Aspergillus ficuum SK004产外切菊粉酶及其酶解菊粉制备高果糖浆的研究
摘要
菊粉是由D-呋喃果糖以β-2,1糖苷键连接的多聚果糖,其还原端连接一个葡萄糖基,呈直链结构,富含于菊芋等多种菊科植物中。菊芋块茎主要成分为菊粉,其含量占干重的70~80%。菊芋的生长对土质要求不严,易种植,产量较高,是制备高果糖浆和低聚果糖的极好原料。菊粉酶是水解菊粉的高效酶,微生物来源的菊粉酶通常是底物诱导性的外切酶,主要通过催化水解菊粉链的非还原性末端的糖苷键,逐一水解释放出果糖,主要产物为果糖。因此筛选高产菌株、提高水解菊粉酶的酶活在高果糖浆工业上有着重要的意义。
本实验室保存的一株Aspergillus ficuum SK004无花果曲霉菌株能产菊粉酶,其混合酶系的I /S为0.43。本研究对SK004菌株进行了发酵条件的优化,确定该菌株主要产胞外酶,且主要为外切菊粉酶。优化条件为(g /L):菊粉25,蛋白胨25,NH_4H_2PO_3 4,NaCl 5,MgSO_4·7H_2O 0.1,ZnSO_4·7H_2O 0.1,pH 6.5,30℃,200 rpm下培养7 d酶活力可稳定在56.7U /ml。
将Aspergillus ficuum SK004菌株所产酶系进行分离纯化,先后通过活性炭脱色、硫酸铵分级沉淀、透析脱盐、DEAE-Sepharose CL-6B离子层析、Sephadex G-75凝胶色谱后,得到一种菊粉酶。经SDS-PAGE分析此菊粉酶为单一谱带,分子量为53665Da。本课题还对纯化后的外切菊粉酶的酶学性质进行了研究。发现该酶的最适反应温度为60℃,最适反应pH为4.5,在50℃保温1 h能保持80%的酶活,在pH 4.0~6.0的范围内常温放置1 h能保持90%以上的酶活。水解菊粉的Km为115.8 mg /mL,Vmax为7.80 mg/ml·min;水解蔗糖的Km为60.06 mg / mL,Vmax为7.69 mg /ml·min。以菊粉为底物,Urea、K~+、Al~(3+)对酶活的影响不大,Zn2+、Mg~(2+)、EDTA、Ca~(2+)、Cu~(2+)和SDS对菊粉酶具有抑制作用,Mn~(2+)、Li~+和Fe~(2+)对菊粉酶有激活作用;以蔗糖为底物,除Al~(3+)有激活作用外,上述其它离子对菊粉酶都具有一定的抑制作用。
通过对影响粗酶液水解速率的温度、pH、加酶量和底物浓度等四个因素的研究,确定粗酶液水解菊粉的最佳反应条件为:菊粉浓度20 g /L,反应体系pH 4.0,反应温度55℃,加酶量为25 U /g菊粉。在最适反应条件下,粗酶液水解4 h,酶解率可以达到88%,水解10 h,酶解率可以达到96%。10 h后的水解反应趋于平缓,单位时间的酶解率增加不多,最终可达到97.55%的酶解率。
提出了菊粉酶酶解菊芋制备高果糖浆的工艺路线,由此路线可以得到果糖含量90%以上的高果糖浆。
Inulin has been defined as a polydisperse carbohydrate material consisting mainly of β-2,1fructosyl-fructose links and end in a glucose unit. Inulin is the main carbohydrate in a variety ofplants. Jerusalem Artichoke contains 70%~80% inulin (dry weight). Because of its ease ofcultivation and harvesting, Jerusalem Artichoke has become the principal source of producinghigh fructose syrup and oligofructose today. Exo-inulinase is 2,1-β-D-fructan fructanohydrolaseswhich convert inulin to fructose by hydrolase the β-2,1 linkage in its unreducing end. Because ofthe hygienical character of high fructose syrup, it is very important to screen outinuinase-producing strain with high enzyme activity in industry.
A strain of Aspergillus ficuum SK004, with a ratio of inuluinase activity to invertase activity(I /S ratio) of 0.43, was stored in our laboratory. The effects of nutrients and other culturalconditions on the exo-inulinase was studied. The optimal medium compositions and culturalconditions were (g /L): inulin 25 , peptone 25, NaCl 5, NH4H2PO4 4, MgSO4·7H2O 0.1,ZnSO4·7H2O 0.1, pH 6.5, temperature 30℃, shaken at 200 rpm with 30 ml medium per flask(250 mL) for 7 d. The maximum enzyme activity was 56.7 U /mL.
The crude inulinase preparation was purified in a sequence of operations includingdecoloration by active carbon, ammonium sulfate fractionation, dialysis, ion exchangechromatography on DEAE-Sepharose CL-6B and column chromatography with Sephadex G-75.One exo-inulinase was obtained. The single band on SDS-PAGE showed that the emzyme washighly purified and its molecular weight was 53665Da.
Properties of the purified exo-inulinase were also studied. The optimal reaction temperatureand pH of exo-inulinase were 60℃ and 4.5 respectively. The enzyme was stable within pH4.0~6.0 and below the temperature 50℃. With inulin as substrate, Km =115.8 mg /mL,Vmax=7.80 mg /ml·min;with sucrose as substrate, Km=60.06 mg / mL,Vmax=7.69 mg /ml·min. Themetal ions (2 mM), with inulin as substrate, the effects on exo-inulinase activity of Urea(10 mM),K+, Al~(3+) were negligible, whereas Mn~(2+), Li~+ and Fe~(2+) exhibited positive effects on exo-inulinaseactivity;Zn~(2+), Mg~(2+), EDTA, Ca~(2+), Cu~(2+) and SDS(40 mM) inhibited activity to different degree.With sucrose as substrate, Al3+ had slight positive effects on inulinase activity;other metals allhad negative effects.
Effects of temperature, pH, enzyme and substrate concentration on the inulin-hydrolyzingdegree were determined. When hydrolyzed in system of 2% inulin solution added crude inulinaseof 25 U/g inulin at pH 4.0, 55℃ for 4 h, the inulin-hydrolyzing degree was 88% ,if hydrolyzedfor 10h, the degree up to 96%. After 10 h, the hydrolyzing degree increased slowly and the totalreducing sugar yield can get to 97.55% at 30h.
A technological line was proposed according to the hydrolysis research. Over 90% fructosesyrup could be obtained by this line.
本实验室保存的一株Aspergillus ficuum SK004无花果曲霉菌株能产菊粉酶,其混合酶系的I /S为0.43。本研究对SK004菌株进行了发酵条件的优化,确定该菌株主要产胞外酶,且主要为外切菊粉酶。优化条件为(g /L):菊粉25,蛋白胨25,NH_4H_2PO_3 4,NaCl 5,MgSO_4·7H_2O 0.1,ZnSO_4·7H_2O 0.1,pH 6.5,30℃,200 rpm下培养7 d酶活力可稳定在56.7U /ml。
将Aspergillus ficuum SK004菌株所产酶系进行分离纯化,先后通过活性炭脱色、硫酸铵分级沉淀、透析脱盐、DEAE-Sepharose CL-6B离子层析、Sephadex G-75凝胶色谱后,得到一种菊粉酶。经SDS-PAGE分析此菊粉酶为单一谱带,分子量为53665Da。本课题还对纯化后的外切菊粉酶的酶学性质进行了研究。发现该酶的最适反应温度为60℃,最适反应pH为4.5,在50℃保温1 h能保持80%的酶活,在pH 4.0~6.0的范围内常温放置1 h能保持90%以上的酶活。水解菊粉的Km为115.8 mg /mL,Vmax为7.80 mg/ml·min;水解蔗糖的Km为60.06 mg / mL,Vmax为7.69 mg /ml·min。以菊粉为底物,Urea、K~+、Al~(3+)对酶活的影响不大,Zn2+、Mg~(2+)、EDTA、Ca~(2+)、Cu~(2+)和SDS对菊粉酶具有抑制作用,Mn~(2+)、Li~+和Fe~(2+)对菊粉酶有激活作用;以蔗糖为底物,除Al~(3+)有激活作用外,上述其它离子对菊粉酶都具有一定的抑制作用。
通过对影响粗酶液水解速率的温度、pH、加酶量和底物浓度等四个因素的研究,确定粗酶液水解菊粉的最佳反应条件为:菊粉浓度20 g /L,反应体系pH 4.0,反应温度55℃,加酶量为25 U /g菊粉。在最适反应条件下,粗酶液水解4 h,酶解率可以达到88%,水解10 h,酶解率可以达到96%。10 h后的水解反应趋于平缓,单位时间的酶解率增加不多,最终可达到97.55%的酶解率。
提出了菊粉酶酶解菊芋制备高果糖浆的工艺路线,由此路线可以得到果糖含量90%以上的高果糖浆。
Inulin has been defined as a polydisperse carbohydrate material consisting mainly of β-2,1fructosyl-fructose links and end in a glucose unit. Inulin is the main carbohydrate in a variety ofplants. Jerusalem Artichoke contains 70%~80% inulin (dry weight). Because of its ease ofcultivation and harvesting, Jerusalem Artichoke has become the principal source of producinghigh fructose syrup and oligofructose today. Exo-inulinase is 2,1-β-D-fructan fructanohydrolaseswhich convert inulin to fructose by hydrolase the β-2,1 linkage in its unreducing end. Because ofthe hygienical character of high fructose syrup, it is very important to screen outinuinase-producing strain with high enzyme activity in industry.
A strain of Aspergillus ficuum SK004, with a ratio of inuluinase activity to invertase activity(I /S ratio) of 0.43, was stored in our laboratory. The effects of nutrients and other culturalconditions on the exo-inulinase was studied. The optimal medium compositions and culturalconditions were (g /L): inulin 25 , peptone 25, NaCl 5, NH4H2PO4 4, MgSO4·7H2O 0.1,ZnSO4·7H2O 0.1, pH 6.5, temperature 30℃, shaken at 200 rpm with 30 ml medium per flask(250 mL) for 7 d. The maximum enzyme activity was 56.7 U /mL.
The crude inulinase preparation was purified in a sequence of operations includingdecoloration by active carbon, ammonium sulfate fractionation, dialysis, ion exchangechromatography on DEAE-Sepharose CL-6B and column chromatography with Sephadex G-75.One exo-inulinase was obtained. The single band on SDS-PAGE showed that the emzyme washighly purified and its molecular weight was 53665Da.
Properties of the purified exo-inulinase were also studied. The optimal reaction temperatureand pH of exo-inulinase were 60℃ and 4.5 respectively. The enzyme was stable within pH4.0~6.0 and below the temperature 50℃. With inulin as substrate, Km =115.8 mg /mL,Vmax=7.80 mg /ml·min;with sucrose as substrate, Km=60.06 mg / mL,Vmax=7.69 mg /ml·min. Themetal ions (2 mM), with inulin as substrate, the effects on exo-inulinase activity of Urea(10 mM),K+, Al~(3+) were negligible, whereas Mn~(2+), Li~+ and Fe~(2+) exhibited positive effects on exo-inulinaseactivity;Zn~(2+), Mg~(2+), EDTA, Ca~(2+), Cu~(2+) and SDS(40 mM) inhibited activity to different degree.With sucrose as substrate, Al3+ had slight positive effects on inulinase activity;other metals allhad negative effects.
Effects of temperature, pH, enzyme and substrate concentration on the inulin-hydrolyzingdegree were determined. When hydrolyzed in system of 2% inulin solution added crude inulinaseof 25 U/g inulin at pH 4.0, 55℃ for 4 h, the inulin-hydrolyzing degree was 88% ,if hydrolyzedfor 10h, the degree up to 96%. After 10 h, the hydrolyzing degree increased slowly and the totalreducing sugar yield can get to 97.55% at 30h.
A technological line was proposed according to the hydrolysis research. Over 90% fructosesyrup could be obtained by this line.
引文
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2. 郑建仙. 功能性食品甜味剂. 北京:中国轻工业出版社,1997.11.
3. 胡莉,何越,谢元. 高果糖浆在饮料中的应用. 中国食品工业网.
4. 张乐兴. 高果糖浆的性质与应用. 广州食品工业科技, 2003,19 (1):44~45.
5. 谢元. 高果糖浆在饮料中的应用. 食品工业,2002,23(2):33~34.
6. 蔡复礼. 果糖的应用特性及其分离. 精细化工,1996,13(2):22~24.
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