CN1958789B - Method for producing laccase through co-culture ferment for fungus - Google Patents

Abstract

This invention discloses a method for producing laccase by fungi co-culture. The method comprises: inoculating the suspension of Trametes sp. AH28-2 into a fermentation culture medium, culturing at 22-37 deg.C for 1-4 days, adding the suspension of Trichoderma sp. ZH1, culturing for 5-10 days, and separating to obtain laccase agent with living laccase content 6,000 U/L. The culture medium is liquid culture medium, and mainly comprises certain amounts of carbon source, nitrogen source, phosphates of potassium and sodium, sulfates of copper and iron, adenine and vitamin B. The method avoids aromatic compounds and heavy metal ions to induce, thus is safe and environmentally friendly, and has high and stable laccase activity.

Description

A kind of method of fermentative production of laccase using fungoid co-cultivated

One, technical field

The present invention relates to a kind of preparation method of industrial enzymes, particularly the preparation method of industrial laccase exactly is a kind of method of fermentative production of laccase using fungoid co-cultivated.

Two, background technology

Laccase (Laccase ECl.10.3.2) is the copper bearing polyphenoloxidase of a class, can the multiple phenols of catalysis and the oxidation of non-phenolic compound, and simultaneous hydrogen reduction Cheng Shui.Laccase extensively is present in higher plant and the higher fungi (particularly basidiomycetes).Laccase effect substrate is extensive, catalytic efficiency is high, has potential significant application value [Hong Yuzhi in fields such as the development of the softening refinement of the degraded of the improvement of the improvement of bio-pulping and bleaching, flavour of food products, feed nutrition, textile dyestuff and conversion, textile fibres, newtype drug exploitation, new bio transmitter and new exploitations of energy resources, Xiao Yazhong, Fang Wei etc. (2005) Trametes sp.AH28-2 laccase A induce synthetic and gene 5 '-clone and the analysis of end control region. biotechnology journal 21:547-552; Xiao YZ, Hong YZ, Li JF et al (2006) Cloning of novel laccase isozyme genes from Trametes sp.AH28-2 andanalyses of their differential expression.Appl Microbiol Biotechnol DOI 10.1007/s00253-005-0188-2], become one of focus of international enzyme technology and the research of environmental science crossing domain in recent years.

At present, fungal laccase adopts artificial synthetic medium's liquid submerged fermentation to be prepared mostly, and this process need interpolation aromatic compounds and/or heavy metal ion have increased cost as inductor; And the introducing of toxicity inductor makes that the fermented liquid post-processing difficulty is big, easily causes environmental pollution, is difficult to carry out large-scale industrial production.

Microorganism is cultivated altogether and is meant beneficial microorganism is added in the substratum, and with the growth that promotes another kind of microorganism strains or improve its metabolic activity, this method is used in a plurality of fields.Utilization is based on the common culture method fermentative production of laccase of the mutual control techniques of microorganism, compares with traditional production method to have two aspect advantages: (1) is with low cost: need not expensive inductor, the fermented liquid aftertreatment is simple; (2) safely cleaning: avoided the use of toxicity fragrant substance and heavy metal ion.

Tentatively carried out the correlative study of common culture method fermentative production of laccase in the world: 1997, German scholar Lang has carried out report the earliest to this, they cultivate in soil with Pleurotus bacterium (pleurotus) etc., the enzyme activity that produces in two months is lower than 7U/g (ABTS method) [Lang E, Eller G and Zadrazil F (1997) Lignocellulose decomposition andproduction of ligninolytic enzymes during interaction of white rot fungi with soil microorganisms.MicrobialEcology 34:1-10]; Calendar year 2001 Denmark scholar Crowe report Pseudomonas fluorescens (Pseudomonas, fluorescens) can induce dry thread Pyrenomycetes (Rhizoctonia solani) to produce a small amount of laccase [Crowe JD and Olsson S (2001) Induction oflaccase activity in Rhizoctonia solani by antagonistic Pseudomonas, fluorescens strains and a range of chemicaltreatments.Appl Environ Microbiol 67:2088-2094]; The same year, France scholar Savoie has studied wooden mould (Trichoderma) to the rotten basidiomycetes of 7 strain leaves, the rotten basidiomycetes laccase synthetic influence of 7 strains wood, enzyme activity all has raising in various degree, and wherein the vigor of mushroom (Lentinula edodes) laccase is brought up to 326.6 μ mol min by 262.6 ^-1L ^-1(ABTS method) [Savoie JM, Mata G and Mamoun M (2001) Variability in brown line formation and extracellular laccase production duringinteraction between white-rot basidiomycetes and Trichoderma harzianum biotype Th2.Mycologia93:243-248]; 2002, laccase is produced bacterium Cerrena maxima to the scholar Koroleva of Russia etc. and manganese peroxidase generation bacterium Cerrena hirsutus cultivates altogether, the laccase vigor increases [Koroleva OV to some extent, Gavrilova VP, Stepanova EV et al (2002) Production of lignin modifying enzymes by co-cultivated white-rot fungi Cerrena maxima andCoriolus hirsutus and characterization of laccase from Cerrena maxima.Enzyme Microb Tech 30:573-580]; Israel scholar Baldrian report in 2004, wood mould (Trichoderma sp.) is cultivated the enzyme work that produces altogether with rainbow conk (Trametes versicolor) and has been improved 40 times than blank, reaches 44.2U/L[Baldrian P (2004) Increase of laccase activityduring interspecific interactions of white-rot fungi.FEMS Microbiol Ecology 50:245-253]; Mexican Mata report in 2005, with the coffee cherry is that matrix is cultivated flat mushroom (Pleurotus spp) and wood mould (Trichoderma spp) altogether, enzyme work rises to l80U/g[Mata G from 150U/g, Hern á ndez D and Andreu L (2005) Changes in lignocellulolyticenzyme activites in six Pleurotus spp strains cultivated on coffee pulp in confrontation with Trichoderma spp.World J Microb Biot21:143-150].

At home, only there is one piece about cultivating the report that influences the laccase expression amount altogether, be that flat mushroom (Pleurotus sp.) carries out solid state fermentation with glossy ganoderma (Ganoderma sp.) mixed culture, enzyme work reaches 2,165U/g (ABTS method), than the vigor summation of single culture improved nearly one times [Huang Huiyan, Zhang Xiaoyu (2004) whiterot fungi mixed culture is the kind reciprocity in the laccase secretion outside born of the same parents. edible fungi of china 23:31-33].

These study explanation, can improve the expression level of laccase by the mutual control techniques that is total to culturing micro-organisms.Yet the output of laccase is all not high in these researchs, is difficult to reach the suitability for industrialized production requirement.On the other hand, related microorganism is cultivated the production technique of producing fungal laccase altogether and is not seen that patent protection is arranged.

Three, summary of the invention

Bolt bacterium AH28-2 (Trametes sp.AH28-2) and wooden mould ZH1 (Trichoderma sp.ZH1) that two kinds of fungal bacterial strains that the present invention uses in being total to cultivation and fermentation are the autonomous seed selection of applicant, wherein the former is the novel laccase enzyme superior strain, and the latter is the microbiological manipulation bacterium.With these two bacterial strains is the fermentative production that material carries out laccase.

This production method comprises that substratum is prepared, kind liquid prepares, sterilizes, inoculates, is total to cultivation and fermentation and enzyme purification, described common cultivation and fermentation is exactly that first bacteria suspension (kind liquid) with bolt bacterium AH28-2 is seeded in the substratum in 22～37 ℃ of cultivations 1～4 day, the bacteria suspension (kind liquid) that adds wooden mould ZH1 then continues to cultivate 5～10 days in 22～37 ℃, separates obtaining the industrial finish zymin at last.If be applied to food, beverage or field of biosensors, then laccase albumen need be further purified.

Described substratum is a liquid nutrient medium, and every liter contains: 10～30g carbon source, 1.0～4.0g nitrogenous source, 0.5～1.5gKH ₂PO ₄, 0.4～0.6g MgSO ₄7H ₂O, 0.05～0.15g Na ₂HPO ₄5H ₂O, 0.005～0.015g CaCl ₂, 0.001～0.003gCuSO ₄5H ₂O, 0.0005～0.0015g FeSO ₄7H ₂O, 0.01～0.04g VITAMIN B4,0.03～0.07g vitamins B, pH4.0～8.0.

Preferred substratum contains for every liter: 20g carbon source, 2.5g nitrogenous source, 1.0g KH ₂PO ₄, 0.5g MgSO ₄7H ₂O, 0.1gNa ₂HPO ₄5H ₂O, 0.01g CaCl ₂, 0.002g CuSO ₄5H ₂O, 0.001g FeSO ₄7H ₂O, 0.0275g VITAMIN B4,0.05g vitamins B, pH4.5～6.5.

Described carbon source is selected from one or more mixed carbon sources of wood sugar, glucose, seminose, semi-lactosi, trehalose, cellobiose, maltose, sucrose, fructose and glycerine, and promptly every liter of substratum contains total carbon source amount 10～30g.Preferred wood sugar.

Described nitrogenous source is selected from Tryptones, peptone, asparagine, glutamine, L-glutamic acid, glycine, L-Ala, phenylalanine, KNO ₃, NH ₄O ₃In one or more mixed nitrogens, promptly every liter of substratum contains total nitrogenous source amount 1.0～4.0g.Preferred Tryptones.

The preparation of described kind of liquid is exactly that bolt bacterium AH28-2 and wooden mould ZH1 are seeded in separately in the above-mentioned substratum in 28 ℃, 100～200rpm and cultivated 1～3 day, and the about 10s of 3000rpm homogenate makes bacteria suspension.

The present invention need not inducing of aromatic compound and heavy metal ion in the laccase production process, the fermented liquid aftertreatment is simple, thereby production technique safety and environmental protection, with low cost.The present invention is fermented the laccase vigor that obtains 6,000 U/L above (guaiacol method), with enzyme quite [the Xiao YZ alive that before efficiently induced generation with aromatic compounds such as Ortho Toluidines, TuXM, WangJ et al (2003) Purification, molecular characterization and reactivity with aromatic compounds of a laccasefrom a basidiomycete Trametes sp.strain AH28-2.Appl Microbiol Biotech 60:700-707].Compare with the chemical induction method, the present invention's laccase vigor that generates that ferments is stable.

The bolt bacterium AH28-2 (Trametes sp.AH28-2) of the autonomous seed selection of applicant send Chinese typical culture collection center preservation on November 22nd, 2005, and preserving number is CCTCCNO:M205134; The mould ZH1 of wood (Trichoderma sp.ZH1) send Chinese typical culture collection center preservation on January 16th, 2006, and preserving number is CCTCCNO:M206014.

Four, embodiment

1, the preparation of bolt bacterium AH28-2 and wooden mould ZH1 bacteria suspension (kind liquid)

At potato substratum (1L substratum: 20% murphy juice, 20g glucose, 3g KH ₂PO ₄, 1.5g MgSO ₄7H ₂O, 0.4g vitamins B add 1.5% agar and make solid medium) the bolt bacterium AH28-2 and the wooden mould ZH1 of 4 ℃ of preservations is inoculated in the kind liquid culture medium of high-temperature sterilization (1L substratum: 15g glucose, 2.5g l-asparagine, 1.0g KH respectively on the inclined-plane ₂PO ₄, 0.5g MgSO ₄7H ₂O, 0.1g Na ₂HPO ₄5H ₂O, 0.01g CaCl ₂, 0.002g CuSO ₄5H ₂O, 0.001gFeSO ₄7H ₂O, 0.0275g VITAMIN B4 and 0.05g vitamins B, natural pH), 28 ℃, 100～200 rpm were cultivated 1～3 day, and 3, the about 10s of 000rpm homogenate makes bacteria suspension.

2, the preparation of cultivation and fermentation substratum altogether

Enzymatic production substratum (1L): 10～30g carbon source, 1.0～4.0g nitrogenous source, 1.0g KH ₂PO ₄, 0.5g MgSO ₄7H ₂O, 0.1g Na ₂HPO ₄5H ₂O, 0.01g CaCl ₂, 0.002g CuSO ₄5H ₂O, 0.001g FeSO ₄7H ₂O, 0.0275g VITAMIN B4 and 0.05g vitamins B, pH4.0～8.0.

3, inoculation and fermentation culture

The bolt bacterium AH28-2 bacteria suspension of preparation is inoculated in the sterilized fermention medium by 1～20% mass fraction, 100～200rpm, 22～37 ℃ cultivated 1～4 day, the bacteria suspension that adds wooden mould ZH1 then by 0.1～10% mass fraction continues to cultivate 5～10 days to producing the enzyme peak.

4, enzyme isolation and purification

Fermentation reaches the laccase peak period, and fermented liquid is separating thallus after filtration, promptly obtains the laccase crude preparation by using.Filtrate is ultrafiltration and concentration or thin up (can be used for association with pulp bleaching, industrial dye decolouring, wastewater treatment, fiber softening, feed processing and other fields) as required.

Crude zyme preparation can obtain pure enzyme by following step successively, and all operations carries out at 4～10 ℃: 1. 6, and the centrifugal 10min of 000rpm, 10～50 times of supernatant liquor ultrafiltration and concentration; 2. supernatant liquor is at 10mmol/L citric acid-Na ₂HPO ₄Damping fluid (pH8.0) dialysis 3～6 hours, then 10, the centrifugal 10min of 000rpm; 3. use dialyzate equilibrated DEAE-Sepharose FF ion exchange column on the supernatant liquor in advance, and carry out linear elution with the ammonium sulfate of 0.3mol/L, dilute with aqua sterilisa (or deionized water) after the active ingredient desalination, can obtain pure zymin (pure zymin can also be used for food and drink industry, biosensor etc. except the Application Areas that can be used for above-mentioned thick enzyme).

5, concrete operations example

(1), distinguishes picking 1cm from the preservation inclined-plane of bolt bacterium AH28-2 and wooden mould ZH1 ²The mycelia piece, be inoculated into separately in the 300mL triangular flask that 100mL kind liquid culture medium is housed, 150rpm, 28 ℃ cultivated respectively 3 days and 1.5 days, 3,000rpm homogenate 10s makes bacteria suspension (facing with existing system).

(2), dress 350mL fermention medium (1L:15g wood sugar, 2.5g Tryptones, 1.0g KH in the 1L triangular flask ₂PO ₄, 0.5g MgSO ₄7H ₂O, 0.1g Na ₂HPO ₄5H ₂O, 0.01g CaCl ₂, 0.002g CuSO ₄5H ₂O, 0.001gFeSO ₄7H ₂O, 0.0275g VITAMIN B4 and 0.05g vitamins B, pH5.0～6.0), inoculation 10mL bolt bacterium AH28-2 bacteria suspension behind the high-temperature sterilization, 130rpm, 28 ℃ of cultivations added wooden mould ZH1 bacteria suspension 2.1mL after 2.5 days, continued to cultivate 7 days.

(3), fermented liquid removes thalline with eight layers of filtered through gauze, filtrate promptly is thick laccase preparation, enzyme work is 6, more than the 000U/L (guaiacol method).So that store, or thin up is directly used in association with pulp bleaching, dye decolored, sewage disposal, fiber softening, feed processing, Alcohol Production etc. to filtrate through the ultrafiltration and concentration freeze-drying.

(4), if be used for food, beverage or biosensor, then be further purified.Handle by the purification step described in " 4, enzyme isolation and purification ", obtain pure laccase preparation.

Claims (4)

1. the method for a fermentative production of laccase using fungoid co-cultivated, comprise that substratum is prepared, kind liquid prepares, sterilizes, inoculates, is total to cultivation and fermentation and enzyme purification, it is characterized in that: described cultivation and fermentation altogether is exactly earlier the bacterial suspension inoculation of bolt bacterium AH28-2 to be cultivated 1～4 day in 22～37 ℃ in the substratum of sterilization, the bacteria suspension that adds wooden mould ZH1 then continues to cultivate 5～10 days in 22～37 ℃, and every liter of substratum contains 10～30g carbon source, 1.0～4.0g nitrogenous source, 0.5～1.5g KH ₂PO ₄, 0.4～0.6g MgSO ₄7H ₂O, 0.05～0.15g Na ₂HPO ₄5H ₂O, 0.005～0.015g CaCl ₂, 0.001～0.003gCuSO ₄5H ₂O, 0.0005～0.0015g FeSO ₄7H ₂O, 0.01～0.04g VITAMIN B4,0.03～0.07g vitamins B, pH4.0～8.0;

Described carbon source is selected from one or more mixed carbon sources of wood sugar, glucose, seminose, semi-lactosi, trehalose, cellobiose, maltose, sucrose, fructose and glycerine;

Described nitrogenous source is selected from peptone, asparagine, glutamine, L-glutamic acid, glycine, L-Ala, phenylalanine, KNO ₃, NH ₄NO ₃In one or more mixed nitrogens.

2. production method according to claim 1 is characterized in that: described every liter of substratum contains 20g carbon source, 2.5g nitrogenous source, 1.0g KH ₂PO ₄, 0.5g MgSO ₄7H ₂O, 0.1g Na ₂HPO ₄5H ₂O, 0.01g CaCl ₂, 0.002gCuSO ₄5H ₂O, 0.001g FeSO ₄7H ₂O, 0.0275g VITAMIN B4,0.05g vitamins B, pH4.5～7.5.

3. production method according to claim 1 and 2 is characterized in that: described carbon source is a wood sugar.

4. production method according to claim 1 and 2 is characterized in that: described nitrogenous source is a Tryptones.