高活力酸性蛋白酶曲霉融合子的选育及其在酱油发酵中的初步应用
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摘要
提高米曲霉酸性蛋白酶活力对于酱油酿造行业具有重要的实际价值。选育食品级高活力酸性蛋白酶米曲霉是提高我国酱油酿造行业原料蛋白利用率和氨基酸态氮转化率的有效途径。以A.oryzae HN3042和A.niger CICC2377为出发菌株,酶解制备原生质体,基于致死损伤互补理论和基因组重组技术选育目标融合子,并初步考查其在高盐稀态酱油发酵中的应用效果。主要研究内容与结果如下:
首先,对亲本原生质体的制备和临界灭活条件进行了系统研究。A.oryzae HN3042和A.niger CICC2377亲本原生质体的制备条件分别是在MPY(麦芽糖-蛋白胨-酵母抽提物)培养基中30℃下100 rpm振荡培养18 h和21 h,菌丝球与总浓度为15 mg/mL的溶壁酶、蜗牛酶和纤维素酶(1:1:1)混合酶液按1g湿菌丝球/10 mL酶液的比例于30℃、80 rpm条件下酶解2.5 h。在此条件下菌丝原生质体的释放量和再生率分别达到107/mL和30 %以上。通过灭活曲线确定A.oryzae HN3042和A.niger CICC2377原生质体的非对称灭活参数分别为15 W紫光灯垂直距离13 cm下照射15 min和65℃热处理10 min,在此剂量下可使双亲原生质体临界灭活。
其次,将灭活后的亲本原生质体在脉冲电场作用下诱导基因组重组。从再生菌落中随机挑选出694株生长较快的融合子,经500 mg/L的樟脑CD培养基和1 mg/L的苯菌灵CD培养基传代培养,获得了稳定的杂核单倍体。经酸性酪蛋白平板初筛和种曲发酵复筛,获得了10株较亲本A.oryzae HN3042在种曲阶段所产酸性蛋白酶活力有50 %以上提高的融合株。其中F76较A.oryzae HN3042提高了82.19 %,且传代稳定,酶系均衡。从菌丝形态学、胞外蛋白表达谱、磷酸酯酶同功酶谱、孢子细胞核倍性和基因组多态性等角度对比分析了融合子和亲本之间的差异性。结果表明融合子在表型和基因型方面与亲本显著不同,证明在亲本原生质体融合过程中发生了基因组重组。
再次,综合运用盐析、离子交换和凝胶层析技术,将F76酸性蛋白酶纯化了17.09倍,酶活回收率为33.56 %,比活力达到4015.49 U/mg。高酶活蛋白峰经SDS-PAGE显示为单一条带,纯化已达电泳纯。将SDS-PAGE和Native-PAGE的单一条带进行比较,二者基本处于同一水平位置,表明F76酸性蛋白酶为单亚基,分子量约为50 kDa。相对于亲本A.oryzae HN3042,F76酸性蛋白酶构象中α-螺旋含量下降了14.3 %,β结构增加了12.8 %,无规则卷曲增加了5.7 %,表明酶的构象较亲本A.oryzae HN3042柔性增强。F76酸性蛋白酶最适温度40℃,在35℃～45℃范围内处理1 h仍保留90 %以上的活力。此酶最适pH为3.5,在pH 3.0～6.5范围内较为稳定。活力受Mn2+的强烈激活,而受Co2+的强烈抑制,10μmol/L的Pepstatin A几乎完全抑制其活力。F76酸性蛋白酶Km值和活化能Ea分别较亲本A.oryzae HN3042下降了27.08 %和0.77 %;热致衰减指数λ较亲本A.oryzae HN3042增加了20.0 %,表明F76酸性蛋白酶热稳定性略有下降。
然后,运用统计学工具对F76酸性蛋白酶产酶条件进行优化。最佳培养基组成为:在基础种曲培养基的基础上,添加1.24 %酵母抽提物、1.15 %葡萄糖、0.05 % MnCl2、0.05 % MgSO4和0.06 % Na2B4O7。F76酸性蛋白酶活力由优化前的1308 U/g(干曲)提高到1703 U/g,提高了30.20 %。最佳培养条件为:接种量8.59×105个孢子/g种曲、培养温度31℃、培养基起始含水量57 %、培养时间86 h。通过优化使种曲培养基酸性蛋白酶活力提高到1936 U/g干曲,在1703 U/g的基础上活力又提高了13.68 %。通过两次统计优化使种曲酸性蛋白酶活力较未优化前提高了48.01 %。
最后,初步考查F76在高盐稀态酱油发酵中的应用效果。发酵至31 d时,F76菌株酱油发酵液中的可溶性无盐固形物、总酸、总氮和氨基酸态氮含量分别较对照菌株A.oryzae HN3042提高了1.8 %、8 %、8.1 %和6.9 %,表明F76发酵酱油的理化指标较A.oryzae HN3042有所改善。发酵至80 d时,F76酱油发酵液中大于10 kDa和5-10 kDa两分子量段的蛋白相对含量分别较A.oryzae HN3042下降了5.56 %和10.65 %,表明F76分泌的蛋白酶系较A.oryzae HN3042有更好的原料蛋白降解性能。发酵至90 d时,F76发酵液中的甜味和鲜味氨基酸相对含量分别为24.98 %和10.03 %,比A.oryzae HN3042的24.63 %和9.86 %略有增加,而苦味氨基酸含量为61.95 %,较A.oryzae HN3042的62.43 %有所降低,表明F76菌株酱油发酵液的风味较A.oryzae HN3042有所改善。风味成分分析表明,F76酱油发酵液中总风味成分含量为79.87 %,而A.oryzae HN3042为73.37 %,明显低于F76发酵液,意味着两菌株发酵产生的酱油在风味上存在着一定的差别。
本研究为F76在酱油发酵工业中的应用奠定了初步基础。
The improvement of acid protease activity in Aspergillus oryzae has practical value for soy sauce brewing industry. Breeding of food-grade strain with high activity of acid protease is an efficient approach to enhancing the protein utilization rate and conversion efficiency of free amino acids in soy sauce fermentation. Based on the theory of asymmetric inactivation and the technique of genome recombination, the protoplasts released from A.oryzae HN3042 and A.niger CICC2377 by enzymatic hydrolysis were induced to fuse by pulse electrofield and subsequent screening of target strains was carried out. Additionaly, the performances of the screened fusant were preliminarily examined in soy sauce fermentation. The main researches and their results are as follows:
Firstly, the parameters of protoplast preparation and inactivation were explored. The conditions for protoplast release from A.oryzae HN3042 and A.niger CICC2377 were as follows: cultivation in a submerged medium consisting of 1.0 % maltose, 1.0 % polytephone and 0.5 % yeast extract for 18 and 21 h respectively at 30 oC and 100 rpm, and then incubating the mycelia with an enzyme cocktail, total concentration of 15 mg/mL and consisting of lywallzyme, cellulose R-10 and snailase with the ratio of 1:1:1, for 2.5 h at 30 oC and 80 rpm, and then regeneration on the high osmotic soybean extract solid medium. Under these conditions, the release and regeneration efficiency of protoplasts from A.oryzae HN3042 and A.niger CICC2377 both reached 107 /mL and above 30 %, respectively. According to the time curse for asymmetric inactivation, the dose of UV irradiation with a 15 W ultraviolet lamp to protoplasts from A. oryzae HN3042 was 15 min under the light with a distance of 13 cm. As for the heating inactivation towards protoplasts from A.niger CICC2377, the parameter was at a water incubation of 65 oC for 10 min. After the asymmetric inactivation, the survival frequency of protoplasts on regeneration plates was nearly zero.
Secondly, genome recombination between the inactivated protoplasts was iuduced by pulse electrofield. 694 strains of fusants with higher growing speed than A.oryzae HN3042 were randomly selected and the diploidization of heterokaryons was conducted with d-camphor with the content of 500 mg per liter Czapek medium. After successive subcultures for 10 generations on camphor medium, subsequent haploidization of the fused diploids was done with benomyl with the content of 1 mg per liter Czapek medium. 10 stable fusants with an enhancement of above 50 % in activity of acid protease than A.oryzae HN3042 were obtained, in which the fusant of F76 showed an improvement of 82.19 %. Furthermore, the analysis of mycelia morphology, SDS-PAGE and esterase isoenzyme profiles, ploids of the conidium nucleus and randomLy amplified polymorphic DNA (RAPD) were applied to identify the fusants through phenotypic and genetic relationship. The results revealed there were considerable differences between the fusants and their parental strains. According to these line evidences, it can be safetly concluded that genome recombination occurred in the process of protoplast fusion.
Thirdly, by salting-out, ion-exchange and molecular sieve chromatography, the acid protease from F76 was purified 17.09 folds, with a yield of 33.56 % and a specific acitivity of 4015.49 U/mg. Purified acid protease moved as single band on SDS-PAGE. Native PAGE ascertained the purified protein monomeric and the molecular weight was deduced to be about 50 kDa by SDS-PAGE. Circular dichroism studies of this enzyme revealed that the contents ofα-helix,β-sheet,β-turns and aperiodic structure in the purified protein were 7.1 %, 39.4 %, 24.7 % and 32.1 %, respectively. Compared with that of A.oryzae HN3042, the content of helix decreased 14.3 % and the content ofβ-structure and aperiodic coil increased 12.8 % and 5.7 %, which indicated that conformation of acid protease from F76 was more flexible than that from A.oryzae HN3042. Acid protease from F76 was stable in the temperature range of 35 oC-45 oC with an optimum temperature for activity of 40 oC and the enzyme was active in the pH range of 3.0-6.5 with an optimum pH of 3.5. Activity of the acid protease from F76 was significantly improved by Mn2+ and inhibited by Co2+, and pepstatin A, a specific inhibitor for aspartic protease, almost completely inhibited the activity of acid protease from F76 with a concentration of 10μmol/L. The value of Km and Ea of the purified acid protease from F76 showed an decrease of 27.08 % and 0.77 % respectively than that of A.oryzae HN3042 and an increasement of 20.0 % in heat-induced attenuation index than that of A.oryzae HN3042 was responsible for the slight decrease of thermostability of the enzyme.
Then, statistical optimization was applied to maximise the production of acid protease from F76. The result showed that optimum medium was the enriched bran and wheat flour medium, with an addition of 1.24 % yeast extract, 1.15 % glucose, 0.05 % MnCl2, 0.05 % MgSO4 and 0.06 % Na2B4O7. Cultured on this enriched medium, F76 produced the activity of acid protease from 1308 U/g to 1703 U/g, an increase of 30.2 %. The optimum conditions for acid protease production were as follows: inoculum volume of 8.59×105 condium per gram of koji-seed medium, incubation temperature of 31 oC, initial mositure content of 57 % and incubation time of 86 h. Under these conditions, the activity of acid protease from F76 reached 1936 U/g from 1703 U/g, a further increase of 13.68 %. By statistical optimization, the activity of acid protease from F76 in koji-seed medium was enhanced 48.01 %.
Finally, the performances of F76 in soy sauce fermentation were preliminarily evaluated. The content of total titration acid, soluble non-salt solids, total soluble nitrogen and amino-type nitrogen in fermented mash at the stage of 31 days was increased by 1.8 %, 8.0 %, 8.1 % and 6.9 % than that of A.oryzae HN3042, respectively, indicative of some improvement in compositions of the fermented mash. At the fermentation stage of 80 days, the content of proteins with molecular weight of above 10 kDa and within 5-10 kDa in fermented mash by F76 was lower 5.56 % and 10.65 % than that by A.oryzae HN3042, respectively, which indicated that F76 had better degradating performance towards soybean proteins during mash fermentation than the parental strain of A.oryzae HN3042. After a 90-day aging period, the content of sweet, umami- and bitter- free amino acids in the fermented mash by F76 was 24.98 %, 10.03 % and 61.95 %, respectively, while the counterparts by A.oryzae HN3042 was 24.63 %, 9.86 % and 62.43 %, respectively. The slight increase in sweet and umami amino acids and remarkable decrease in bitter amino acids showed that the flavor of fermented mash by F76 was more pleasant than that by A.oryzae HN3042. Volatile compouds analysis showed that the content of total volatile compouds in fermentated mash by F76 was 79.87 % while the counterparts by Aoryzae HN3042 was only 73.37 %, indicating the difference in flavor of fermented soy sauce between F76 and A.oryzae HN3042.
This study provides a preliminary foundation for the application of F76 in soy sauce fermentation.
首先,对亲本原生质体的制备和临界灭活条件进行了系统研究。A.oryzae HN3042和A.niger CICC2377亲本原生质体的制备条件分别是在MPY(麦芽糖-蛋白胨-酵母抽提物)培养基中30℃下100 rpm振荡培养18 h和21 h,菌丝球与总浓度为15 mg/mL的溶壁酶、蜗牛酶和纤维素酶(1:1:1)混合酶液按1g湿菌丝球/10 mL酶液的比例于30℃、80 rpm条件下酶解2.5 h。在此条件下菌丝原生质体的释放量和再生率分别达到107/mL和30 %以上。通过灭活曲线确定A.oryzae HN3042和A.niger CICC2377原生质体的非对称灭活参数分别为15 W紫光灯垂直距离13 cm下照射15 min和65℃热处理10 min,在此剂量下可使双亲原生质体临界灭活。
其次,将灭活后的亲本原生质体在脉冲电场作用下诱导基因组重组。从再生菌落中随机挑选出694株生长较快的融合子,经500 mg/L的樟脑CD培养基和1 mg/L的苯菌灵CD培养基传代培养,获得了稳定的杂核单倍体。经酸性酪蛋白平板初筛和种曲发酵复筛,获得了10株较亲本A.oryzae HN3042在种曲阶段所产酸性蛋白酶活力有50 %以上提高的融合株。其中F76较A.oryzae HN3042提高了82.19 %,且传代稳定,酶系均衡。从菌丝形态学、胞外蛋白表达谱、磷酸酯酶同功酶谱、孢子细胞核倍性和基因组多态性等角度对比分析了融合子和亲本之间的差异性。结果表明融合子在表型和基因型方面与亲本显著不同,证明在亲本原生质体融合过程中发生了基因组重组。
再次,综合运用盐析、离子交换和凝胶层析技术,将F76酸性蛋白酶纯化了17.09倍,酶活回收率为33.56 %,比活力达到4015.49 U/mg。高酶活蛋白峰经SDS-PAGE显示为单一条带,纯化已达电泳纯。将SDS-PAGE和Native-PAGE的单一条带进行比较,二者基本处于同一水平位置,表明F76酸性蛋白酶为单亚基,分子量约为50 kDa。相对于亲本A.oryzae HN3042,F76酸性蛋白酶构象中α-螺旋含量下降了14.3 %,β结构增加了12.8 %,无规则卷曲增加了5.7 %,表明酶的构象较亲本A.oryzae HN3042柔性增强。F76酸性蛋白酶最适温度40℃,在35℃～45℃范围内处理1 h仍保留90 %以上的活力。此酶最适pH为3.5,在pH 3.0～6.5范围内较为稳定。活力受Mn2+的强烈激活,而受Co2+的强烈抑制,10μmol/L的Pepstatin A几乎完全抑制其活力。F76酸性蛋白酶Km值和活化能Ea分别较亲本A.oryzae HN3042下降了27.08 %和0.77 %;热致衰减指数λ较亲本A.oryzae HN3042增加了20.0 %,表明F76酸性蛋白酶热稳定性略有下降。
然后,运用统计学工具对F76酸性蛋白酶产酶条件进行优化。最佳培养基组成为:在基础种曲培养基的基础上,添加1.24 %酵母抽提物、1.15 %葡萄糖、0.05 % MnCl2、0.05 % MgSO4和0.06 % Na2B4O7。F76酸性蛋白酶活力由优化前的1308 U/g(干曲)提高到1703 U/g,提高了30.20 %。最佳培养条件为:接种量8.59×105个孢子/g种曲、培养温度31℃、培养基起始含水量57 %、培养时间86 h。通过优化使种曲培养基酸性蛋白酶活力提高到1936 U/g干曲,在1703 U/g的基础上活力又提高了13.68 %。通过两次统计优化使种曲酸性蛋白酶活力较未优化前提高了48.01 %。
最后,初步考查F76在高盐稀态酱油发酵中的应用效果。发酵至31 d时,F76菌株酱油发酵液中的可溶性无盐固形物、总酸、总氮和氨基酸态氮含量分别较对照菌株A.oryzae HN3042提高了1.8 %、8 %、8.1 %和6.9 %,表明F76发酵酱油的理化指标较A.oryzae HN3042有所改善。发酵至80 d时,F76酱油发酵液中大于10 kDa和5-10 kDa两分子量段的蛋白相对含量分别较A.oryzae HN3042下降了5.56 %和10.65 %,表明F76分泌的蛋白酶系较A.oryzae HN3042有更好的原料蛋白降解性能。发酵至90 d时,F76发酵液中的甜味和鲜味氨基酸相对含量分别为24.98 %和10.03 %,比A.oryzae HN3042的24.63 %和9.86 %略有增加,而苦味氨基酸含量为61.95 %,较A.oryzae HN3042的62.43 %有所降低,表明F76菌株酱油发酵液的风味较A.oryzae HN3042有所改善。风味成分分析表明,F76酱油发酵液中总风味成分含量为79.87 %,而A.oryzae HN3042为73.37 %,明显低于F76发酵液,意味着两菌株发酵产生的酱油在风味上存在着一定的差别。
本研究为F76在酱油发酵工业中的应用奠定了初步基础。
The improvement of acid protease activity in Aspergillus oryzae has practical value for soy sauce brewing industry. Breeding of food-grade strain with high activity of acid protease is an efficient approach to enhancing the protein utilization rate and conversion efficiency of free amino acids in soy sauce fermentation. Based on the theory of asymmetric inactivation and the technique of genome recombination, the protoplasts released from A.oryzae HN3042 and A.niger CICC2377 by enzymatic hydrolysis were induced to fuse by pulse electrofield and subsequent screening of target strains was carried out. Additionaly, the performances of the screened fusant were preliminarily examined in soy sauce fermentation. The main researches and their results are as follows:
Firstly, the parameters of protoplast preparation and inactivation were explored. The conditions for protoplast release from A.oryzae HN3042 and A.niger CICC2377 were as follows: cultivation in a submerged medium consisting of 1.0 % maltose, 1.0 % polytephone and 0.5 % yeast extract for 18 and 21 h respectively at 30 oC and 100 rpm, and then incubating the mycelia with an enzyme cocktail, total concentration of 15 mg/mL and consisting of lywallzyme, cellulose R-10 and snailase with the ratio of 1:1:1, for 2.5 h at 30 oC and 80 rpm, and then regeneration on the high osmotic soybean extract solid medium. Under these conditions, the release and regeneration efficiency of protoplasts from A.oryzae HN3042 and A.niger CICC2377 both reached 107 /mL and above 30 %, respectively. According to the time curse for asymmetric inactivation, the dose of UV irradiation with a 15 W ultraviolet lamp to protoplasts from A. oryzae HN3042 was 15 min under the light with a distance of 13 cm. As for the heating inactivation towards protoplasts from A.niger CICC2377, the parameter was at a water incubation of 65 oC for 10 min. After the asymmetric inactivation, the survival frequency of protoplasts on regeneration plates was nearly zero.
Secondly, genome recombination between the inactivated protoplasts was iuduced by pulse electrofield. 694 strains of fusants with higher growing speed than A.oryzae HN3042 were randomly selected and the diploidization of heterokaryons was conducted with d-camphor with the content of 500 mg per liter Czapek medium. After successive subcultures for 10 generations on camphor medium, subsequent haploidization of the fused diploids was done with benomyl with the content of 1 mg per liter Czapek medium. 10 stable fusants with an enhancement of above 50 % in activity of acid protease than A.oryzae HN3042 were obtained, in which the fusant of F76 showed an improvement of 82.19 %. Furthermore, the analysis of mycelia morphology, SDS-PAGE and esterase isoenzyme profiles, ploids of the conidium nucleus and randomLy amplified polymorphic DNA (RAPD) were applied to identify the fusants through phenotypic and genetic relationship. The results revealed there were considerable differences between the fusants and their parental strains. According to these line evidences, it can be safetly concluded that genome recombination occurred in the process of protoplast fusion.
Thirdly, by salting-out, ion-exchange and molecular sieve chromatography, the acid protease from F76 was purified 17.09 folds, with a yield of 33.56 % and a specific acitivity of 4015.49 U/mg. Purified acid protease moved as single band on SDS-PAGE. Native PAGE ascertained the purified protein monomeric and the molecular weight was deduced to be about 50 kDa by SDS-PAGE. Circular dichroism studies of this enzyme revealed that the contents ofα-helix,β-sheet,β-turns and aperiodic structure in the purified protein were 7.1 %, 39.4 %, 24.7 % and 32.1 %, respectively. Compared with that of A.oryzae HN3042, the content of helix decreased 14.3 % and the content ofβ-structure and aperiodic coil increased 12.8 % and 5.7 %, which indicated that conformation of acid protease from F76 was more flexible than that from A.oryzae HN3042. Acid protease from F76 was stable in the temperature range of 35 oC-45 oC with an optimum temperature for activity of 40 oC and the enzyme was active in the pH range of 3.0-6.5 with an optimum pH of 3.5. Activity of the acid protease from F76 was significantly improved by Mn2+ and inhibited by Co2+, and pepstatin A, a specific inhibitor for aspartic protease, almost completely inhibited the activity of acid protease from F76 with a concentration of 10μmol/L. The value of Km and Ea of the purified acid protease from F76 showed an decrease of 27.08 % and 0.77 % respectively than that of A.oryzae HN3042 and an increasement of 20.0 % in heat-induced attenuation index than that of A.oryzae HN3042 was responsible for the slight decrease of thermostability of the enzyme.
Then, statistical optimization was applied to maximise the production of acid protease from F76. The result showed that optimum medium was the enriched bran and wheat flour medium, with an addition of 1.24 % yeast extract, 1.15 % glucose, 0.05 % MnCl2, 0.05 % MgSO4 and 0.06 % Na2B4O7. Cultured on this enriched medium, F76 produced the activity of acid protease from 1308 U/g to 1703 U/g, an increase of 30.2 %. The optimum conditions for acid protease production were as follows: inoculum volume of 8.59×105 condium per gram of koji-seed medium, incubation temperature of 31 oC, initial mositure content of 57 % and incubation time of 86 h. Under these conditions, the activity of acid protease from F76 reached 1936 U/g from 1703 U/g, a further increase of 13.68 %. By statistical optimization, the activity of acid protease from F76 in koji-seed medium was enhanced 48.01 %.
Finally, the performances of F76 in soy sauce fermentation were preliminarily evaluated. The content of total titration acid, soluble non-salt solids, total soluble nitrogen and amino-type nitrogen in fermented mash at the stage of 31 days was increased by 1.8 %, 8.0 %, 8.1 % and 6.9 % than that of A.oryzae HN3042, respectively, indicative of some improvement in compositions of the fermented mash. At the fermentation stage of 80 days, the content of proteins with molecular weight of above 10 kDa and within 5-10 kDa in fermented mash by F76 was lower 5.56 % and 10.65 % than that by A.oryzae HN3042, respectively, which indicated that F76 had better degradating performance towards soybean proteins during mash fermentation than the parental strain of A.oryzae HN3042. After a 90-day aging period, the content of sweet, umami- and bitter- free amino acids in the fermented mash by F76 was 24.98 %, 10.03 % and 61.95 %, respectively, while the counterparts by A.oryzae HN3042 was 24.63 %, 9.86 % and 62.43 %, respectively. The slight increase in sweet and umami amino acids and remarkable decrease in bitter amino acids showed that the flavor of fermented mash by F76 was more pleasant than that by A.oryzae HN3042. Volatile compouds analysis showed that the content of total volatile compouds in fermentated mash by F76 was 79.87 % while the counterparts by Aoryzae HN3042 was only 73.37 %, indicating the difference in flavor of fermented soy sauce between F76 and A.oryzae HN3042.
This study provides a preliminary foundation for the application of F76 in soy sauce fermentation.
引文
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