extracellular glucans from dietary sucrose, contribute to dental plaque formation byStreptococcus gordoniiandStreptococcus mutans. The alpha-amylase-binding protein A (AbpA) ofS. gordonii, an early colonizing bacterium in dental plaque, interacts with salivary amylase and may influence dental plaque formation by this organism. We examined the interaction of amylase and recombinant AbpA (rAbpA), together with Gtfs ofS. gordoniiandS. mutans. 相似文献17.
The aim of this study is to characterize extracellular phospholipase, proteinase, and esterase activities of Candida parapsilosis and C. metapsilosis isolated from clinical sources. Using PCR-restriction fragment length polymorphism (PCR–RFLP) of the secondary alcohol dehydrogenase
(SADH) gene fragment, we identified 20 as C. parapsilosis and 11 as C. metapsilosis from 31 isolates of C. parapsilosis species complex. No C. orthopsilosis was identified. A significantly high isolation frequency of C. metapsilosis (35.5%) was observed. Subsequent evaluation of enzymatic profile showed that 90.5% of C. parapsilosis and 91.7% of C. metapsilosis isolates were phospholipase producers. No difference in phospholipase activity was observed between two species. In terms
of proteinase, 81.0% of C. parapsilosis and 83.3% of C. metapsilosis isolates were positive. A higher level of proteinase activity was detected in C. parapsilosis. A remarkably high proportion of both C. parapsilosis and C. metapsilosis isolates exhibited strong phospholipase and proteinase activities, suggesting that the production of these two enzymes might
be common for them. On the other hand, both species similarly displayed rare esterase activity, with only one C. parapsilosis and two C. metapsilosis isolates being positive. Our data may further add to the confusion concerning the hydrolytic enzymatic activities of the
C. parapsilosis complex, and a wider collection of isolates and standardized methods may help to address the issue. 相似文献
18.
The effects of aqueous leachate of Nicotiana plumbaginifolia Viv. on germination, seedling growth, amylase activity, sugar and starch contents of germinated seeds of maize (Zea mays L. cv. Uttam) were examined. Effects of leachate on photosynthetic pigments, protein content, activities of nitrate reductase
and some antioxidants were also studied. Higher concentration of aqueous leachate of N. plumbaginifolia reduced the germination rate (GR). However, final germination percentage remained almost unaffected. Lower concentration
of leachate stimulated the amylase activity and resulted in higher sugar content and GR. The increasing concentrations of
leachate inhibited the conversion of starch into sugars. Allelochemicals decreased the amount of chlorophyll a, chlorophyll
b, carotenoids, protein and nitrate reductase activity (NRA). The leachate of lower concentrations stimulated the activity
of peroxidase but slight decrease was recorded in higher concentration. Superoxide dismutase and catalase exhibited concentration
dependent increase except in seedlings treated with 100% concentration of leachate. Impairment of various metabolic activities
due to leachate resulted in decreased root and shoot length. 相似文献
19.
Niu Q Huang X Zhang L Lian L Li Y Li J Yang J Zhang K 《Applied microbiology and biotechnology》2007,75(1):141-148
Bacillus nematocida is a Gram-positive bacterium capable of killing nematodes. Our recent studies identified an extracellular serine protease
Bace16 in B. nematocida as a candidate of pathogenic factor in the infection against nematodes, which displayed a high similarity with the serine
protease family subtilisin BPN’, and the MEROPS ID is S08.034. To further confirm the roles that bace16 played in the mechanism of nematocidal pathogenesis, recombinant mature Bace16 (rm-Bace16) was expressed in Escherichia coli strain BL21 using pET-30 vector system. Bioassay experiments demonstrated that the purified recombinant protease had the
ability to degrade nematode cuticles and kill nematodes. In addition, a bace16 knockout mutant of B. nematocida constructed by homologous recombination showed considerably lower proteolytic activity and less than 50% nematocidal activity
than the wild-type strain. These results confirmed that Bace16 could serve as an important virulence factor during the infectious
process.
Qiuhong Niu and Xiaowei Huang contributed equally to this work. 相似文献
20.
The serine protease gene from a thermophilic fungus Thermoascus aurantiacus var. levisporus, was cloned, sequenced, and expressed in Pichia pastoris and the recombinant protein was characterized. The full-length cDNA of 2,592 bp contains an ORF of 1,482 bp encoding 494
amino acids. Sequence analysis of the deduced amino acid sequence revealed high homology with subtilisin serine proteases.
The putative enzyme contained catalytic domain with active sites formed by three residues of Aspl83, His215, and Ser384. The
molecular mass of the recombinant enzyme was estimated to be 59.1 kDa after overexpression in P. pastoris. The activity of recombinant protein was 115.58 U/mg. The protease exhibited its maximal activity at 50°C and pH 8.0 and
kept thermostable at 60°C, and retained 60% activity after 60 min at 70° C. The protease activity was found to be inhibited
by PMSF, but not by DTT or EDTA. The enzyme has broad substrate specificity such as gelatin, casein and pure milk, and exhibiting
highest activity towards casein. 相似文献