微生物发酵生产耐酸性α-淀粉酶的研究
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摘要
随着我国淀粉质原料深加工工业的发展,工艺条件的改变,要求酶制剂工业不断更新和完善淀粉酶的种类以满足工业生产的需求。目前,国内外市场中的常用的淀粉酶为中温、高温以及碱性α—淀粉酶,其适用pH范围一般为6.0～10.0,在酸性条件下其酶活性明显降低,已不能满足一些酸性条件下淀粉原料的深加工工艺的要求。因此,开发新型耐酸性α—淀粉酶制剂便显得愈发迫切。
本研究采用微生物分离和诱变育种技术获得一株可产生耐酸性α—淀粉酶菌株;进一步采用生物化学分析方法及统计学方法对该菌株产酶的最适发酵条件、及其所产耐酸性α-淀粉酶的酶学性质和其酶的分离提取工艺条件进行了研究。所得研究结果如下:
1.筛选得到一株产量较高的黑曲霉耐酸性α-淀粉酶生产菌株Tx78,以其为出发菌株进行了紫外线诱变及亚硝基胍诱变,结果得到了一株产酶量可达1120U/mL的高产突变株UNTx78-358,较出发菌提高了86.7%。而且该菌株遗传性能稳定,具有工业化生产的潜力。
2.研究了UNTx78-358所产耐酸性α-淀粉酶的酶学性质,该酶为胞外酶,其最适反应温度为70℃,最适反应pH为4.0,K_m值为7.89g/L,当Ca~(2+)浓度为7mmol/L时对该酶活性具有显著的促进作用;该酶具有良好的耐酸性和热稳定性,薄层层析表明该酶制剂水解淀粉最终产物为麦芽糖和葡萄糖。能够实现在酸性条件下淀粉液化和糖化的同步进行的生产过程,从而简化淀粉加工工艺,节约成本。
3.确定了菌株UNTx78-358液体发酵产酶的最佳培养基组成为:淀粉6.23g/L;蛋白胨12g/L;CaCl_2 0.287g/L,以及1‰的植酸;最适发酵条件为:培养基pH为4.0,摇瓶装量为100mL/300mL,接种量为10%,30℃培养48h后补料20mL继续培养至96h。其产酶量平均达到2400U/mL以上,达到目前国内同比先进水平。
4.研究确定了该菌株液体发酵生产耐酸性α-淀粉酶的提取工艺条件为:发酵液体经8层纱布(或200筛)过滤得到粗酶液,粗酶液经调节pH=6.0,于55℃下浓缩后;加入3.5倍的冷藏无水乙醇,收集沉淀于-50℃冷冻干燥即得耐酸性α-淀粉酶的粗酶粉剂,其收率达89.3%,纯化倍数为2.8倍,粗酶得率达到8g/L以上。再经二次离子交换层析纯化后,SDS-PAGE电泳验证该酶的分子量约为74kD。通过对该酶的N-末端氨基酸序列进行测定,发现该酶的N-末端的10个氨基酸序列为Leu,Ser,Ala,Ala,Glu,Thr,Arg,Thr,Gln,Ser,Ile,Tyr与文献报道的A.niger所产耐酸性α-淀粉酶的N-末端氨基酸序列一致。
本研究旨在通过选育具有高产耐酸性-α淀粉酶能力的菌株,并通过对其酶学性质进行系统研究,以及该菌株的液体发酵产酶条件的优化和下游分离提取技术的研究。为我国耐酸性α-淀粉酶制剂的开发提供技术支持。在此基础上还将进一步进行大规模发酵工艺及下游提取工艺的研究,争取早日实现产业化丌发。因此,本研究的实施不仅可以填补我国在耐酸性淀粉水解酶类的生产上的空白,具有较高的学术价值,如能进一步深入研究还可以产生巨大的经济效益和社会效益。
Recently, as our country's starch processing industry development so quickly, the process technology has been changed. More and more factory needs the new kinds of amylase to be suit for the new methods. Now, in the national and native markets people can only find theα-amylase is functional in high or low temperature and also the alkaline conditions, its suitable pH is from 6-10. Under the acidity conditions, its major activity will be lost, so can not do the starch processing under the acidity conditions. Then the needs of a novelα-amylase can tolerance the acidity condition are more urgent. So our research is a systemic work about how to produce the acid-stableα-amylase by the microbial fermentation. It includes: breeding the acid-stableα-amylase producing strains, enzymology characteristic research of the acid-stableα-amylase, conditions optimization of the liquid fermentation of the enzyme producing strain, isolated and purified of the acid-stableα-amylase. The results are as follows:
1. The high acid-stableα-amylase productivity strain Aspergillus niger Tx-78 was isolated from mold culture. After the UV and NG mutation treatment, the mutant strain can produce the target enzyme 1120U/mL. It is higher than the originate strain 86.7%. And also this mutant has a nice genetic stability.
2. By the enzymology characteristic research of the acid-stableα-amylase produced from Mutant strain UNTx78-358, the enzyme was identified as an exoenzyme. Its reaction optimum temperature and pH is 70℃and 4.0 respectively. The kinetics research shown its Km is 7.89g/L. And Ca~(2+) cans accelerant the activity of this enzyme remarkable, especially when the consistency of Ca~(2+) is 7mmol/L. The results of TLC demonstrated that the products from starch catalyzed by the enzyme were mainly maltose and glucose. The results show this enzyme has a good acid tolerant and thermo stability, which can achieve the liquefaction and saccharification of the starch spontaneously. So it will have a good application prospect.
3. By the optimum fermentation medium research of UNTx78-358. The results show: the optimum culture conclude starch 6.23g/L, peptone 12g/L, CaCl20.287g/L respectively, and phytic acid 1‰. The optimum fermentation conditions are as follows: 100ml/300mL per flask, 10% inoculums concentration, 48h cultivate on 30℃. After that, replenish 20ml culture then continue cultivate until 96h, and the pH is 4.0. By this experiment, the production of the enzyme can be 2400/mL, is higher than the original production 114.3%.
4. The isolated processing of the enzyme producing strain is as follows: The crude enzyme fraction can get by 8 layer gauze filter or 200 seive mesh . Adjust the pH to 6.0 of the crude enzyme fraction then concentrate it by rotary evaporator under the 0.09Mpa, 55℃. Then concentrate the enzyme fraction by 3.5 times ethanol under 4℃. Collect the precipitate and freeze drying it under -50℃, 24h. After that, the enzyme can be isolated. Its activity can remain 89.3%, purified times is 2.8, yield is 8g/L. The enzyme was purified by CM52 and DE52 cellulose ion-exchange chromatography and SDS-PAGE. The results show the mass of this enzyme is about 74kD. And its N-terminal amino acid sequence is Leu, Ser, Ala, Ala, Glu, Thr, Arg, Thr, Gln, Ser, Ile, Tyr, as same as the sequence of acid-stableα-amylase product from A. niger in other reports.
The aim of this reaserch is provide the useful statistics for the development of the acid-stableα-amylase in our country by the isolation and screening high yield strain, enzymology characteristic research, optimum fermentation conditions design and isolated processing research. For that reasons, this research not only can optimize for maximum profit of our countries production of acid-stableα-amylase, But also will be taking higher position in terms of academic and economic values
本研究采用微生物分离和诱变育种技术获得一株可产生耐酸性α—淀粉酶菌株;进一步采用生物化学分析方法及统计学方法对该菌株产酶的最适发酵条件、及其所产耐酸性α-淀粉酶的酶学性质和其酶的分离提取工艺条件进行了研究。所得研究结果如下:
1.筛选得到一株产量较高的黑曲霉耐酸性α-淀粉酶生产菌株Tx78,以其为出发菌株进行了紫外线诱变及亚硝基胍诱变,结果得到了一株产酶量可达1120U/mL的高产突变株UNTx78-358,较出发菌提高了86.7%。而且该菌株遗传性能稳定,具有工业化生产的潜力。
2.研究了UNTx78-358所产耐酸性α-淀粉酶的酶学性质,该酶为胞外酶,其最适反应温度为70℃,最适反应pH为4.0,K_m值为7.89g/L,当Ca~(2+)浓度为7mmol/L时对该酶活性具有显著的促进作用;该酶具有良好的耐酸性和热稳定性,薄层层析表明该酶制剂水解淀粉最终产物为麦芽糖和葡萄糖。能够实现在酸性条件下淀粉液化和糖化的同步进行的生产过程,从而简化淀粉加工工艺,节约成本。
3.确定了菌株UNTx78-358液体发酵产酶的最佳培养基组成为:淀粉6.23g/L;蛋白胨12g/L;CaCl_2 0.287g/L,以及1‰的植酸;最适发酵条件为:培养基pH为4.0,摇瓶装量为100mL/300mL,接种量为10%,30℃培养48h后补料20mL继续培养至96h。其产酶量平均达到2400U/mL以上,达到目前国内同比先进水平。
4.研究确定了该菌株液体发酵生产耐酸性α-淀粉酶的提取工艺条件为:发酵液体经8层纱布(或200筛)过滤得到粗酶液,粗酶液经调节pH=6.0,于55℃下浓缩后;加入3.5倍的冷藏无水乙醇,收集沉淀于-50℃冷冻干燥即得耐酸性α-淀粉酶的粗酶粉剂,其收率达89.3%,纯化倍数为2.8倍,粗酶得率达到8g/L以上。再经二次离子交换层析纯化后,SDS-PAGE电泳验证该酶的分子量约为74kD。通过对该酶的N-末端氨基酸序列进行测定,发现该酶的N-末端的10个氨基酸序列为Leu,Ser,Ala,Ala,Glu,Thr,Arg,Thr,Gln,Ser,Ile,Tyr与文献报道的A.niger所产耐酸性α-淀粉酶的N-末端氨基酸序列一致。
本研究旨在通过选育具有高产耐酸性-α淀粉酶能力的菌株,并通过对其酶学性质进行系统研究,以及该菌株的液体发酵产酶条件的优化和下游分离提取技术的研究。为我国耐酸性α-淀粉酶制剂的开发提供技术支持。在此基础上还将进一步进行大规模发酵工艺及下游提取工艺的研究,争取早日实现产业化丌发。因此,本研究的实施不仅可以填补我国在耐酸性淀粉水解酶类的生产上的空白,具有较高的学术价值,如能进一步深入研究还可以产生巨大的经济效益和社会效益。
Recently, as our country's starch processing industry development so quickly, the process technology has been changed. More and more factory needs the new kinds of amylase to be suit for the new methods. Now, in the national and native markets people can only find theα-amylase is functional in high or low temperature and also the alkaline conditions, its suitable pH is from 6-10. Under the acidity conditions, its major activity will be lost, so can not do the starch processing under the acidity conditions. Then the needs of a novelα-amylase can tolerance the acidity condition are more urgent. So our research is a systemic work about how to produce the acid-stableα-amylase by the microbial fermentation. It includes: breeding the acid-stableα-amylase producing strains, enzymology characteristic research of the acid-stableα-amylase, conditions optimization of the liquid fermentation of the enzyme producing strain, isolated and purified of the acid-stableα-amylase. The results are as follows:
1. The high acid-stableα-amylase productivity strain Aspergillus niger Tx-78 was isolated from mold culture. After the UV and NG mutation treatment, the mutant strain can produce the target enzyme 1120U/mL. It is higher than the originate strain 86.7%. And also this mutant has a nice genetic stability.
2. By the enzymology characteristic research of the acid-stableα-amylase produced from Mutant strain UNTx78-358, the enzyme was identified as an exoenzyme. Its reaction optimum temperature and pH is 70℃and 4.0 respectively. The kinetics research shown its Km is 7.89g/L. And Ca~(2+) cans accelerant the activity of this enzyme remarkable, especially when the consistency of Ca~(2+) is 7mmol/L. The results of TLC demonstrated that the products from starch catalyzed by the enzyme were mainly maltose and glucose. The results show this enzyme has a good acid tolerant and thermo stability, which can achieve the liquefaction and saccharification of the starch spontaneously. So it will have a good application prospect.
3. By the optimum fermentation medium research of UNTx78-358. The results show: the optimum culture conclude starch 6.23g/L, peptone 12g/L, CaCl20.287g/L respectively, and phytic acid 1‰. The optimum fermentation conditions are as follows: 100ml/300mL per flask, 10% inoculums concentration, 48h cultivate on 30℃. After that, replenish 20ml culture then continue cultivate until 96h, and the pH is 4.0. By this experiment, the production of the enzyme can be 2400/mL, is higher than the original production 114.3%.
4. The isolated processing of the enzyme producing strain is as follows: The crude enzyme fraction can get by 8 layer gauze filter or 200 seive mesh . Adjust the pH to 6.0 of the crude enzyme fraction then concentrate it by rotary evaporator under the 0.09Mpa, 55℃. Then concentrate the enzyme fraction by 3.5 times ethanol under 4℃. Collect the precipitate and freeze drying it under -50℃, 24h. After that, the enzyme can be isolated. Its activity can remain 89.3%, purified times is 2.8, yield is 8g/L. The enzyme was purified by CM52 and DE52 cellulose ion-exchange chromatography and SDS-PAGE. The results show the mass of this enzyme is about 74kD. And its N-terminal amino acid sequence is Leu, Ser, Ala, Ala, Glu, Thr, Arg, Thr, Gln, Ser, Ile, Tyr, as same as the sequence of acid-stableα-amylase product from A. niger in other reports.
The aim of this reaserch is provide the useful statistics for the development of the acid-stableα-amylase in our country by the isolation and screening high yield strain, enzymology characteristic research, optimum fermentation conditions design and isolated processing research. For that reasons, this research not only can optimize for maximum profit of our countries production of acid-stableα-amylase, But also will be taking higher position in terms of academic and economic values
引文
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