解淀粉芽孢杆菌C1-6固体发酵生产γ--多聚谷氨酸的工艺及技术研究
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摘要
γ多聚谷氨酸(poly-γ-glutamic acid,γ-PGA)是微生物利用D-和/或L-谷氨酸通过α-氨基与γ-羧基之间形成的γ-酰胺键聚合而成的一种阴离子型高分子聚合物,具有水溶性、可生物降解、可食用等特点,对人体和环境安全。其分子内含有大量游离羧基,对养分具有较大的亲和力,同时又具有较强的保湿和吸水性,近年来在食品工业、化妆品生产、制药业、污水处理和农业等方面具有广泛的应用。本论文将γ-PGA的微生物合成与固体废弃物资源化处理相结合,通过固体发酵生产γ-PGA,并以此开发出一种新型的生物有机肥,对产γ-PGA菌株的筛选鉴定及诱变选育、固体发酵参数及培养基的优化、γ-PGA及固体发酵产品的生物学效应、固体发酵体系内微生物群落变化、固体发酵体系内细菌总数和功能菌株的数量变化、γ-PGA降解酶基因ywtD的克隆与表达及YwtD的降解特性等方面进行了研究。主要研究结果如下：
1.从菜园土中分离到39株菌落粘稠的的菌株,并从中筛选到一株细菌C1,其液体发酵产物的最大吸收波长为209nm,分子量大于130kDa,水解产物98%以上为谷氨酸,因此确定该产物为γ-PGA。经氨基酸分析仪测定与计算,菌株C1液体发酵γ-PGA的产量为18.4g/L。依据细胞形态、生理生化特征及16S rRNA基因序列分析将该菌株鉴定为一株解淀粉芽孢杆菌,并命名为Bacillus amyloliquefaciens C1。
2.以B. amyloliquefaciens C1为出发菌株,对其进行紫外线-亚硝基胍复合诱变,选育到一株正突变菌株C1-6。该突变株液体发酵生产γ-PGA的产量为24.2g/L,比原始菌株提高了31.52%,且转接8次后产量仍能保持稳定。利用γ-PGA水溶液浸泡黄瓜种子,在缺水条件下,能够促进黄瓜种子发芽后根的生长,且这种促进作用随着γ-PGA浓度的提高而愈加显著。水培实验结果表明低营养条件下添加γ-PGA能够显著促进玉米幼苗的生长(株高和生物量),提高玉米幼苗叶绿素相对含量、根系活力和N、P、K累积量。
3.以工农业生产的固体废弃物为主要基质,利用单因子实验和响应曲面法分别对菌株C1-6固体发酵生产γ-PGA的参数及培养基配方进行优化。结果显示,在小规模的发酵条件下(20g发酵基质,初始含水量50%,初始pH7.0),菌株C1-6在成分为5.51g腐熟牛粪,1.91g豆粕,0.57g玉米粉,2.15g味精粕,1.5g麸皮,0.5g菜饼,0.1g柠檬酸,0.05gMgSO4·7H2O和0.03gMnSO4·H2O的优化培养基中,37℃下静置发酵48小时可以生产4.37%(w/w)的γ-PGA,分子量大于130kDa。以此发酵产物作为生物有机肥(CBIOF)进行盆栽实验,结果表明,施用普通有机肥(OF)和CBIOF均能够提高玉米苗期的生物量、株高、叶长、叶宽和茎粗、可溶性蛋白、可溶性糖含量和根系活性。同等施肥量条件下,施用CBIOF对玉米幼苗的促生效果显著优于施用OF的处理。
4.将优化培养基成分按等比例放大,进行室外大规模发酵实验。整个固体发酵过程按温度变化可以分成3个阶段：常温预备期(1～2天)、高温发酵期(3～29天)、降温成熟期(30～33天)。发酵20天后,γ-PGA的产量达到0.60%(w/w),在此之后有所下降,并在发酵结束时,维持在0.57%。PCR-DGGE结果显示,固体发酵体系内微生物主要分为6大目:Firmicutes Bacillales; Firmicutes Lactobacillales; Actinobacteria Actinobacteridae; Proteobacteria Alphaprobacteria; Proteobacteria Deltaprobacteria; Proteobacteria Gammaprobacteria.随着固体发酵的进行,发酵体系中微生物群落多样性有所减少。在固体发酵的常温阶段,Proteobacteria是发酵基质中微生物群落的主要组成成分；然而在高温阶段,Proteobacteria逐渐被Firmicutes和Actinobacteria取代。在固体发酵的成熟阶段,微生物群落逐渐趋于稳定。接种的功能菌株B.amyloliquefaciens C1-6始终是整个固体发酵过程中的主要微生物。利用PCR-DGGE手段检测固体发酵过程中微生物群落的变化能够在大规模生产中指示发酵的成熟进程。
5.固体发酵过程中细菌数量和发酵功能菌株数量对固体发酵进程、发酵产物特点具有决定性的作用。利用SYBR Green real-time PCR方法对y-PGA固体发酵体系中细菌总数量进行检测,同时设计针对Bamyloliquefaciens的特异性引物(pgsB726-f/pgsB791-r)和探针(pgsB-probe)并利用TaqMan real-time PCR技术对固体发酵体系中功能菌株B. amyloliquefaciens C1-6的数量进行检测。结果显示,在固体发酵初始阶段,固体发酵体系内细菌总量为2.20×10916S rRNA gene copies/g样品,B.amyloliquefaciens数量为2.62×106pgsB gene copies/g样品。随着固体发酵的进行细菌总量和B. amyloliquefaciens菌体数量逐渐增加。在固体发酵结束时,细菌总量为3.95R10916S rRNA gene copies/g样品,相对于固体发酵开始时变化较小。而B.amyloliquefaciens数量为2.48×108pgsB gene copies/g样品,较固体发酵开始时增长了两个数量级,约占整个发酵体系中细菌总量的5.17%,成为发酵体系中的优势菌群。
6.将B. amyloliquefaciens C1-6编码γ-PGA降解酶的基因ywtD克隆到表达载体pET29a(+)获得重组质粒pET29α-ywtD,并转化至E. coli BL21感受态细胞。所得的重组转化子通过IPTG诱导能够表达成熟的YwtD蛋白。SDS-PAGE结果显示YwtD的分子量为46.6kDa。将YwtD通过金属亲和层析的方法纯化,并在细胞外进行该酶的降解特性研究。结果显示,该蛋白以内切酶形式降解γ-PGA,并能够在较广的温度(25～45℃)和pH(4.0～8.0)范围内降解γ-PGA.在最适降解条件下(30℃和pH4.0),YwtD经过16h可以将分子量为1,800kDa的γ-PGA降解为375.2kDa的片段,降解效率高达79.2%。Zn2+能够显著抑制YwtD的活性,而Ca2+、Fe3+、Mn2+、Ni2+、Mg2+对YwtD降解活性均没有显著影响。
Poly-y-glutamic acid (y-PGA), a naturally occurring anionic high molecular weight biopolymer, was connected by y-amide bonds between y-carboxyl and a-amino groups of D-and/or L-glutamic acid by certain microbial species. y-PGA is water-soluble, biodegradable, edible and nontoxic to human and environment. In addition, it has a good affinity to nutrients and a strong ability to preserve moisture and retain water because there are large amounts of free carboxyl in its intramolecules. In recent years, y-PGA has been used for various applications in foods, cosmetics, medicine, waste water treatments and agriculture. The objective of this dissertation is to develop a new type of bio-organic fertilizer by solid-state fermentation (SSF). The present work consists of:(1) isolation and identification of the strain producing y-PGA;(2) mutation breeding of the y-PGA productive strain;(3) optimization of the parameters and medium of SSF for producing y-PGA;(4) the effects of y-PGA on germination of cucumber seeds and the effects of the products of SSF on growth of maize seedlings repectively;(5) the dynamics shifts of microbial communities during SSF by denaturing gradient gel electrophoresis (DGGE);(6) quantification of the total amount of bacteria and B. amyloliquefaciens in the solid-state fermentation by real-time PCR assay;(7) cloning and heterologous expression of y-PGA degradation gene, ywtD. The main results obtained in this study were summarized as below:
1.39strains with viscous colonies on plates were isolated from vegetable soil, among which a strain that can produce y-PGA with a yield of18.4g/L was screened. The product of C1produced by liquid fermentation had a maximum absorption wavelength at209nm and its molecular weight was more than130kDa. With the analysis of colony morphology, physiological and biochemical characteristics, as well as the phylogenetic analysis of16S rRNA gene sequence, Cl was identified as a Bacillus amyloliquefaciens, which was named as Bacillus amyloliquefaciens C1.
2. B. amyloliquefaciens C1was treated with an ultraviolet-nitrosoguanidine composite mutation. The resulted mutant strain C1-6was screened with a y-PGA productivity of24.2g/L, which was increased by31.52%compared with the wild strain and had a good genetic stability after8streaks. Soaking cucumber seeds with y-PGA could promote the growth of germinative cucumber seeds under water shortage condition. In contrast with the treatment of full-strength nutrient solution, remarkable increase of root and shoot biomass, height and accumulation of N, P and K of the maize seedlings were obtained under low-strength nutrient solution treatments. In addition, all y-PGA treatments increased the relative chlorophyll content (SPAD) of maize seedlings.
3. The parameters and medium components of SSF for the production of y-PGA by B. amyloliquefaciens C1-6were optimized by single-factor experiments and response surface methodology (RSM) using agro-industrial organic wastes as basic substrate respectively. The optimal SSF medium (20g substrates with50%initial moisture and initial pH7.0) was determined to contain5.51g dairy manure compost,1.91g soybean cake,0.57g corn flour,2.15g monosodium glutamate production residues (MGPR),1.5g wheat bran,0.5g rapeseed cake,0.1g citric acid,0.05g MgSO4·7H2O and0.03g MnSO4·H2O, in which C1-6produced y-PGA up to4.37%when fermented for48h at37℃.SDS-PAGE showed that the molecular weight of the y-PGA produced by SSF was more than130kDa. The SSF product was applied as bio-organic fertilizer (CBIOF) in pot experiments, which showed that the biomass and height of maize seedlings, length and width of leaf, and stem thickness were significantly increased. Besides, the contents of soluble protein, soluble sugar, and root activity were all increased in the CBIOF treatment. At equal fertilization level, the promotion effects on maize seedlings by applied with CBIOF was much better than applied with regular organic fertilizer (OF).
4. Scale-up SSF experiments based on the result of optimized medium in flask were carried out outdoor. The whole SSF process could be divided into three phases:an initial short mesophilic period (1～2days), followed by a long thermophilic period (3-29days) and a curing period with a decrease in temperature (30-33days). The production of y-PGA reached a maximum of0.6%after20days fermentation, and then it decreased a little to0.57%at the end of the fermentation. PCR-DGGE profile showed that the microbes in the substrate of SSF could be classed into6orders:Firmicutes Bacillales; Firmicutes Lactobacillales; Actinobacteria Actinobacteridae; Proteobacteria Alphaprobacteria; Proteobacteria Deltaprobacteria; Proteobacteria Gammaprobacteria. A remarkable reduction of microbial diversity was detected during the fermentation process, while the inoculum, B. amyloliquefaciens C1-6, was detected as the dominant organism through the whole process. In the mesophilic phase of SSF, Proteobacteria was the dominant microbe, which was replaced by Firmicutes and Actinobacteria in the thermophilic phase. In the mature stage of fermentation, the microbial community gradually tended to be stable. The molecular analysis of the bacterial diversity has significant potential for instructing the maturing process of SSF to produce y-PGA at a large-scale level, which could be a benefit in the production of high quality and stable SSF products.
5. The total number of bacteria and the starting functional strain played an important role in controlling the process of solid-state fermentation and characterize of the fermentation products. SYBR Green real-time PCR and TaqMan real-time PCR were employed to quantify the total number of bacteria and the starting functional strain B. amyloliquefaciens respectively in solid-state fermentation that produce y-PGA. For the development of the methodology based on TaqMan probe, the primer pair pgsB726-f/pgsB791-r and the pgsB-probe were designed from one of the y-PGA synthetase gene (pgsB) of B. amyloliquefaciens. SYBR Green real-time PCR showed the amount of total bacteria reached3.95×10916S rRNA gene copies/g sample after30days, which was about only twice compared with that at the beginning. TaqMan real-time PCR revealed that the number of B. amyloliquefaciens was2.62×106pgsB gene copies/g sample at the beginning of the fermentation, while it increased to2.482×108pgsB gene copies/g sample at the end of the fermentation. B. amyloliquefaciens growed well and became the dominant strain in the fermentation product thus increased the y-PGA production.
6. A key property of y-PGA required for practical applications is molecular weight. The ywtD gene, which expresses an enzyme that degrades y-PGA, was cloned from B. amyloliquefaciens Cl-6and ligated to an expression vector pET29a(+) to obtain pET29a-ywtD). The recombinant plasmid was then transformed into the competence cell of E. coli BL21. Histidine-tagged YwtD was induced by1mM IPTG and purified by Ni-chelating affinity chromatography from sonicated cells of the positive transformant. YwtD, which had a molecular weight of46.6kDa detected by SDS-PAGE, was proved to be an endo-hydrolase enzyme and exhibited a remarkable activity in y-PGA degradation at a wide range of temperature (25～45℃) and pH (pH4.0～8.0). The optimal condition for YwtD was at30℃and pH4.0, under which y-PGA with a molecular weight of1,800kDa was degraded to375.2kDa. The activity of YwtD enzyme was found to be inhibited by Zn2+, while Ca2+, Fe3+, Mn2+, Ni2+and Mg2+showed no effects on the activity of YwtD.
1.从菜园土中分离到39株菌落粘稠的的菌株,并从中筛选到一株细菌C1,其液体发酵产物的最大吸收波长为209nm,分子量大于130kDa,水解产物98%以上为谷氨酸,因此确定该产物为γ-PGA。经氨基酸分析仪测定与计算,菌株C1液体发酵γ-PGA的产量为18.4g/L。依据细胞形态、生理生化特征及16S rRNA基因序列分析将该菌株鉴定为一株解淀粉芽孢杆菌,并命名为Bacillus amyloliquefaciens C1。
2.以B. amyloliquefaciens C1为出发菌株,对其进行紫外线-亚硝基胍复合诱变,选育到一株正突变菌株C1-6。该突变株液体发酵生产γ-PGA的产量为24.2g/L,比原始菌株提高了31.52%,且转接8次后产量仍能保持稳定。利用γ-PGA水溶液浸泡黄瓜种子,在缺水条件下,能够促进黄瓜种子发芽后根的生长,且这种促进作用随着γ-PGA浓度的提高而愈加显著。水培实验结果表明低营养条件下添加γ-PGA能够显著促进玉米幼苗的生长(株高和生物量),提高玉米幼苗叶绿素相对含量、根系活力和N、P、K累积量。
3.以工农业生产的固体废弃物为主要基质,利用单因子实验和响应曲面法分别对菌株C1-6固体发酵生产γ-PGA的参数及培养基配方进行优化。结果显示,在小规模的发酵条件下(20g发酵基质,初始含水量50%,初始pH7.0),菌株C1-6在成分为5.51g腐熟牛粪,1.91g豆粕,0.57g玉米粉,2.15g味精粕,1.5g麸皮,0.5g菜饼,0.1g柠檬酸,0.05gMgSO4·7H2O和0.03gMnSO4·H2O的优化培养基中,37℃下静置发酵48小时可以生产4.37%(w/w)的γ-PGA,分子量大于130kDa。以此发酵产物作为生物有机肥(CBIOF)进行盆栽实验,结果表明,施用普通有机肥(OF)和CBIOF均能够提高玉米苗期的生物量、株高、叶长、叶宽和茎粗、可溶性蛋白、可溶性糖含量和根系活性。同等施肥量条件下,施用CBIOF对玉米幼苗的促生效果显著优于施用OF的处理。
4.将优化培养基成分按等比例放大,进行室外大规模发酵实验。整个固体发酵过程按温度变化可以分成3个阶段：常温预备期(1～2天)、高温发酵期(3～29天)、降温成熟期(30～33天)。发酵20天后,γ-PGA的产量达到0.60%(w/w),在此之后有所下降,并在发酵结束时,维持在0.57%。PCR-DGGE结果显示,固体发酵体系内微生物主要分为6大目:Firmicutes Bacillales; Firmicutes Lactobacillales; Actinobacteria Actinobacteridae; Proteobacteria Alphaprobacteria; Proteobacteria Deltaprobacteria; Proteobacteria Gammaprobacteria.随着固体发酵的进行,发酵体系中微生物群落多样性有所减少。在固体发酵的常温阶段,Proteobacteria是发酵基质中微生物群落的主要组成成分；然而在高温阶段,Proteobacteria逐渐被Firmicutes和Actinobacteria取代。在固体发酵的成熟阶段,微生物群落逐渐趋于稳定。接种的功能菌株B.amyloliquefaciens C1-6始终是整个固体发酵过程中的主要微生物。利用PCR-DGGE手段检测固体发酵过程中微生物群落的变化能够在大规模生产中指示发酵的成熟进程。
5.固体发酵过程中细菌数量和发酵功能菌株数量对固体发酵进程、发酵产物特点具有决定性的作用。利用SYBR Green real-time PCR方法对y-PGA固体发酵体系中细菌总数量进行检测,同时设计针对Bamyloliquefaciens的特异性引物(pgsB726-f/pgsB791-r)和探针(pgsB-probe)并利用TaqMan real-time PCR技术对固体发酵体系中功能菌株B. amyloliquefaciens C1-6的数量进行检测。结果显示,在固体发酵初始阶段,固体发酵体系内细菌总量为2.20×10916S rRNA gene copies/g样品,B.amyloliquefaciens数量为2.62×106pgsB gene copies/g样品。随着固体发酵的进行细菌总量和B. amyloliquefaciens菌体数量逐渐增加。在固体发酵结束时,细菌总量为3.95R10916S rRNA gene copies/g样品,相对于固体发酵开始时变化较小。而B.amyloliquefaciens数量为2.48×108pgsB gene copies/g样品,较固体发酵开始时增长了两个数量级,约占整个发酵体系中细菌总量的5.17%,成为发酵体系中的优势菌群。
6.将B. amyloliquefaciens C1-6编码γ-PGA降解酶的基因ywtD克隆到表达载体pET29a(+)获得重组质粒pET29α-ywtD,并转化至E. coli BL21感受态细胞。所得的重组转化子通过IPTG诱导能够表达成熟的YwtD蛋白。SDS-PAGE结果显示YwtD的分子量为46.6kDa。将YwtD通过金属亲和层析的方法纯化,并在细胞外进行该酶的降解特性研究。结果显示,该蛋白以内切酶形式降解γ-PGA,并能够在较广的温度(25～45℃)和pH(4.0～8.0)范围内降解γ-PGA.在最适降解条件下(30℃和pH4.0),YwtD经过16h可以将分子量为1,800kDa的γ-PGA降解为375.2kDa的片段,降解效率高达79.2%。Zn2+能够显著抑制YwtD的活性,而Ca2+、Fe3+、Mn2+、Ni2+、Mg2+对YwtD降解活性均没有显著影响。
Poly-y-glutamic acid (y-PGA), a naturally occurring anionic high molecular weight biopolymer, was connected by y-amide bonds between y-carboxyl and a-amino groups of D-and/or L-glutamic acid by certain microbial species. y-PGA is water-soluble, biodegradable, edible and nontoxic to human and environment. In addition, it has a good affinity to nutrients and a strong ability to preserve moisture and retain water because there are large amounts of free carboxyl in its intramolecules. In recent years, y-PGA has been used for various applications in foods, cosmetics, medicine, waste water treatments and agriculture. The objective of this dissertation is to develop a new type of bio-organic fertilizer by solid-state fermentation (SSF). The present work consists of:(1) isolation and identification of the strain producing y-PGA;(2) mutation breeding of the y-PGA productive strain;(3) optimization of the parameters and medium of SSF for producing y-PGA;(4) the effects of y-PGA on germination of cucumber seeds and the effects of the products of SSF on growth of maize seedlings repectively;(5) the dynamics shifts of microbial communities during SSF by denaturing gradient gel electrophoresis (DGGE);(6) quantification of the total amount of bacteria and B. amyloliquefaciens in the solid-state fermentation by real-time PCR assay;(7) cloning and heterologous expression of y-PGA degradation gene, ywtD. The main results obtained in this study were summarized as below:
1.39strains with viscous colonies on plates were isolated from vegetable soil, among which a strain that can produce y-PGA with a yield of18.4g/L was screened. The product of C1produced by liquid fermentation had a maximum absorption wavelength at209nm and its molecular weight was more than130kDa. With the analysis of colony morphology, physiological and biochemical characteristics, as well as the phylogenetic analysis of16S rRNA gene sequence, Cl was identified as a Bacillus amyloliquefaciens, which was named as Bacillus amyloliquefaciens C1.
2. B. amyloliquefaciens C1was treated with an ultraviolet-nitrosoguanidine composite mutation. The resulted mutant strain C1-6was screened with a y-PGA productivity of24.2g/L, which was increased by31.52%compared with the wild strain and had a good genetic stability after8streaks. Soaking cucumber seeds with y-PGA could promote the growth of germinative cucumber seeds under water shortage condition. In contrast with the treatment of full-strength nutrient solution, remarkable increase of root and shoot biomass, height and accumulation of N, P and K of the maize seedlings were obtained under low-strength nutrient solution treatments. In addition, all y-PGA treatments increased the relative chlorophyll content (SPAD) of maize seedlings.
3. The parameters and medium components of SSF for the production of y-PGA by B. amyloliquefaciens C1-6were optimized by single-factor experiments and response surface methodology (RSM) using agro-industrial organic wastes as basic substrate respectively. The optimal SSF medium (20g substrates with50%initial moisture and initial pH7.0) was determined to contain5.51g dairy manure compost,1.91g soybean cake,0.57g corn flour,2.15g monosodium glutamate production residues (MGPR),1.5g wheat bran,0.5g rapeseed cake,0.1g citric acid,0.05g MgSO4·7H2O and0.03g MnSO4·H2O, in which C1-6produced y-PGA up to4.37%when fermented for48h at37℃.SDS-PAGE showed that the molecular weight of the y-PGA produced by SSF was more than130kDa. The SSF product was applied as bio-organic fertilizer (CBIOF) in pot experiments, which showed that the biomass and height of maize seedlings, length and width of leaf, and stem thickness were significantly increased. Besides, the contents of soluble protein, soluble sugar, and root activity were all increased in the CBIOF treatment. At equal fertilization level, the promotion effects on maize seedlings by applied with CBIOF was much better than applied with regular organic fertilizer (OF).
4. Scale-up SSF experiments based on the result of optimized medium in flask were carried out outdoor. The whole SSF process could be divided into three phases:an initial short mesophilic period (1～2days), followed by a long thermophilic period (3-29days) and a curing period with a decrease in temperature (30-33days). The production of y-PGA reached a maximum of0.6%after20days fermentation, and then it decreased a little to0.57%at the end of the fermentation. PCR-DGGE profile showed that the microbes in the substrate of SSF could be classed into6orders:Firmicutes Bacillales; Firmicutes Lactobacillales; Actinobacteria Actinobacteridae; Proteobacteria Alphaprobacteria; Proteobacteria Deltaprobacteria; Proteobacteria Gammaprobacteria. A remarkable reduction of microbial diversity was detected during the fermentation process, while the inoculum, B. amyloliquefaciens C1-6, was detected as the dominant organism through the whole process. In the mesophilic phase of SSF, Proteobacteria was the dominant microbe, which was replaced by Firmicutes and Actinobacteria in the thermophilic phase. In the mature stage of fermentation, the microbial community gradually tended to be stable. The molecular analysis of the bacterial diversity has significant potential for instructing the maturing process of SSF to produce y-PGA at a large-scale level, which could be a benefit in the production of high quality and stable SSF products.
5. The total number of bacteria and the starting functional strain played an important role in controlling the process of solid-state fermentation and characterize of the fermentation products. SYBR Green real-time PCR and TaqMan real-time PCR were employed to quantify the total number of bacteria and the starting functional strain B. amyloliquefaciens respectively in solid-state fermentation that produce y-PGA. For the development of the methodology based on TaqMan probe, the primer pair pgsB726-f/pgsB791-r and the pgsB-probe were designed from one of the y-PGA synthetase gene (pgsB) of B. amyloliquefaciens. SYBR Green real-time PCR showed the amount of total bacteria reached3.95×10916S rRNA gene copies/g sample after30days, which was about only twice compared with that at the beginning. TaqMan real-time PCR revealed that the number of B. amyloliquefaciens was2.62×106pgsB gene copies/g sample at the beginning of the fermentation, while it increased to2.482×108pgsB gene copies/g sample at the end of the fermentation. B. amyloliquefaciens growed well and became the dominant strain in the fermentation product thus increased the y-PGA production.
6. A key property of y-PGA required for practical applications is molecular weight. The ywtD gene, which expresses an enzyme that degrades y-PGA, was cloned from B. amyloliquefaciens Cl-6and ligated to an expression vector pET29a(+) to obtain pET29a-ywtD). The recombinant plasmid was then transformed into the competence cell of E. coli BL21. Histidine-tagged YwtD was induced by1mM IPTG and purified by Ni-chelating affinity chromatography from sonicated cells of the positive transformant. YwtD, which had a molecular weight of46.6kDa detected by SDS-PAGE, was proved to be an endo-hydrolase enzyme and exhibited a remarkable activity in y-PGA degradation at a wide range of temperature (25～45℃) and pH (pH4.0～8.0). The optimal condition for YwtD was at30℃and pH4.0, under which y-PGA with a molecular weight of1,800kDa was degraded to375.2kDa. The activity of YwtD enzyme was found to be inhibited by Zn2+, while Ca2+, Fe3+, Mn2+, Ni2+and Mg2+showed no effects on the activity of YwtD.
引文
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