几丁质脱乙酰酶产生菌的筛选
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摘要
几丁质脱乙酰酶(Chitin deacetylase,简称CDA)是能够将几丁质分子中的乙酰基直接脱除而生成壳聚糖的酶,而几丁质作为酶的底物,难溶于水,因此CDA酶活力的测定非常复杂和困难,使得CDA的相关研究进展缓慢。本论文研究探讨了以对硝基乙酰苯胺作为CDA的底物、以对硝基苯胺作为CDA催化脱乙酰产物测定CDA活力的可能性,并建立了一种简便、高效的酶活力测定方法。实验考察了对硝基乙酰苯胺和对硝基苯胺的特征吸收峰;通过考察不同缓冲液体系的紫外可见吸收波谱排除了缓冲液对酶活力测定的干扰;模拟酶解体系测定结果表明,建立的酶活测定方法具有较好的稳定性和重现性。
以几丁质为唯一碳源,以对硝基乙酰苯胺为颜色指示剂,采用平板变色圈法,从15份土样中筛选获得459株菌,通过变色滤纸条检验法从459株菌株中得到使滤纸条变色较快、较深的菌株268株进行摇瓶发酵,测定发酵液的CDA酶活力,获得一株产酶活力较高且性能稳定的CDA产生菌11-3。测得菌株粗酶对胶体几丁质作用产物证实有乙酸产生,说明该菌株产酶对胶体几丁质具有脱乙酰作用。通过BIOLOG细菌鉴定系统分析和16SrDNA序列分析,结合菌株11-3的培养特征及生理生化特征,鉴定菌株11-3为红球菌属(Rhodococcus sp.),迄今为止未见该类菌产生CDA的研究报道。
研究确定了菌株11-3产酶的较优培养基组成和培养条件。菌株11-3发酵产酶培养基碳源以白砂糖最佳,氮源以蛋白胨最佳,并需要添加硫酸铵和玉米浆,乙酸钠、磷酸二氢钾对产酶有促进作用,而乙酸铵、硫酸亚铁对产酶则有抑制作用,硫酸镁、氯化钙、硫酸锰对产酶影响不大。产酶最佳培养基组成(g/L):蛋白胨2.5,白砂糖7.0,硫酸铵2.5,磷酸二氢钾1.5,乙酸钠2.0,玉米浆5.0。最佳培养条件为:250mL三角瓶装液量30mL～35mL,发酵初始pH为7.5,种龄24h,接种量5%,培养温度30℃,摇床转速180r/min,发酵周期60h。产酶条件优化后,菌株11-3产酶水平提高了近100倍,且性能稳定,说明菌株11-3是一支具有开发潜力的CDA生产菌株。
研究了菌株11-3的部分酶学性质。菌株11-3粗酶具有较好的热稳定性,45℃保温1h仍能保持90%以上的活力,酶的最适作用温度为50℃;最适作用pH为7.0,在pH7.0—10.0之间有较高的活性,Ag~+对该酶有较强的抑制作用。
本论文建立了一种简便、高效的CDA酶活力测定方法,并利用该方法筛选获得一支性能稳定、产CDA活力较高、热稳定性好的红球菌11-3,为进一步开展相关研究奠定了基础。
Chitin deacetylase(CDA) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-D-glucosamine in chitin,convering it to chitosan.As the substrate of the enzyme mentioned above,chitin is slightly soluble,which makes it difficult to determine the enzyme activity of CDA, thus it hampered the progress in CDA research.A simple and effective method of determining the enzyme activity was put forward with p-nitroacetanilide as substrate and paranitroanilinum as production.The FAP(Features Absorption Peak) of p-nitroacetanilide and paranitroanilinum were taken into account.
By analyzing absorption spectrum of various buffer solution,interference of buffer on the enzyme activity were excluded.The stability and repeatability of absorption values in the enzyme reaction system was assured by simulating system.In comparing to the traditional method,the determination was greatly simplified with identical outcome.
Using culture media with chitin as sole carbon source and addition of p-nitroacetanilide as color indicator,we isolated 459 strains with yellow circle surrounding from 15 soil samples.Then,268 strains was isolated after second screening by discolorating filter paper chips at faster speed with darker color. Screened after being incubated by shake flasks together with the method mentioned above,a strain with CDA chitin deacetylase productive ability together with optimal stabilization,named strain 11-3,was finally obtained. Gas chromatography showed acetic acid existed in the production of reaction between crude enzyme and colloidal chitin,which displayed the deacetylation characteristic of the enzyme.The result of physiological and biochemical characteristics conbined,studies indicate that strain 11-3 may be members of genus Rhodococcus sp.There's no report on this genus culture with the ability of producing CDA so far.
Influence factors on the enzyme production of Strain 11-3 were acquired by researching on various factors.The result was as follows:It could utilize many kinds of materials as carbon resource;Saccharose was the best of all. When came to Nitrogen resource,peptone was the best with the addition of ammonium sulfate and corn slurry.Sodium acetate promoted enzyme production while ammonium acetate was to the contrary.FeSO_4 strains had a stronger effect on inhibiting enzyme production;KH_2PO_4 promoted enzyme production,the impact of MgSO_4,CaCl_2,and MnSO_4 on enzyme production was insignificant.The optimal medium composition(g/L) was:sucrose 5, corn pulp 5,ammonium sulfate 2,peptone 2,KH_2PO_4 1.5,sodium acetate 1, pH 7.5.The optimal culture conditions were:broth volume 30-35 mL in 250 mL Erlenmeyer flasks,inoculation time 24 h,inoculation volume 5%, agitation speed 180 r/min at 30℃,fermentation time 60 hours.After optimization of enzyme production conditions,enzyme production increased from the 57 U/mL initially to 5035 U/mL,nearly 100 times concerning the initial yield,which accounted for the great potential of the strain 11-3.
Part of the enzyme characteristic of strain 11-3 was investigated and excellent thermal stability displayed:when treated in 45℃for 1h,more than 90%activity remained.The best temperature was 50℃and the optimal pH was 7.0.Higher enzyme activity remained with pH variation was 7.0-10.0. Ag~+ was of effective inhibition to it.
A simple and effective method of determining the enzyme activity was put forward in the thesis.Rhodococcus 11-3 with stable performance,higher production CDA vitality and excellent thermal stability was acquired. Foundation was laid for further research.
以几丁质为唯一碳源,以对硝基乙酰苯胺为颜色指示剂,采用平板变色圈法,从15份土样中筛选获得459株菌,通过变色滤纸条检验法从459株菌株中得到使滤纸条变色较快、较深的菌株268株进行摇瓶发酵,测定发酵液的CDA酶活力,获得一株产酶活力较高且性能稳定的CDA产生菌11-3。测得菌株粗酶对胶体几丁质作用产物证实有乙酸产生,说明该菌株产酶对胶体几丁质具有脱乙酰作用。通过BIOLOG细菌鉴定系统分析和16SrDNA序列分析,结合菌株11-3的培养特征及生理生化特征,鉴定菌株11-3为红球菌属(Rhodococcus sp.),迄今为止未见该类菌产生CDA的研究报道。
研究确定了菌株11-3产酶的较优培养基组成和培养条件。菌株11-3发酵产酶培养基碳源以白砂糖最佳,氮源以蛋白胨最佳,并需要添加硫酸铵和玉米浆,乙酸钠、磷酸二氢钾对产酶有促进作用,而乙酸铵、硫酸亚铁对产酶则有抑制作用,硫酸镁、氯化钙、硫酸锰对产酶影响不大。产酶最佳培养基组成(g/L):蛋白胨2.5,白砂糖7.0,硫酸铵2.5,磷酸二氢钾1.5,乙酸钠2.0,玉米浆5.0。最佳培养条件为:250mL三角瓶装液量30mL～35mL,发酵初始pH为7.5,种龄24h,接种量5%,培养温度30℃,摇床转速180r/min,发酵周期60h。产酶条件优化后,菌株11-3产酶水平提高了近100倍,且性能稳定,说明菌株11-3是一支具有开发潜力的CDA生产菌株。
研究了菌株11-3的部分酶学性质。菌株11-3粗酶具有较好的热稳定性,45℃保温1h仍能保持90%以上的活力,酶的最适作用温度为50℃;最适作用pH为7.0,在pH7.0—10.0之间有较高的活性,Ag~+对该酶有较强的抑制作用。
本论文建立了一种简便、高效的CDA酶活力测定方法,并利用该方法筛选获得一支性能稳定、产CDA活力较高、热稳定性好的红球菌11-3,为进一步开展相关研究奠定了基础。
Chitin deacetylase(CDA) is an enzyme that catalyzes the hydrolysis of acetamine groups of N-acetyl-D-glucosamine in chitin,convering it to chitosan.As the substrate of the enzyme mentioned above,chitin is slightly soluble,which makes it difficult to determine the enzyme activity of CDA, thus it hampered the progress in CDA research.A simple and effective method of determining the enzyme activity was put forward with p-nitroacetanilide as substrate and paranitroanilinum as production.The FAP(Features Absorption Peak) of p-nitroacetanilide and paranitroanilinum were taken into account.
By analyzing absorption spectrum of various buffer solution,interference of buffer on the enzyme activity were excluded.The stability and repeatability of absorption values in the enzyme reaction system was assured by simulating system.In comparing to the traditional method,the determination was greatly simplified with identical outcome.
Using culture media with chitin as sole carbon source and addition of p-nitroacetanilide as color indicator,we isolated 459 strains with yellow circle surrounding from 15 soil samples.Then,268 strains was isolated after second screening by discolorating filter paper chips at faster speed with darker color. Screened after being incubated by shake flasks together with the method mentioned above,a strain with CDA chitin deacetylase productive ability together with optimal stabilization,named strain 11-3,was finally obtained. Gas chromatography showed acetic acid existed in the production of reaction between crude enzyme and colloidal chitin,which displayed the deacetylation characteristic of the enzyme.The result of physiological and biochemical characteristics conbined,studies indicate that strain 11-3 may be members of genus Rhodococcus sp.There's no report on this genus culture with the ability of producing CDA so far.
Influence factors on the enzyme production of Strain 11-3 were acquired by researching on various factors.The result was as follows:It could utilize many kinds of materials as carbon resource;Saccharose was the best of all. When came to Nitrogen resource,peptone was the best with the addition of ammonium sulfate and corn slurry.Sodium acetate promoted enzyme production while ammonium acetate was to the contrary.FeSO_4 strains had a stronger effect on inhibiting enzyme production;KH_2PO_4 promoted enzyme production,the impact of MgSO_4,CaCl_2,and MnSO_4 on enzyme production was insignificant.The optimal medium composition(g/L) was:sucrose 5, corn pulp 5,ammonium sulfate 2,peptone 2,KH_2PO_4 1.5,sodium acetate 1, pH 7.5.The optimal culture conditions were:broth volume 30-35 mL in 250 mL Erlenmeyer flasks,inoculation time 24 h,inoculation volume 5%, agitation speed 180 r/min at 30℃,fermentation time 60 hours.After optimization of enzyme production conditions,enzyme production increased from the 57 U/mL initially to 5035 U/mL,nearly 100 times concerning the initial yield,which accounted for the great potential of the strain 11-3.
Part of the enzyme characteristic of strain 11-3 was investigated and excellent thermal stability displayed:when treated in 45℃for 1h,more than 90%activity remained.The best temperature was 50℃and the optimal pH was 7.0.Higher enzyme activity remained with pH variation was 7.0-10.0. Ag~+ was of effective inhibition to it.
A simple and effective method of determining the enzyme activity was put forward in the thesis.Rhodococcus 11-3 with stable performance,higher production CDA vitality and excellent thermal stability was acquired. Foundation was laid for further research.
引文
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