反式茴脑微生物降解和转化合成茴香醛和茴香酸
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摘要
八角是广西盛产的天然香料资源,八角茴香油(简称茴油)是从八角果、枝和叶中提取的一种芳香精油,其中反式茴脑含量为80%-90%。反式茴脑是具有丙烯基苯结构的化合物,通常作为化学法合成香料的起始原料。微生物在降解丙烯基苯化合物的过程中产生的中间产物大部分都是具有很高经济价值的芳香族化合物。利用微生物转化反式茴脑极有可能获得茴香醛、茴香酸等高附加值的天然生物香料。因此本课题选择反式茴脑为底物,筛选能降解并转化反式茴脑合成茴香醛或茴香酸的微生物；同时对影响转化的因素、生物降解中间产物的分离鉴定以及反式茴脑的生物降解途径进行了探讨。
一、反式茴脑生物降解和转化体系的分析方法建立
建立了分析反式茴脑降解和转化体系中反式茴脑和产物茴香醛、茴香酸的薄层层析法(TLC法)、薄层层析-紫外分光光度法(TLC-UV法)、反相高效液相色谱法(RP-HPLC法)和硫代巴比妥酸分光光度法(TBA法)。TLC法选用的适宜展开剂配方为石油醚(bp.60℃～90℃)：氯仿：乙酸乙酯：甲酸(V/V/V/V)=25：10：3：0.2,该法能同时定性和半定量分析反式茴脑、茴香醛和茴香酸,操作简单,检测快速,适合于生物降解和转化反式茴脑菌株的快速筛选。TLC-UV法和RP-HPLC法可同时定量分析反式茴脑、茴香醛和茴香酸三元混合体系,RP-HPLC法测定的准确性高于TLC-UV法。适宜的RP-HPLC法条件为：采用Kromasil-100A C18色谱柱(250 mm×4.6mmx5μm),流动相为V(乙腈)：V(水)：V(冰醋酸)=70：30：0.02,等度洗脱,流速0.8 mL·min-1,检测波长为260 nm,进样量5μL,柱温室温。此外,还可根据需要采用硫代巴比妥酸分光光度法定量测定样液中的茴香醛,反式茴脑和茴香酸对茴香醛的测定几乎没有影响。
二、生物降解反式茴脑的微生物筛选及鉴定
通过从广西高峰林场八角种植区土样、八角植物内生菌、八角加工车间废液中筛选耐受高浓度茴油的微生物,并检验菌株降解反式茴脑合成茴香醛或茴香酸的能力。用逐级添加茴油的富集培养方法从土样中获得了一株能够在1%(V/V)茴油浓度下生长的菌株BT-13,确定其在改良M9培养基上,一定的转化条件下,具有高转化反式茴脑的能力,转化产物以茴香酸为主。菌株BT-13根据其形态特征观察、部分生理生化特性和16S rDNA序列同源性比较,鉴定为假单胞菌属(Pseudomonas sp.)。从新鲜八角果、枝、叶中分离到87株内生菌,其中真菌69株,细菌18株。有一株内生细菌BZ-15对反式茴脑降解能力较强,可检测到茴香酸的生成。该菌经16SrDNA序列同源性比较,鉴定为恶臭假单胞菌(Pseudomonas putida)。从八角加工车间废液中筛选获得了一株具有较强降解反式茴脑能力并转化合成少量茴香醛的真菌菌株ZJ-9,通过对其形态特征、培养特征的观察,对照《真菌鉴定手册》,鉴定该真菌为黑曲霉。
三、微生物转化反式茴脑合成茴香醛和茴香酸工艺研究
研究了假单胞菌BT-13在有机溶剂-水两相体系中生物转化反式茴脑合成茴香醛的条件。首先考察了在摇瓶条件下,有机溶剂极性和加入量、培养基配方、转化时间、转化温度、摇床转速、装液量以及pH值等条件对游离细胞转化合成茴香醛的影响。结果表明,适宜有机溶剂为乙酸乙酯,加入量控制在10%(V/V)。适宜的培养基为改良马丁氏培养基,培养方式为摇床培养。在装液量为20 mL/150 mL三角瓶,培养基初始pH控制在6.5,温度30℃,转速150 r·min-1,转化时间30 h,茴香醛的摩尔生成率为7.7%。接着在此两相体系中采用海藻酸钙固定化细胞对反式茴脑进行转化,茴香醛的摩尔生成率可提高至12.6%。为实现转化液中未转化的反式茴脑和茴香醛的分离,转化液首先用等体积的乙酸乙酯萃取,取上层有机相,接着在有机相中加入饱和亚硫酸氢钠将茴香醛反萃到水相,再将水相茴香醛加成物酸化成醛的形式,最后用乙酸乙酯萃取可得到茴香醛。
开展了假单胞菌BT-13在改良M9培养基上转化反式茴脑合成茴香酸的条件优化,考察了培养基配方、底物加入量、摇床转速、温度和培养基初始pH值对茴香酸生成的影响。结果表明：在改良M9培养基中添加碳源的浓度高于1 g·L-’时,明显抑制反式茴脑的转化。生成茴香酸的优化条件是,培养基配方为麦芽糖0.5 g·L-1, NH4Cl 0.5 g·L-1, FeSO4·7H2O 0.01 g·L-1, MgSO4·7H2O2.0g·L-1, NaCl 0.5 g·L-1, Na2HPO4 6.8 g·L-1, KH2PO4 3.0 g·L-1, CaCl2 0.02 g·L-1;反式茴脑加入量为9.83 g-L-’,摇床转速为200 r-min-1,转化温度为30℃,转化培养基的初始pH为7.0。在优化条件下,茴香酸积累的浓度为3.49 g·L-1,摩尔生成率为34.6%,比优化前结果提高了92.8%。假单胞菌BT-13转化反式茴脑的主要产物为茴香酸,还含有茴香醛、茴香二醇等中间产物。利用5L机械搅拌发酵罐进行放大试验,茴香酸浓度最高可达3.57 g·L-1,摩尔生成率为36.1%。转化液经酸化、乙酸乙酯萃取、真空浓缩和结晶可得到针状的茴香酸晶体。
四、反式茴脑在假单胞菌BT-13和黑曲霉ZJ-9作用下的可能降解途径
采用高效液相色谱和气相色谱/质谱联用等手段分析假单胞菌BT-13和黑曲霉ZJ-9降解反式茴脑的中间产物,通过与标准物质的比对或GC-MS图谱分析确定了假单胞菌BT-13降解反式茴脑四种中间产物,分别为茴香环氧化物、茴香二醇、茴香醛和茴香酸；黑曲霉ZJ-9降解反式茴脑五种中间产物,分别为茴香环氧化物、茴香二醇、茴香醇、茴香醛和茴香酸,且在一定条件下,茴香醇有一定程度的积累。这些中间产物中茴香醇、茴香醛和茴香酸在香料和医药产业中是高附加值的芳香族化合物。根据中间产物的生成情况,推测了这两种菌降解反式茴脑的可能途径。假单胞菌BT-13和黑曲霉ZJ-9降解反式茴脑的共同可能途径：反式茴脑首先在丙烯基侧链双键处加氧,形成茴香环氧化物,环氧化物水解后形成茴香二醇,进一步氧化生成茴香醛、茴香酸。在黑曲霉ZJ-9中,茴香醛还可以被还原生成茴香醇。两种菌都通过形成茴香二醇途径实现对反式茴脑的降解,但在某些中间产物积累上存在差异。
假单胞菌BT-13和黑曲霉ZJ-9胞内酶中检测到过氧化物酶(POD)和过氧化氢酶(CAT)的活性。假单胞菌BT-13胞内检测的POD和CAT酶活最高分别为122 U·mL-1和625 U-mL-1,黑曲霉ZJ-9胞内检测的POD和CAT酶活最高分别为4.8 U·mL-1和42.5 U·mL-1,明显低于假单胞菌BT-13。从检测的酶活结果和两株菌降解反式茴脑中间产物的积累情况,得出黑曲霉ZJ-9和假单胞菌BT-13降解反式茴脑的关键酶及酶的调控存在差异。以假单胞菌BT-13粗酶液转化反式茴脑的转化液中检测到茴香环氧化物、茴香醛和茴香酸。适量的添加H202对酶催化反式茴脑合成茴香酸有促进作用,转化适宜pH=8。
Illicium verum Hook.f. is a kind of local rich natural flavor in Guangxi. Anise oil is extracted from the fruits, branches and leaves of Illicium verum Hook.f. by steam-distillation extraction, in which trans-anethole content of 80% to 90%.trans-Anethole, a type of propenylbenzene compound, is usually used as starting material to synthesize fragrants in flavor and fragrance industry. Most valuable aromatic compounds are produced as intermediates in the biodegradation pathways of propenylbenzene compounds. Thus, it is likely that microorganisms capable of utilizing trans-anethole will produce high value-added natural biological spices, such as anisaldehyde, anisic acid etc. Therefore,trans-anethole was selected as substrate. Microorganisms that can degrade and transfor trans-anethole to anisaldehyde or anisic acid were isolated. Meanwhile, the factors that affected biotransformation, isolation and identification of intermediate products and trans-anethole biodegradation pathways were studied.
1. Establishment of methods for analyzing trans-anethole, anisaldehyde and anisic acid in trans-anethole degradation and transformation system
A thin layer chromatography method (TLC), thin layer chromatography-UV spectrophotometry method (TLC-UV), reversed phase high performance liquid chromatography method (RP-HPLC) and thiobarbituric acid spectrophotometric method (TBA) for the analysis of trans-anethole, anisaldehyde and anisic acid in trans-anethole biodegradation and transformation system were developed. The suitable developing solvent of TLC was petroleum ether (bp.60℃-90℃): chloroform:ethyl acetate:formic acid (V/V/V/V)= 25:10:3:0.2. TLC was found to be simple and rapid for qualitative and semiquantitative analysis of trans-anethole, anisaldehyde and anisic acid. The method was suitable for rapid screening of trans-anethole degrading microorganisms. TLC-UV and RP-HPLC method can be simultaneous quantitative analysis of trans-anethole, anisaldehyde and anisic acid. RP-HPLC method was accurate above TLC-UV method. RP-HPLC conditions were as follow:The separation was performed on Kromasil-100A C18 column (250 mm×4.6 mm×5μm) using V(acetonitrile):V(water):V(acetic acid)=70:30:0.02 as the mobile phase. The flow rate was 0.8 mL·min-1. The detection wave length was 260 nm. The injection volume was 5μL, and the column temperature was room temperature. The method showed good linear relationship, precision and repeatability. In addition, anisaldehyde in transformation liquids could also be detected by TBA method.trans-Anethole and anisic acid don't interfered with the determination of anisaldehyde.
2. Isolation and identification of trans-anethole degrading microorganisms
Isolation of trans-anethole degrading microorganisms which can grow on high concentration of anise oil from the soil under aniseed tree, the fruits、branches and leaves of fresh aniseed, waste residue of aniseed workshop. Then biotransformation of trans-anethole to anisaldehyde or anisic acid with these strains were carried out by fermentation. Bacteria were isolated from soil under aniseed trees by enrichment culture. Bacterial strain BT-13, which utilizes 1%(V/V) anise oil as the sole source of carbon and energy in modified M9 media, was screened out base on its good capability to biotransform trans-anethole to anisic acid. This strain was identified as Pseudomonas sp. according to visual observation, physiological and biochemical experiments and 16S rDNA sequence analysis. Eighty-seven endophytes were isolated from the fruits、branches and leaves of fresh aniseed. It includes sixty-nine strains of endophytic fungus, eighteen strains of endophytic bacteria. A endophytic bacterium designated BZ-15 was newly isolated based on its ability to degrade trans-anethole. Anisic acid was detected in the culture.This strain was identified as Pseudomonas putida according to its 16S rDNA sequence analysis. A fungal strain ZJ-9 was newly isolated from waste residue in aniseed workshop based on its ability to degrade trans-anethole. A small amount of anisaldehyde was detected in the culture. Strain ZJ-9 was identified as Aspergillus niger according to its morphological characteristics, culture characteristics and "fungal identification manual".
3. Biodegradation and biotransformation technology of trans-anethole to anisaldehyde and anisic acid
The biotransformation process of trans-anethole to anisaldehyde by Pseudomonas sp. BT-13 was carried out in aqueous-organic solvents biphasic systems. The effects of some key factors such as organic solvent polarity and content, the component of culture medium, conversion time, temperature, medium capacity, rotate speed of rotary shaker and medium initial pH were investigated on the biotransformation of trans-anethole to anisaldehyde by free cell. The results showed that the suitable organic solvent was ethyl acetate, ethyl acetate content was 10%(V/V), the suitable medium was the modified Martin's medium, medium capacity was 20 mL/150 mL flask, medium initial pH 6.5, temperature 30℃, rotate speed of rotary shaker at 150 r·min-1, conversion time 30 h. On the optimum conditions, the molar generation ratio of anisaldehyde was increased to 12.6% by immobilized cells with calcium alginate compared with 7.7% of free cells. In order to separate trans-anethole and anisaldehyde, trans-anethole and anisaldehyde were extracted from biotransformation liquids by equal volume ethyl acetate at first, then trans-anethole was stripped to water phase via saturation sodium bisulfite solution. Because trans-anethole was almost insoluble in water, this reactive-extraction also resulted in anisaldehyde purification. Finally anisaldehyde was obtained from acidic water phase by ethyl acetate.
The optimization of conversion conditons for the biotransformation of trans-anethole to anisic acid by Pseudomonas sp. BT-13 was carried out in modified M9 medium, on which the effects of culture medium components, substrate concentration, shaking speed, temperature and initial medium pH were studied. The results showed that the carbon source concentration of above 1 g·L-1 in modified M9 medium could marketdly inhibit the transformation of trans-anethole. The optimal medium composition was obtained:maltose 0.5 g·L-1,NH4Cl 0.5 g·L-1,FeSO4·7H2O 0.01 g·L-1, MgSO4·7H2O 2.0 g·L-1,NaCl 0.5 g·L-1, Na2HPO4 6.8 g·L-1, KH2PO4 3.0 g·L-1 CaCl2 0.02 g·L-1. Under an optimal condition of trans-anethole concentration of 9.83 g·L-1, shaking speed 200 r·min-1, temperature 30℃, pH 7.0, the cumulative concentration of anisic acid could reach to 3.49 g·L-1 with a molar generation ratio of 34.6%, which increased 92.8% than that under the unoptimized conditions. Anisic acid was proved to be the leading product of this biotransformation, while some other intermediates such as anethole epoxide, anisaldehyde,t-anethole-diol etc could also be detected in the culture. When the biotransformation was processed in 5 L fermentor, the anisic acid of 3.57 g·L-1 was obtained with a molar generation ratio of 36.1%. Crystals of anisic acid was obtained from biotransformation liquid afer acidified, extracted with ethyl acetate, vacuum concentration and crystallization.
4. Proposed degradation pathway of trans-anethole by Pseudomonas sp. BT-13 and Aspergillus niger ZJ-9
The degradation pathway of trans-anethole in Pseudomonas sp. BT-13 and Aspergillus niger ZJ-9 were studied by analyzing intermediates using HPLC and GC-MS. Some main intermediates were identified by comparison with standard sample or GC-MS spectra analysis. Four intermediates that is anethole epoxide, anethyl diol, anisaldehyde and anisic acid were produced in the degradation of trans-anethole by Pseudomonas sp. BT-13; Five intermediates that is anethole epoxide, anethyl diol, anisyl alcohol, anisaldehyde and anisic acid were produced in the degradation of trans-anethole by Aspergillus niger ZJ-9. Under certain conditions, anisyl alcohol was accumulated. These intermediate products are all high value-added aromatic compounds in the perfume and pharmaceutical industries. According to the generation of intermediates, possible degradation pathways of trans-anethole by Pseudomonas sp. BT-13 and Aspergillus niger ZJ-9 were proposed:First the 1,2-CC double bond in the trans-anethole side chain is epoxidized and anethole epoxide is produced.Then the epoxide is hydrolysed, forming anethyl diol which is subsequently oxidized to anisaldehyde. Anisaldehyde can continue to be oxidized to anisic acid. In Aspergillus niger ZJ-9, anisaldehyde also can continue to be reduced to anisyl alcohol. In two strains,trans-anethole was degraded through anethyl diol pathway, but the accumulation of some intermediate products was different.
Peroxidase (POD) and catalase (CAT) activity were detected in intracellular enzyme extracted from Pseudomonas sp. BT-13 and Aspergillus niger ZJ-9. POD and CAT activity of Pseudomonas sp. BT-13 were 122 U·mL-1 and 625 U·mL-1, but POD and CAT activity of Aspergillus niger ZJ-9 were 4.8 U·mL-1 and 42.5 U·mL-1, significantly lower than Pseudomonas sp. BT-13. According to the testing enzymes activity and the accumulation of the intermediate products, there were some differences among the key enzymes and enzyme regulation in biodegradation of trans-anethole by Aspergillus niger ZJ-9 and Pseudomonas sp. BT-13. Anethole epoxide, anisaldehyde, and anisic acid were also detected in the biotransformation liquids of trans-anethole by Pseudomonas sp. BT-13 crude enzyme. Adding amount of H2O2 could promote the biotransformation of trans-anethole to anisic acid. Enzymatic transforming suitable pH was 8.
一、反式茴脑生物降解和转化体系的分析方法建立
建立了分析反式茴脑降解和转化体系中反式茴脑和产物茴香醛、茴香酸的薄层层析法(TLC法)、薄层层析-紫外分光光度法(TLC-UV法)、反相高效液相色谱法(RP-HPLC法)和硫代巴比妥酸分光光度法(TBA法)。TLC法选用的适宜展开剂配方为石油醚(bp.60℃～90℃)：氯仿：乙酸乙酯：甲酸(V/V/V/V)=25：10：3：0.2,该法能同时定性和半定量分析反式茴脑、茴香醛和茴香酸,操作简单,检测快速,适合于生物降解和转化反式茴脑菌株的快速筛选。TLC-UV法和RP-HPLC法可同时定量分析反式茴脑、茴香醛和茴香酸三元混合体系,RP-HPLC法测定的准确性高于TLC-UV法。适宜的RP-HPLC法条件为：采用Kromasil-100A C18色谱柱(250 mm×4.6mmx5μm),流动相为V(乙腈)：V(水)：V(冰醋酸)=70：30：0.02,等度洗脱,流速0.8 mL·min-1,检测波长为260 nm,进样量5μL,柱温室温。此外,还可根据需要采用硫代巴比妥酸分光光度法定量测定样液中的茴香醛,反式茴脑和茴香酸对茴香醛的测定几乎没有影响。
二、生物降解反式茴脑的微生物筛选及鉴定
通过从广西高峰林场八角种植区土样、八角植物内生菌、八角加工车间废液中筛选耐受高浓度茴油的微生物,并检验菌株降解反式茴脑合成茴香醛或茴香酸的能力。用逐级添加茴油的富集培养方法从土样中获得了一株能够在1%(V/V)茴油浓度下生长的菌株BT-13,确定其在改良M9培养基上,一定的转化条件下,具有高转化反式茴脑的能力,转化产物以茴香酸为主。菌株BT-13根据其形态特征观察、部分生理生化特性和16S rDNA序列同源性比较,鉴定为假单胞菌属(Pseudomonas sp.)。从新鲜八角果、枝、叶中分离到87株内生菌,其中真菌69株,细菌18株。有一株内生细菌BZ-15对反式茴脑降解能力较强,可检测到茴香酸的生成。该菌经16SrDNA序列同源性比较,鉴定为恶臭假单胞菌(Pseudomonas putida)。从八角加工车间废液中筛选获得了一株具有较强降解反式茴脑能力并转化合成少量茴香醛的真菌菌株ZJ-9,通过对其形态特征、培养特征的观察,对照《真菌鉴定手册》,鉴定该真菌为黑曲霉。
三、微生物转化反式茴脑合成茴香醛和茴香酸工艺研究
研究了假单胞菌BT-13在有机溶剂-水两相体系中生物转化反式茴脑合成茴香醛的条件。首先考察了在摇瓶条件下,有机溶剂极性和加入量、培养基配方、转化时间、转化温度、摇床转速、装液量以及pH值等条件对游离细胞转化合成茴香醛的影响。结果表明,适宜有机溶剂为乙酸乙酯,加入量控制在10%(V/V)。适宜的培养基为改良马丁氏培养基,培养方式为摇床培养。在装液量为20 mL/150 mL三角瓶,培养基初始pH控制在6.5,温度30℃,转速150 r·min-1,转化时间30 h,茴香醛的摩尔生成率为7.7%。接着在此两相体系中采用海藻酸钙固定化细胞对反式茴脑进行转化,茴香醛的摩尔生成率可提高至12.6%。为实现转化液中未转化的反式茴脑和茴香醛的分离,转化液首先用等体积的乙酸乙酯萃取,取上层有机相,接着在有机相中加入饱和亚硫酸氢钠将茴香醛反萃到水相,再将水相茴香醛加成物酸化成醛的形式,最后用乙酸乙酯萃取可得到茴香醛。
开展了假单胞菌BT-13在改良M9培养基上转化反式茴脑合成茴香酸的条件优化,考察了培养基配方、底物加入量、摇床转速、温度和培养基初始pH值对茴香酸生成的影响。结果表明：在改良M9培养基中添加碳源的浓度高于1 g·L-’时,明显抑制反式茴脑的转化。生成茴香酸的优化条件是,培养基配方为麦芽糖0.5 g·L-1, NH4Cl 0.5 g·L-1, FeSO4·7H2O 0.01 g·L-1, MgSO4·7H2O2.0g·L-1, NaCl 0.5 g·L-1, Na2HPO4 6.8 g·L-1, KH2PO4 3.0 g·L-1, CaCl2 0.02 g·L-1;反式茴脑加入量为9.83 g-L-’,摇床转速为200 r-min-1,转化温度为30℃,转化培养基的初始pH为7.0。在优化条件下,茴香酸积累的浓度为3.49 g·L-1,摩尔生成率为34.6%,比优化前结果提高了92.8%。假单胞菌BT-13转化反式茴脑的主要产物为茴香酸,还含有茴香醛、茴香二醇等中间产物。利用5L机械搅拌发酵罐进行放大试验,茴香酸浓度最高可达3.57 g·L-1,摩尔生成率为36.1%。转化液经酸化、乙酸乙酯萃取、真空浓缩和结晶可得到针状的茴香酸晶体。
四、反式茴脑在假单胞菌BT-13和黑曲霉ZJ-9作用下的可能降解途径
采用高效液相色谱和气相色谱/质谱联用等手段分析假单胞菌BT-13和黑曲霉ZJ-9降解反式茴脑的中间产物,通过与标准物质的比对或GC-MS图谱分析确定了假单胞菌BT-13降解反式茴脑四种中间产物,分别为茴香环氧化物、茴香二醇、茴香醛和茴香酸；黑曲霉ZJ-9降解反式茴脑五种中间产物,分别为茴香环氧化物、茴香二醇、茴香醇、茴香醛和茴香酸,且在一定条件下,茴香醇有一定程度的积累。这些中间产物中茴香醇、茴香醛和茴香酸在香料和医药产业中是高附加值的芳香族化合物。根据中间产物的生成情况,推测了这两种菌降解反式茴脑的可能途径。假单胞菌BT-13和黑曲霉ZJ-9降解反式茴脑的共同可能途径：反式茴脑首先在丙烯基侧链双键处加氧,形成茴香环氧化物,环氧化物水解后形成茴香二醇,进一步氧化生成茴香醛、茴香酸。在黑曲霉ZJ-9中,茴香醛还可以被还原生成茴香醇。两种菌都通过形成茴香二醇途径实现对反式茴脑的降解,但在某些中间产物积累上存在差异。
假单胞菌BT-13和黑曲霉ZJ-9胞内酶中检测到过氧化物酶(POD)和过氧化氢酶(CAT)的活性。假单胞菌BT-13胞内检测的POD和CAT酶活最高分别为122 U·mL-1和625 U-mL-1,黑曲霉ZJ-9胞内检测的POD和CAT酶活最高分别为4.8 U·mL-1和42.5 U·mL-1,明显低于假单胞菌BT-13。从检测的酶活结果和两株菌降解反式茴脑中间产物的积累情况,得出黑曲霉ZJ-9和假单胞菌BT-13降解反式茴脑的关键酶及酶的调控存在差异。以假单胞菌BT-13粗酶液转化反式茴脑的转化液中检测到茴香环氧化物、茴香醛和茴香酸。适量的添加H202对酶催化反式茴脑合成茴香酸有促进作用,转化适宜pH=8。
Illicium verum Hook.f. is a kind of local rich natural flavor in Guangxi. Anise oil is extracted from the fruits, branches and leaves of Illicium verum Hook.f. by steam-distillation extraction, in which trans-anethole content of 80% to 90%.trans-Anethole, a type of propenylbenzene compound, is usually used as starting material to synthesize fragrants in flavor and fragrance industry. Most valuable aromatic compounds are produced as intermediates in the biodegradation pathways of propenylbenzene compounds. Thus, it is likely that microorganisms capable of utilizing trans-anethole will produce high value-added natural biological spices, such as anisaldehyde, anisic acid etc. Therefore,trans-anethole was selected as substrate. Microorganisms that can degrade and transfor trans-anethole to anisaldehyde or anisic acid were isolated. Meanwhile, the factors that affected biotransformation, isolation and identification of intermediate products and trans-anethole biodegradation pathways were studied.
1. Establishment of methods for analyzing trans-anethole, anisaldehyde and anisic acid in trans-anethole degradation and transformation system
A thin layer chromatography method (TLC), thin layer chromatography-UV spectrophotometry method (TLC-UV), reversed phase high performance liquid chromatography method (RP-HPLC) and thiobarbituric acid spectrophotometric method (TBA) for the analysis of trans-anethole, anisaldehyde and anisic acid in trans-anethole biodegradation and transformation system were developed. The suitable developing solvent of TLC was petroleum ether (bp.60℃-90℃): chloroform:ethyl acetate:formic acid (V/V/V/V)= 25:10:3:0.2. TLC was found to be simple and rapid for qualitative and semiquantitative analysis of trans-anethole, anisaldehyde and anisic acid. The method was suitable for rapid screening of trans-anethole degrading microorganisms. TLC-UV and RP-HPLC method can be simultaneous quantitative analysis of trans-anethole, anisaldehyde and anisic acid. RP-HPLC method was accurate above TLC-UV method. RP-HPLC conditions were as follow:The separation was performed on Kromasil-100A C18 column (250 mm×4.6 mm×5μm) using V(acetonitrile):V(water):V(acetic acid)=70:30:0.02 as the mobile phase. The flow rate was 0.8 mL·min-1. The detection wave length was 260 nm. The injection volume was 5μL, and the column temperature was room temperature. The method showed good linear relationship, precision and repeatability. In addition, anisaldehyde in transformation liquids could also be detected by TBA method.trans-Anethole and anisic acid don't interfered with the determination of anisaldehyde.
2. Isolation and identification of trans-anethole degrading microorganisms
Isolation of trans-anethole degrading microorganisms which can grow on high concentration of anise oil from the soil under aniseed tree, the fruits、branches and leaves of fresh aniseed, waste residue of aniseed workshop. Then biotransformation of trans-anethole to anisaldehyde or anisic acid with these strains were carried out by fermentation. Bacteria were isolated from soil under aniseed trees by enrichment culture. Bacterial strain BT-13, which utilizes 1%(V/V) anise oil as the sole source of carbon and energy in modified M9 media, was screened out base on its good capability to biotransform trans-anethole to anisic acid. This strain was identified as Pseudomonas sp. according to visual observation, physiological and biochemical experiments and 16S rDNA sequence analysis. Eighty-seven endophytes were isolated from the fruits、branches and leaves of fresh aniseed. It includes sixty-nine strains of endophytic fungus, eighteen strains of endophytic bacteria. A endophytic bacterium designated BZ-15 was newly isolated based on its ability to degrade trans-anethole. Anisic acid was detected in the culture.This strain was identified as Pseudomonas putida according to its 16S rDNA sequence analysis. A fungal strain ZJ-9 was newly isolated from waste residue in aniseed workshop based on its ability to degrade trans-anethole. A small amount of anisaldehyde was detected in the culture. Strain ZJ-9 was identified as Aspergillus niger according to its morphological characteristics, culture characteristics and "fungal identification manual".
3. Biodegradation and biotransformation technology of trans-anethole to anisaldehyde and anisic acid
The biotransformation process of trans-anethole to anisaldehyde by Pseudomonas sp. BT-13 was carried out in aqueous-organic solvents biphasic systems. The effects of some key factors such as organic solvent polarity and content, the component of culture medium, conversion time, temperature, medium capacity, rotate speed of rotary shaker and medium initial pH were investigated on the biotransformation of trans-anethole to anisaldehyde by free cell. The results showed that the suitable organic solvent was ethyl acetate, ethyl acetate content was 10%(V/V), the suitable medium was the modified Martin's medium, medium capacity was 20 mL/150 mL flask, medium initial pH 6.5, temperature 30℃, rotate speed of rotary shaker at 150 r·min-1, conversion time 30 h. On the optimum conditions, the molar generation ratio of anisaldehyde was increased to 12.6% by immobilized cells with calcium alginate compared with 7.7% of free cells. In order to separate trans-anethole and anisaldehyde, trans-anethole and anisaldehyde were extracted from biotransformation liquids by equal volume ethyl acetate at first, then trans-anethole was stripped to water phase via saturation sodium bisulfite solution. Because trans-anethole was almost insoluble in water, this reactive-extraction also resulted in anisaldehyde purification. Finally anisaldehyde was obtained from acidic water phase by ethyl acetate.
The optimization of conversion conditons for the biotransformation of trans-anethole to anisic acid by Pseudomonas sp. BT-13 was carried out in modified M9 medium, on which the effects of culture medium components, substrate concentration, shaking speed, temperature and initial medium pH were studied. The results showed that the carbon source concentration of above 1 g·L-1 in modified M9 medium could marketdly inhibit the transformation of trans-anethole. The optimal medium composition was obtained:maltose 0.5 g·L-1,NH4Cl 0.5 g·L-1,FeSO4·7H2O 0.01 g·L-1, MgSO4·7H2O 2.0 g·L-1,NaCl 0.5 g·L-1, Na2HPO4 6.8 g·L-1, KH2PO4 3.0 g·L-1 CaCl2 0.02 g·L-1. Under an optimal condition of trans-anethole concentration of 9.83 g·L-1, shaking speed 200 r·min-1, temperature 30℃, pH 7.0, the cumulative concentration of anisic acid could reach to 3.49 g·L-1 with a molar generation ratio of 34.6%, which increased 92.8% than that under the unoptimized conditions. Anisic acid was proved to be the leading product of this biotransformation, while some other intermediates such as anethole epoxide, anisaldehyde,t-anethole-diol etc could also be detected in the culture. When the biotransformation was processed in 5 L fermentor, the anisic acid of 3.57 g·L-1 was obtained with a molar generation ratio of 36.1%. Crystals of anisic acid was obtained from biotransformation liquid afer acidified, extracted with ethyl acetate, vacuum concentration and crystallization.
4. Proposed degradation pathway of trans-anethole by Pseudomonas sp. BT-13 and Aspergillus niger ZJ-9
The degradation pathway of trans-anethole in Pseudomonas sp. BT-13 and Aspergillus niger ZJ-9 were studied by analyzing intermediates using HPLC and GC-MS. Some main intermediates were identified by comparison with standard sample or GC-MS spectra analysis. Four intermediates that is anethole epoxide, anethyl diol, anisaldehyde and anisic acid were produced in the degradation of trans-anethole by Pseudomonas sp. BT-13; Five intermediates that is anethole epoxide, anethyl diol, anisyl alcohol, anisaldehyde and anisic acid were produced in the degradation of trans-anethole by Aspergillus niger ZJ-9. Under certain conditions, anisyl alcohol was accumulated. These intermediate products are all high value-added aromatic compounds in the perfume and pharmaceutical industries. According to the generation of intermediates, possible degradation pathways of trans-anethole by Pseudomonas sp. BT-13 and Aspergillus niger ZJ-9 were proposed:First the 1,2-CC double bond in the trans-anethole side chain is epoxidized and anethole epoxide is produced.Then the epoxide is hydrolysed, forming anethyl diol which is subsequently oxidized to anisaldehyde. Anisaldehyde can continue to be oxidized to anisic acid. In Aspergillus niger ZJ-9, anisaldehyde also can continue to be reduced to anisyl alcohol. In two strains,trans-anethole was degraded through anethyl diol pathway, but the accumulation of some intermediate products was different.
Peroxidase (POD) and catalase (CAT) activity were detected in intracellular enzyme extracted from Pseudomonas sp. BT-13 and Aspergillus niger ZJ-9. POD and CAT activity of Pseudomonas sp. BT-13 were 122 U·mL-1 and 625 U·mL-1, but POD and CAT activity of Aspergillus niger ZJ-9 were 4.8 U·mL-1 and 42.5 U·mL-1, significantly lower than Pseudomonas sp. BT-13. According to the testing enzymes activity and the accumulation of the intermediate products, there were some differences among the key enzymes and enzyme regulation in biodegradation of trans-anethole by Aspergillus niger ZJ-9 and Pseudomonas sp. BT-13. Anethole epoxide, anisaldehyde, and anisic acid were also detected in the biotransformation liquids of trans-anethole by Pseudomonas sp. BT-13 crude enzyme. Adding amount of H2O2 could promote the biotransformation of trans-anethole to anisic acid. Enzymatic transforming suitable pH was 8.
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