CN103146583B - High-yield viscosity-reduction enzyme system strain and use thereof - Google Patents

High-yield viscosity-reduction enzyme system strain and use thereof Download PDF

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CN103146583B
CN103146583B CN201210234738.5A CN201210234738A CN103146583B CN 103146583 B CN103146583 B CN 103146583B CN 201210234738 A CN201210234738 A CN 201210234738A CN 103146583 B CN103146583 B CN 103146583B
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reduction enzyme
viscosity reduction
viscosity
enzyme
enzyme system
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CN103146583A (en
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赵海
靳艳玲
方扬
黄玉红
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Chengdu Institute of Biology of CAS
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Chengdu Institute of Biology of CAS
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

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Abstract

The invention relates to a high-yield viscosity-reduction enzyme system strain and a use thereof. The high-yield viscosity-reduction enzyme system strain is named as CMC-37, forms a blue-green bacterial colony, and has hypha transverse septa and brush-shaped conidiophores subjected to multiple branching processes. The high-yield viscosity-reduction enzyme system strain has a category name of Penicillium ochrochloron Biourge and has a preservation number of CBS131817. Through agricultural waste corncobs, the high-yield viscosity-reduction enzyme system strain realizes high-yield production of a viscosity-reduction enzyme system. Through the high-yield viscosity-reduction enzyme system strain, fresh sweet potato viscosity is reduced by 91.26%. The high-yield viscosity-reduction enzyme system strain has obvious effects and a low cost, effectively solves that potato raw material fermentation in China has a high cost, and greatly improves feasibility and economy of potato raw material fermentation in China.

Description

Bacterial classification and application thereof that a kind of high yield viscosity reduction enzyme is
Technical field
The invention belongs to microbial technology field, relate to bacterial classification and application thereof that a kind of high yield viscosity reduction enzyme is.
Background technology
Along with oil is exhausted and problem of environmental pollution is day by day serious, the development of biofuel ethanol has been subject to general concern and the attention of countries in the world.As first-generation alcohol fuel, the food crop such as corn (U.S.) and sugarcane (Brazil) are main raw material (Srichuwong S et al, 2009), have obtained development rapidly in the past few decades.But and some developing countries crisis in food in short supply in some land resourcess are uneconomical also unpractical strategy taking grain as raw material production alcohol fuel.Therefore research (Kumar et al., 2008 have widely been obtained with agricultural, forestry and trade waste as the Mierocrystalline celluloses such as straw and hemicellulose raw material production alcohol fuel s-generation biofuel ethanol; Sukumaran et al., 2009; Yang et al., 2011).But biomass resistance (biomass recalcitrance) (Himmel et al., 2007) cause the structural polysaccharide in vegetable fibre to be difficult to be utilized by microorganism, if do not improve kinetic parameter and transformation efficiency that cellulose degradation is fermentability carbohydrate, do not solve the chemical structure characteristic of these raw material hard degradations, whole production process is also very difficult and expensive.The output of biofuel ethanol is brought up to 1.05 hundred million tons by American plan to 2011 year, and 1/4 the fuel that European Union plans the transport trade of the year two thousand thirty European Union will be replaced by biofuel.China 12 in the works, the output of alcohol fuel will be brought up to 5,000,000 tons.In these planning, economical and produce efficiently alcohol fuel to also have very large space.
The tubers non-grain raw materials such as sweet potato have high starch and fermentable sugar, and China is sweet potato largest production state in the world, be comparatively desirable 1.5 generation alcohol fuel raw material.But the fermentation liquid viscosity of these tuberss is very large, causes mash difficulty of transportation, heat conduction efficiency in technical process and the activity (Zhang et al, 2010) of yeast are reduced.The zymotechnique that employing stream adds and interpolation fermentation water can not solving practical problems.Therefore in the full fermenting raw materials process such as sweet potato, develop a kind of technique of adding viscosity reduction enzyme system, added the commercial enzymes such as zytase, cellulase, polygalacturonase, dextranase and reach the effect of quick viscosity reduction.But commodity viscosity reduction enzyme is expensive, being applied to industrial production has still increased production cost.
Therefore Cost Problems become 1.5 generations and 2 generation fuel ethanol production common problem, reduce production costs and must deal with problems from root.The report that for independent development viscosity reduction enzyme is not also at present, therefore, the present invention discloses bacterial classification and the application thereof that a kind of high yield viscosity reduction enzyme is, be the screening of bacterial strain and the optimization of fermentating enzyme-producing condition by viscosity reduction enzyme, utilize its fermentation crude enzyme liquid, by simple operation operation, can reduce potato raw material viscosity, reach good pretreating effect.
Summary of the invention
Object of the present invention is exactly in order to overcome the above problems, and provides a kind of bacterial strain green ochre mould Penicillium ochrochloron that produces viscosity reduction enzyme and can effectively reduce the viscosity of sweet potato fermentation liquid, to reduce Fuel Ethanol Production Cost.
For achieving the above object, the present invention has adopted following technical scheme:
The invention discloses a kind of green ochre mould Penicillium ochrochloron, preserving number is: CBS131817.This bacterial strain is that a strain viscosity reduction enzyme is superior strain, called after CMC-37, and this bacterial strain bacterium colony is blue-greenish colour, and mycelia has tabula, and its conidiophore, through branch repeatedly, forms broom shape body.
About microbial preservation information:
Strain name: green ochre mould Penicillium ochrochloron
Preservation date: 27/01/2012
Depositary institution: Dutch microbial strains preservation center
Address: Dutch Utrecht 3584 zip code, blue No. 8 of Uppsala, Royal Netherlands Academy of Arts and Sciences-Dutch microbial strains preservation center (CBS-KNAW) fungal organism center of diversity (Centraalbureau voor Schimmelcultures, FungalBiodiversity Centre, Institute of the RoyalNetherlands Academy of Arts and Sciences, CBS-KNAW Fungal Biodiversity Centre, Uppsalalaan8,3584CT Utrecht, The Netherlands)
Preserving number: CBS131817
The invention also discloses above-mentioned bacterial strains green ochre mould Penicillium ochrochloron screening source;
Particularly, described screening derives from the sweet potato of rotting.
The invention also discloses above-mentioned bacterial strains green ochre mould Penicillium ochrochloron screening screening process;
Particularly, described screening process, the sweet potato of rotting is through 10 -1, 10 -2, 10 -3, 10 -4, 10 -5, 10 -6, 10 -7after dilution, get respectively 100 μ L10 -5, 10 -6, 10 -7concentration bacteria suspension is coated in screening culture medium, cultivates after 3 days for 30 DEG C, and picking list bacterium colony, in PDA slant medium, dull and stereotypedly dyes with Congo red dye liquor, selects the large and obvious bacterial strain of yellow transparent circle.
Described screening culture medium is (g/L): CMC10, yeast extract paste 4.5, (NH 4) 2sO 44.5, NaCl5.0, K 2hPO 42.0, MgSO 4.7H 2o0.4, agar15.
Described Congo red dye liquor staining procedure is: after bacterium colony picking, utilize 1% Congo red dye liquor dyeing 10min, outwell staining fluid and use 1M NaCl rinse three times.
The invention also discloses above-mentioned bacterial strains green ochre mould Penicilium ochrochloron produces viscosity reduction enzyme and is applied to the pre-treatment of tubers alcohol fuel fermentation viscosity reduction purposes for fermenting;
Particularly, preferably fresh sweet potato of described tubers.
The invention also discloses the fermention medium that produces viscosity reduction enzyme with above-mentioned green ochre mould Penicillium ochrochloron;
Particularly, described fermention medium is (g/L): corn cob 20, KH 2pO 42.0, NH 4nO 34.5, MgSO 40.15, CaCl 20.3, FeSO 4.7H 2o0.0005, ZnSO 4.7H 2o0.0014, MnSO 4.H 2o0.0016, COCl 2.6H 2o0.0016, Tween803.3, pH6.5.
The invention also discloses the fermentation condition that green ochre mould Penicillium ochrochloron produces viscosity reduction enzyme;
Particularly, described fermentation condition is 30 DEG C, 130rpm, 5 days.
The invention also discloses the viscosity reducing process that produces viscosity reduction enzyme with above-mentioned green ochre mould Penicillium ochrochloron;
Particularly, described viscosity reducing process comprises:
(1). making beating: sweet potato stem tuber raw material is cleaned, making beating.
(2). liquefaction: in sweet potato slurry, add α-amylase according to 90～150KNU/kg starch, 80～90 DEG C liquefy to Iod R be reddish-brown, add tap water by material-water ratio 3:1 after being cooled to room temperature.
(3). pyroprocessing: after liquefaction, sweet potato slurry is acted on to 20min under 115 DEG C of high temperature.
(4). pre-treatment: when temperature is reduced to 30～50 DEG C, add 40mL viscosity reduction enzyme in per kilogram sweet potato slurry, 50 DEG C of effect 2h.
Described α-amylase standard enzyme vigor is 90KNU/g, and enzyme work is defined as at 37 DEG C, and when pH5.6, the enzyme amount of 5.26 grams of starch of hydrolysis per hour is 1 KNU.
After described viscosity reduction enzyme is fermented by green ochre mould Penicillium ochrochloron, through centrifugal, supernatant liquor filters and obtains through 0.22 μ m strainer again.
Described viscosimetric analysis instrument is the digital rotational viscosimeter of RDV-2+PRO type that Shanghai Nirun Intelligent Technology Co., Ltd. produces.
The present invention has obtained the bacterial strain green ochre mould Penicillium ochrochloron of a strain product viscosity reduction enzyme by screening, this bacterial strain utilizes agricultural wastes corn cob for main raw material fermentation generation viscosity reduction enzyme, agricultural wastes are effectively utilized, with low cost, this viscosity reduction enzyme is applied to taking non-grain sweet potato stem tuber as raw material production alcohol fuel in fermentation viscosity reduction pretreatment technology, viscosity reducing effect is obvious, and the one-tenth that greatly reduces alcohol fuel produces cost.
Embodiment
With specific embodiment, technical scheme of the present invention is described below; but be not the restriction to technical scheme of the present invention; those skilled in the art should understand that; still can modify or be equal to replacement invention; and not departing from any modification or partial replacement of the spirit and scope of the present invention, it all should be encompassed among protection scope of the present invention.
Embodiment 1
The screening and culturing basal growth that green ochre mould Penicillium ochrochloron can be sole carbon source at Xylo-Mucine (CMC), cultivates after 3 days for 30 DEG C, and Congo red dye liquor experiment shows larger yellow transparent circle.
Embodiment 2
Green ochre mould Penicillium ochrochloron is inoculated in without after the fermention medium of optimizing, 30 DEG C, 130rpm cultivated after 4 days, 50g liquefaction the slurry of the sweet potato after high temperature add 2mL to filter and obtain enzyme liquid through centrifugal and 0.22 μ m strainer, and after 50 DEG C of effect 2h, viscosity degradation rate is 75.78%.
Embodiment 3
Green ochre mould Penicillium ochrochloron is inoculated in after the fermention medium of 50mL through optimizing, 30 DEG C, 130rpm cultivated after 4 days, 50g liquefaction the slurry of the sweet potato after high temperature add 2mL to filter and obtain enzyme liquid through centrifugal and 0.22 μ m strainer, and after 50 DEG C of effect 2h, viscosity degradation rate is 84.10%.
Embodiment 4
Green ochre mould Penicillium ochrochloron is inoculated in after the fermention medium of 50mL through optimizing, 30 DEG C, 130rpm cultivated after 5 days, 50g liquefaction the slurry of the sweet potato after high temperature add 2mL to filter and obtain enzyme liquid through centrifugal and 0.22 μ m strainer, and after 50 DEG C of effect 2h, viscosity degradation rate is 89.09%.
Embodiment 5
Green ochre mould Penicillium ochrochloron is inoculated in after the fermention medium of 200mL through optimizing, 30 DEG C, 130rpm cultivated after 5 days, 100g liquefaction the slurry of the sweet potato after high temperature add 4mL to filter and obtain enzyme liquid through centrifugal and 0.22 μ m strainer, and after 50 DEG C of effect 2h, viscosity degradation rate is 84.96%.
Embodiment 6
Green ochre mould Penicillium ochrochloron is inoculated in after the fermention medium of 20L through optimizing, 30 DEG C, 130rpm cultivated after 5 days, and 150g liquefaction the slurry of the sweet potato after high temperature add 3L through centrifugal acquisition enzyme liquid, and after 50 DEG C of effect 2h, viscosity degradation rate is 89.95%.
Embodiment 7
Green ochre mould Penicillium ochrochloron is inoculated in after the fermention medium of 20L through optimizing, 30 DEG C, 130rpm cultivated after 5 days, and 300g liquefaction the slurry of the sweet potato after high temperature add 6L through centrifugal acquisition enzyme liquid, and after 50 DEG C of effect 2h, viscosity degradation rate is 91.26%.
Wherein described in embodiment 3-7, the described fermention medium through optimizing is (g/L): corn cob 20, KH 2pO 42.0, NH 4nO 34.5, MgSO 40.15, CaCl 20.3, FeSO 4.7H 2o0.0005, ZnSO 4.7H 2o0.0014, MnSO 4.H 2o0.0016, COCl 2.6H 2o0.0016, Tween803.3, pH6.5.

Claims (3)

1. viscosity reduction enzyme is a bacterial strain CMC-37, it is characterized in that, its Classification And Nomenclature be green ochre mould ( penicillium ochrochloron), being preserved in Dutch microbial strains preservation center, preserving number is: CBS131817.
2. viscosity reduction enzyme claimed in claim 1 is bacterial strain CMC-37 produces viscosity reduction enzyme purposes for fermenting.
3. be the method that bacterial strain CMC-37 produces viscosity reduction enzyme viscosity reduction with viscosity reduction enzyme claimed in claim 1, it is characterized in that described method comprises:
(1) making beating: sweet potato stem tuber raw material is cleaned, making beating;
(2) liquefaction: in sweet potato slurry, add α-amylase according to 90～150KNU/kg starch, 80～90 DEG C liquefy to Iod R be reddish-brown, add tap water by required material-water ratio after being cooled to room temperature; Enzyme work is defined as at 37 DEG C, and when pH5.6, the enzyme amount of 5.26 grams of starch of hydrolysis per hour is 1 KNU;
(3) pyroprocessing: after liquefaction, sweet potato slurry is acted on to 20min under 115 DEG C of high temperature;
(4) pre-treatment: when temperature is reduced to 30～50 DEG C, add 40mL viscosity reduction enzyme in per kilogram sweet potato slurry, 50 DEG C of effect 2h;
Wherein, viscosity reduction enzyme be viscosity reduction enzyme claimed in claim 1 be after bacterial strain CMC-37 fermentation through centrifugal, supernatant liquor filters and obtains through 0.22 μ m strainer again;
Described viscosity reduction enzyme is that the fermention medium of bacterial strain CMC-37 fermentation is g/L: corn cob 20, KH 2pO 42.0, NH 4nO 34.5, MgSO 40.15, CaCl 20.3, FeSO 47H 2o 0.0005, ZnSO 47H 2o 0.0014, MnSO 4h 2o 0.0016, CoCl 26H 2o 0.0016, Tween80 3.3, pH6.5;
Described viscosity reduction enzyme is that the fermentation condition of bacterial strain CMC-37 fermentation is 30 DEG C, 130rpm, 5 days.
CN201210234738.5A 2012-07-09 2012-07-09 High-yield viscosity-reduction enzyme system strain and use thereof Active CN103146583B (en)

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CN103525709B (en) * 2013-09-27 2015-09-09 中国科学院成都生物研究所 A kind of common Fusarium, the method preparing viscosity-reduction enzyme and application thereof
CN103614299B (en) * 2013-10-20 2015-09-09 中国科学院成都生物研究所 A kind of volume branch Mucor, the method preparing viscosity-reduction enzyme and application thereof

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CN101717728B (en) * 2009-12-28 2013-04-10 华东理工大学 Penicillium and application thereof in catalyzing and hydrolyzing lignocellulose
CN102174411B (en) * 2010-12-06 2013-01-23 中国农业科学院农业资源与农业区划研究所 Penicillium ochrochloron Y5 and microbial inoculum thereof for degrading straw
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