JP5848128B2 - 共有結合的に固定化した酵素及びその製造方法 - Google Patents
共有結合的に固定化した酵素及びその製造方法 Download PDFInfo
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- JP5848128B2 JP5848128B2 JP2011529064A JP2011529064A JP5848128B2 JP 5848128 B2 JP5848128 B2 JP 5848128B2 JP 2011529064 A JP2011529064 A JP 2011529064A JP 2011529064 A JP2011529064 A JP 2011529064A JP 5848128 B2 JP5848128 B2 JP 5848128B2
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- JUWGUJSXVOBPHP-UHFFFAOYSA-B titanium(4+);tetraphosphate Chemical compound [Ti+4].[Ti+4].[Ti+4].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O JUWGUJSXVOBPHP-UHFFFAOYSA-B 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 229910001930 tungsten oxide Inorganic materials 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000010457 zeolite Substances 0.000 description 1
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Description
調製後の日数 材料の活性(SU/g)
0 30.39
1 27.28
2 32.17
5 32.71
6 32.19
8 33.37
12 32.88
15 29.1
20 31.38
29 41.33
36 29.05
51 39.07
68 30.06
82 34.81
103 36.08
126 39.27
159 32.7
294 48.1
394 37
460 40
試薬
硫酸アンモニアストック溶液(2000mg% NH3−N):H2Oに94.34グラムの(NH4)2SO4を溶解する。濃H2SO4を1滴添加する。H2Oで1リットルにする。
フェノール試薬:H2Oに50グラムのフェノールと0.25グラムのニトロプルシドナトリウム(ニトロフェリシアン化ナトリウム)とを溶解し、H2Oで1リットルにする。
次亜塩素酸試薬:約500mlのH2Oに25グラムのNaOHを溶解する。20mlの10%次亜塩素酸ナトリウム漂白剤を添加し、H2Oで1リットルにする。
NH3−Nスタンダード(10mg%、15mg%及び20mg%):0.5ml、0.75ml及び1mlの硫酸アンモニアストック溶液(2000mg%)をそれぞれ100ml容のメスフラスコにピペッティングし、H2Oで一定容量にする。
手順
スダンダード、試料及びブランクを全て三連で行う。
1mlのフェノール試薬を全ての試験管に加える。
20ulのH2Oをブランクとしてそれぞれの試験管に加える。
20ulのスタンダートをそれぞれの試験管に加える。
混合する。
1mlの次亜塩素酸試薬を添加し、ボルテックスする。
室温で20分間静置する。
8mlのH2Oを添加し、3回ボルテックスする。
参照キュベットにおいてブランクを使用して635nmで吸光度を読み取る(規定通り)。
試料の吸光度をスタンダードの吸光度と比較することにより、試料中のNH3−N(mg%)を算出する。
酸を撹拌中のポリマーに非常にゆっくりと(液滴で)添加することにより、10gのBPEIを25mLの5M HCl中に溶解した。これには約30分〜45分かかった。全ての酸を添加したら、ポリマー溶液を室温に冷却されるまで撹拌した。これには約10分かかった。
スタンダード 1 2 3 平均 標準偏差
10 0.317 0.312 0.318 0.316 0.003515 0.479 0.489 0.485 0.484 0.007020 0.661 0.633 0.658 0.651 0.0198
スタンダード 1 2 3 平均 標準偏差
バッチ15 1.052 1.043 1.036 1.044 0.0080
材料の活性:30.39SU/g
635nmでの吸光度。
3.5mLのEGDGEを使用する以外は実施例1のバッチ調製に従った。架橋密度を算出すると、およそ20%であった。
スタンダード 1 2 3 平均 標準偏差
10 0.327 0.321 0.322 0.323 0.0042 15 0.489 0.491 0.495 0.492 0.0014 20 0.66 0.666 0.661 0.662 0.0042
スタンダード 1 2 3 平均 標準偏差
バッチ13 1.548 1.584 1.563 1.565 0.0180
材料の活性:23.44SU/g
635nmでの吸光度。
7mLのEGDGEを使用する以外は実施例1のバッチ調製に従った。架橋密度を算出すると、およそ40%であった。
スタンダード 1 2 3 平均 標準偏差
10 0.314 0.306 0.312 0.31 0.005715 0.491 0.492 0.494 0.492 0.000720 0.632 0.671 0.676 0.652 0.0276
スタンダード 1 2 3 平均 標準偏差
バッチ11 0.469 0.462 0.468 0.466 0.0038
材料の活性:7.25SU/g
635nmでの吸光度。
2.0gのBPEI/EGDGE/ウレアーゼ/炭素の組成物を撹拌バーの入った100mL容のメスフラスコに秤量した。10mLの酢酸ナトリウム/塩化ナトリウムバブラー(bubber)溶液をフラスコに加えて、撹拌を始めた。
ウレアーゼ−ストリップ溶液を、pH7.0でウシアルブミンを300mLのリン酸緩衝溶液に添加することにより調製した。アルブミンを最終濃度が150mg%になるように添加した。
算出:
溶液Bの活性=NH4 +(mg%)/100mL
溶液Aの活性=(溶液Bの活性)×10mL/2.0gの材料
ウレアーゼの漏出=(溶液Aの活性)/100mL
結果:
漏出=0.00148SU/g/mL
ウレアーゼストリップ溶液(リン酸緩衝液+150mg%のアルブミン)中に1.5gの尿素を溶解することによりリン酸尿素溶液を調製した。
スタンダード 1 2 3 平均 標準偏差
10 0.305 0.308 0.301 0.305 0.002115 0.477 0.468 0.472 0.472 0.006420 0.637 0.627 0.633 0.632 0.0070
スタンダード 1 2 3 平均 標準偏差
バッチ13 1.548 1.584 1.563 1.565 0.0181
バッチ13試料におけるNH3(mg%):14.51
材料の活性:7.25SU/g
これらの一連の実施例において、実施例1を言及された量の各構成要素を除いて本質的に繰り返し、さらに使用するウレアーゼはSigma-Aldrichから入手し、約2700SU/g〜5400SU/gの酵素活性を有することを特徴とし得る精製形態であった。共有結合した巨大分子の形成体中に少なくとも1つの酵素と、少なくとも1つのポリマーと、少なくとも1つの架橋剤とを有する組成物の効果を測定するために、この反応で使用する架橋剤の量、及び/又はアミン含有ポリマー、及び/又は含水量を変えた。以下の結果で分かるように、精製形態のウレアーゼは、調製後何日も経った後でも依然として優れた酵素安定性を維持していた。精製ウレアーゼが粗ウレアーゼよりもかなり安定性が低いと考えられ、反応又は修飾、例えば架橋の際に失活しやすい可能性があるので、このことは精製酵素、例えば精製ウレアーゼを使用する場合に特に重要である。これらの実験で、酵素活性は5日超、10日超、15日超、30日超、45日超等の期間にわたり得ることができ、かつ安定であり得ることが分かった。結果を以下で要約する。
試薬:10gのBPEI、25mlの5N HCl、0.1gのウレアーゼ、5滴のDI H2O、1.75mlのEGDGE
結果:
調製後の日数 材料の活性(SU/g)
1 3.4
9 3
16 2.2
22 4.7
29 4.7
37 3.4
57 2.7
試薬:10gのBPEI、25mlの5N HCl、0.3gのウレアーゼ、12mlのDI H2O、1.75mlのEGDGE
結果:
調製後の日数 材料の活性(SU/g)
1 6.3
7 7.2
14 6.9
22 7.4
42 5.6
試薬:10gのBPEI、25mlの5N HCl、0.3gのウレアーゼ、7.5mlのDI H2O、1.75mlのEGDGE
結果:
調製後の日数 材料の活性(SU/g)
1 7.2
5 7.5
8 9.3
28 6.9
試薬:10gのBPEI、25mlの5N HCl、0.3gのウレアーゼ、4.0mlのDI H2O、1.75mlのEGDGE
結果:
調製後の日数 材料の活性(SU/g)
1 4.5
2 5.1
22 2.9
試薬:8gのBPEI、20mlの5N HCl、0.3gのウレアーゼ、6.0mlのDI H2O、1.40mlのEGDGE
結果:
調製後の日数 材料の活性(SU/g)
1 13.6
2 14
7 12.4
30 25.3
47 13.3
試薬:8gのBPEI、20mlの5N HCl、0.3gのウレアーゼ、3.0mlのDI H2O、1.40mlのEGDGE
結果:
調製後の日数 材料の活性(SU/g)
2 13.4
25 19.5
42 12.9
試薬:6gのBPEI、15mlの5N HCl、0.3gのウレアーゼ、4.0mlのDI H2O、1.05mlのEGDGE
結果:
調製後の日数 材料の活性(SU/g)
5 19.7
11 16.3
Claims (5)
- 共有結合的に固定化した酵素を含み、酵素活性を有する、乾燥した固体組成物であって、共有結合した巨大分子の形成体中に(i)少なくとも1つの酵素と、(ii)少なくとも1つのポリマーと、(iii)少なくとも1つの架橋剤との反応生成物を含み、酵素が乾燥状態、周囲温度及び圧力で、少なくとも90日間保存される場合、製造時の酵素活性と比較して90%を超える酵素活性を保持し、
さらに活性炭を含み、並びに、
該酵素がタチナタマメに由来する粗ウレアーゼ、該ポリマーがポリ(エチレンイミン)、及び該架橋剤がエチレングリコールジグリシジルエーテルである、組成物。
- 酵素が架橋剤及びポリマーと共有結合する、請求項1に記載の組成物。
- 共有結合した巨大分子の形成体中に固定化ウレアーゼと、少なくとも1つのポリマーと、少なくとも1つの架橋剤とを含む、請求項1又は2に記載の組成物。
- 吸着カートリッジ中に層として請求項1〜3のいずれか1に記載の組成物を含む、吸着カートリッジ。
- 組成物に隣接したアルミナの存在を排除する、請求項4に記載の吸着カートリッジ。
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