CN113718002B - 一种利用玉米芯碱抽提剩余物制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法 - Google Patents
一种利用玉米芯碱抽提剩余物制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法 Download PDFInfo
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- galactomannan
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- 229920000926 Galactomannan Polymers 0.000 title claims abstract description 86
- OMDQUFIYNPYJFM-XKDAHURESA-N (2r,3r,4s,5r,6s)-2-(hydroxymethyl)-6-[[(2r,3s,4r,5s,6r)-4,5,6-trihydroxy-3-[(2s,3s,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyoxan-2-yl]methoxy]oxane-3,4,5-triol Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)[C@H](O)[C@H](O)[C@H](O)O1 OMDQUFIYNPYJFM-XKDAHURESA-N 0.000 title claims abstract description 59
- 239000003513 alkali Substances 0.000 title claims abstract description 53
- 238000000605 extraction Methods 0.000 title claims abstract description 44
- 229920001542 oligosaccharide Polymers 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 23
- 102000004190 Enzymes Human genes 0.000 claims abstract description 73
- 108090000790 Enzymes Proteins 0.000 claims abstract description 73
- 239000007788 liquid Substances 0.000 claims abstract description 32
- -1 galactomannan oligosaccharide Chemical class 0.000 claims abstract description 23
- 241000499912 Trichoderma reesei Species 0.000 claims abstract description 11
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 10
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 31
- 230000000694 effects Effects 0.000 claims description 28
- 239000006228 supernatant Substances 0.000 claims description 27
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 238000006243 chemical reaction Methods 0.000 claims description 15
- 239000012153 distilled water Substances 0.000 claims description 13
- 150000003384 small molecules Chemical class 0.000 claims description 13
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- 108010030291 alpha-Galactosidase Proteins 0.000 claims description 11
- 102000005840 alpha-Galactosidase Human genes 0.000 claims description 11
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- 239000002244 precipitate Substances 0.000 claims description 10
- 239000000758 substrate Substances 0.000 claims description 10
- 235000019441 ethanol Nutrition 0.000 claims description 9
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- 239000000243 solution Substances 0.000 description 31
- 240000008042 Zea mays Species 0.000 description 16
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 16
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 16
- 235000005822 corn Nutrition 0.000 description 16
- 108010055059 beta-Mannosidase Proteins 0.000 description 15
- 102100032487 Beta-mannosidase Human genes 0.000 description 9
- BTJIUGUIPKRLHP-UHFFFAOYSA-N 4-nitrophenol Chemical compound OC1=CC=C([N+]([O-])=O)C=C1 BTJIUGUIPKRLHP-UHFFFAOYSA-N 0.000 description 8
- 239000001913 cellulose Substances 0.000 description 7
- 229920002678 cellulose Polymers 0.000 description 7
- 235000010980 cellulose Nutrition 0.000 description 7
- 238000002835 absorbance Methods 0.000 description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 238000005227 gel permeation chromatography Methods 0.000 description 6
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- 229920000168 Microcrystalline cellulose Polymers 0.000 description 4
- 239000007857 degradation product Substances 0.000 description 4
- 235000021255 galacto-oligosaccharides Nutrition 0.000 description 4
- 150000003271 galactooligosaccharides Chemical class 0.000 description 4
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 4
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- 229940016286 microcrystalline cellulose Drugs 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
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- 239000010902 straw Substances 0.000 description 3
- HNSDLXPSAYFUHK-UHFFFAOYSA-N 1,4-bis(2-ethylhexyl) sulfosuccinate Chemical compound CCCCC(CC)COC(=O)CC(S(O)(=O)=O)C(=O)OCC(CC)CCCC HNSDLXPSAYFUHK-UHFFFAOYSA-N 0.000 description 2
- 229920000742 Cotton Polymers 0.000 description 2
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 240000007594 Oryza sativa Species 0.000 description 2
- 235000007164 Oryza sativa Nutrition 0.000 description 2
- 241000209140 Triticum Species 0.000 description 2
- 235000021307 Triticum Nutrition 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- HEBKCHPVOIAQTA-NGQZWQHPSA-N d-xylitol Chemical compound OC[C@H](O)C(O)[C@H](O)CO HEBKCHPVOIAQTA-NGQZWQHPSA-N 0.000 description 2
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- 238000001704 evaporation Methods 0.000 description 2
- 239000000446 fuel Substances 0.000 description 2
- HYBBIBNJHNGZAN-UHFFFAOYSA-N furfural Chemical compound O=CC1=CC=CO1 HYBBIBNJHNGZAN-UHFFFAOYSA-N 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
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- 235000009566 rice Nutrition 0.000 description 2
- 238000002390 rotary evaporation Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 241000219146 Gossypium Species 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- 229920000161 Locust bean gum Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 239000003153 chemical reaction reagent Substances 0.000 description 1
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 description 1
- 238000002485 combustion reaction Methods 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 235000013325 dietary fiber Nutrition 0.000 description 1
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- 238000005516 engineering process Methods 0.000 description 1
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- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
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- 238000011081 inoculation Methods 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- SURQXAFEQWPFPV-UHFFFAOYSA-L iron(2+) sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Fe+2].[O-]S([O-])(=O)=O SURQXAFEQWPFPV-UHFFFAOYSA-L 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 239000000711 locust bean gum Substances 0.000 description 1
- 235000010420 locust bean gum Nutrition 0.000 description 1
- WRUGWIBCXHJTDG-UHFFFAOYSA-L magnesium sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Mg+2].[O-]S([O-])(=O)=O WRUGWIBCXHJTDG-UHFFFAOYSA-L 0.000 description 1
- 229940061634 magnesium sulfate heptahydrate Drugs 0.000 description 1
- OQTQHQORDRKHFW-UHFFFAOYSA-L manganese(2+);sulfate;heptahydrate Chemical compound O.O.O.O.O.O.O.[Mn+2].[O-]S([O-])(=O)=O OQTQHQORDRKHFW-UHFFFAOYSA-L 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 238000010299 mechanically pulverizing process Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 150000002772 monosaccharides Chemical class 0.000 description 1
- 238000001728 nano-filtration Methods 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
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- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Chemical class 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
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- 230000001681 protective effect Effects 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 241000894007 species Species 0.000 description 1
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- 239000004753 textile Substances 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
- 229920001221 xylan Polymers 0.000 description 1
- 150000004823 xylans Chemical group 0.000 description 1
- RZLVQBNCHSJZPX-UHFFFAOYSA-L zinc sulfate heptahydrate Chemical compound O.O.O.O.O.O.O.[Zn+2].[O-]S([O-])(=O)=O RZLVQBNCHSJZPX-UHFFFAOYSA-L 0.000 description 1
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- C12P19/00—Preparation of compounds containing saccharide radicals
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2465—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
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- C12N9/2477—Hemicellulases not provided in a preceding group
- C12N9/2488—Mannanases
- C12N9/2491—Beta-mannosidase (3.2.1.25), i.e. mannanase
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Abstract
本申请公开了一种利用玉米芯碱抽提剩余物制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法,以玉米芯碱抽提剩余物为碳源,以里氏木霉为产酶菌株,进行发酵产酶,获得酶液;直接利用该酶液对含半乳甘露聚糖的原料进行酶解,获得小分子半乳甘露聚糖和半乳甘露低聚糖。该酶液无需纯化即可直接水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖。从而实现对工业生产废弃物的再利用,提高其附加值,降低半乳甘露低聚糖的生产成本,具有非常好的应用前景。
Description
技术领域
本发明属于生物活性物质制备技术领域,具体涉及一种利用玉米芯碱抽提剩余物制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法。
背景技术
木质纤维素是地球上产量最多、分布最广的可再生资源。木质纤维素主要由纤维素(28%~50%)、半纤维素(20%~30%)、木质素(18%~30%)以及少部分灰分构成。资料显示全世界木质纤维素每年产量达到1500亿吨,我国光农作物秸秆年产量就有大约11亿吨,由此可知木质纤维素资源的丰富程度。我国农作物秸秆种类众多,主要包括水稻、小麦、玉米、薯类、棉花、甘蔗。玉米是全世界总产量最高的农作物。玉米在国内的种植面积和总产量仅次于水稻和小麦,是我国三大主要农作物之一。玉米芯是玉米果穗去籽脱粒后的穗轴,一般占玉米穗重量20.0%~30.0%,玉米年产量高达千亿公斤,其中玉米芯约占玉米10%,产量达千万吨以上。目前玉米芯是我国各类秸秆资源中高值化利用率较高的种类。以前玉米芯主要作为生活燃料,如今随着技术的发展,玉米芯的用处也与之前大有不同。现在主要利用玉米芯制备生物乙醇、糠醛、丁醇、低聚木糖等高附加值物质。实现了原料化、基料化、饲料化及能源化的综合利用。
但是玉米芯利用完的剩余物是个比较大的麻烦,如玉米芯碱抽提物。玉米芯碱抽提物是玉米芯在制备低聚木糖过程中产生的残渣。一般都当做废弃物丢弃,或者作为生活燃料。但是玉米芯碱抽提剩余物中含有大量的碱,在进行填埋处理时会造成严重的土地污染,燃烧时也会污染空气。另外,玉米芯碱抽提物剩余物中含有丰富的纤维素,纤维素是自然界中分布最广、含量最多的一种多糖,虽然不能够被人体直接利用,却是重要的膳食纤维。而且纤维素的应用非常广泛,主要应用于纺织、印染、石油钻探、造纸、陶瓷、合成洗涤、日用化工等工业。如果将碱抽提物中纤维素进行利用,将会带来额外的经济效益和生态效益。
里氏木霉是多细胞的真核微生物,是生产甘露聚糖酶的菌种之一。里氏木霉利用微晶纤维素可产甘露聚糖酶,产酶效果较其他底物相比好。但微晶纤维素价格较高,约为1万元/吨。由于玉米芯碱抽提物剩余物中含有大量的纤维素,可作为产酶的底物制备甘露聚糖酶,若以玉米芯碱抽提物剩余物以底物制备甘露聚糖酶,可以降低生产成本,同时实现玉米芯碱抽提剩余物由低附加值物到高附加值物的转变。
发明内容
针对现有技术中存在的生产成本问题,本发明要解决的一个技术问题在于提供一种利用玉米芯碱抽提剩余物制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法,具有操作简单,降低生产成本,目标产物得率高等优势。
为了解决上述问题,本发明所采用的技术方案如下:
一种利用玉米芯碱抽提剩余物制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法,以玉米芯碱抽提剩余物为碳源,以里氏木霉为产酶菌株,进行发酵产酶,获得酶液;直接利用该酶液对含半乳甘露聚糖的原料进行酶解,获得小分子半乳甘露聚糖和半乳甘露低聚糖。
所述利用玉米芯碱抽提剩余物制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法,玉米芯碱抽提剩余物由以下方法获得:取玉米芯,以1∶20的固液比加入10%的氢氧化钠溶液混合,在100℃下抽提1小时,抽提结束后过滤,获得的固体残渣为玉米芯碱抽提剩余物。
所述利用玉米芯碱抽提剩余物制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法,获得的玉米芯碱抽提剩余物,在使用前,采用蒸馏水对剩余物进行多次洗涤,干燥备用。
所述利用玉米芯碱抽提剩余物制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法,玉米芯碱抽提剩余物水洗涤获得的废水在经过蒸发浓缩过滤后形成高浓度的碱液,重复用于玉米芯碱抽提,实现碱液的循环利用。
所述利用玉米芯碱抽提剩余物制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法,以玉米芯碱抽提剩余物为碳源时,玉米芯碱抽提剩余物的浓度为15~30g/L。
所述利用玉米芯碱抽提剩余物制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法,所得酶液中β-甘露聚糖酶酶活力不低于1.5U/mL,α-半乳糖苷酶活力不高于为0.06U/mL,β-甘露聚糖酶活力不高于为0.03U/mL。
所述利用玉米芯碱抽提剩余物制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法,含半乳甘露聚糖的原料为田菁种子,经机械粉碎至20~100目,按1∶50固液比加入蒸馏水,于50℃抽提24h后,于10000转/分条件下离心10min获得上清液,并向上清液中加入无水乙醇,所得沉淀物经真空干燥得到半乳甘露聚糖粉状固体,用于酶解制备小分子半乳甘露聚糖和半乳甘露低聚糖。
所述利用玉米芯碱抽提剩余物制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法,取半乳甘露聚糖粉状固体,直接加入酶液、蒸馏水、1mol/L柠檬酸缓冲液,充分混合均匀,于底物浓度2%、酶加量20U/g半乳甘露聚糖、pH值4.8、50℃条件下反应24h;酶水解反应结束后,将酶水解物置于100℃下处理10min从而使酶失活,失活的酶解液于10000转/分条件下离心10min,上清液即为含小分子半乳甘露聚糖和半乳甘露低聚糖的酶解液。
所述利用玉米芯碱抽提剩余物制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法,步骤如下:
1)碱抽提玉米芯获得剩余物,用蒸馏水对剩余物进行多次洗涤,回收洗涤水,浓缩后循环用于碱抽提玉米芯,获得的剩余物干燥备用;
2)以步骤1)处理后的剩余物为碳源配置发酵培养基,按10%的接种量接入里氏木霉孢子,置于28-30℃、170转/分的恒温摇床中培养4天,进行发酵产酶,获得酶液;其中,剩余物的浓度为15~30g/L,所得酶液中β-甘露聚糖酶酶活力不低于1.5U/mL,α-半乳糖苷酶活力不高于为0.06U/mL,β-甘露聚糖酶活力不高于为0.03U/mL。
3)取田菁种子,经机械粉碎至20~100目,按1∶50固液比加入蒸馏水,于50℃抽提24h后,于10000转/分条件下离心10min获得上清液,并向上清液中加入无水乙醇,所得沉淀物经真空干燥得到半乳甘露聚糖粉状固体;直接利用步骤2)制备的酶液对半乳甘露聚糖粉状固体进行酶解,底物浓度2%、酶加量20U/g半乳甘露聚糖、pH值4.8、50℃条件下反应24h;酶水解反应结束后,将酶水解物置于100℃下处理10min从而使酶失活,失活的酶解液于10000转/分条件下离心10min,获得上清液,采用乙醇分级沉淀,即获得小分子半乳甘露聚糖和半乳甘露低聚糖,其中,半乳甘露低聚糖的分子量小于3000Da,小分子半乳甘露聚糖的分子量在3000到20000Da。
一种制备β-甘露聚糖酶的方法,以玉米芯碱抽提剩余物为碳源,以里氏木霉为产酶菌株,进行发酵产酶,获得酶液;所得酶液中β-甘露聚糖酶酶活力不低于1.5U/mL,α-半乳糖苷酶活力不高于为0.06U/mL,β-甘露聚糖酶活力不高于为0.03U/mL。
有益效果:与现有的技术相比,本发明的优点包括:
(1)本申请以废弃物玉米芯碱抽提剩余物为原料,采用里氏木霉为产酶菌株,发酵产生富含β-甘露聚糖酶的酶液;同时,酶液中β-甘露糖苷酶含量(活力)不高于0.02U/mL,α-半乳糖苷酶活力低,可直接用于制备小分子半乳甘露聚糖和半乳甘露低聚糖,有效降低了发酵用酶的制备成本。
(2)本申请制备的酶液,无需纯化除去β-甘露糖苷酶即可直接水解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖,并可有效提高小分子半乳甘露聚糖和半乳甘露低聚糖的得率,尤其是分子量低于10000Da的产物,该产物活性高,应用范围广,具有更大的商业应用价值。
(3)由于玉米芯碱抽提剩余物含有较多的碱,无法通过填埋处理,会造成环境污染,本申请将玉米芯碱抽提剩余物作为产酶底物可以实现其由低附加值物到高附加值物的转变,有效解决玉米芯碱抽提剩余物处理难的问题。同时,该酶液可用于制备小分子半乳甘露聚糖和半乳甘露低聚糖,从而降低生产成本,具有非常好的应用前景。
具体实施方式
为使本发明的上述目的、特征和优点能够更加明显易懂,下面结合具体实施例对本发明的具体实施方式做详细的说明。
对以下实施例中使用的产品性能测试如下:
(1)小分子半乳甘露聚糖和半乳甘露低聚糖的分子量分布采用凝胶渗透色谱法(GPC)测定:
色谱条件如下:色谱仪:安捷伦高效液相色谱仪1260,色谱柱:WatersUltrahydrogel TM 2000(7.8×300mm)、Waters Ultrahydrogel TM 250(7.8×300mm)和Waters Ultrahydrogel TM 120(7.8×300mm)三柱依次串联,保护柱:WatersUltrahydrogel TM Guard Column(6×40mm),检测器:示差检测器,流动相:水,流动相流速:0.60mL/min,柱温:65℃,进样体积:10.0μL,采用聚乙二醇作为标准样品进行分子量测定。
(2)小分子半乳甘露聚糖和半乳甘露低聚糖的糖含量采用酸水解法和离子色谱法测定:
测定方法如下:取小分子半乳甘露聚糖和半乳甘露低聚糖样品0.3g置于水解瓶中,加入87mL4%H2SO4于121℃下反应1h,反应结束后取1mL液体用50%NaOH调节反应液pH至中性,并离心(10000转/分,5min)取上清液,最后用ICS-5000离子交换色谱仪测定反应液中甘露糖和半乳糖浓度。
离子色谱测试条件如下:色谱仪:戴安离子色谱仪ICS-5000,色谱柱:2x250mmDionex AminoPac PA10,保护柱:2×50mm Dionex AminoPac PA10,检测器:电导检测器,流动相:3mmol氢氧化钠;流速:0.20mL/min;柱温:30℃;进样体积:10.0μL,外标法测定。则样品中小分子半乳甘露聚糖和半乳甘露低聚糖的纯度计算如下:
(3)β-甘露聚糖酶活力测定:
在25mL刻度试管中加入0.9mL 5g/L洋槐豆胶底物溶液,50℃预热5min,加入0.1mL适当稀释的酶液,于50℃下反应30min,立即加入3.0mL DNS试剂终止反应,随后沸水浴中处理7min,冷却后定容到25mL,充分摇匀,于540nm下测定反应混合物的吸光度,并根据吸光度与还原糖的相关关系,计算反应生成的还原糖的浓度。1个β-甘露聚糖酶活力单位(U)以每分钟水解底物产生1μmol还原糖(以甘露糖计)所需β-甘露聚糖酶的酶量进行计算。
(4)α-半乳糖苷酶活力测定:
于15mL试管中加入0.1mL适当稀释的酶液和0.9mL 1mmol/L pNPG(对硝基苯酚-α-D-吡喃半乳糖苷)溶液于50℃下保温10min,立即加入2.0mL 1mol/L的Na2CO3溶液终止反应,加入10mL蒸馏水,充分摇匀,于400nm下测定反应混合物的吸光度,并依据吸光度与对硝基苯酚的相关关系,计算反应生成的对硝基苯酚的浓度。1个α-半乳糖苷酶活力单位(U)以每分钟水解pNPG释放1μmol对硝基苯酚所需α-半乳糖苷酶的酶量进行计算。
(5)β-甘露糖苷酶活力测定:
于15mL试管中加入0.1mL适当稀释的酶液和0.9mL 1mmol/L pNPM(对硝基苯酚-β-D-吡喃甘露糖苷)溶液,于50℃下保温10min,立即加入2.0mL 1mol/L的Na2CO3溶液终止反应,加入10mL蒸馏水,充分摇匀,于400nm下测定反应混合物的吸光度,并依据吸光度与对硝基苯酚的相关关系,计算酶水解反应过程生成的对硝基苯酚的浓度。1个β-甘露糖苷酶酶活力单位(U)以每分钟水解pNPM释放1μmol对硝基苯酚所需β-甘露糖苷酶的酶量进行计算。
以下实施例采用的原料玉米芯碱抽提剩余物,为常规碱抽提的剩余物,典型的制备方法可以为:取500g玉米芯,以50g/L的添加量加入100g/L的氢氧化钠溶液混合,在100℃下抽提1小时,抽提结束后过滤,滤液为木聚糖溶液,固体残渣为玉米芯碱抽提剩余物。
实施例1
以玉米芯碱抽提剩余物为碳源发酵产酶,包括以下步骤:
(1)玉米芯碱抽提剩余物的前处理
取200g玉米芯碱抽提剩余物置于2L烧杯中,加入1L蒸馏水,搅拌30分钟后,过滤,将过滤物转移至另一个2L烧杯中,加入1L蒸馏水,搅拌30分钟,如此循环3次后,将玉米芯碱抽提剩余物置于40℃烘箱干燥。将3次循环的滤液混合蒸发浓缩,制得高浓度的氢氧化钠溶液,循环用于步骤(1)的玉米芯碱抽提。
(2)产酶培养基(g/L)
葡萄糖1.0,玉米芯碱抽提剩余物20.0,硫酸铵4.72,尿素2.15,磷酸二氢钾2.0,无水氯化钙0.3,七水合硫酸镁0.3,七水合硫酸亚铁0.005,七水合硫酸锰0.0016,七水合硫酸锌0.0014,氯化钴0.002。加入50mL 1mol/L的柠檬酸钠缓冲液调节培养基的pH至4.8。
以微晶纤维素取代玉米芯碱抽提剩余物作为碳源,作为对照产酶培养基。
(3)发酵产酶
将上述50mL培养基置于250mL带棉塞的三角瓶中,按10%的接种量接入里氏木霉孢子,置于28-30℃、170转/分的恒温摇床中培养4天。培养结束后,培养液于3000转/分下离心10min,取上清液(酶液)分别测定α-半乳糖苷酶、β-甘露糖苷酶和β-甘露聚糖酶的酶活。
结果表明,里氏木霉以玉米芯碱抽提剩余物为碳源发酵产酶,所得酶液(记为酶液I)中β-甘露聚糖酶酶活力为1.517U/mL,α-半乳糖苷酶活力为0.049U/mL,β-甘露聚糖酶活力为0.02U/mL。
多为对照,里氏木霉以微晶纤维素为碳源发酵产酶,所得酶液(记为酶液II)中β-甘露聚糖酶酶活力为3.92U/mL,α-半乳糖苷酶活力为0.1U/mL,β-甘露聚糖酶活力为0.02U/mL。
实施例2
酶解半乳甘露聚糖制备小分子半乳甘露聚糖和半乳甘露低聚糖,步骤如下:
(1)半乳甘露聚糖定向酶水解
含半乳甘露聚糖的豆科种子(田菁)经机械粉碎至20~100目,按添加量为20g/L加入蒸馏水,于50℃抽提24h后,于10000转/分条件下离心10min获得上清液,并向上清液中加入无水乙醇,所得沉淀物经真空干燥得到半乳甘露聚糖粉状固体。
称取上述半乳甘露聚糖粉状固体20.0g于2L酶反应罐中,加入蒸馏水、实施例1中得到的酶液(酶液I或酶液II)、1mol/L柠檬酸缓冲液使反应液体积为1000mL,充分混合均匀,于底物浓度2%、酶加量20U/g半乳甘露聚糖、pH值4.8、50℃条件下反应24h。酶水解反应结束后,将酶水解物置于100℃下处理10min从而使酶失活,失活的酶解液于10000转/分条件下离心10min,上清液即为含小分子半乳甘露聚糖和半乳甘露低聚糖的酶解液。
(2)取步骤(1)中的含小分子半乳甘露聚糖和半乳甘露低聚糖的酶解液上清1000mL,在搅拌条件下加入无水乙醇,使体系中乙醇浓度为40%(v/v),于10000转/分条件下离心10min得到上清液以及沉淀。沉淀用40%(v/v)的乙醇水溶液洗涤3次、离心(10000转/分、10min)、冷冻干燥得到组分,并将之命名为GalM40,采用凝胶色谱法测定小分子半乳甘露聚糖组分GalM40的分子量,并采用酸水解法和离子色谱法测定其半乳甘露聚糖降解产物含量。上清液继续用于下一级的分级分离。
(3)取步骤(2)中固液分离后的上清液,在搅拌条件下加入无水乙醇,使体系中乙醇浓度为50%(v/v),于10000转/分条件下离心10min得到上清液以及沉淀。沉淀用50%(v/v)的乙醇水溶液洗涤3次、离心(10000转/分、10min)、冷冻干燥得到组分,并将之命名为GalM50,采用凝胶色谱法测定小分子半乳甘露聚糖组分GalM50的分子量,并采用酸水解法和离子色谱法测定其半乳甘露聚糖降解产物含量。上清液继续用于下一级的分级分离。
(4)取步骤(3)中固液分离后的上清液,在搅拌条件下加入无水乙醇,使体系中乙醇浓度为65%(v/v),于10000转/分条件下离心10min得到上清液以及沉淀。沉淀用65%(v/v)的乙醇水溶液洗涤3次、离心(10000转/分、10min)、冷冻干燥得到组分,并将之命名为GalM65,采用凝胶色谱法测定小分子半乳甘露聚糖组分GalM65的分子量,并采用酸水解法和离子色谱法测定其半乳甘露聚糖降解产物含量。上清液继续用于下一级的分级分离。
(5)取步骤(4)中固液分离后的上清液,于70℃、160mbar下减压旋转蒸发除去其中的乙醇,取一部分上清液采用酸水解法和离子色谱法测定其半乳甘露聚糖降解产物含量,其余液体通过纳滤(200Da)除去其中的单糖,然后将截留液于70℃、160mbar下减压旋转蒸发浓缩,获得的浓缩液经干燥得到组分GalMOS,采用凝胶色谱法测定半乳甘露低聚糖组分GalMOS的分子量。
结果如表1所示,与对照(酶液II)相比,采用本申请的酶液(酶液I)制备的3种小分子半乳甘露聚糖GalM40、GalM50和GalM65中,GalM40得率低于对照,GalM50和GalM65的得率远高于对照,半乳甘露低聚糖GalMOS得率高于对照13.76%。可见本申请的方法,特别使用用于制备分子量低于10000Da的小分子半乳甘露聚糖和半乳甘露低聚糖。
表1小分子半乳甘露聚糖得率及平均分子量(重均分子量)
产物 | 得率(酶液I) | 得率(酶液II) | 平均分子量 |
GalM40 | 30.81% | 40.81% | 12600Da |
GalM50 | 8.98% | 3.98% | 8120Da |
GalM65 | 9.74% | 5.74% | 3910Da |
GalMOS | 13.76% | 9.76% | 1590Da |
Claims (1)
1.一种利用玉米芯碱抽提剩余物制备小分子半乳甘露聚糖和半乳甘露低聚糖的方法,其特征在于,步骤如下:
1)取玉米芯,以1:20的固液比加入10%的氢氧化钠溶液混合,在100℃下抽提1小时,抽提结束后过滤,获得的固体残渣为玉米芯碱抽提剩余物,用蒸馏水对剩余物进行多次洗涤,回收洗涤水,浓缩后循环用于碱抽提玉米芯,获得的剩余物干燥备用;
2)以步骤1)处理后的剩余物为碳源配置发酵培养基,按10%的接种量接入里氏木霉孢子,置于28~30℃、170转/分的恒温摇床中培养4天,进行发酵产酶,获得酶液;其中,剩余物的浓度为15~30g/L,所得酶液中β-甘露聚糖酶酶活力不低于1.5U/mL,α-半乳糖苷酶活力不高于为0.06U/mL,β-甘露糖苷酶活力不高于为0.03U/mL;
3)取田菁种子,经机械粉碎至20~100目,按1∶50固液比加入蒸馏水,于50℃抽提24h后,于10000转/分条件下离心10min获得上清液,并向上清液中加入无水乙醇,所得沉淀物经真空干燥得到半乳甘露聚糖粉状固体;直接利用步骤2)制备的酶液对半乳甘露聚糖粉状固体进行酶解,底物浓度2%、酶加量20U/g半乳甘露聚糖、pH值4.8、50℃条件下反应24h;酶水解反应结束后,将酶水解物置于100℃下处理10min从而使酶失活,失活的酶解液于10000转/分条件下离心10min,获得上清液,采用乙醇分级沉淀,即获得小分子半乳甘露聚糖和半乳甘露低聚糖,其中,半乳甘露低聚糖的分子量小于3000Da,小分子半乳甘露聚糖的分子量在3000到20000Da。
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