摘要
葡萄糖耐量因子(GTF)是一种小分子的铬结合蛋白(肽),三价有机铬是其重要活性成分,含有半胱氨酸、天门冬氨酸、甘氨酸和谷氨酸。本项研究从本试验室收藏的7株啤酒酵母入手,探索一条通过航天诱变、高产菌株的选育、GTF的分离纯化以及功能特性研究为主要线索的微生物发酵高产GTF的途径。本论文的主要试验结果如下:
利用GTF可溶于氨水的性质,完善其提取参数,优化消解工艺,确定GTF有效提取方法和测定方法。结果表明:0.1g的菌体悬浮于10mL的0.1moL/L氨水溶液于37℃、200r/min提取3h,5000r/min离心10min,收集上清液,上火焰原子吸收光谱法测定其铬含量。验证试验结果显示该方法可有效的测定和评价菌体中GTF的含量。该方法可靠,处理时间短、耗酸量少,其回收率在95%～102.5%之间,相对偏差小于4%。
利用航天诱变技术对本实验是收藏的7株啤酒酵母进行诱变处理。通过梯度含铬平板、液体培养和筛选得到一株遗传性能稳定、GTF高产菌株YS-3。其有机铬产量为1258μg/g.dcw,总铬含量为1926μg/g.dcw,有机铬率达65.31%,生物量40 g/L。相比于出发菌株Y-3,YS-3有机铬产量提高了53%。
铬离子为800μg/mL时,严重抑制菌体的生长;400μg/mL时,对菌体有刺激生长作用。与无铬离子培养相比,400μg/mL的铬离子延长了菌体延迟期,发酵6 h后进入对数生长期,生物量增长超过无铬离子培养。有机铬富集主要发生在对数生长期(6 h～44 h);菌体最快的有机铬富集速度主要出现在延迟期(0 h～6 h),此时单位菌体的富铬量最大。通过二次加铬培养,摇瓶培养YS-3富集有机铬量可提高一倍,有机铬为2644μg/g.dcw左右,菌体生物量无显著性降低。发酵罐扩大规模培养,有机铬量为1930μg/g.dcw,生物量为33 g/L。
采用中红外分析了其蛋白结合铬前后的二级结构的变化。大分子蛋白结合铬后,部分α-螺旋结构和无规则卷曲向β-转角结构转变,β-折叠结构几乎无变化;小分子蛋白结合铬后,部分β-折叠结构转向无规则卷曲和α-螺旋,β-转角结构几乎无变化。
通过HPLC-ICP-MS/AES对Sephadex G-75凝胶层析Peak1、Peak2中金属铬和蛋白的分布进行微量分析和评估,对大、小含铬蛋白组分采用不同的分离纯化路线:小分子含铬蛋白:采用三次超滤(100kD、50kD、10KD)、Sephadex G25、Sephadex G15凝胶过滤层析脱盐处理获得峰形对称的含铬峰PP1。大分子含铬蛋白采用Sephadex G75、高效液相层析处理。
对峰PP1的结构分析表明其为一个含铬的小肽类物质,由天门冬氨酸、谷氨酸、甘氨酸、半胱氨酸组成;其中的氨基酸有可能并非以肽键相连,或以配位键与金属结合。峰PP1中含有两个m/z比值为712.2535、769.2747离子峰,并根据其一、二级质谱图推测其氨基酸组成比例。通过LC-ESI-MS对peak1中第8管进行测定,分析该含铬大分子蛋白有可能为HBT1蛋白或3-磷酸甘油醛脱氢酶或核蛋白体蛋白。
糖尿病动物试验结果显示富铬酵母菌体对糖尿病有一定的改善作用,具有一定的降脂、降糖作用(糖基化血红蛋白),对胰腺组织和β细胞具有一定的保护和恢复作用。
从本文结果可以看出,通过航天诱变、筛选得到的YS-3,在优化其发酵条件基础上,通过二次补铬工艺,有机铬产量达至1930μg/g.dcw,生物量为33 g/L。发酵所产的GTF的氨基酸组成与文献报道一致,通过动物饲喂试验证明其对糖尿病有一定改善作用,具有降脂、降血糖、保护胰腺组织的功能。因此通过微生物高产GTF、使生物铬补充剂工业化具有广阔的研究价值和发展前景。
Glucose Tolerance Factor (GTF) is a low molecular weight Cr binding polypetide, consisted of glycine, cysteine, glutamic acid and aspartic acid and Cr3+ being its essential active center. GTF has been proven to play an important role in normal glucose homeostasis by many studies and it has become the focus of studies. The aim of this paper is to carry out comprehensive research in the field of a high yield of GTF by yeast and the structure and function of GTF. The main results of this paper are as follows:
According to the property of ammonia-solubility of GTF, develop an effective ammonia extraction method of organic chromium and determination method by perfect and optimize its extraction and digestion parameters. 0.1g yeast suspended in 10mL 0.1mol/L NH4OH, shook with 200r/min for 3h at 37℃, collected the supernatant with centrifugation, microwave digested and oxidized the supernatant. Determining the content of the organic chromium of GTF, residual chromium in the yeast after extraction and the total chromium in the yeast with flame atomic absorption spectrometry. The experiment result showed that the theoretical residual chromium content meet with determination result closely. This method is suitable for determining GTF in the yeast.
Seven strains yeast is mutagenized by piggyback experiment. Using chromium chloride hexahydrate as the chromium source, A strain of high-biomass, producing glucose tolerance factor yeast strain was screened through gradient concentration of chromium plates cultivation and liquid fermentation cultivation. The biomass of it was 40 g/L. the content of organic Cr was 1287μg/g.dcw and that of total Cr was 1917μg/g.dcw. Compared with the original strain, the organic chromium of YS-3 was improved by 53% with same biomass and pH during cultivation.
YPD is fitter for YS-3 to produce more GTF compared with wort. Growth of YS-3 in media with 800μg/mL Cr3+ was strongly inhibited, and was stimulated with 400μg/mL Cr3+. In the presence of Cr3+400μg/mL, the lag phase is prolonged and after initial six hours the concentration of cell increased sharply and exceeded that without Cr3+. The accumulation of chromium ions into yeast cells was mainly completed in the exponential growth(6 h～44 h) and the fastest speed of accumulating chromium mainly occurred in the lag phase(0 h～6 h). The amount of organic chromium doubled through supplementation of chromium, reaching 2644μg/g.dcw without significant decrease in biomass. With pilot-scale fed-batch fermentation, the organic chromium reached 1930μg/g.dcw and biomass 33g/L.
Through bioaccumulation and biotransformation, chromium was uptake into the yeast to bind on the protein, which had a great impact on the protein secondary structure. When chromium binding on the big molecule weight protein, the secondary structure changed fromα-helix and random-coil toβ-turn,β-turn increased,α-helix increased, random-coil decreased a little andβ-sheet changed nothing. When chromium binded on the small molecule weight protein, the secondary structure changed fromβ-sheet toα-helix and random-coil,β-sheet decreased greatly, random-coil increased greatly,α-helix increased a little andβ-turn remain intact.
The distribution of chromium and protein were analyzed in Peak1 and Peak2 through HPLC-ICP-MS/AES, different purification method was adopted to purify two groups of chromium containing protein. In the case of low molecule weight protein, ammonia extract was purified by ultra-filtration(100kD、50kD、10KD), condense, gel filtration column chromatography (Sephadex G25), gel filtration column chromatography (Sephadex G15), achieving PP1 containing chromium. In the case of big molecule weight protein, ammonia extract was purified by gel filtration column chromatography (Sephadex G75), HPLC, achieving the eighth tube containing chromium.
Ultraviolet wavelength scan and amino acid composition analysis showed that PP1 was a polypeptide containing chromium, consisted of glycine, cysteine, glutamic acid and aspartic acid. N-termianl sequence analysis showed that amino acids in PP1 were possibily connected by coordination bond, not by the amide bond. The peptide mass fingerprints produced by PP1 were studied by MALDI-TOF-TOF-MS and PP1 was analyzed by HPLC-ICP-MS/AES. The result showed that protein in peak PP1 belong to clusters of similar protein with high purity. There are only two quasi-molecular ions with high ion abundance in first order mass spectrum, m/z ratio of ions 712.2535, 769.2747. Make a speculation on the proportion of amino acid composition of the two quasi-molecular ions according to its second order mass spectrum and the result of amino acid composition of peak PP1.
The peptide mass fingerprints produced by the eighth tube in Peak1 was studied by HPLC-ESI-MS. Data from HPLC-ESI-MS showed that the big molecule weight containing chromium possibly is Glyceraldehyde-3-phosphate dehydrogenase 3 or Ribosomal protein L2A or Protein HBT1.
Diabetes mellitus models induced by streptozotocin were treated with high chromium brewer’s yeast (250～1000μg/kg.dCr) for a period of 3 months. It showed that organic chromium have an improvement on diabetes mellitus. TCHO decreased very significantly in low group and middle group and TG decreased significantly in low group. Serum insulin increased significantly in low group and high group. GHb decreased significantly in high group; Pathological examination of pancrease islet revealed that organic chromium had a certain protective effect on theβ-cell.
Above all, the organic chromium yield of YS-3, mutagenized by space piggyback, reached 1930μg/g .dcw and biomass of it reached 33g/L, through supplement chromium twice. The composition of GTF isolated from YS-3 was in accord with other reports. Furthermore effect of high chromium yeast on diabetes mice showed that it have improvement on blood glucose, blood lipids and impaired islet andβcells. We could draw a conclusion that GTF from yeast should be paid more attention to and studied further.
引文
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