摘要
合成、半合成抗生素的研究,是探索和开发新抗生素的最重要的途径之一。而D-对羟基苯甘氨酸(D-pHPG)是半合成p-内酰胺类抗生素的重要前体,是目前制药行业竞争激烈的产品之一,化学法制备D-pHPG带来严重的污染和毒害,而酶法是较好的方法。酶法是利用D-海因酶(DHase)将苯海因(DL-pHPH)转化为N-氨甲酰基D-对羟基苯甘氨酸(NC-D-pHPG),N-氨甲酰基-D-氨基酸酰胺水解酶(DCase)则继续将NC-D-pHPG转化为D-pHPG。但现有的菌种产酶活力不高,故选育高产菌株提高酶活以及采用新技术提高酶利用率具有重要的研究价值。
首先对出发菌株Spj0104的酶活水平进行了验证,随之开展低能N+离子注入技术的研究,对出发菌株运用单因子实验以确定最佳能量与剂量,采用正交法对低能离子注入的参数进行了优化,并在最优条件下对于一次注入后诱变选育获得的优良菌株Spi03和经过微波诱变后的优良菌株Sp902进行了二次离子注入。最后对于筛选到的高产菌株Sp208进行了发酵条件的优化和遗传稳定性检测。以下是整个实验结果：
(1)在显微镜下Spj0104呈短杆状,生长周期比较长,在0-21h为生长延滞期；从21h开始,OD600迅速增加,直到57h,不再增大,且保持相对稳定的状态,此为对数生长期；57-69h OD值相对稳定,处于稳定生长期。通过HPLC法测定经DHase和DCase催化后生成的终产物D-pHPG的含量,测定出该菌株的酶活为0.118955 u/mL。
(2)在低能离子注入的准备工作中先要对待诱变的菌液风干处理。在30min时,菌膜完全干燥,至50min时,菌膜彻底干燥。随着时间的增加,菌株由于缺乏水分和养料,致使存活率越来越低,所以当菌膜吹至30min时即可进行离子注入处理。
(3)对低能N+离子注入的能量和剂量分别在固定了脉冲和间隔时间,多次不同剂量重复的前提下作了单因子实验,确定了最适合的能量为30KeV。在30KeV能量条件下,设计了多梯度的剂量,不同剂量下的存活率曲线为“马鞍形曲线”。在马鞍形曲线的“马鞍”两侧选取了60×1014ions/cm2,80×1014ions/cm2, 100×1014ions/cm2,120×1014ions/cm2,140×1014ions/cm2这几个剂量,试验结果综合考虑酶活、存活率等几个参数,确定最佳剂量为60×1014ions/cm2。在确定了最佳能量和剂量的基础上,采用了四因素三水平的正交实验,对能量、剂量、脉冲和间隔时间进行正交优化设计。得出了最适诱变参数为：能量20KeV,剂量40×1014ions/cm2,脉冲时间15S,间隔时间15S。
(4)一次离子注入后筛选到一株优良菌株Spi03,酶活达到了0.2422 u/mL。再将其在最佳离子注入条件下进行二次离子注入诱变,同时将经过微波诱变筛选到的菌株Sp902号进行离子注入处理,分别筛选到了Sp208酶活为0.3271u/mL和Sp201酶活为0.2382u/mL,分别比原来提高了35.53%和28.41%,
经过微波和离子注入复合诱变的菌株在形态上变化很大,负突变株较多。从众多的菌株中还筛选到一株菌株Sp206酶活为0.1536,虽然总酶活降低,但是DHase的酶活有较大的提高。
(5)对发酵培养基成分作了正交实验和分析,得出了优化的培养基配方为：玉米浆3.0%；葡萄糖1.5%；诱导剂3.0%；氯化钠0.3%;(NH4)SO40.1%;MgSO4 0.05%;CoCl2 0.01%。对于影响发酵单位的发酵条件温度、初始pH、装量和发酵周期安排了正交实验设计,得到了最佳的发酵条件为温度30℃,初始PH7.0,装量40mL,发酵周期22h。发酵周期较原来提前了2h,缩短了发酵周期。Sp208菌株在最佳的发酵培养基成分和发酵条件下培养,酶活达到了0.402u/mL,提高了22.89%。传代5次,遗传稳定性较好。
Study on synthetic and semi synthetic antibiotic is one of the important ways to explore and develop new antibiotic. D-hydroxyphenylglycine (D-pHPG) is the most important precursor used for the synthesis of semi synthetic lactam types of antibiotic and the product attracting keen competition in the field of pharmacy. Enzyme method is rather better than the chemical one which brings severe pollution and poison. In enzyme method, P-hydroxyphenyldantoin (p-HPH) was transformed into N-Carbamoyl-D-P-hydroxy-phenyldantoin (NC-D-pHPG) by Dhydantoinase (DHase), and then NC-D -pHPG was transformed into D-pHPG by Ncarbamyl Danimo-acid amidohydrolase (DCase). Since the enzyme activity of the existing strain was too low, it was very significant to improve the enzyme activity of the strain by breeding high-yield strain and increasing the enzyme using rate with new techniques.
The enzyme activity of the starting strain Spj0104 was validated at first. Then low energy ion implantation and single factor experiments were used to find the optimal energy and dosage. With the reference parameters of energy and dosage orthogonal optimization plan was designed and carried out. Under the optimal condition Spi03 selected from the strains under first ion implantation and Sp902 (one of the MW mutants) were arranged new ion implantation. High-yield strain was screened out among all the mutants. Moreover by orthogonal optimization the culture medium and culture condition were investigated, and then the result of optimization was used to fermentation. In the end genetic stability was conformed. The results of all experiments show as follow:
(1) Spj0104 appears like short bacilliform under microscope. Its growth period was rather too long with delaying time from 0 to 21 h. In the logarithm time,21h to 57h, the value of OD6oo increased quickly. While in the stable time,57h to 69h, it kept relatively steady. The enzyme activity was 0.118 955 u/mL determined by HPLC method.
(2) In the preparation work of low energy ion implantation, the material should be dried with asepsis air. At 30 min, velum was dry and at 50 min, it was completely dry. The more time increased, the lower the living rate of the strain was. Therefore,30min was the optimal time of drying.
(3) Single factor experiments were made basing on fixed pulse, fixed interval time and different dosages of repetition.30KeV was confirmed as the optimal energy. At 30KeV, the curve of the living rates of the strains under variable dosages in the experiment looked like a saddle. According to the papers reported, several dosages (60×1014ions/cm2, 80×1014ions/cm2,100×1014ions/cm2,120×1014ions/cm2,14×1014ions/cm2) that at the two sides of "the saddle" were chosen to investigate the changes of the mutants. Taking enzyme activity, living rate,etc. into consideration,60×1014ions/cm2 was the best dosage. On the basis of optimal energy and dosage, orthogonal optimization was made on energy, dosage, pulse and interval. The results showed the optimal condition was 20KeV,40x1014ions/cm2,15S pulse and 15S interval。
(4) Good strain Spi03 whose enzyme activity was 0.2422u/mL, was a mutant strain suffered from ion implantation and Sp902 was a mutant strain had been treated by MW.Both of them were treated with low energy ion implantation. Two good strains Sp208 and Sp201 were screened out. Their enzyme activities are 0.327lu/mL and 0.2382u/ml respectively, and increasing by 35.53 and 28.41 percent.
The mutant strains treated by MW and ion implantation changed a lot in forms and many of them were low enzyme producing strain. Strain Sp206, enzyme activity was 0.1536 u/mL. Though total enzyme activity descended, DHase activity increased a lot.
(5) The optimal fermentation medium listed below:Corn steep liquor 3.0%, Glucose 1.5%; Inducement 3.0%;Nacl 0.3%;(NH4) SO40.1%; MgSO40.05%;CoCl20.01%.The optimal fermentation conditions were temperature of 30℃, initial pH of 7.0, capacity of 40mL and fermentation time of 22h. The fermentation time was 2h shorter than the original. Being cultured under optimal medium and fermentation conditions, the strain Sp208's enzyme activity reached 0.402u/mL, increasing by 22.89 percent. Five generations'of culture showed good genetic stability.
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