木聚糖酶产生菌的培养条件优化及部分酶学性质研究
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摘要
对本实验室保藏的绿色木霉(Trichoderma viride)菌株。进行液态发酵，并对产酶发酵条件作了优化。优化的液态发酵条件如下：以10％玉米芯粉为碳源，0.5％的蛋白胨为氮源，100mL三角瓶20％装液量，2％接种量，培养基初始pH为4.0，30℃，180rpm条件下培养72h。
将在此条件下培养得到的发酵液离心，加入50％饱和度的(NH_4)_2SO_4盐析，得到粗酶液。经测定，酶活为449U／mL，比活184．9U／mg。对此酶液进行部分基本酶学性质研究。测得该酶的最适反应温度为60℃；最适pH为4.0；有一定的热稳定性，40℃下能较长时间地保持较高活性，当高于40℃时，酶活迅速下降，作用20min后，相对酶活仅为4.5％；此酶在酸性环境下有较强的稳定性，但随着pH值的上升，酶的稳定性迅速下降，当pH值达到6.0时几乎检测不到酶活。考察了部分金属离子对该酶酶活的影响：当加入较低浓度金属离子时，Cu~(2+)对木聚糖酶有较强的抑制作用，其抑制率达到了41％。Al~(3+)次之，此外CO~(2+)、Cr~(3+)在此浓度对酶也有不同程度的抑制作用。随着金属离子浓度的增加，先前起抑制作用的金属离子对酶活力的抑制减弱，其中Cu~(2+)对木聚糖酶的抑制率从41％降低到8％。Al~(3+)、CO~(2+)、Cr~(3+)对木聚糖酶的抑制率也有不同程度的降低。其它金属离子K~+、Ba~(2+)、Ca~(2+)、Mn~(2+)、Fe~(3+)、Mg~(2+)等对酶活力无明显影响。
此外，考察了将木聚糖酶与不同浓度保护剂：蔗糖、麦芽糖和葡萄糖作用后，木聚糖酶热稳定性和酸稳定性的变化。发现木聚糖酶与蔗糖和麦芽糖作用后，其热稳定性和酸稳定性均有不同程度的提高。其中，蔗糖的效果相对较好，当蔗糖浓度达到3％时，木聚糖酶的残余酶活达到最大值。此后。保护剂浓度的增大对酶活影响己不明显。葡萄糖对酶的保护作用极小。
Optimized the fermentation conditions of the Trichoderma viride collected in our lab.The optimization result through experiment is described as below: 10%corncob-powder of total medium volume as the carbon source, 0.5% peptone asthe nitrogen source, 20% triangle flask bulk for medium volume, 2% inoculatingvolume, initializing medium pH to 4.0, 30℃, 180 rpm to ferment for 72 hours.The fermentation broth was purified through centrifuging and salting out with 50%ammonium sulfate. The activity and the specific activity of the obtained crudxylanase is 449U/mL and 184.9U/mg respectively. Basic enzymatic characteristicsof obtained enzyme were studied. The optimal temperature is 60℃, and it showsrelatively good thermal stability under 40℃; The optimal pH condition is 4.0, andit is stable under acid environment but as the ascensus of pH value its stabilitydecrease quickly. The influences of some metal ions on xylanase activity were alsostudied. Lower concentration of the ions such as Cu~(2+)、Al~(3+)、Co~(2+) and Cr~(3+) inhibitthe enzyme activity. Especially the Cu~(2+), it has a relatively strong inhibit on theenzyme and it inhibited 41% of the activity. As the ascensus of the concentration,the inhibition attenuated. The inhibition of 2mmol/L Cu~(2+) degraded to 8 %. Theinfluences of other metal ions such as K~+、Ba~(2+)、Ca~(2+)、Mn~(2+)、Fe~(3+)、Mg~(2+)on xylanaseare not obvious Besides, we investigated the thermal and acid stability of xylanase after itsincubation with different concentration of protecttive solute(sucrose、maltose andglucose). The protective solute can improve the thermal and acid stability ofxylanase. The effects of sucrose are the best, it can retain most of the activity at theconcentration of 3％. At higher concentration the effects are not obvious any more.
将在此条件下培养得到的发酵液离心，加入50％饱和度的(NH_4)_2SO_4盐析，得到粗酶液。经测定，酶活为449U／mL，比活184．9U／mg。对此酶液进行部分基本酶学性质研究。测得该酶的最适反应温度为60℃；最适pH为4.0；有一定的热稳定性，40℃下能较长时间地保持较高活性，当高于40℃时，酶活迅速下降，作用20min后，相对酶活仅为4.5％；此酶在酸性环境下有较强的稳定性，但随着pH值的上升，酶的稳定性迅速下降，当pH值达到6.0时几乎检测不到酶活。考察了部分金属离子对该酶酶活的影响：当加入较低浓度金属离子时，Cu~(2+)对木聚糖酶有较强的抑制作用，其抑制率达到了41％。Al~(3+)次之，此外CO~(2+)、Cr~(3+)在此浓度对酶也有不同程度的抑制作用。随着金属离子浓度的增加，先前起抑制作用的金属离子对酶活力的抑制减弱，其中Cu~(2+)对木聚糖酶的抑制率从41％降低到8％。Al~(3+)、CO~(2+)、Cr~(3+)对木聚糖酶的抑制率也有不同程度的降低。其它金属离子K~+、Ba~(2+)、Ca~(2+)、Mn~(2+)、Fe~(3+)、Mg~(2+)等对酶活力无明显影响。
此外，考察了将木聚糖酶与不同浓度保护剂：蔗糖、麦芽糖和葡萄糖作用后，木聚糖酶热稳定性和酸稳定性的变化。发现木聚糖酶与蔗糖和麦芽糖作用后，其热稳定性和酸稳定性均有不同程度的提高。其中，蔗糖的效果相对较好，当蔗糖浓度达到3％时，木聚糖酶的残余酶活达到最大值。此后。保护剂浓度的增大对酶活影响己不明显。葡萄糖对酶的保护作用极小。
Optimized the fermentation conditions of the Trichoderma viride collected in our lab.The optimization result through experiment is described as below: 10%corncob-powder of total medium volume as the carbon source, 0.5% peptone asthe nitrogen source, 20% triangle flask bulk for medium volume, 2% inoculatingvolume, initializing medium pH to 4.0, 30℃, 180 rpm to ferment for 72 hours.The fermentation broth was purified through centrifuging and salting out with 50%ammonium sulfate. The activity and the specific activity of the obtained crudxylanase is 449U/mL and 184.9U/mg respectively. Basic enzymatic characteristicsof obtained enzyme were studied. The optimal temperature is 60℃, and it showsrelatively good thermal stability under 40℃; The optimal pH condition is 4.0, andit is stable under acid environment but as the ascensus of pH value its stabilitydecrease quickly. The influences of some metal ions on xylanase activity were alsostudied. Lower concentration of the ions such as Cu~(2+)、Al~(3+)、Co~(2+) and Cr~(3+) inhibitthe enzyme activity. Especially the Cu~(2+), it has a relatively strong inhibit on theenzyme and it inhibited 41% of the activity. As the ascensus of the concentration,the inhibition attenuated. The inhibition of 2mmol/L Cu~(2+) degraded to 8 %. Theinfluences of other metal ions such as K~+、Ba~(2+)、Ca~(2+)、Mn~(2+)、Fe~(3+)、Mg~(2+)on xylanaseare not obvious Besides, we investigated the thermal and acid stability of xylanase after itsincubation with different concentration of protecttive solute(sucrose、maltose andglucose). The protective solute can improve the thermal and acid stability ofxylanase. The effects of sucrose are the best, it can retain most of the activity at theconcentration of 3％. At higher concentration the effects are not obvious any more.
引文
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