Research Article Received: 5 March 2014
Revised: 1 August 2014
Accepted article published: 8 August 2014
Published online in Wiley Online Library: 2 September 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6868
Purification and characterization of 𝜶-acetolactate decarboxylase (ALDC) from newly isolated Lactococcus lactis DX Yuxing Guo,a Daodong Pan,a,b* Haibing Ding,a Zhen Wu,a Yangying Sunb and Xiaoqun Zengb Abstract BACKGROUND: Diacetyl (2,3-butanedione) is a common flavor aroma from fermented dairy products. There is a need to screen new microorganisms that can efficiently produce large amounts of diacetyl. RESULTS: A new lactic acid bacterium that produced high concentrations of diacetyl was identified based on Gram staining, microscopic examination and 16S rDNA sequence analysis as Lactococcus lactis DX. Its 𝜶-acetolactate decarboxylase (ALDC) was purified using 0.45 g mL−1 ammonium sulfate precipitation, Sephacryl S-300 and S-200 HR and native-PAGE. The purified ALDC displayed a monomer structure and had a molecular mass of about 73.1 kDa, which was estimated using SDS-PAGE. IR analysis showed that the ALDC had a typical protein structure. The optimal temperature and pH for ALDC activity were 40 ∘ C and 6.5 respectively. The ALDC of L. lactis DX was activated by Fe2+ , Zn2+ , Mg2+ , Ba2+ and Ca2+ , while Cu2+ significantly inhibited ALDC activity. Leucine, valine and isoleucine activated the ALDC. CONCLUSION: A strain that had high ability to produce diacetyl was identified as L. lactis DX. The difference in diacetyl production may be due to the ALDC, which is different from other ALDCs. © 2014 Society of Chemical Industry Keywords: 𝛼-acetolactate decarboxylase; diacetyl; purification; characterization
INTRODUCTION
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resulted in an increased concentration of diacetyl. Metabolic engineering methods (e.g. inactivating certain enzymes) have produced large quantities of diacetyl.7 Therefore it is crucial to better understand the characterization of ALDC during the metabolism of diacetyl in order to improve ALDC production. The objective of this study was to purify and characterize ALDC of a high-diacetyl-producing strain so as to understand the characterization of ALDC during the metabolism of diacetyl. A strain demonstrating high productivity of diacetyl was identified by Gram staining, microscopic examination and 16S rDNA sequence analysis. The ALDC of the selected strain was purified using ammonium sulfate precipitation and Sephacryl HR chromatography and then recovered by native polyacrylamide gel electrophoresis (native-PAGE). The ALDC was characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and infrared (IR) analysis.
∗
Correspondence to: Daodong Pan, Department of Food Science and Technology, Jinling College, Nanjing Normal University, Nanjing, 210097, China. E-mail: [email protected]
a Department of Food Science and Technology, Jinling College, Nanjing Normal University, Nanjing, 210097, China b Food Science & Technology Department, Marine Science School, Ningbo University, Ningbo, 315211, Zhejiang, China
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Diacetyl is a common flavor aroma from yogurt, sour cream and other fermented dairy products. Diacetyl helps determine the quality of yogurt and its acceptance by consumers.1 Since consumer demand for diacetyl is increasing, there is growing interest in alternative methods of producing it.2 Some citrate-utilizing lactic acid bacteria such as Lactobacillus, Lactococcus lactis subsp. lactis and Leuconostoc sp. can produce diacetyl.3 However, Aymes et al.3 reported that most of these strains produced only small amounts of diacetyl (∼0.05 mmol L−1 ). Therefore there is a need for new microorganisms that can efficiently produce large amounts of diacetyl. Diacetyl production involves four enzymes: citrate lyase, 𝛼-acetolactate synthetase, 𝛼-acetolactate decarboxylase (ALDC) and diacetyl reductase. 𝛼-Acetolactate is produced from two molecules of pyruvate via the activity of 𝛼-acetolactate synthase.4 𝛼-Acetolactate can be transformed into diacetyl by spontaneous oxidative decarboxylation and into acetoin by spontaneous non-oxidative decarboxylation or by ALDC (EC 4.1.1.5).3 In yoghurt the concentration of acetoin is often higher than that of diacetyl. Le Bars and Yvon5 reported that the aroma of diacetyl is 100-fold more powerful than that of acetoin. ALDC is a key enzyme in producing large amounts of diacetyl. Some researchers have focused on ALDC to improve diacetyl production. Rattray et al.6 impaired the ALDC activity of Leuconostoc pseudomesenteroides, which
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MATERIALS AND METHODS Materials A 16S rDNA bacterial identification polymerase chain reaction (PCR) kit was purchased from Takara Biotechnology (Dalian, China). Sephacryl S-300, Sephacryl S-200 and a molecular weight electrophoresis calibration kit for SDS electrophoresis were purchased from Amersham Biosciences (Uppsala, Sweden). Coomassie Blue R 250, o-phenylenediamine, 𝛼-naphthol, creatine and bovine serum albumin were purchased from Sigma (St Louis, MO, USA). Strain screening and identification Preliminary screening Ten strains were isolated from fermented dairy products and preserved in our laboratory (Nanjing Normal University, Nanjing, China). They were cultured in 0.11 g mL−1 reconstituted skim milk powder at 37 ∘ C for 24 h. Trichloroacetic acid solution
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(0.16 g mL−1 ) was added to the yogurt, which was then was centrifuged at 3056 × g for 20 min. The supernatant was used to measure the diacetyl content in yogurt. Diacetyl was derivatized with o-phenylenediamine, producing 2,3-dimethylquinoxaline with absorbance at 335 nm. A 10 mL aliquot of supernatant was mixed with 0.5 mL of 0.01 g mL−1 o-phenylenediamine and placed in darkness for 30 min. To stop the reaction, 4 mol L−1 HCl solution was added. The absorbance was measured at 335 nm. Diacetyl content was calculated by the equation8 c = 4(1.2E − 0.01), where c (mg L−1 ) is the content of diacetyl in yogurt, E is the absorbance, 1.2 is the conversion coefficient of absorbance and diacetyl content and 0.01 is the adjustment coefficient. Strain identification After preliminary screening, the strain that produced the highest content of diacetyl was named DX. Then strain DX was identified
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(b)
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Figure 1. (a) 16S rDNA of strain DX. Lanes 1–4: PCR amplicon of 16S rDNA gene. (b) Phylogenetic tree based on 16S rDNA sequences showing position of strain DX among closely related organisms. The scale bar represents 0.01 nucleotide substitution per position.
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Purification and characterization of ALDC
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Table 1. Flavor in yogurt fermented by Lactococcus lactis DX Retention time (min) 2.86 3.38 4.10 5.00 7.10 10.40 11.10 11.77 12.59 14.25 14.38 14.75 15.85 16.02 16.42 16.63 16.87 17.17 17.66 18.26 18.67 19.85 20.81 21.62 23.08 25.22 25.38
Compound Acetone Butanone Ethanol Diacetyl Acetyl propionyl Heptanone 3-Methylcrotonaldehyde Methyl butanol Glutaraldehyde Amyl alcohol 2-Methyl-3-pentanol 2-Nonanone Acetic acid 2-Furaldehyde 2-Ethylhexanol 2-Cyclopentene-1,4-dione Benzaldehyde Linalool formate 2,2-Dimethylpropionic acid Butyric acid Furanmethanol 4-Methoxybenzaldehyde oxime Hexanoic acid 2-Phenylethanol Octanoic acid Decanoic acid 2,4-di-tert-Butylphenol
Relative content (%) 1.36 1.56 3.10 8.38 2.70 1.90 0.32 0.69 0.78 0.65 0.85 0.45 6.51 1.94 0.37 0.36 1.62 0.46 0.21 5.94 8.82 4.75 13.63 0.59 10.15 1.37 0.77
Content (mg L−1 ) 3.633 4.167 8.282 22.383 7.212 5.075 0.856 1.843 2.083 1.736 2.270 1.202 17.382 5.182 0.988 0.962 4.325 1.229 0.561 15.866 22.358 12.687 36.405 1.576 27.111 3.659 2.057
using Gram staining, microscopic examination and 16S rDNA sequence analysis. The total DNA of the DX strain was extracted. The strain’s 16S rDNA gene was amplified using PCR with primers K1: 5′ -AACTGAAGAGTTTGATCCTGGCTC-3′ and K2: 5′ -TACGGTTACC TTGTTACGACTT-3′ . The PCR procedure was as follows: 94 ∘ C for 5 min; 30 cycles of 94 ∘ C for 1 min, 56 ∘ C for 45 s and 72 ∘ C for 2 min; 72 ∘ C for 10 min.9 The PCR products were sequenced and then searched in the NCBI BLAST (National Center for Biotechnology Information, MD, USA) sequence database. The phylogenetic tree was established using Clustalx1.8 (UCD Conway Institute, Dublin, Ireland) and Mega 3.1 (The Biodesign Institute, AZ, USA).
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Purification of ALDC Preparation of crude ALDC Lactococcus lactis DX was cultured in MRS medium at 37 ∘ C for 24 h. The fermentation broth was centrifuged (3056 × g, 20 min, 4 ∘ C) to harvest the cells, which were then washed three times with 50 mmol L−1 Tris-HCl buffer (pH 7). The washed cells were ultrasonicated (output power 300 W, work time 3 s, pause time 5 s, 200 times). The clear supernatant was collected by centrifugation (5433 × g, 20 min, 4 ∘ C) and used as crude ALDC.12 Ammonium sulfate precipitation Ammonium sulfate (450 g L−1 ) was added to the crude ALDC. The mixture was centrifuged (5433 × g, 20 min, 4 ∘ C) to collect the precipitate. The collected precipitate was dialyzed against 50 mmol L−1 Tris-HCl buffer (pH 7) overnight at 4 ∘ C. Sephacryl HR chromatography The concentrated crude ALDC solution from the previous step was applied to a Sephacryl S-300 HR column (1 cm × 40 cm) equilibrated with 50 mmol L−1 Tris-HCl (pH 7.8). Proteins were eluted with the same buffer at a flow rate of 25 mL h−1 . The fractions were collected and the ALDC activity was measured. The fractions that contained ALDC activity were pooled and concentrated using polyethylene glycol 20 000. Then the collected ALDC fraction was applied to a Sephacryl S-200 HR column (1 cm × 40 cm). The elution condition was the same as that used for the Sephacryl S-300 HR column. The fractions that contained ALDC activity were pooled and concentrated again. Recovery of ALDC using native-PAGE The active fractions after Sephacryl S-200 HR were examined using native-PAGE with 50 g L−1 acrylamide stacking gel and 100 g L−1 acrylamide running gel. Two gels were prepared. One (gel 1) was stained with Coomassie Blue R 250, while the other (gel 2) was not stained. The protein band (on gel 2) confirmed by gel 1 was excised. The excised gel piece was placed in a microfuge tube and 0.5–1 mL of elution buffer (50 mmol L−1 Tris-HCl, 150 mmol L−1 NaCl, 0.1 mmol L−1 EDTA, pH 7.5) was added. The gel pieces were pulverized and incubated overnight at 4 ∘ C. The mixture was centrifuged (3773 × g, 15 min, 4 ∘ C) to obtain the supernatant, which was measured for ALDC activity. Molecular mass measurement The band that exhibited ALDC activity was recovered using the above method. The molecular mass of the purified ALDC was determined under denaturing conditions by SDS-PAGE with 50 g L−1 acrylamide stacking gel and 100 g L−1 acrylamide running gel. Proteins in the gels were stained with Coomassie Blue R 250. Measurement of ALDC activity ALDC can transform 𝛼-acetolactate into acetoin. The 𝛼-acetolactate was transformed into acetoin by incubation with the obtained ALDC at 45 ∘ C for 30 min in the presence of HCl.13 The acetoin reacts with a mixture of 𝛼-naphthol and creatine, resulting in the formation of a red-colored product. The absorbance of the red product was measured at 522 nm. One unit of ALDC activity corresponds to the formation of 1 μg acetoin min−1 at 45 ∘ C.13
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Measurement of yogurt flavor using gas chromatography/mass spectrometry (GC/MS) An 8 mL yogurt sample was placed in a 15 mL solid phase microextraction (SPME) vial. A 75 μm carboxen/polydimethylsiloxane fiber was inserted into the SPME vial and maintained for 30 min at 50 ∘ C. After analyte extraction, the fiber was thermally desorbed at 250 ∘ C for 3 min into the glass liner of the gas chromatograph (GC) injection port. A Varian 3800 GC (Walnut Creek, CA, USA) equipped with an ion trap mass detector Varian Saturn 2200 mass spectrometer (MS) was used. GC analysis was carried out on a Varian FactorFour VF-5MS fused silica column (30 m × 0.25 mm i.d., 0.25 μm film thickness). Helium was employed as carrier gas at a flow rate of 1 mL min−1 . The procedure followed was as reported previously.10 External standard calibration was used to obtain
yogurt flavor content.11 Diacetyl (Sigma) was used as analytical standard.
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Table 2. Purification of ALDC Purification step
Total protein(mg)
Cell-free extract Ammonium sulfate precipitation Sephacryl HR Native-PAGE
82.50 20.97 2.10 0.12
Specific activity(U mg−1 )
Total activity(U) 673.29 305.38 216.73 34.37
Protein quantification Protein concentrations were assessed with Coomassie protein assay reagent using the method described by Bradford.14 Bovine serum albumin was used as standard protein.
8.16 14.56 103.20 281.70
Recovery(%) 100.00 51.20 32.18 5.10
Purification(fold) 1.00 1.78 12.65 34.52
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IR analysis of ALDC The IR spectrum of ALDC was measured according to a previously described method15 using a Fourier transform infrared spectrophotometer (Nexus 5 DXC FTIR, Thermo Nicolet, Madison, WI, USA). The ALDC was mixed with KBr powder, ground and pressed into 1 mm pellets for IR measurement in the frequency range 4000–500 cm−1 .2 Characterization of ALDC Effect of pH and temperature on ALDC activity ALDC was pre-incubated for 30 min at 37 ∘ C in phosphate-buffered saline (PBS) of varying pH (6, 6.5, 7, 7.5, 8 and 8.5). ALDC activity was measured. ALDC was dissolved in PBS (pH 6) and pre-incubated for 30 min at different temperatures (20, 25, 30, 35, 40 and 45 ∘ C). ALDC activity was measured. Effect of metal ions on ALDC activity Eight metal ions (Ba2+ , Zn2+ , Mn2+ , Co2+ , Mg2+ , Cu2+ , Ca2+ and Fe2+ ) were added separately at 1 mmol L−1 to ALDC solutions. The mixtures were pre-incubated for 30 min at 37 ∘ C and the ALDC activity was estimated.
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Effect of amino acids on ALDC activity Three amino acids (leucine, isoleucine and valine) were added separately at 1 mmol L−1 to ALDC solutions. The mixtures were pre-incubated for 30 min at 37 ∘ C and the ALDC activity was estimated. Statistical analysis All experiments were carried out in triplicate. All data are presented as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 17.0 (AsiaAnalytics, Shanghai, China).
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Strain screening and identification Ten strains collected in the laboratory were used to make yogurt. The diacetyl content of each yogurt was measured. The strain that produced the highest levels of diacetyl was named DX and was identified as a Gram-positive bacterium using Gram staining and microscopic examination (data not shown). The 16S rDNA sequence of the DX strain was 1392 bp (Fig. 1a). The phylogenetic tree (Fig. 1b) was established using Clustalx 1.8 and Mega 3.1. According to the phylogenetic tree, the 16S rDNA of the DX strain
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Figure 2. (a) Sephacryl S-300 HR chromatography of crude ALDC obtained by ammonium sulfate precipitation. (b) Sephacryl S-200 HR chromatography of fraction 2 obtained from Sephacryl S-300 HR chromatography. (c) Native-PAGE (1) and SDS-PAGE (2) of fractions during purification of ALDC. Lane M: molecular weight standards.
was identified (99%) as L. lactis. The strain that produced the greatest amount of diacetyl in yogurt was identified as L. lactis DX. The flavor of the yogurt fermented by L. lactis DX was measured by GC/MS. Diacetyl exhibited a retention time of 5 min (Table 1). The content of diacetyl in yogurt fermented by L. lactis DX was 22.39 mg L−1 . The concentration of diacetyl in fresh cultured
© 2014 Society of Chemical Industry
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Figure 3. (a) IR analysis of ALDC. (b) Effect of temperature on ALDC activity. (c) Effect of pH on ALDC activity. (d) Effect of metal ions and amino acids on ALDC activity.
buttermilk is typically in the range 2–4 mg L−1 .7 These results show that L. lactis DX has the ability to produce a significant amount of diacetyl.
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Purification of ALDC Ammonium sulfate (450 g L−1 ) was used to fractionate crude ALDC. Total ALDC activity was 305.38 U, while the specific activity was 14.56 U mg−1 (Table 2). The purified ALDC was applied to a Sephacryl S-300 HR column, which led to separation into three fractions. The second fraction
(peak 2) had the highest ALDC activity (Fig. 2a). Peak 2 was collected and applied to a Sephacryl S-200 HR column, which led to two peaks. The second peak (peak 2) had the highest ALDC activity (Fig. 2b). After Sephacryl HR chromatography, total ALDC activity was 216.73 U and the specific activity was 103.20 U mg−1 (Table 2). Native-PAGE of the ALDC obtained from Sephacryl S-200 HR chromatography led to the recovery of four protein bands (Fig. 2c(1)), whose ALDC activities were measured. The results showed that band 2 exhibited ALDC activity. The total activity of band 2 was 34.37 U, while the specific activity was 281.70 U mg−1
www.soci.org (Table 2). The specific enzyme activity after each purification step is given in Table 2. The enzyme was purified about 34.52-fold. The recovery activity was about 5.10%. SDS-PAGE showed one band of ALDC, which had a molecular mass of 73.1 kDa (Fig. 2c(2)).
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the addition of an ALDC inhibitor, which can increase diacetyl production.
CONCLUSION IR analysis of ALDC IR spectroscopy provides information about the secondary structure content of proteins. The IR analysis of purified ALDC is shown in Fig. 3a. The bands at 3430 cm−1 suggest hydroxyl stretching vibrations, while the bands in the 2891 cm−1 region correspond to C-H stretching vibrations. The absorptions in the 1600–1700 cm−1 region suggest amide I bands, which lead to stretching vibrations of the C=O bond of the amide. The absorptions in the region of 1200–1360 cm−1 suggest amide III bands, while the absorptions at 1094 cm−1 indicate C-O stretching vibrations.
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Characterization of ALDC of L. lactis DX Diacetyl originates from the chemical oxidative decarboxylation of 𝛼-acetolactate, while acetoin originates from the decarboxylation of 𝛼-acetolactate by ALDC.7 ALDC is a critical enzyme in the production of diacetyl. Strains that have low ALDC activity internally accumulate 𝛼-acetolactate, which then leads to the accumulation of diacetyl. Therefore the ALDC of L. lactis DX was purified and characterized to find its differences from other reported ALDCs. In our research the optimal temperature and pH for the ALDC from L. lactis DX were 40 ∘ C (Fig. 3b) and 6.5 (Fig. 3c) respectively. The effects of various compounds on ALDC activity are summarized in Fig. 3d. The ALDC was activated by Fe2+ , Zn2+ , Mg2+ , Ba2+ and Ca2+ , while Cu2+ caused significant inhibition of ALDC activity. The purified ALDC probably consisted of a single subunit, because ALDC appeared to be a single band when analyzed using SDS-PAGE and native-PAGE. The molecular weight estimated by SDS-PAGE was 73.1 kDa. There are several other reports on ALDCs. The ALDC from Leuconostoc lactis NCW1 is a dimer of 49.0 kDa subunits with an optimal pH of 6. The ALDC activity from Ln lactis NCW1 is independent of metals or branched-chain amino acids.16 Rasmussen et al.17 reported the molecular weight of ALDC from Lactobcillus casei DSM 2547 as 48.0 kDa with an optimal pH of 4.5–5 and an optimal temperature of 40 ∘ C. The enzyme could be partially reactivated by Zn2+ . The ALDC from L. lactis subsp. lactis NCDO 2118 is composed of six identical subunits of 265.0 kDa.18 The ALDC from Bacillus brevis has a molecular weight of 29.1 kDa.19 The ALDC of Brevibacterium acetylicum was activated by Zn2+ . The molecular weight of the native enzyme was 62.0 kDa and the subunit molecular weight was 31.0 kDa when measured by SDS-PAGE.20 Three amino acids (leucine, valine and isoleucine) were able separately to activate the purified ALDC 5.6-, 4.0- and 4.1-fold respectively (Fig. 3d). Phalip et al.18 also reported that the ALDC from L. lactis subsp. lactis NCDO 2118 was greatly stimulated by the addition of leucine, valine and isoleucine. Monnet et al.21 reported that 𝛼-acetolactate was a precursor of branched-chain amino acids and that when they were present in excess the flux of 𝛼-acetolactate was diverted towards acetoin owing to an allosteric activation of the ALDC and to a translational regulation of ALDC synthesis. From these reports we can see that the ALDC of L. lactis DX differs from the ALDCs of other strains. Lactococcus lactis DX had significant ability to produce diacetyl. Further study should continue into the ALDC of L. lactis DX and certain methods, such as controlling the fermentation pH and temperature, as well as
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The strain that had the greatest ability to produce diacetyl was identified as L. lactis DX. The ALDC of L. lactis DX was purified by ammonium sulfate precipitation and Sephacryl S-300 and S-200 HR chromatography and recovered by native-PAGE. The purified ALDC differed from other ALDCs obtained from reported strains. The purified ALDC had a monomer structure and a molecular mass of about 73.1 kDa. The ALDC had a typical protein structure as shown by IR analysis. The optimal temperature and pH for the ALDC were 40 ∘ C and 6.5 respectively. The activity of the ALDC was influenced by metal ions and was greatly stimulated by the addition of leucine, valine and isoleucine. The results of this study help elucidate the characterization of ALDC and further illuminate the processes behind the biosynthesis of diacetyl.
ACKNOWLEDGEMENTS This work was supported by the Natural Science Foundation of China (41276121, 31101314, 31471598), the Natural Science Foundation of Jiangsu Province (BK2011787, BK20141447), the Natural Science Fund for Colleges and Universities in Jiangsu Province (13KJB550013), the Natural Science Foundation of Zhejiang Province (2012C12016-1) and the Projects of Ningbo Science and Technology Bureau (2012A610145).
REFERENCES 1 Hernández EJG, Estepa RG and Rivas IR, Analysis of diacetyl in yogurt by two new spectrophotometric and fluorimetric methods. Food Chem 53:315–319 (1995). 2 Liu Y, Zhang S, Yong Y-C, Ji Z, Ma X, Xu Z, et al., Efficient production of acetoin by the newly isolated Bacillus licheniformis strain MEL09. Process Biochem 46:390–394 (2011). 3 Aymes F, Monnet C and Corrieu G, Effect of 𝛼-acetolactate decarboxylase inactivation on 𝛼-acetolactate and diacetyl production by Lactococcus lactis subsp. lactis biovar diacetylactis. J Biosci Bioeng 87:87–92 (1999). 4 Christ P, Jeroen H, Marjo S, Van A and Vos W, Metabolic engineering of Lactococcus lactis: influence of the overproduction of 𝛼-acetolactate synthase in strains deficient in lactate dehydrogenase as a function of culture conditions. Appl Environ Microbiol 61:3967–3971 (1995). 5 Le Bars D and Yvon M, Formation of diacetyl and acetoin by Lactococcus lactis via aspartate catabolism. J Appl Microbiol 104:171–177 (2008). 6 Rattray FP, Walfridsson M and Nilsson D, Purification and characterization of a diacetyl reductase from Leuconostoc pseudomesenteroides. Int Dairy J 10:781–789 (2000). 7 Boumerdassi H, Monnet C, Desmazeaud M and Corrieu G, Isolation and properties of Lactococcus lactis subsp. lactis biovar diacetylactis CNRZ 483 mutants producing diacetyl and acetoin from glucose. Appl Environ Microbiol 63:2293–2299 (1997). 8 Wang W, Zhang L and Li Y, Study on the change of aldehydes and diacetyls in fermented milk. China Dairy Ind 34:50–53 (2006). 9 Zeng XQ, Pan DD and Guo YX, The probiotic properties of Lactobacillus buchneri P2. J Appl Microbiol 108:2059–2066 (2010). 10 Lara-Gonzalo A, Sánchez-Uría JE, Segovia-García E and Sanz-Medel A, Critical comparison of automated purge and trap and solid-phase microextraction for routine determination of volatile organic compounds in drinking waters by GC–MS. Talanta 74:1455–1462 (2008). 11 Pandya GH, Gavane AG and Kondawar VK, Assessment of occupational exposure to VOCs at the Gantry gasoline terminal. J Environ Sci Eng 48:175–182 (2006).
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12 Guo Y, Pan D and Tanokura M, Optimisation of hydrolysis conditions for the production of the angiotensin-I converting enzyme (ACE) inhibitory peptides from whey protein using response surface methodology. Food Chem 114:328–333 (2009). 13 Phalip V, Schmitt P and Diviès C, Purification and characterization of the catabolic 𝛼-acetolactate synthase from Leuconostoc mesenteroides subsp. cremoris. Curr Microbiol 31:316–321 (1995). 14 Bradford MM, A rapid and sensitive method for the quantitation of protein using the principle of protein–dye binding. Anal Biochem 72:248–254 (1976). 15 Chen Y, Mao W, Tao H, Zhu W, Qi X, Chen Y, et al., Structural characterization and antioxidant properties of an exopolysaccharide produced by the mangrove endophytic fungus Aspergillus sp. Y16. Bioresour Technol 102:8179–8184 (2011). 16 O’Sullivan SM, Condon S, Cogan TM and Sheehan D, Purification and characterisation of acetolactate decarboxylase from Leuconostoc lactis NCW1. FEMS Microbiol Lett 194:245–249 (2001).
17 Rasmussen AM, Gibson RM, Godtfredsen SE and Ottesen M, Purification of 𝛼-acetolactate decarboxylase from Lactobacillus casei DSM 2547. Carlsberg Res Commun 50:73–82 (1987). 18 Phalip V, Monnet C, Schmitt P, Renault P, Godon J-J and Diviès C, Purification and properties of the 𝛼-acetolactate decarboxylase from Lactococcus lactis subsp. lactis NCDO 2118. FEBS Lett 351:95–99 (1994). 19 Svendsen I, Jensen BR and Ottesen M, Complete amino acid sequence of 𝛼-acetolactate decarboxylase from Bacillus brevis. Carlsberg Res Commun 54:157–163 (1989). 20 Vincent P, Philippe C and Charles D, Purification and characterization of 𝛼-acetolactate decarboxylase from Brevibacterium acetylicum. Agric Biol Chem 53:1913–1918 (1989). 21 Monnet C, Nardi M, Hols P, Gulea M, Corrieu G and Monnet V, Regulation of branched-chain amino acid biosynthesis by 𝛼-acetolactate decarboxylase in Streptococcus thermophilus. Lett Appl Microbiol 36:399–405 (2003).
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Revised: 1 August 2014
Accepted article published: 8 August 2014
Published online in Wiley Online Library: 2 September 2014
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6868
Purification and characterization of 𝜶-acetolactate decarboxylase (ALDC) from newly isolated Lactococcus lactis DX Yuxing Guo,a Daodong Pan,a,b* Haibing Ding,a Zhen Wu,a Yangying Sunb and Xiaoqun Zengb Abstract BACKGROUND: Diacetyl (2,3-butanedione) is a common flavor aroma from fermented dairy products. There is a need to screen new microorganisms that can efficiently produce large amounts of diacetyl. RESULTS: A new lactic acid bacterium that produced high concentrations of diacetyl was identified based on Gram staining, microscopic examination and 16S rDNA sequence analysis as Lactococcus lactis DX. Its 𝜶-acetolactate decarboxylase (ALDC) was purified using 0.45 g mL−1 ammonium sulfate precipitation, Sephacryl S-300 and S-200 HR and native-PAGE. The purified ALDC displayed a monomer structure and had a molecular mass of about 73.1 kDa, which was estimated using SDS-PAGE. IR analysis showed that the ALDC had a typical protein structure. The optimal temperature and pH for ALDC activity were 40 ∘ C and 6.5 respectively. The ALDC of L. lactis DX was activated by Fe2+ , Zn2+ , Mg2+ , Ba2+ and Ca2+ , while Cu2+ significantly inhibited ALDC activity. Leucine, valine and isoleucine activated the ALDC. CONCLUSION: A strain that had high ability to produce diacetyl was identified as L. lactis DX. The difference in diacetyl production may be due to the ALDC, which is different from other ALDCs. © 2014 Society of Chemical Industry Keywords: 𝛼-acetolactate decarboxylase; diacetyl; purification; characterization
INTRODUCTION
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resulted in an increased concentration of diacetyl. Metabolic engineering methods (e.g. inactivating certain enzymes) have produced large quantities of diacetyl.7 Therefore it is crucial to better understand the characterization of ALDC during the metabolism of diacetyl in order to improve ALDC production. The objective of this study was to purify and characterize ALDC of a high-diacetyl-producing strain so as to understand the characterization of ALDC during the metabolism of diacetyl. A strain demonstrating high productivity of diacetyl was identified by Gram staining, microscopic examination and 16S rDNA sequence analysis. The ALDC of the selected strain was purified using ammonium sulfate precipitation and Sephacryl HR chromatography and then recovered by native polyacrylamide gel electrophoresis (native-PAGE). The ALDC was characterized using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and infrared (IR) analysis.
∗
Correspondence to: Daodong Pan, Department of Food Science and Technology, Jinling College, Nanjing Normal University, Nanjing, 210097, China. E-mail: [email protected]
a Department of Food Science and Technology, Jinling College, Nanjing Normal University, Nanjing, 210097, China b Food Science & Technology Department, Marine Science School, Ningbo University, Ningbo, 315211, Zhejiang, China
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Diacetyl is a common flavor aroma from yogurt, sour cream and other fermented dairy products. Diacetyl helps determine the quality of yogurt and its acceptance by consumers.1 Since consumer demand for diacetyl is increasing, there is growing interest in alternative methods of producing it.2 Some citrate-utilizing lactic acid bacteria such as Lactobacillus, Lactococcus lactis subsp. lactis and Leuconostoc sp. can produce diacetyl.3 However, Aymes et al.3 reported that most of these strains produced only small amounts of diacetyl (∼0.05 mmol L−1 ). Therefore there is a need for new microorganisms that can efficiently produce large amounts of diacetyl. Diacetyl production involves four enzymes: citrate lyase, 𝛼-acetolactate synthetase, 𝛼-acetolactate decarboxylase (ALDC) and diacetyl reductase. 𝛼-Acetolactate is produced from two molecules of pyruvate via the activity of 𝛼-acetolactate synthase.4 𝛼-Acetolactate can be transformed into diacetyl by spontaneous oxidative decarboxylation and into acetoin by spontaneous non-oxidative decarboxylation or by ALDC (EC 4.1.1.5).3 In yoghurt the concentration of acetoin is often higher than that of diacetyl. Le Bars and Yvon5 reported that the aroma of diacetyl is 100-fold more powerful than that of acetoin. ALDC is a key enzyme in producing large amounts of diacetyl. Some researchers have focused on ALDC to improve diacetyl production. Rattray et al.6 impaired the ALDC activity of Leuconostoc pseudomesenteroides, which
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MATERIALS AND METHODS Materials A 16S rDNA bacterial identification polymerase chain reaction (PCR) kit was purchased from Takara Biotechnology (Dalian, China). Sephacryl S-300, Sephacryl S-200 and a molecular weight electrophoresis calibration kit for SDS electrophoresis were purchased from Amersham Biosciences (Uppsala, Sweden). Coomassie Blue R 250, o-phenylenediamine, 𝛼-naphthol, creatine and bovine serum albumin were purchased from Sigma (St Louis, MO, USA). Strain screening and identification Preliminary screening Ten strains were isolated from fermented dairy products and preserved in our laboratory (Nanjing Normal University, Nanjing, China). They were cultured in 0.11 g mL−1 reconstituted skim milk powder at 37 ∘ C for 24 h. Trichloroacetic acid solution
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(0.16 g mL−1 ) was added to the yogurt, which was then was centrifuged at 3056 × g for 20 min. The supernatant was used to measure the diacetyl content in yogurt. Diacetyl was derivatized with o-phenylenediamine, producing 2,3-dimethylquinoxaline with absorbance at 335 nm. A 10 mL aliquot of supernatant was mixed with 0.5 mL of 0.01 g mL−1 o-phenylenediamine and placed in darkness for 30 min. To stop the reaction, 4 mol L−1 HCl solution was added. The absorbance was measured at 335 nm. Diacetyl content was calculated by the equation8 c = 4(1.2E − 0.01), where c (mg L−1 ) is the content of diacetyl in yogurt, E is the absorbance, 1.2 is the conversion coefficient of absorbance and diacetyl content and 0.01 is the adjustment coefficient. Strain identification After preliminary screening, the strain that produced the highest content of diacetyl was named DX. Then strain DX was identified
(a)
(b)
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Figure 1. (a) 16S rDNA of strain DX. Lanes 1–4: PCR amplicon of 16S rDNA gene. (b) Phylogenetic tree based on 16S rDNA sequences showing position of strain DX among closely related organisms. The scale bar represents 0.01 nucleotide substitution per position.
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© 2014 Society of Chemical Industry
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Table 1. Flavor in yogurt fermented by Lactococcus lactis DX Retention time (min) 2.86 3.38 4.10 5.00 7.10 10.40 11.10 11.77 12.59 14.25 14.38 14.75 15.85 16.02 16.42 16.63 16.87 17.17 17.66 18.26 18.67 19.85 20.81 21.62 23.08 25.22 25.38
Compound Acetone Butanone Ethanol Diacetyl Acetyl propionyl Heptanone 3-Methylcrotonaldehyde Methyl butanol Glutaraldehyde Amyl alcohol 2-Methyl-3-pentanol 2-Nonanone Acetic acid 2-Furaldehyde 2-Ethylhexanol 2-Cyclopentene-1,4-dione Benzaldehyde Linalool formate 2,2-Dimethylpropionic acid Butyric acid Furanmethanol 4-Methoxybenzaldehyde oxime Hexanoic acid 2-Phenylethanol Octanoic acid Decanoic acid 2,4-di-tert-Butylphenol
Relative content (%) 1.36 1.56 3.10 8.38 2.70 1.90 0.32 0.69 0.78 0.65 0.85 0.45 6.51 1.94 0.37 0.36 1.62 0.46 0.21 5.94 8.82 4.75 13.63 0.59 10.15 1.37 0.77
Content (mg L−1 ) 3.633 4.167 8.282 22.383 7.212 5.075 0.856 1.843 2.083 1.736 2.270 1.202 17.382 5.182 0.988 0.962 4.325 1.229 0.561 15.866 22.358 12.687 36.405 1.576 27.111 3.659 2.057
using Gram staining, microscopic examination and 16S rDNA sequence analysis. The total DNA of the DX strain was extracted. The strain’s 16S rDNA gene was amplified using PCR with primers K1: 5′ -AACTGAAGAGTTTGATCCTGGCTC-3′ and K2: 5′ -TACGGTTACC TTGTTACGACTT-3′ . The PCR procedure was as follows: 94 ∘ C for 5 min; 30 cycles of 94 ∘ C for 1 min, 56 ∘ C for 45 s and 72 ∘ C for 2 min; 72 ∘ C for 10 min.9 The PCR products were sequenced and then searched in the NCBI BLAST (National Center for Biotechnology Information, MD, USA) sequence database. The phylogenetic tree was established using Clustalx1.8 (UCD Conway Institute, Dublin, Ireland) and Mega 3.1 (The Biodesign Institute, AZ, USA).
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Purification of ALDC Preparation of crude ALDC Lactococcus lactis DX was cultured in MRS medium at 37 ∘ C for 24 h. The fermentation broth was centrifuged (3056 × g, 20 min, 4 ∘ C) to harvest the cells, which were then washed three times with 50 mmol L−1 Tris-HCl buffer (pH 7). The washed cells were ultrasonicated (output power 300 W, work time 3 s, pause time 5 s, 200 times). The clear supernatant was collected by centrifugation (5433 × g, 20 min, 4 ∘ C) and used as crude ALDC.12 Ammonium sulfate precipitation Ammonium sulfate (450 g L−1 ) was added to the crude ALDC. The mixture was centrifuged (5433 × g, 20 min, 4 ∘ C) to collect the precipitate. The collected precipitate was dialyzed against 50 mmol L−1 Tris-HCl buffer (pH 7) overnight at 4 ∘ C. Sephacryl HR chromatography The concentrated crude ALDC solution from the previous step was applied to a Sephacryl S-300 HR column (1 cm × 40 cm) equilibrated with 50 mmol L−1 Tris-HCl (pH 7.8). Proteins were eluted with the same buffer at a flow rate of 25 mL h−1 . The fractions were collected and the ALDC activity was measured. The fractions that contained ALDC activity were pooled and concentrated using polyethylene glycol 20 000. Then the collected ALDC fraction was applied to a Sephacryl S-200 HR column (1 cm × 40 cm). The elution condition was the same as that used for the Sephacryl S-300 HR column. The fractions that contained ALDC activity were pooled and concentrated again. Recovery of ALDC using native-PAGE The active fractions after Sephacryl S-200 HR were examined using native-PAGE with 50 g L−1 acrylamide stacking gel and 100 g L−1 acrylamide running gel. Two gels were prepared. One (gel 1) was stained with Coomassie Blue R 250, while the other (gel 2) was not stained. The protein band (on gel 2) confirmed by gel 1 was excised. The excised gel piece was placed in a microfuge tube and 0.5–1 mL of elution buffer (50 mmol L−1 Tris-HCl, 150 mmol L−1 NaCl, 0.1 mmol L−1 EDTA, pH 7.5) was added. The gel pieces were pulverized and incubated overnight at 4 ∘ C. The mixture was centrifuged (3773 × g, 15 min, 4 ∘ C) to obtain the supernatant, which was measured for ALDC activity. Molecular mass measurement The band that exhibited ALDC activity was recovered using the above method. The molecular mass of the purified ALDC was determined under denaturing conditions by SDS-PAGE with 50 g L−1 acrylamide stacking gel and 100 g L−1 acrylamide running gel. Proteins in the gels were stained with Coomassie Blue R 250. Measurement of ALDC activity ALDC can transform 𝛼-acetolactate into acetoin. The 𝛼-acetolactate was transformed into acetoin by incubation with the obtained ALDC at 45 ∘ C for 30 min in the presence of HCl.13 The acetoin reacts with a mixture of 𝛼-naphthol and creatine, resulting in the formation of a red-colored product. The absorbance of the red product was measured at 522 nm. One unit of ALDC activity corresponds to the formation of 1 μg acetoin min−1 at 45 ∘ C.13
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Measurement of yogurt flavor using gas chromatography/mass spectrometry (GC/MS) An 8 mL yogurt sample was placed in a 15 mL solid phase microextraction (SPME) vial. A 75 μm carboxen/polydimethylsiloxane fiber was inserted into the SPME vial and maintained for 30 min at 50 ∘ C. After analyte extraction, the fiber was thermally desorbed at 250 ∘ C for 3 min into the glass liner of the gas chromatograph (GC) injection port. A Varian 3800 GC (Walnut Creek, CA, USA) equipped with an ion trap mass detector Varian Saturn 2200 mass spectrometer (MS) was used. GC analysis was carried out on a Varian FactorFour VF-5MS fused silica column (30 m × 0.25 mm i.d., 0.25 μm film thickness). Helium was employed as carrier gas at a flow rate of 1 mL min−1 . The procedure followed was as reported previously.10 External standard calibration was used to obtain
yogurt flavor content.11 Diacetyl (Sigma) was used as analytical standard.
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Table 2. Purification of ALDC Purification step
Total protein(mg)
Cell-free extract Ammonium sulfate precipitation Sephacryl HR Native-PAGE
82.50 20.97 2.10 0.12
Specific activity(U mg−1 )
Total activity(U) 673.29 305.38 216.73 34.37
Protein quantification Protein concentrations were assessed with Coomassie protein assay reagent using the method described by Bradford.14 Bovine serum albumin was used as standard protein.
8.16 14.56 103.20 281.70
Recovery(%) 100.00 51.20 32.18 5.10
Purification(fold) 1.00 1.78 12.65 34.52
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IR analysis of ALDC The IR spectrum of ALDC was measured according to a previously described method15 using a Fourier transform infrared spectrophotometer (Nexus 5 DXC FTIR, Thermo Nicolet, Madison, WI, USA). The ALDC was mixed with KBr powder, ground and pressed into 1 mm pellets for IR measurement in the frequency range 4000–500 cm−1 .2 Characterization of ALDC Effect of pH and temperature on ALDC activity ALDC was pre-incubated for 30 min at 37 ∘ C in phosphate-buffered saline (PBS) of varying pH (6, 6.5, 7, 7.5, 8 and 8.5). ALDC activity was measured. ALDC was dissolved in PBS (pH 6) and pre-incubated for 30 min at different temperatures (20, 25, 30, 35, 40 and 45 ∘ C). ALDC activity was measured. Effect of metal ions on ALDC activity Eight metal ions (Ba2+ , Zn2+ , Mn2+ , Co2+ , Mg2+ , Cu2+ , Ca2+ and Fe2+ ) were added separately at 1 mmol L−1 to ALDC solutions. The mixtures were pre-incubated for 30 min at 37 ∘ C and the ALDC activity was estimated.
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Effect of amino acids on ALDC activity Three amino acids (leucine, isoleucine and valine) were added separately at 1 mmol L−1 to ALDC solutions. The mixtures were pre-incubated for 30 min at 37 ∘ C and the ALDC activity was estimated. Statistical analysis All experiments were carried out in triplicate. All data are presented as mean ± standard deviation (SD). Statistical analysis was performed using SPSS 17.0 (AsiaAnalytics, Shanghai, China).
RESULTS AND DISCUSSION
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Strain screening and identification Ten strains collected in the laboratory were used to make yogurt. The diacetyl content of each yogurt was measured. The strain that produced the highest levels of diacetyl was named DX and was identified as a Gram-positive bacterium using Gram staining and microscopic examination (data not shown). The 16S rDNA sequence of the DX strain was 1392 bp (Fig. 1a). The phylogenetic tree (Fig. 1b) was established using Clustalx 1.8 and Mega 3.1. According to the phylogenetic tree, the 16S rDNA of the DX strain
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Figure 2. (a) Sephacryl S-300 HR chromatography of crude ALDC obtained by ammonium sulfate precipitation. (b) Sephacryl S-200 HR chromatography of fraction 2 obtained from Sephacryl S-300 HR chromatography. (c) Native-PAGE (1) and SDS-PAGE (2) of fractions during purification of ALDC. Lane M: molecular weight standards.
was identified (99%) as L. lactis. The strain that produced the greatest amount of diacetyl in yogurt was identified as L. lactis DX. The flavor of the yogurt fermented by L. lactis DX was measured by GC/MS. Diacetyl exhibited a retention time of 5 min (Table 1). The content of diacetyl in yogurt fermented by L. lactis DX was 22.39 mg L−1 . The concentration of diacetyl in fresh cultured
© 2014 Society of Chemical Industry
J Sci Food Agric 2015; 95: 1655–1661
Purification and characterization of ALDC
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Figure 3. (a) IR analysis of ALDC. (b) Effect of temperature on ALDC activity. (c) Effect of pH on ALDC activity. (d) Effect of metal ions and amino acids on ALDC activity.
buttermilk is typically in the range 2–4 mg L−1 .7 These results show that L. lactis DX has the ability to produce a significant amount of diacetyl.
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Purification of ALDC Ammonium sulfate (450 g L−1 ) was used to fractionate crude ALDC. Total ALDC activity was 305.38 U, while the specific activity was 14.56 U mg−1 (Table 2). The purified ALDC was applied to a Sephacryl S-300 HR column, which led to separation into three fractions. The second fraction
(peak 2) had the highest ALDC activity (Fig. 2a). Peak 2 was collected and applied to a Sephacryl S-200 HR column, which led to two peaks. The second peak (peak 2) had the highest ALDC activity (Fig. 2b). After Sephacryl HR chromatography, total ALDC activity was 216.73 U and the specific activity was 103.20 U mg−1 (Table 2). Native-PAGE of the ALDC obtained from Sephacryl S-200 HR chromatography led to the recovery of four protein bands (Fig. 2c(1)), whose ALDC activities were measured. The results showed that band 2 exhibited ALDC activity. The total activity of band 2 was 34.37 U, while the specific activity was 281.70 U mg−1
www.soci.org (Table 2). The specific enzyme activity after each purification step is given in Table 2. The enzyme was purified about 34.52-fold. The recovery activity was about 5.10%. SDS-PAGE showed one band of ALDC, which had a molecular mass of 73.1 kDa (Fig. 2c(2)).
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the addition of an ALDC inhibitor, which can increase diacetyl production.
CONCLUSION IR analysis of ALDC IR spectroscopy provides information about the secondary structure content of proteins. The IR analysis of purified ALDC is shown in Fig. 3a. The bands at 3430 cm−1 suggest hydroxyl stretching vibrations, while the bands in the 2891 cm−1 region correspond to C-H stretching vibrations. The absorptions in the 1600–1700 cm−1 region suggest amide I bands, which lead to stretching vibrations of the C=O bond of the amide. The absorptions in the region of 1200–1360 cm−1 suggest amide III bands, while the absorptions at 1094 cm−1 indicate C-O stretching vibrations.
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Characterization of ALDC of L. lactis DX Diacetyl originates from the chemical oxidative decarboxylation of 𝛼-acetolactate, while acetoin originates from the decarboxylation of 𝛼-acetolactate by ALDC.7 ALDC is a critical enzyme in the production of diacetyl. Strains that have low ALDC activity internally accumulate 𝛼-acetolactate, which then leads to the accumulation of diacetyl. Therefore the ALDC of L. lactis DX was purified and characterized to find its differences from other reported ALDCs. In our research the optimal temperature and pH for the ALDC from L. lactis DX were 40 ∘ C (Fig. 3b) and 6.5 (Fig. 3c) respectively. The effects of various compounds on ALDC activity are summarized in Fig. 3d. The ALDC was activated by Fe2+ , Zn2+ , Mg2+ , Ba2+ and Ca2+ , while Cu2+ caused significant inhibition of ALDC activity. The purified ALDC probably consisted of a single subunit, because ALDC appeared to be a single band when analyzed using SDS-PAGE and native-PAGE. The molecular weight estimated by SDS-PAGE was 73.1 kDa. There are several other reports on ALDCs. The ALDC from Leuconostoc lactis NCW1 is a dimer of 49.0 kDa subunits with an optimal pH of 6. The ALDC activity from Ln lactis NCW1 is independent of metals or branched-chain amino acids.16 Rasmussen et al.17 reported the molecular weight of ALDC from Lactobcillus casei DSM 2547 as 48.0 kDa with an optimal pH of 4.5–5 and an optimal temperature of 40 ∘ C. The enzyme could be partially reactivated by Zn2+ . The ALDC from L. lactis subsp. lactis NCDO 2118 is composed of six identical subunits of 265.0 kDa.18 The ALDC from Bacillus brevis has a molecular weight of 29.1 kDa.19 The ALDC of Brevibacterium acetylicum was activated by Zn2+ . The molecular weight of the native enzyme was 62.0 kDa and the subunit molecular weight was 31.0 kDa when measured by SDS-PAGE.20 Three amino acids (leucine, valine and isoleucine) were able separately to activate the purified ALDC 5.6-, 4.0- and 4.1-fold respectively (Fig. 3d). Phalip et al.18 also reported that the ALDC from L. lactis subsp. lactis NCDO 2118 was greatly stimulated by the addition of leucine, valine and isoleucine. Monnet et al.21 reported that 𝛼-acetolactate was a precursor of branched-chain amino acids and that when they were present in excess the flux of 𝛼-acetolactate was diverted towards acetoin owing to an allosteric activation of the ALDC and to a translational regulation of ALDC synthesis. From these reports we can see that the ALDC of L. lactis DX differs from the ALDCs of other strains. Lactococcus lactis DX had significant ability to produce diacetyl. Further study should continue into the ALDC of L. lactis DX and certain methods, such as controlling the fermentation pH and temperature, as well as
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The strain that had the greatest ability to produce diacetyl was identified as L. lactis DX. The ALDC of L. lactis DX was purified by ammonium sulfate precipitation and Sephacryl S-300 and S-200 HR chromatography and recovered by native-PAGE. The purified ALDC differed from other ALDCs obtained from reported strains. The purified ALDC had a monomer structure and a molecular mass of about 73.1 kDa. The ALDC had a typical protein structure as shown by IR analysis. The optimal temperature and pH for the ALDC were 40 ∘ C and 6.5 respectively. The activity of the ALDC was influenced by metal ions and was greatly stimulated by the addition of leucine, valine and isoleucine. The results of this study help elucidate the characterization of ALDC and further illuminate the processes behind the biosynthesis of diacetyl.
ACKNOWLEDGEMENTS This work was supported by the Natural Science Foundation of China (41276121, 31101314, 31471598), the Natural Science Foundation of Jiangsu Province (BK2011787, BK20141447), the Natural Science Fund for Colleges and Universities in Jiangsu Province (13KJB550013), the Natural Science Foundation of Zhejiang Province (2012C12016-1) and the Projects of Ningbo Science and Technology Bureau (2012A610145).
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12 Guo Y, Pan D and Tanokura M, Optimisation of hydrolysis conditions for the production of the angiotensin-I converting enzyme (ACE) inhibitory peptides from whey protein using response surface methodology. Food Chem 114:328–333 (2009). 13 Phalip V, Schmitt P and Diviès C, Purification and characterization of the catabolic 𝛼-acetolactate synthase from Leuconostoc mesenteroides subsp. cremoris. Curr Microbiol 31:316–321 (1995). 14 Bradford MM, A rapid and sensitive method for the quantitation of protein using the principle of protein–dye binding. Anal Biochem 72:248–254 (1976). 15 Chen Y, Mao W, Tao H, Zhu W, Qi X, Chen Y, et al., Structural characterization and antioxidant properties of an exopolysaccharide produced by the mangrove endophytic fungus Aspergillus sp. Y16. Bioresour Technol 102:8179–8184 (2011). 16 O’Sullivan SM, Condon S, Cogan TM and Sheehan D, Purification and characterisation of acetolactate decarboxylase from Leuconostoc lactis NCW1. FEMS Microbiol Lett 194:245–249 (2001).
17 Rasmussen AM, Gibson RM, Godtfredsen SE and Ottesen M, Purification of 𝛼-acetolactate decarboxylase from Lactobacillus casei DSM 2547. Carlsberg Res Commun 50:73–82 (1987). 18 Phalip V, Monnet C, Schmitt P, Renault P, Godon J-J and Diviès C, Purification and properties of the 𝛼-acetolactate decarboxylase from Lactococcus lactis subsp. lactis NCDO 2118. FEBS Lett 351:95–99 (1994). 19 Svendsen I, Jensen BR and Ottesen M, Complete amino acid sequence of 𝛼-acetolactate decarboxylase from Bacillus brevis. Carlsberg Res Commun 54:157–163 (1989). 20 Vincent P, Philippe C and Charles D, Purification and characterization of 𝛼-acetolactate decarboxylase from Brevibacterium acetylicum. Agric Biol Chem 53:1913–1918 (1989). 21 Monnet C, Nardi M, Hols P, Gulea M, Corrieu G and Monnet V, Regulation of branched-chain amino acid biosynthesis by 𝛼-acetolactate decarboxylase in Streptococcus thermophilus. Lett Appl Microbiol 36:399–405 (2003).
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