1、碳源对 Bacillus cereus CZ 氨肽酶产量及糖代谢酶活性的影响现代食品科技Modern Food Science and Technology 2014, Vol.30, No.6Effects of Carbon Sources on Production of LeucineAminopeptidase and Activities of Sugar Metabolic EnzymesProduced by Bacillus cereus CZCUI Tang-bing, JIANG LI-xiang, HAN Qian, LI Jun-xiaAbstract: Leucin
2、e aminopeptidase (LAP) produced by Bacillus cereus CZ was investigated that after 48 h fermentation. For the sugar metabolic enzymes, D-galactose was used.Key words: leucine aminopeptidase; Bacillus cereus.Article No.: 1673-9078(2014)6-158-167碳源对 CZ 氨肽酶产量及510006）摘要：生产亮氨酸氨肽酶（LAP）进行了研究。结果表明，L-阿拉伯糖最利于
3、LAP 生产，D-半乳糖和 LAP 产生。L-阿拉伯糖和单糖混合、乳糖或半乳糖与葡萄糖混合 2%的乳糖、半乳糖或阿拉伯糖作为碳源，发酵 48 h 时 LAP 产量并没有达到 -D-半乳糖则强烈抑制该酶的活性，半乳糖激酶受 D-半乳糖强烈诱导。L-L- 阿拉伯糖和半乳糖的浓度为 4%时，LAP 产量达到最高。胞外半乳糖激酶随 D-半乳糖L-阿拉伯糖异构酶活性随 L-阿拉伯糖浓度增加而增加。当 L-阿拉伯糖为 4%时， LAP 产量在发酵 56 h4%时，LAP 产量随时间延长而增加。关键词：; - 半乳糖苷酶；半乳糖激酶；L-阿拉伯糖异构酶；蜡样芽孢杆菌Proteolytic enzymes a
4、re ubiquitous in occurrence,being found in all living organisms 1. ProteolyticReceived date：2014-01-09Corresponding author: CUI Tang-bing (1967-), male, associate professor,ph.D, Engaged in the work of teaching and scientific research inbioengineering and microbial fermentation. enzymes include two
5、kinds of enzymes, exopeptidase (a group of enzymes that catalyze the hydrolysis of single amino acids from the end of a polypeptide chain) and endopeptidase (a group of enzymes that catalyze the cleavage of internal bonds in a polypeptide or protein) 2. Aminopeptidases (APases) are a large and diver
6、se group 158现代食品科技 Modern Food Science and Technology132014, Vol.30, No.6of enzymes that catalyze the cleavage of amino acids from the amino terminal of proteins or peptide substrates. APases play important roles in the maintenance of cell metabolism, such as participate in the presence of MHC I, af
7、fect the formation of bioactive peptides, guarantee the normal cycle of amino acids, combine with DNA to regulate transcription and control the biosynthesis of pyrimidine, alginate and cholera toxins, act an important role in meiosis and so on 3.According to the different optimum reaction substrates
8、, aminopeptidases are divided into leucine aminopeptidase, valine aminopeptidase, alanine aminopeptidase, phenylalanine aminopeptidase, proline ammonia peptide enzymes and so on. Leucine aminopeptidases (LAPs), a class of exoproteases that preferably catalyze the hydrolysis of leucine residues from
9、the amino-termini of proteins or peptides 4, usually show broad substrate specificity. In view of their specificity, structure and wide distribution, LAP has become the most typical representative of the family aminopeptidases.Several LAPs from many tissues and organs have been isolated 5 and charac
10、terized and a substantial amount of knowledge about bacterial aminopeptidases ibelong to a class of zinc-requiring Some LAPs are exceptional enzymes ions for full enzyme activity 7for LAP also have been their amino primary sequences bestatin enzyme kinetics 9. are evolutionarily conserved regions .B
11、. cereus and growing plants, and it is also well adapted for growing in the intestinal tract of insects and mammals 11, B. cereus produces several toxins, so often considered to be a kind of pathogenic bacteria 12, but some strains of B. cereus not involved in foodborne illness outbreaks have been i
12、solated from foods. B. cereus can be used as antagonistic bacteria in biological control, thereby having caused widespread attention. B. cereus itself can beprepared as micro-ecological agents and microbial fertilizers, the European Union has authorized B.cereus var. toyoi strain NCIMB 40112 (Toyoce
13、rin) as feed additive for swine, bovine, poultry and rabbit 14. B. cereus can also produce a varieties of bioactive substances, including cereins 15,16, peptide antibiotics 17,18, enzymes19-22, insecticidal exotoxins 23,24 and hemolysin 25.Few studies were reported about LAP from B. cereus. We have
14、selected a B. cereusa probiotics product used as feed This paper 1 1.1 B.cereus. An (6 g/L), K2HPO4 (2 g/L) and used to activate the strain. The seed 7.0) was composed of peptone (10 g/L), (5 g/L) and NaCl (10 g/L). The basal fermentation medium (pH 7.0) contained glucose (20 4 (1 g/L), yeast extrac
15、t (10 g/L), peptone (10 g/L) and K2HPO4 (2 g/L). Fermentation was performed under the following conditions: 50 mL of fermentation medium with 3% inoculation volume, 37 , 200 r/min. The carbon source was varied during fermentation.1.2 LAP production with different carbon sources and in fermentation t
16、ime courseTo evaluate the effect of different carbon sources on LAP production, glucose, lactose, D-galactose, maltose, soluble starch, sodium bicarbonate and sodium citrate were used. The concentrations of carbon sources were all 20 g/L. After fermentation, the activity of LAP was measured. Each fe
17、rmentation was performed in three replicates. Fermentation flasks containing different carbon sources were sampled every 8 h. OD600, the activities of LAP and the metabolic key enzymes of different sugars were determined respectively.1.3 Effect of mixed sugars on LAP productionTwo kinds of sugars we
18、re mixed at a ratio of 1:1. LAP production and the metabolic key enzymes of159现代食品科技 Modern Food Science and Technology 2014, Vol.30, No.6different sugars were compared under the mixed sugars and four single sugars in the fermentation media. 1.4 LAP production with different concentrations of sugars
19、 and in fermentation time courseThe better two of all mixed and single sugars for LAP production were selected. Varying concentrations (10 g/L, 20 g/L, 30 g/L, 40 g/L, 50 g/L) of the two carbon sources were prepared in three parallel samples for LAP production. Fermentation flasks containing one of
20、the best two carbon sources were sampled every 8 h. OD600, the activities of LAP and the metabolic key enzymes of these two sugars were determined. 1.5 Aminopeptidase activity assayFermentation broth was collected and centrifuged for 15 min at 5000 r/min and 4 . The supernatant was used for enzyme a
21、ctivity assay. The reaction mixture contained 0.5 mL supernatant, 6 mL Tris-HCl buffer and 0.5 mL 26 mmol/L LNA(L-leucine-p-nitroanilide) ethanol solution, incubated for 10 min at 37 , then placed in ice bath for 5 min. OD405 of the reaction solution was measured. One unit of enzyme activity was def
22、ined as the amount of enzyme that catalyzed LNA to produce 1 mol of p1.6 Activity assay of key enzymes pathway of sugarFor extracellularenzymesONP per minute 26,27.Activity assay of Galactokinase. The reaction system (100 L) was composed of 50 mmol/L Tris-HCl (pH 8.0), 10 mmol/L ATP, 8 mmol/L D-Gal
23、(D-galactose), 5 mmol/L MgC12, 10 L supernatant and 50 L ddH2O, incubated for 3 h at 45 and added 3 mL DNS (3,5-Dinitrosalicylic acid) reagents to stop the reaction, after treated in boiling water bath for 10 min, put into ice bath for 2 min, and then OD575TP to Activity of The reaction mixture with
24、 appropriate 0.9 0.5 D-galactose solution, h , then boiled for 10 2in 30 min at 25 and OD540 value as the amount of enzyme that catalyzed produced 1 g tagatose per minute 30.2 LAP production with different carbon sourcesmin at 5000 r/min and 4 enzyme activity assayactivity assay, fermentation for 15
25、 min at 5000 4 , the pellet was suspended disrupted by by for 40 min, the -galactosidase. The reaction mixture mL supernatant with the appropriate 0.7 mL 0.6% ONPG (ortho- nitrophenyl-beta-D-galactopyranoside) solution, after incubated for 10 min at 37 , the reaction solution was added by 0.8 mL 5%
26、Na2CO3 for the color development, then the above solution was added by 6.4 mL phosphate buffer (pH 7.0), shaked evenly and then OD420 value was measured. One unit of activity was defined as the amount of enzyme that catalyzed ONPG to produce 1 mol of160Fig.1 LAP production with different carbon sour
27、ces Note: The concentration of carbon sources was 2%, fermentation time was 48 h. Data are the mean of three replicates, and the error bars represent the standard deviation.To determine the optimal carbon source for LAP production, eight different carbon sources were used. Among various carbon sourc
28、es, L-arabinose showed the highest LAP production (202.77 1.19 U/mL), followed by D-galactose, lactose and sodium citrate, but glucose,现代食品科技 Modern Food Science and Technology 2014, Vol.30, No.6maltose, sucrose, and sodium bicarbonate exhibited very low LAP production (Fig. 1). The same finding is
29、reported by Yang 31, they find that protease production by Bacillus subtilis is greatly enhanced by addition of lactose or arabinose into the medium and that 1% (m/V) arabinose is the most effective substrate for protease production. In our study, glucose showed carbon catabolite repression on LAP p
30、roduction (Fig. 1). Similarly, for Marine Aspergillus flavus, the minimum aminopeptidase activity was found to be produced with the use of glucose, but arabinose, lactose, sucrose inhibited LAP production, the maximum aminopeptidase activity was with the use of maltose 32. Transcription of the yeast
31、 vacuolar aminopeptidase yscI-encoding gene (APE1) is regulated by the carbon source, responding to glucose catabolite repression 33. On the contrary, there are reports, which show that glucose stimulates the aminopeptidase production from Lactococcus lactis subsp. lactis and cremoris 34, thermophil
32、ic Streptomyces strains 35 and the marine bacterium Vibrio SA1 36. After 48 h of 202.77 1.19 U/mL of LAP was obtained, and the level of LAP production was close to so far the highest LAPproduction (376.9 by Li Bin et al. 37.reached to the highest value (73.49 9.21 U/mL) at 48 h, but the extracellula
33、r enzyme activity did not appear until 32 h, only 15.81 1.47 U/mL at 48 h (Fig. 2b), which maybe come from cell autolysis .2.2 activities of their metabolic enzymesIn this experiment, lactose for better LAP used as sources. In LAP production and the of three sugars, of galactokinase and LAP producti
34、on trend 40 h, but fast sugars as carbon optimal carbon source for glucose strong inhibitory effect on LAP-galactosidase, one of three enzymes encoded by lactose operon, cleaves -linked galactose residues from various compounds and is commonly used to cleave lactose into galactose and glucose 38. -g
35、alactosidase showed mainly intracellular enzyme activity, and increased quickly from 8 h to 40 h, and rapidly from 40 h to 48 h. The intracellular -galactosidase productionFig.2 LAP production, cell biomass and activities of carbonmetabolic enzymes in time courseNoet: The concentrarion of sugars was
36、 2%, fermentation time was 48 h; (a) LAP production and cell biomass. Real lines standed for cell biomass, broken lines standed for LAP production; ()161现代食品科技 Modern Food Science and Technology 2014, Vol.30, No.6glucose, () lactose, () D-galactose, () L-arabinose; (b) the activities of galactosidas
37、e. () extracelluar activities, () intracelluar activities; (c) the activities of galactokinase. () extracelluar activities, ( ) intracelluar activities; (d) the activities of L-arabinoseisomerase. ( ) extracelluar activities, () intracelluar activities. Data are the mean of three replicates, and the
38、 error bars represent the standard deviation.biomass attained the highest with lactose + glucose as carbon sources, followed by glucose, lactose + L-arabinose, D-galactose + glucose, L-arabinose + glucose, D-galactose + L-arabinose, L-arabinose, OD600 values were all above 10. But lactose, lactose+D
39、-galactose, especially D-galactose seemed disadvantageous to cellgrowth.Galactokinase, one of three enzymes encoded by galactose operon, is an ATP-dependent enzyme that catalyzes the phosphorylation of galactose to form galactose-1-phosphate. The conversion of D-galactose to the more metabolically u
40、seful glucose-1-phosphate is accomplished by the action of four enzymes in the Leloir pathway 39. B. cereus CZ produced both intracellular and extracellular galactokinase with very low activities. Galactokinase production increased rapidly from 0 h to 8 h, the intracellular part remained stable from
41、 8 h to 48 h, the extracellular part remained stable from 8 h to 32 h, but extracellular part was one time higher than that of the intracellular part (Fig. 2c).L-arabinoseisomerase is one of encoded by arabinose operon. B. cereusactivity of L-arabinoseisomerase. The from 0 h to 48 h, reached 175.11
42、CZ. LAP better carbon source D-galactose and the highest at 24 h 2.3 B. cereus CZ reached the highest amount as carbon source, whereas displayed the lowest amount (only 4.20 0.05 U/mL) with glucose as carbon source. Compared with L-arabinose alone, L-arabinose mixed with glucose, lactose or galactos
43、e reduced largely LAP production. Especially, L-arabinose mixed with glucose resulted in about seven times reduction, lactose and galactose mixed respectively with glucose had a similar impact on LAP production. Cell162Fig.3 LAP production, cell biomass and activities of carbon metabolic enzymes wit
44、h simple sugars and mixed sugars现代食品科技 Modern Food Science and Technology 2014, Vol.30, No.6Note: The total concentration of sugars was 2%, mixed sugars contained two simple sugars (according to the ratio of 1:1), fermentation time was 48 h. (a) LAP production and cell biomass. Line chart standed fo
45、r cell biomass, bar chart standed for LAP production; (b) the activities of galactosidase. Black column chart standed for extracellular activities, blank column chart standed for intracellular activities; (c) the activities of galactokinase. Black column chart standed for extracellular activities, b
46、lank column chart standed for intracellular activities; (d) the activities of L-arabinoseisomerase. Black column chart standed for extracellular activities, blank column chart standed for intracellular activities. Data are the mean of three replicates, and the error bars represent the standard devia
47、tion.the Leloir pathway, a metabolic pathway has been found in most organisms for the catabolism of -D-galactose to glucose 1-phosphate 46. In our study, galactokinase was induced strongly by D-galactose, reached 0.26 0.02 U/mL (the extracellular activity 0.14 0.02 U/mL, the intracellular activity 0.13 0.00 U/mL). L-arabinose, lactose + D-galactose, glucose + D-galactose and L-arabinose + D-galactose had similar inducing effects on galactokinase
