果胶酶高产菌种的筛选及其酶学性质的研究
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摘要
果胶酶是分解果胶质酶类的总称。通常包括聚半乳糖醛酸酶(PG)、果胶酯酶(PE)、果胶裂解酶(PL)等主要组分。果胶酶是一种重要的工业酶制剂,广泛应用于果汁、果酒加工、蔬菜汁提取、酒精发酵、饲料、木材防腐、麻类脱胶、棉织物精练加工等领域。我国果胶酶生产大多采用固体发酵法,因该方法比较粗放,产品质量与国外产品差距较大,果胶酶市场基本被国外产品垄断。目前国内对果胶酶发酵生产的研究大多局限于单一聚半乳糖醛酸组分,而对发酵过程中同时考察三种果胶酶系(PG、PE、PL)的研究相对较少,本论文采用产果胶酶的优良菌种—黑曲霉SS9,通过研究培养条件对该菌所产三种酶组分的活力影响,获得最佳产酶培养基和条件,并通过对国外酶产品与自制酶的酶系组份活力比对,尝试分析国外酶与国产酶应用效果差距的内在原因。
从本实验室保存的产果胶酶的黑曲霉、根霉入手,设计了三种不同类型的筛选培养基,对其进行自然分离复壮筛选,筛选出产酶能力较强的菌株—黑曲霉SS9,通过实验对比,发现溴甲酚蓝变色圈法是一种有效的分离筛选方法,并采用该筛选方法从自然界采集的10份土样中,分离筛选出12株产果胶酶菌株,验证了所设计的筛选方法是有效可行的,为果胶酶菌种的分离提供了一种较可靠有效的筛选方法。
在确定各种因素对产酶影响的基础上,对黑曲霉液体发酵产果胶酶进行条件的优化,得出了适合三种酶组分产生的综合培养基:桔皮粉3 g、(NH4 ) 2 SO4 2 g、麸皮12 g、尿素0.075 g、CaCl2 0.03 g、KH2 PO4 3.8 g、K2HPO4·3H2O 0.2 g、乙二醇15 mL、水100 mL,pH 6.0,产酶最高可达PG 32.82万U/mL、PE 31862 U/mL,PL 0.716 U/mL。在实验过程中,对产PL进行特别的研究,对产PL的培养基特别是复合氮源,进行了优化,得到产PL的适合培养基:桔皮粉4 g、NaNO3 0.4 g、麸皮20 g、尿素0.075 g、CaCl2 0.03 g、MgSO4 0.05 g、KH2 PO4 3.8 g、K2HPO4·3H2O 0.2 g、乙二醇15 mL、水100 mL,pH 6.0,产酶最高可达到1.267 U/mL。并测定了三种不同酶系的发酵曲线。
通过对果胶酶各组分酶学特性的研究,了解果胶酶各组分的最适pH(PG 4.0、PE 6.0、PL 6.0),酶稳定的pH范围(PG 2.0～8.0、PE 4.0～7.0、PL 5.0～8.0),最适温度(PG 50℃、PE 50℃、PL 45℃),稳定的温度范围(PG在55℃以下、PE和PL在50℃以下)。
开展了发酵粗酶液提高苹果出汁率的应用研究。通过优化,得到最佳的工艺参数为:pH 3.5、温度40℃、果胶酶的用量为0.5﹪,处理时间90 min,苹果的出汁率达到最高。并对发酵粗酶液与国外品牌商品酶制剂的酶活力和出汁率进行分析比较,其主要差距是PL的活力偏低,对应的出汁率也偏低,经分析,在苹果出汁方面,除了PG的作用外,PL也有着重要的作用,为果胶酶的应用研究提供了参考依据。
The pectinase is the total call of the things that can break up the pectin.It’s category usually conclude the PG,PE and PL.Traditionally the pectinase is used to the processing of the syrup and the ratafee.Now the application extend to the fields including the extract of the vegetable juice,antisepsis of the wood, ethanol fermentation, feedstuff,colloid remove of the hemp,refining and processing of the cotton weave.In our country the solid fermentation is common.Beacause this method is extensive and it’s product is hard to reach the request of some industries especially the food industry,the pectinase is limited greatly to apply in the industry producing.Now the researches in our country is most about the sigle pectinase and the complex study on the PG,PE and PL in the process of the fermentation is less.The study is based on a excellence strain-Asperillus niger SS9 and get the optimum condition of the pectinase producing through the enzyme activity analyse of the three pectinase.We can find the distance between our production and the oversea’s through the enzyme activity contrast.
The experiment is started with Asperillus niger and Rhizopus strains selecting which is conserved in the lab.For the variety of the selecting methods the experiment choosed three different mediums,and through the contrast a effective path has been concerned .A strain which has the great ability to break up the pectin come true.On the study we can confirm that the 3th selecting medium is useful and effective.To learn the live character and offer the reference to the next experiment,we meature the strain’s growth speed.We have got twelve strains which has the activity from ten soil swatches.The result is the confirmation od the selecting method we desired and this will be a effective and dependable way for the pectinase strain selecting.
Through a series of researchs some factors which can influence the pectinase production have been researched and comfirmed. We made the cultrue optimized and the result studying is indicated: in initial pH5-7,temperature 28℃-30℃,the best sonstitue of the culture is citrus peel powder 3 g, bran 12 g , urea 0.075 g, (NH4)2SO4 2 g,CaCl2 0.03 g, KH2PO4 3.8 g, K2HPO4·3H2O 0.2 g,glycol 15 mL, H2O 100 mL , pH 6.0, Output is : PG 32,820,000 U/ mL, PE 31862 U/ mL, PL 0.716 U/ mL.In the process we study the culture optimization of the PL especially.Based on the complex nitrogen researchs we made the culture that can produce PL conformably: citrus peel powder 4g, bran 20 g , urea 0.075 g, NaNO3 0.4 g,CaCl2 0.03 g, MgSO4 0.05 g,KH2PO4 3.8 g, K2HPO4·3H2O 0.2 g,glycol 15 mL, H2O 100 mL , pH 6.0,the PL can reach 1.267 U/ mL.And we made the ferment curve of the three enzymes.
Through the research on the character of the different pectinase,we learned that the optimal pH(PG 4.0、PE 6.0、PL 6.0); the pH stability of the three pectinase(PG 2.0～8.0、PE 4.0～7.0、PL 5.0～8.0);the optimal temperatur(ePG 50℃、PE 50℃、PL 45℃); the temperature stability of pectinase(PG below 55℃、PE and PL below 50℃).
The pectinase liguid of the fermentation is applied to the research that improve the rate of the apple juice lixiviated.Through a series of the optimization a fit condition for the experiment is that:the initial pH is 3.5,the temperature is 40℃,the quantity of the pectinase used is 0.5 percent,the time of the reaction is ninty minutes,the rate of the juice lixiviated is the highest.We can find the difference between the pectinase we produced and the enzyme product overseas through the contrast of the component analysed.Through the research we can discover that the activity of our production is lower and the juice rate is also lower.The result is not only the PG but also the PL is important to the apple juice.The factors influence the juice lixiviated can also be analysed and this research will be useful for the next study.
从本实验室保存的产果胶酶的黑曲霉、根霉入手,设计了三种不同类型的筛选培养基,对其进行自然分离复壮筛选,筛选出产酶能力较强的菌株—黑曲霉SS9,通过实验对比,发现溴甲酚蓝变色圈法是一种有效的分离筛选方法,并采用该筛选方法从自然界采集的10份土样中,分离筛选出12株产果胶酶菌株,验证了所设计的筛选方法是有效可行的,为果胶酶菌种的分离提供了一种较可靠有效的筛选方法。
在确定各种因素对产酶影响的基础上,对黑曲霉液体发酵产果胶酶进行条件的优化,得出了适合三种酶组分产生的综合培养基:桔皮粉3 g、(NH4 ) 2 SO4 2 g、麸皮12 g、尿素0.075 g、CaCl2 0.03 g、KH2 PO4 3.8 g、K2HPO4·3H2O 0.2 g、乙二醇15 mL、水100 mL,pH 6.0,产酶最高可达PG 32.82万U/mL、PE 31862 U/mL,PL 0.716 U/mL。在实验过程中,对产PL进行特别的研究,对产PL的培养基特别是复合氮源,进行了优化,得到产PL的适合培养基:桔皮粉4 g、NaNO3 0.4 g、麸皮20 g、尿素0.075 g、CaCl2 0.03 g、MgSO4 0.05 g、KH2 PO4 3.8 g、K2HPO4·3H2O 0.2 g、乙二醇15 mL、水100 mL,pH 6.0,产酶最高可达到1.267 U/mL。并测定了三种不同酶系的发酵曲线。
通过对果胶酶各组分酶学特性的研究,了解果胶酶各组分的最适pH(PG 4.0、PE 6.0、PL 6.0),酶稳定的pH范围(PG 2.0～8.0、PE 4.0～7.0、PL 5.0～8.0),最适温度(PG 50℃、PE 50℃、PL 45℃),稳定的温度范围(PG在55℃以下、PE和PL在50℃以下)。
开展了发酵粗酶液提高苹果出汁率的应用研究。通过优化,得到最佳的工艺参数为:pH 3.5、温度40℃、果胶酶的用量为0.5﹪,处理时间90 min,苹果的出汁率达到最高。并对发酵粗酶液与国外品牌商品酶制剂的酶活力和出汁率进行分析比较,其主要差距是PL的活力偏低,对应的出汁率也偏低,经分析,在苹果出汁方面,除了PG的作用外,PL也有着重要的作用,为果胶酶的应用研究提供了参考依据。
The pectinase is the total call of the things that can break up the pectin.It’s category usually conclude the PG,PE and PL.Traditionally the pectinase is used to the processing of the syrup and the ratafee.Now the application extend to the fields including the extract of the vegetable juice,antisepsis of the wood, ethanol fermentation, feedstuff,colloid remove of the hemp,refining and processing of the cotton weave.In our country the solid fermentation is common.Beacause this method is extensive and it’s product is hard to reach the request of some industries especially the food industry,the pectinase is limited greatly to apply in the industry producing.Now the researches in our country is most about the sigle pectinase and the complex study on the PG,PE and PL in the process of the fermentation is less.The study is based on a excellence strain-Asperillus niger SS9 and get the optimum condition of the pectinase producing through the enzyme activity analyse of the three pectinase.We can find the distance between our production and the oversea’s through the enzyme activity contrast.
The experiment is started with Asperillus niger and Rhizopus strains selecting which is conserved in the lab.For the variety of the selecting methods the experiment choosed three different mediums,and through the contrast a effective path has been concerned .A strain which has the great ability to break up the pectin come true.On the study we can confirm that the 3th selecting medium is useful and effective.To learn the live character and offer the reference to the next experiment,we meature the strain’s growth speed.We have got twelve strains which has the activity from ten soil swatches.The result is the confirmation od the selecting method we desired and this will be a effective and dependable way for the pectinase strain selecting.
Through a series of researchs some factors which can influence the pectinase production have been researched and comfirmed. We made the cultrue optimized and the result studying is indicated: in initial pH5-7,temperature 28℃-30℃,the best sonstitue of the culture is citrus peel powder 3 g, bran 12 g , urea 0.075 g, (NH4)2SO4 2 g,CaCl2 0.03 g, KH2PO4 3.8 g, K2HPO4·3H2O 0.2 g,glycol 15 mL, H2O 100 mL , pH 6.0, Output is : PG 32,820,000 U/ mL, PE 31862 U/ mL, PL 0.716 U/ mL.In the process we study the culture optimization of the PL especially.Based on the complex nitrogen researchs we made the culture that can produce PL conformably: citrus peel powder 4g, bran 20 g , urea 0.075 g, NaNO3 0.4 g,CaCl2 0.03 g, MgSO4 0.05 g,KH2PO4 3.8 g, K2HPO4·3H2O 0.2 g,glycol 15 mL, H2O 100 mL , pH 6.0,the PL can reach 1.267 U/ mL.And we made the ferment curve of the three enzymes.
Through the research on the character of the different pectinase,we learned that the optimal pH(PG 4.0、PE 6.0、PL 6.0); the pH stability of the three pectinase(PG 2.0～8.0、PE 4.0～7.0、PL 5.0～8.0);the optimal temperatur(ePG 50℃、PE 50℃、PL 45℃); the temperature stability of pectinase(PG below 55℃、PE and PL below 50℃).
The pectinase liguid of the fermentation is applied to the research that improve the rate of the apple juice lixiviated.Through a series of the optimization a fit condition for the experiment is that:the initial pH is 3.5,the temperature is 40℃,the quantity of the pectinase used is 0.5 percent,the time of the reaction is ninty minutes,the rate of the juice lixiviated is the highest.We can find the difference between the pectinase we produced and the enzyme product overseas through the contrast of the component analysed.Through the research we can discover that the activity of our production is lower and the juice rate is also lower.The result is not only the PG but also the PL is important to the apple juice.The factors influence the juice lixiviated can also be analysed and this research will be useful for the next study.
引文
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2.郑寿亭,高培基等,酶与食品加工,北京:轻工出版社,1991,111-112
3. T.Sakai,Y Ozaki. Protopectin solubilizing enzyme that does not catalyse the degrada- tion of polygalacturonic acid.Agric.Biol.Chem,1988,52(4):1091-1093
4. Tyagi,R;Gupta,M.N. Purification and immobilization of Aspergillus niger pectinase on magnetic latex beads, Biocatalysis and Biotransformation,1995,12(4): 293-298
5. Szaniawski,Andrzej R; Spencer,H.Garth,Effect of pectin concentration and crossflow velocity on permeability in the microfiltration of dilute pectin solutions by macroporous titania membranes containing immobilized pectinase.,Biotechnology Progress, 1996,12(4):403-405
6. RONALD P. DE VRIES JAAP VISSER.Aspergillus Enzymes involved in degradation of plant cell wall polysaccharides.MICROBIOLOGY AND MOLECULAR BIOLOGY REVIEWS,2001,.497-522
7.王璋.食品酶学.北京:轻工业出版社,1990,164-175
8. Ros,J.M.Production of pectic enzymes from Rhizopus nigicans cultures with different sources of carbon.J.Ann. Microbiol.Enzimol,1993,43:71-6
9. Reginald H.Waiter.The Chemistry and technology of pectin.Academic Press. San Diego,1991
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