商品脂肪酶降解壳聚糖机理研究
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摘要
商品脂肪酶具有非专一性降解壳聚糖的活力,但对于脂肪酶降解壳聚糖的机理在国内外均未见报道。本论文研究商品脂肪酶对壳聚糖的降解特性以及作用机理,这对于阐明非专一性酶水解壳聚糖机理具有重要的理论价值和学术意义,同时对于指导低分子量壳聚糖(LMWC)和甲壳低聚糖(COS)的工业化酶法生产也具有重要的现实意义。
首先从四种不同来源的商品脂肪酶中选择了一种来源于Aspergillus Oryzae的具有较强壳聚糖水解活力的脂肪酶,研究了该酶降解壳聚糖的特性,并对该酶降解壳聚糖的产物进行了分析。脂肪酶对不同脱乙酰度(DD)的壳聚糖均有显著的水解作用,作用于DD为64%、73%、82%和90%的壳聚糖的最适pH值分别为4.2、4.4、4.6和5.0,最适温度均为60℃。脂肪酶水解不同DD壳聚糖的反应均遵循Michaelis-Meten方程,动力学分析结果表明DD值为73%和82%的壳聚糖是脂肪酶较容易水解的底物。脂肪酶催化壳聚糖降解可生成各种聚合度(DP = 2~6)的低聚糖,但在反应的最后,产物仅剩下氨基葡萄糖(GlcN),这表明在脂肪酶中存在外切酶。脂肪酶催化不同DD壳聚糖水解生成的产物完全相同。
采用超滤、DEAE-Sepharose CL-6B离子交换层析、Phenyl Sepharose CL-4B疏水作用层析和Sephacryl S-200凝胶过滤层析等一系列分离纯化操作,从脂肪酶中分离得到一个具有壳聚糖酶活力但不具有脂肪酶活力的酶组分。HPLC结果表明纯化酶的纯度达到99%以上,SDS-PAGE结果表明纯化酶已经达到均一。由于分离纯化过程中其他组分均不具备壳聚糖酶活力,因此说明脂肪酶的壳聚糖酶活力是由纯化酶的水解作用引起的。SDS-PAGE测得纯化酶的分子量约为74 kDa,而在非还原SDS-PAGE条件下纯化酶条带出现在130 kDa附近,表明纯化酶由两个相对分子质量相同的亚基组成,而且这两个亚基通过二硫键连接在一起。
利用氨基酸自动分析仪测定了纯化酶的氨基酸组成,结果表明天冬氨酸和谷氨酸含量较高,组氨酸、精氨酸和赖氨酸等碱性氨基酸的含量较低;含硫氨基酸的含量都非常低,而丝氨酸、甘氨酸、苏氨酸和丙氨酸等中性氨基酸的含量都较高。采用Edman降解法测得纯化酶N-末端12个氨基酸的序列为Ala– Leu– Arg– Leu– Asn– Ser– Pro– Asn– Asn– Ile– Ala– Val。在非冗余蛋白质序列( Non-redundant protein sequences)数据库中采用NCBI Blastp2程序对测得的N-末端氨基酸序列进行相似性比较,发现该酶与来自米曲霉的一个蛋白质具有很高的相似性,12个氨基酸序列的区域一致性达到100%。
采用DEPC、NBS、Ch-T、EDAC、PMSF、CHD、NAI、DTNB和DTT等化学修饰剂对纯化酶的氨基酸残基进行修饰,研究了纯化酶中必需基团的组成。DEPC对酶的修饰结果表明有1分子组氨酸残基位于纯化酶的活性中心;利用NBS修饰酶蛋白,结果表明色氨酸残基处于酶的活性中心,且至少有1分子色氨酸残基位于酶活性中心的底物结合部位;Ch-T和EDAC对酶的修饰结果表明甲硫氨酸残基和羧基也是酶的必需基团。利用PMSF、CHD、NAI、DTNB和DTT分别对酶分子中的羟基、精氨酸残基、酪氨酸残基、巯基和二硫键进行修饰,结果表明这些修饰剂在较高的浓度范围内均没有引起酶活的显著降低,因此羟基、精氨酸残基、酪氨酸残基、巯基和二硫键都不是维持酶活的必需基团。
研究了纯化酶的酶学性质,结果表明纯化酶在pH 4.6时的活力较高,在pH4.5 ~ 9.5之间具有较好的稳定性;在60℃时活力达到最高,在低于60℃的温度下时具有较高的热稳定性。Ni2+、Co2+和Mn2+等金属离子对酶的活力具有明显的激活作用,而Fe3+、Sn2+、Pb2+和Hg2+则能强烈抑制酶活,其中Hg2+的抑制能力最强,Na+、K+、Mg2+、Zn2+和Cd2+对酶活的影响较小。纯化酶对于DD为73%、81%和82%的壳聚糖显示了较高的水解活力,而对DD为64%和90%的壳聚糖的水解活力较低。
通过TLC法和HPLC法分析纯化酶降解已知结构甲壳低聚糖和乙酰甲壳低聚糖的产物,研究了纯化酶的作用模式。结果表明纯化酶以外切的形式作用于甲壳低聚糖,并将单糖GlcN顺次从甲壳低聚糖的末端释放出来,这与氨基葡萄糖苷酶(GlcNase)的作用模式是一致的;同时纯化酶以外切酶的形式作用于乙酰甲壳低聚糖的末端,并依次将乙酰甲壳二糖[(GlcNAc)2]释放出来,这是乙酰甲壳二糖苷酶典型的作用模式。因此该酶同时具有GlcNase活力和乙酰甲壳二糖苷酶活力,既能水解壳聚糖中GlcN-GlcN之间的糖苷键,也能水解GlcNAc-GlcNAc之间的糖苷键。
本论文通过超滤、凝胶过滤层析、疏水作用层析和离子交换层析等分离纯化技术,从脂肪酶中得到了一种具有壳聚糖水解活力但不具有脂肪酶活力的酶组分,该酶具有GlcNase和乙酰甲壳二糖苷酶双重活力,说明该酶的水解作用是脂肪酶降解壳聚糖的主要原因,从而阐明了商品脂肪酶非专一性降解壳聚糖的机理。
Commercial lipases exhibit non-specific activity on chitosan depolymerization, however, no research on mechanicsm of chitosan deploymerization by lipase has been reported at home and abroad. In this study, characteristics and mechanism of chitosan hydrolysis by a commercial lipase were investigated, which is of important theoretical and academic value in elucidating mechanism of chitosan hydrolysis by non-specific enzymes and of practical significance in instructing industrial enzymatic production of low molecular chitosan and chitooligosaccharides (COS).
Characteristics of chitosan hydrolysis by a commercial lipase from Aspergillus oryzae were investigated and hydrolysis products were analyzed. The lipase showed obvious hydrolytic activity on chitosans with different degrees of deacetylation (DD). The optimum pH values of the lipase on chitosans with DD of 64%, 73%, 82%, and 90% were 4.2, 4.4, 4.6, and 5.0, respectively, and optimum temperatures were all 60℃. The hydrolysis reactions of the lipase on different chitosans obeyed Michaelis-Meten equation, and kinetic parameters indicate that chitosans with DD of 73% and 82% were susceptible to be hydrolyzed. Products of chitosan hydrolysis by lipase included chitosan-oligomers with degree of polymerization (DP) 2~6, but the final product was glucosamine (GlcN), indicating some hydrolase with exo-mode action existing in the lipase. Products of hydrolysis of chitosans with different DD catalyzed by the lipase were identical.
A hydrolase with chitosanolytic activity but no lipolytic activity was purified from lipase by using a combination of ultrafiltration, DEAE-Sepharose CL-6B ion exchange chromatography, Phenyl-Sepharose CL-4B hydrophobic interaction chromatography, and Sephacryl S-200 gel filtration chromatography. Results of HPLC and SDS-PAGE show that the hydrolase had been purified to homogeneity. Because no other components of the lipase showed chitosanolytic activity during purification process, chitosanolytic activity the lipase exhibited was caused by the purified enzyme. Molecular mass of the purified enzyme estimated by SDS-PAGE and non-reducing SDS-PAGE was about 74 kDa and 130 kDa, respectively, indicating that the enzyme was composed of two identical subunits bound together with disulfide bonds.
Amino acids composition of the purified enzyme was analyzed by auto-amino acids analyzer. The results show that concentrations of Asp and Glu were high, those of His, Arg, and Lys were low, while those of sulfur-containing amino acids were extremely low. The N-terminal sequence containing 12 amino acids of the purified enzyme determined by using the Edman degradation technique was Ala– Leu– Arg– Leu– Asn– Ser– Pro– Asn– Asn– Ile– Ala– Val. The similarity of the N-terminal sequence was blasted in the Non-redundant Protein Sequences database using the NCBI Blastp2 program, and it was found that this sequence was similar to that of a protein from Aspergillus oryzae, and the identity of the 12 amino acids was 100%.
The purified enzyme was modified by DEPC, NBS, Ch-T, EDAC, PMSF, CHD, NAI, DTNB, and DTT. Modification of the purified enzyme by DEPC shows that 1 mole of histidine residue existed in the active site of the enzyme. Modification by NBS shows that tryptophan residues existed in the active site and there is at least 1 mole of tryptophan residue in the substrate binding site. Results of modification by Ch-T and EDAC indicate that methionine residues and carbosyl groups were essential groups of the purified enzyme. In addition, hydroxyl groups, arginine residues, tyrosine residues, sulfhydryl groups, and disulfide bonds were not essential groups of the purified enzyme.
The enzyme showed the optimum action pH value and temperature were 4.6 and 60?C, respectively, and it was stable in pH range of 4.5~9.5 and at temperatures lower than 60?C. Metal ions such as Ni2+, Co2+, and Mn2+ had obvious activation effects on the enzymatic activity, while Fe3+, Sn2+, Pb2+, and Hg2+ inactivated the enzyme, and Na+, K+, Mg2+, Zn2+, and Cd2+ had no obvious effect on the purified enzyme. The enzyme exhibited higher chitosanolytic activity toward chitosans which were 73%, 81% and 82% deacetylated and lower activity to chitosans with DD of 64% and 90%.
The action mode of the purified enzyme was studied. TLC method and HPLC method were used to analyze the hydrolysis products of standard compounds (chitosan-oligomers and chitin-oligomers) catalyzed by the purified enzyme. The results show that the purified enzyme acted in an exo-mode and released GlcN residues successively from chitosan-oligomers, which is the characteristic of exo-β-D-glucosaminidase (GlcNase). In addition, the enzyme acted in an exo-mode on and released (GlcNAc)2 successively from chitin– oligomers, which is the characteristic of chitobiosidase. Therefore, the purified enzyme exhibited both GlcNase activity and chitobiosidase activity, and split glycoside bonds between GlcN-GlcN and GlcNAc-GlcNAc.
In this study, a hydrolase with chitosanolytic activity but no lipolytic activity was purified from a commercial lipase to homogeneity by using a combination of ion exchange chromatography, hydrophobic interaction chromatography, and gel filtration chromatography. The purified enzyme exhibited both GlcNase activity and chitobiosidase activity, therefore, the chitosanolytic activity the lipase exhibited was caused by the purified enzyme.
首先从四种不同来源的商品脂肪酶中选择了一种来源于Aspergillus Oryzae的具有较强壳聚糖水解活力的脂肪酶,研究了该酶降解壳聚糖的特性,并对该酶降解壳聚糖的产物进行了分析。脂肪酶对不同脱乙酰度(DD)的壳聚糖均有显著的水解作用,作用于DD为64%、73%、82%和90%的壳聚糖的最适pH值分别为4.2、4.4、4.6和5.0,最适温度均为60℃。脂肪酶水解不同DD壳聚糖的反应均遵循Michaelis-Meten方程,动力学分析结果表明DD值为73%和82%的壳聚糖是脂肪酶较容易水解的底物。脂肪酶催化壳聚糖降解可生成各种聚合度(DP = 2~6)的低聚糖,但在反应的最后,产物仅剩下氨基葡萄糖(GlcN),这表明在脂肪酶中存在外切酶。脂肪酶催化不同DD壳聚糖水解生成的产物完全相同。
采用超滤、DEAE-Sepharose CL-6B离子交换层析、Phenyl Sepharose CL-4B疏水作用层析和Sephacryl S-200凝胶过滤层析等一系列分离纯化操作,从脂肪酶中分离得到一个具有壳聚糖酶活力但不具有脂肪酶活力的酶组分。HPLC结果表明纯化酶的纯度达到99%以上,SDS-PAGE结果表明纯化酶已经达到均一。由于分离纯化过程中其他组分均不具备壳聚糖酶活力,因此说明脂肪酶的壳聚糖酶活力是由纯化酶的水解作用引起的。SDS-PAGE测得纯化酶的分子量约为74 kDa,而在非还原SDS-PAGE条件下纯化酶条带出现在130 kDa附近,表明纯化酶由两个相对分子质量相同的亚基组成,而且这两个亚基通过二硫键连接在一起。
利用氨基酸自动分析仪测定了纯化酶的氨基酸组成,结果表明天冬氨酸和谷氨酸含量较高,组氨酸、精氨酸和赖氨酸等碱性氨基酸的含量较低;含硫氨基酸的含量都非常低,而丝氨酸、甘氨酸、苏氨酸和丙氨酸等中性氨基酸的含量都较高。采用Edman降解法测得纯化酶N-末端12个氨基酸的序列为Ala– Leu– Arg– Leu– Asn– Ser– Pro– Asn– Asn– Ile– Ala– Val。在非冗余蛋白质序列( Non-redundant protein sequences)数据库中采用NCBI Blastp2程序对测得的N-末端氨基酸序列进行相似性比较,发现该酶与来自米曲霉的一个蛋白质具有很高的相似性,12个氨基酸序列的区域一致性达到100%。
采用DEPC、NBS、Ch-T、EDAC、PMSF、CHD、NAI、DTNB和DTT等化学修饰剂对纯化酶的氨基酸残基进行修饰,研究了纯化酶中必需基团的组成。DEPC对酶的修饰结果表明有1分子组氨酸残基位于纯化酶的活性中心;利用NBS修饰酶蛋白,结果表明色氨酸残基处于酶的活性中心,且至少有1分子色氨酸残基位于酶活性中心的底物结合部位;Ch-T和EDAC对酶的修饰结果表明甲硫氨酸残基和羧基也是酶的必需基团。利用PMSF、CHD、NAI、DTNB和DTT分别对酶分子中的羟基、精氨酸残基、酪氨酸残基、巯基和二硫键进行修饰,结果表明这些修饰剂在较高的浓度范围内均没有引起酶活的显著降低,因此羟基、精氨酸残基、酪氨酸残基、巯基和二硫键都不是维持酶活的必需基团。
研究了纯化酶的酶学性质,结果表明纯化酶在pH 4.6时的活力较高,在pH4.5 ~ 9.5之间具有较好的稳定性;在60℃时活力达到最高,在低于60℃的温度下时具有较高的热稳定性。Ni2+、Co2+和Mn2+等金属离子对酶的活力具有明显的激活作用,而Fe3+、Sn2+、Pb2+和Hg2+则能强烈抑制酶活,其中Hg2+的抑制能力最强,Na+、K+、Mg2+、Zn2+和Cd2+对酶活的影响较小。纯化酶对于DD为73%、81%和82%的壳聚糖显示了较高的水解活力,而对DD为64%和90%的壳聚糖的水解活力较低。
通过TLC法和HPLC法分析纯化酶降解已知结构甲壳低聚糖和乙酰甲壳低聚糖的产物,研究了纯化酶的作用模式。结果表明纯化酶以外切的形式作用于甲壳低聚糖,并将单糖GlcN顺次从甲壳低聚糖的末端释放出来,这与氨基葡萄糖苷酶(GlcNase)的作用模式是一致的;同时纯化酶以外切酶的形式作用于乙酰甲壳低聚糖的末端,并依次将乙酰甲壳二糖[(GlcNAc)2]释放出来,这是乙酰甲壳二糖苷酶典型的作用模式。因此该酶同时具有GlcNase活力和乙酰甲壳二糖苷酶活力,既能水解壳聚糖中GlcN-GlcN之间的糖苷键,也能水解GlcNAc-GlcNAc之间的糖苷键。
本论文通过超滤、凝胶过滤层析、疏水作用层析和离子交换层析等分离纯化技术,从脂肪酶中得到了一种具有壳聚糖水解活力但不具有脂肪酶活力的酶组分,该酶具有GlcNase和乙酰甲壳二糖苷酶双重活力,说明该酶的水解作用是脂肪酶降解壳聚糖的主要原因,从而阐明了商品脂肪酶非专一性降解壳聚糖的机理。
Commercial lipases exhibit non-specific activity on chitosan depolymerization, however, no research on mechanicsm of chitosan deploymerization by lipase has been reported at home and abroad. In this study, characteristics and mechanism of chitosan hydrolysis by a commercial lipase were investigated, which is of important theoretical and academic value in elucidating mechanism of chitosan hydrolysis by non-specific enzymes and of practical significance in instructing industrial enzymatic production of low molecular chitosan and chitooligosaccharides (COS).
Characteristics of chitosan hydrolysis by a commercial lipase from Aspergillus oryzae were investigated and hydrolysis products were analyzed. The lipase showed obvious hydrolytic activity on chitosans with different degrees of deacetylation (DD). The optimum pH values of the lipase on chitosans with DD of 64%, 73%, 82%, and 90% were 4.2, 4.4, 4.6, and 5.0, respectively, and optimum temperatures were all 60℃. The hydrolysis reactions of the lipase on different chitosans obeyed Michaelis-Meten equation, and kinetic parameters indicate that chitosans with DD of 73% and 82% were susceptible to be hydrolyzed. Products of chitosan hydrolysis by lipase included chitosan-oligomers with degree of polymerization (DP) 2~6, but the final product was glucosamine (GlcN), indicating some hydrolase with exo-mode action existing in the lipase. Products of hydrolysis of chitosans with different DD catalyzed by the lipase were identical.
A hydrolase with chitosanolytic activity but no lipolytic activity was purified from lipase by using a combination of ultrafiltration, DEAE-Sepharose CL-6B ion exchange chromatography, Phenyl-Sepharose CL-4B hydrophobic interaction chromatography, and Sephacryl S-200 gel filtration chromatography. Results of HPLC and SDS-PAGE show that the hydrolase had been purified to homogeneity. Because no other components of the lipase showed chitosanolytic activity during purification process, chitosanolytic activity the lipase exhibited was caused by the purified enzyme. Molecular mass of the purified enzyme estimated by SDS-PAGE and non-reducing SDS-PAGE was about 74 kDa and 130 kDa, respectively, indicating that the enzyme was composed of two identical subunits bound together with disulfide bonds.
Amino acids composition of the purified enzyme was analyzed by auto-amino acids analyzer. The results show that concentrations of Asp and Glu were high, those of His, Arg, and Lys were low, while those of sulfur-containing amino acids were extremely low. The N-terminal sequence containing 12 amino acids of the purified enzyme determined by using the Edman degradation technique was Ala– Leu– Arg– Leu– Asn– Ser– Pro– Asn– Asn– Ile– Ala– Val. The similarity of the N-terminal sequence was blasted in the Non-redundant Protein Sequences database using the NCBI Blastp2 program, and it was found that this sequence was similar to that of a protein from Aspergillus oryzae, and the identity of the 12 amino acids was 100%.
The purified enzyme was modified by DEPC, NBS, Ch-T, EDAC, PMSF, CHD, NAI, DTNB, and DTT. Modification of the purified enzyme by DEPC shows that 1 mole of histidine residue existed in the active site of the enzyme. Modification by NBS shows that tryptophan residues existed in the active site and there is at least 1 mole of tryptophan residue in the substrate binding site. Results of modification by Ch-T and EDAC indicate that methionine residues and carbosyl groups were essential groups of the purified enzyme. In addition, hydroxyl groups, arginine residues, tyrosine residues, sulfhydryl groups, and disulfide bonds were not essential groups of the purified enzyme.
The enzyme showed the optimum action pH value and temperature were 4.6 and 60?C, respectively, and it was stable in pH range of 4.5~9.5 and at temperatures lower than 60?C. Metal ions such as Ni2+, Co2+, and Mn2+ had obvious activation effects on the enzymatic activity, while Fe3+, Sn2+, Pb2+, and Hg2+ inactivated the enzyme, and Na+, K+, Mg2+, Zn2+, and Cd2+ had no obvious effect on the purified enzyme. The enzyme exhibited higher chitosanolytic activity toward chitosans which were 73%, 81% and 82% deacetylated and lower activity to chitosans with DD of 64% and 90%.
The action mode of the purified enzyme was studied. TLC method and HPLC method were used to analyze the hydrolysis products of standard compounds (chitosan-oligomers and chitin-oligomers) catalyzed by the purified enzyme. The results show that the purified enzyme acted in an exo-mode and released GlcN residues successively from chitosan-oligomers, which is the characteristic of exo-β-D-glucosaminidase (GlcNase). In addition, the enzyme acted in an exo-mode on and released (GlcNAc)2 successively from chitin– oligomers, which is the characteristic of chitobiosidase. Therefore, the purified enzyme exhibited both GlcNase activity and chitobiosidase activity, and split glycoside bonds between GlcN-GlcN and GlcNAc-GlcNAc.
In this study, a hydrolase with chitosanolytic activity but no lipolytic activity was purified from a commercial lipase to homogeneity by using a combination of ion exchange chromatography, hydrophobic interaction chromatography, and gel filtration chromatography. The purified enzyme exhibited both GlcNase activity and chitobiosidase activity, therefore, the chitosanolytic activity the lipase exhibited was caused by the purified enzyme.
引文
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