CN103981168B - 一种提高液体酶稳定性的组合物 - Google Patents

一种提高液体酶稳定性的组合物 Download PDF

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CN103981168B
CN103981168B CN201410246814.3A CN201410246814A CN103981168B CN 103981168 B CN103981168 B CN 103981168B CN 201410246814 A CN201410246814 A CN 201410246814A CN 103981168 B CN103981168 B CN 103981168B
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王凤超
王海
徐雪梅
占志平
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Wuxi Weilan Biotechnology Co ltd
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Abstract

本发明提供一种能够提高液体酶稳定性的组合物,该组合物包含有丙二醇、蔗糖、麦芽糊精和氯化钙;其中蔗糖和麦芽糊精占组合物总重量的15%-55%,蔗糖与麦芽糊精的质量比为1:2-4。本发明提供的组合物能有效提高纤维素酶、木聚糖酶、脂肪酶等液体酶制剂的稳定性,使液体酶制剂在37℃条件下储存8个月后,仍能保持80%以上的酶活,稳定性优于市售国外酶制剂产品。本发明所述组合物中各组分之间产生了协同促进作用,添加组合物的酶制剂的残余酶活比单独添加等量丙二醇、蔗糖、麦芽糊精或氯化钙的酶制剂的最高残余酶活还要高出10%-17%,取得了意料不到的技术效果。本发明所述组合物能有效延长液体酶制剂的储存期限,因此有利于液体酶的推广和使用。

Description

一种提高液体酶稳定性的组合物
技术领域
本发明属于酶制剂技术领域,具体涉及一种提高液体酶稳定性的组合物及其应用方法。
背景技术
随着生物科技的不断发展,酶制剂在工业、食品及饲料行业中的应用越来越广泛。其中液体酶是酶制剂的主要剂型,其占有的市场份额不断扩大。酶是一种具有生物活性的蛋白质,只有维持其特定的空间结构,才能具有活性。而酶的三维结构是由疏水作用、静电作用、氢键及二硫键维持的,在液体状态下酶分子处于舒展的状态,活性中心裸露,很容易受化学、物理和生物等因素的影响,发生氧化、水解等反应而使酶分子的空间结构改变,失去活性,造成酶蛋白的聚集沉淀,既导致产品酶活下降,也影响产品外观,从而限制了液体酶制剂的市场推广。
目前市售的大多数液体酶制剂产品主要采用如下方法来解决稳定性的问题:1)通过对酶蛋白分子进行一定的化学修饰提高其稳定性,例如:利用一些小分子物质(糖类、多羟基醇类等)与酶蛋白分子表面的-NH、-OH或-COOH等基团发生反应,改变侧链的结构,防止氧化、水解等反应的发生,从而提高酶分子的稳定性;2)选择合适种类的稳定剂及合适的浓度提高酶的稳定性,能够提供适宜的渗透压,使酶分子处于相对脱水的状态,多为无机盐类或多羟基醇类;3)确定液体酶的最适pH范围,pH是影响液体酶稳定性的关键因素之一。
目前市售的液体酶制剂产品多数在6个月内较稳定,但储存期限超过6个月后,酶活降低得很快,严重限制了液体酶的推广和使用。
发明内容
本发明为解决现有技术问题,提供了一种能够提高液体酶稳定性的组合物及其应用方法,从而满足液体酶长期储存的需要。
本发明一方面提供了一种组合物,该组合物包含有丙二醇、蔗糖、麦芽糊精和氯化钙;其中蔗糖和麦芽糊精占组合物总重量的15%-55%,蔗糖与麦芽糊精的质量比为1:2-4。
所述组合物,各组分及其占组合物总重量的百分比分别为:
所述组合物,各组分及其占组合物总重量的百分比分别为:
本发明另一方面提供了上述组合物在液体酶中的应用。
所述组合物在液体酶中的添加比例为0.15-0.35g/mL。
本发明提供的组合物能有效提高纤维素酶、木聚糖酶、脂肪酶等液体酶制剂的稳定性,使液体酶制剂在37℃条件下储存8个月后,仍能保持80%以上的酶活,稳定性优于市售国外酶制剂产品。本发明所述组合物中各组分之间产生了协同促进作用,添加组合物的酶制剂的残余酶活比单独添加等量丙二醇、蔗糖、麦芽糊精或氯化钙的酶制剂的最高残余酶活还要高出10%-17%,取得了意料不到的技术效果。本发明所述组合物能有效延长液体酶制剂的储存期限,因此有利于液体酶的推广和使用。
具体实施方式
申请人在长期的研究中发现,蔗糖和麦芽糊精的含量对本发明所述组合物的稳定效果影响较大,在相同添加量的情况下,当组合物中蔗糖和麦芽糊精的含量在15%-55%的范围内时,组合物对酶制剂的稳定效果明显,当蔗糖和麦芽糊精的含量低于15%或高于55%时,酶制剂的残余酶活显著降低;且当蔗糖和麦芽糊精的质量比在1:2-4范围内时,组合物的稳定效果最佳,尤其是蔗糖和麦芽糊精的质量比为1:3时,添加该组合物的酶制剂在相同条件下储存8个月,残余酶活仍高达85%以上,取得了意料不到的效果。
下面结合具体的实施方案对本发明进行更详细的描述。但本发明并不受所述实施例的限制。提供实施方案的目的是为了使说明书全面而透彻,并向本领域的技术人员全面传达发明的范围。除另定义外,本发明所使用的所有技术和科学术语均具有作为本发明所属技术领域中普通技术人员通常理解的同样的含义。
本发明所用仪器设备为行业内常用设备,所用原料和试剂可选自市售任意一种,例如:丙二醇、蔗糖、麦芽糊精和氯化钙均可选购自国药集团;实施例4所述市售国外的纤维素酶、木聚糖酶和脂肪酶产品均可购自杰能科生物工程有限公司。
实施例1
一种组合物,各组分及其占组合物总重量的百分比分别为:
将上述的各个组分混合均匀制成组合物。
实施例2
一种组合物,各组分及其占组合物总重量的百分比分别为:
实施例3
一种组合物,各组分及其占组合物总重量的百分比分别为:
实施例4液体酶制剂稳定性测试
将上述组合物按0.15-0.35g/mL比例添加到液体酶(未添加稳定剂)中。
1)纤维素酶的初始酶活为8000u/ml,实施例1-3所述组合物的添加量为0.15g/mL;
2)木聚糖酶的初始酶活为50000u/ml,实施例1-3所述组合物的添加量为0.35g/mL;
3)脂肪酶的初始酶活为10000u/ml,实施例1-3所述组合物的添加量为0.25g/mL。
将上述液体酶置于37℃培养箱内储存,每30天检测一次酶活,计算酶活残留率。以未添加组合物的液体酶作为空白对照,同时与具有相同酶活水平的市售液体酶产品(含稳定剂)做效果比较,结果如表1-3所示。
表1:纤维素酶产品稳定性分析结果
空白对照 市售产品 实施例1 实施例2 实施例3
起始 100% 100% 100% 100% 100%
30天 70.5% 92.5% 92.7% 95.6% 94.5%
60天 65.8% 87.2% 87.6% 93.4% 92.1%
90天 58.1% 84.7% 84.5% 91.6% 91.5%
120天 45.6% 82.9% 83.5% 91.0% 90.8%
150天 32.4% 80.7% 82.3% 89.2% 88.9%
180天 20.8% 74.8% 80.2% 88.5% 86.7%
210天 9.1% 48.1% 75.8% 85.9% 83.3%
240天 5.4% 36.9% 71.0% 82.1% 80.2%
表2:木聚糖酶产品稳定性分析结果
空白对照 市售产品 实施例1 实施例2 实施例3
起始 100% 100% 100% 100% 100%
30天 75.9% 91.2% 91.8% 96.6% 95.7%
60天 65.7% 84.5% 84.7% 94.1% 92.2%3 -->
90天 56.2% 82.2% 83.1% 93.6% 89.8%
120天 33.7% 80.6% 81.2% 91.5% 87.1%
150天 28.5% 78.1% 78.6% 90.4% 85.9%
180天 18.2% 71.0% 75.5% 88.3% 83.2%
210天 8.5% 47.2% 73.5% 86.2% 81.5%
240天 4.1% 32.5% 70.2% 85.1% 80.1%
表3:脂肪酶产品稳定性分析结果
空白对照 市售产品 实施例1 实施例2 实施例3
起始 100% 100% 100% 100% 100%
30天 80.8% 92.6% 93.5% 96.8% 96.1%
60天 75.7% 87.3% 87.8% 95.6% 94.2%
90天 60.5% 85.1% 85.5% 92.1% 91.9%
120天 54.2% 82.2% 83.6% 90.9% 90.3%
150天 46.1% 79.2% 79.4% 89.5% 89.0%
180天 30.5% 72.4% 75.1% 88.2% 87.8%
210天 11.2% 49.8% 72.8% 84.1% 83.2%
240天 6.9% 39.4% 70.1% 81.5% 81.0%
从表1-3的数据可以看出,与空白对照组相比,市售的含稳定剂的液体酶产品和添加了本发明实施例1-3所述组合物的液体酶,其残余酶活水平均显著高于空白对照组。与市售国外液体酶产品相比,添加本发明实施例1-3所述组合物的液体酶各阶段的残余酶活均高于国外产品,尤其是在储存6个月后,国外液体酶产品的残余酶活迅速降低至50%以下,而添加本发明所述组合物的液体酶的酶活则下降缓慢,37℃储存8个月后,残余酶活仍高于70%。其中实施例2和3所述组合物对液体酶的稳定效果最佳,使液体酶在37℃储存8个月后,残余酶活仍高于80%,远远优于市售国外产品。从而说明实施例2和3所述组合物中各组分间实现了最优配比,取得了意料不到的效果。
实施例5组分间协同促进作用验证试验
分别将丙二醇、蔗糖、麦芽糊精、氯化钙和实施例3所述组合物这5种稳定剂按0.15g/mL的比例添加到液体纤维素酶(未加任何稳定剂成分)中,其初始酶活为8000u/ml。将该酶置于37℃培养箱内储存,每30天检测一次酶活,计算酶活残留率。以未添加任何稳定剂成分的液体酶作为空白对照。结果如表4所示:
表4:纤维素酶产品稳定性分析结果
空白对照 丙二醇 蔗糖 麦芽糊精 氯化钙 实施例3
起始 100% 100% 100% 100% 100% 100%
30天 70.5% 85.5% 84.7% 86.6% 82.8% 94.5%
60天 65.8% 82.2% 82.6% 84.4% 81.1% 92.1%
90天 58.1% 80.7% 80.5% 82.6% 79.4% 91.5%4 -->
120天 45.6% 78.9% 79.4% 81.1% 78.6% 90.8%
150天 32.4% 77.1% 76.6% 79.8% 75.8% 88.9%
180天 20.8% 74.8% 72.1% 76.7% 74.2% 86.7%
210天 9.1% 71.1% 69.8% 70.9% 67.2% 83.3%
240天 5.4% 66.1% 63.0% 66.4% 64.2% 80.2%
从表4的数据可以看出,与空白对照组相比,单独添加丙二醇、蔗糖、麦芽糊精或氯化钙均可明显提高纤维素酶的稳定性;但与实施例3所述组合物相比,上述单个组分的作用效果远低于四种组分组合在一起的效果。在储存期内,添加实施例3所述组合物的纤维素酶的残余酶活比单独添加等量丙二醇、蔗糖、麦芽糊精或氯化钙的纤维素酶的最高残余酶活还要高出10%-17%,从而说明本发明所述组合物中各组分之间产生了协同促进作用,取得了意料不到的技术效果。
申请人在脂肪酶、木聚糖酶等其他酶制剂中也进行了上述实验,经证实能达到同样的效果。
实施例6蔗糖和麦芽糊精总含量对组合物稳定效果的影响
1.试验样品
样品1:丙二醇85%,(蔗糖+麦芽糊精)10%,氯化钙5%;
样品2:丙二醇80%,(蔗糖+麦芽糊精)15%,氯化钙5%;(实施例3)
样品3:丙二醇65%,(蔗糖+麦芽糊精)32%,氯化钙3%;
样品4:丙二醇52%,(蔗糖+麦芽糊精)45%,氯化钙3%;
样品5:丙二醇42%,(蔗糖+麦芽糊精)55%,氯化钙3%;
样品6:丙二醇38%,(蔗糖+麦芽糊精)60%,氯化钙2%;
样品7:丙二醇33%,(蔗糖+麦芽糊精)65%,氯化钙2%;
上述样品中蔗糖与麦芽糊精的质量比均为1:2。
2.实验过程
分别将样品1-6按0.35g/mL的比例添加到液体木聚糖酶(未加任何稳定剂成分)中,其初始酶活为50000u/ml。将该酶置于37℃培养箱内储存,每30天检测一次酶活,计算酶活残留率。以未添加任何稳定剂成分的液体酶作为空白对照。结果如表5所示:
表5木聚糖酶产品稳定性分析
从表5的数据可以看出,与空白对照组相比,样品1-7所述组合物均能提高木聚糖酶的稳定性;当组合物中蔗糖和麦芽糊精的含量在15%-55%范围内,对酶的稳定效果最好,储存6个月后仍能保持80%以上的残余酶活,其中添加样品3的木聚糖酶储存8个月后,残余酶活仍高于80%;而当组合物中蔗糖和麦芽糊精的含量低于15%或高于55%时,其稳定效果显著降低,添加样品1、6、7的木聚糖酶在相同条件下储存6个月后,残余酶活低于55%。从而说明蔗糖和麦芽糊精的总含量对本发明所述组合物的酶稳定效果具有意料不到的影响。
实施例7蔗糖与麦芽糊精的配比对组合物稳定效果的影响
试验样品
丙二醇65%,(蔗糖+麦芽糊精)32%,氯化钙3%;其中:
样品1:蔗糖:麦芽糊精=1:1;
样品2:蔗糖:麦芽糊精=1:2;
样品3:蔗糖:麦芽糊精=1:3;(实施例2)
样品4:蔗糖:麦芽糊精=1:4;
样品5:蔗糖:麦芽糊精=1:5;
样品6:蔗糖:麦芽糊精=1:6;
2.实验过程
分别将样品1-6按0.25g/mL的比例添加到液体木聚糖酶(未加任何稳定剂成分)中,其初始酶活为50000u/ml。将该酶置于37℃培养箱内储存,每30天检测一次酶活,计算酶活残留率。以未添加任何稳定剂成分的液体酶作为空白对照。结果如表6所示:
表6:木聚糖酶产品稳定性分析结果
空白对照 样品1 样品2 样品3 样品4 样品5 样品6
起始 100% 100% 100% 100% 100% 100% 100%
30天 75.9% 90.2% 93.1% 96.6% 93.8% 90.5% 90.0%
60天 65.7% 88.6% 92.0% 94.1% 91.1% 89.1% 88.2%
90天 56.2% 86.1% 90.7% 93.6% 90.4% 85.1% 85.9%
120天 33.7% 84.8% 88.2% 91.5% 88.5% 84.7% 84.1%
150天 28.5% 82.9% 86.1% 90.4% 86.8% 81.9% 81.5%
180天 18.2% 80.6% 85.3% 88.3% 85.7% 80.1% 81.0%6 -->
210天 8.5% 73.5% 82.1% 86.2% 83.4% 74.1% 74.7%
240天 4.1% 70.2% 80.9% 85.1% 81.4% 71.5% 72.2%
从表6的数据可以看出,与空白对照组相比,样品1-6所述组合物均能提高木聚糖酶的稳定性;当组合物中蔗糖与麦芽糊精的质量比在1:2-4范围内,对酶的稳定效果最好,储存8个月后仍能保持80%以上的残余酶活,其中添加样品3(蔗糖与麦芽糊精的质量比为1:3)的木聚糖酶,在相同条件下储存8个月后,残余酶活高达85%;而当组合物中蔗糖与麦芽糊精的质量比高于1:2,或低于1:4时,其稳定效果显著降低,添加样品1、5、6的木聚糖酶在相同条件下储存8个月后,残余酶活较样品2-4降低了10-15%。从而说明蔗糖和麦芽糊精的配比对本发明所述组合物的酶稳定效果具有意料不到的影响。

Claims (5)

1.一种组合物,其特征在于,所述的组合物中的组分及其占组合物总重量的百分比如下:丙二醇42-80%、蔗糖5-11%、麦芽糊精10-44%、氯化钙3-5%;且蔗糖和麦芽糊精占组合物总重量的15%-55%,蔗糖与麦芽糊精的质量比为1:2-4。
2.如权利要求1所述的组合物,其特征在于,所述的组分及其占组合物总重量的百分比分别为:丙二醇65%、蔗糖8%、麦芽糊精24%、氯化钙3%。
3.权利要求1-2任一项所述的组合物作为液体酶稳定剂的应用。
4.一种液体酶,其特征在于,所述的液体酶中添加有权利要求1-2任一项所述的组合物。
5.如权利要求4所述的液体酶,其特征在于,所述的组合物在液体酶中的添加比例为0.15-0.35g/mL。
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