CN1443841A - Lipasegenous bactenia, its screening method and industrial application - Google Patents
Lipasegenous bactenia, its screening method and industrial application Download PDFInfo
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Abstract
一种脂肪酶产生菌及其筛选方法和产业化应用，属于食品行业中有机相中芳香酯合成用脂肪酶产生菌技术领域。该菌株是从中国大曲中分离得到的一种华根霉，命名为Rhizopus chinensis CCTCC M201021。其筛选方法是取曲样或酒醅。经过稀释涂布平板，单菌落移接斜面培养基，摇瓶培养，离心，上清液注初筛平板孔，挑取透明圈和/或荧光圈较大的菌进行摇瓶发酵复筛，再复筛。菌体冷冻干燥制得全细胞华根霉脂肪酶酶制剂。制得的脂肪酶酶制剂用于有机相中芳香酯的转化合成。
The invention relates to a lipase-producing bacterium, its screening method and industrial application, and belongs to the technical field of lipase-producing bacteria for synthesis of aromatic esters in organic phases in the food industry. The strain is a Rhizopus sinensis isolated from Daqu in China, named Rhizopus chinensis CCTCC M201021. The screening method is to take koji samples or fermented grains. After dilution and coating on the plate, single colonies were transferred to the slant medium, cultured in shake flasks, centrifuged, the supernatant was poured into the wells of the primary screening plate, and the bacteria with larger transparent circles and/or fluorescent circles were picked for re-screening in shake flask fermentation. Double screen. Whole-cell Rhizopus chinensis lipase enzyme preparation was obtained by freeze-drying the bacteria. The prepared lipase enzyme preparation is used for conversion synthesis of aromatic ester in organic phase.
Description
Technical field
The present invention relates to a kind of lipase and produce bacterium and screening method and commercial application, belong to synthetic aroma ester technical field in the food service industry lipase organic phase.
Background technology
Aromatic ester is the short-chain fatty acid ester, is natural fruit fragrance, is the important component of essence, spices, is widely used in brewageing, in the daily living article such as food, beverage, medicine and makeup.In China, only drink industry just reaches about three kilotons with the ethyl hexanoate annual requirement.The production of present global aromatic ester is the extraction, to be by traditional chemical synthesis production all from natural phant except that the only a few ester, and the purity of product and security are difficult to guarantee that some is returned environment and brings many negative impacts.Although at present the chemosynthesis fatty acid ester is also both economical, along with people to the pursuit of green food and the favor of natural product, directly from plant, extract and can't satisfy growing demand again, people begin sight is invested the method for bio-transformation and produce.
Daqu is the saccharifying agent that China's brewing wine is used, and starter also is simultaneously to give birth to pastil, is the source of Daqu distiller's yeast perfume (or spice), wherein contains a large amount of microorganisms, particularly is rich in the microorganism that produces ester perfume (or spice).Most of aromatic esters are the important flavour substancess in the liquor, therefore might contain synthetic these aromatic ester synthetic lipase generation bacterium of catalysis in organic solvent efficiently and stably in the Daqu.
Summary of the invention
(1) technical problem that will solve
The purpose of this invention is to provide a kind of lipase and produce bacterium and screening method and commercial application, be used for lipase organic phase synthetic aroma ester.
(2) technical scheme
From Chinese Daqu, separate and obtain a strain zhizopchin.This bacterial strain pure growth has been preserved in Wuhan China typical culture collection center (called after Rhizopus chinensis CCTCCM201021) June 24 calendar year 2001.Its on morphological specificity more near zhizopchin, but on physiological and biochemical property, relatively go back some difference with selected zhizopchin (Rhizopus chinensis Saito AS3.1166) as standard, classification of fungi system according to Arnsworth, belong to Zygomycotina, zygomycetes, mucorales, Mucoraceae, Rhizopus, zhizopchin kind (Rhizopus chinensis);
The screening of Rhizopus chinensis CCTCC M201021 bacterial strain
Earlier adopting short-chain aliphatic ester and/or long chain fatty acid ester is substrate, is the primary dcreening operation index with the ability of microbiological degradation short-chain aliphatic ester and/or long chain fatty acid ester; Serves as to sieve index for the first time again with microorganism to the tolerance performance of organic solvent; Again with the ability of microorganism Acrawax in organic phase for sieving index more again; Make up above three kinds of screening methods from bent sample of the traditional Daqu of China or wine unstrained spirits, through the dilution spread plate, single bacterium colony moves and connects slant medium, shake-flask culture, centrifugal, supernatant liquor is annotated the primary dcreening operation plate well, the bigger bacterial strain of picking transparent circle or fluorescent ring adds and contains in 1～10% heptane solvent environment shake-flask culture and measure the solvent environment lipase activity and carry out multiple sieve the first time, centrifugal with passing through behind the microorganism shake-flask culture that multiple sieve obtains for the first time, it is index sieve again again that the thalline lyophilize makes after the zymin with organic synthesis fatty acid ester ability, and the thalline lyophilize makes full cellular enzymes preparation.
Get bent sample of 0.5～5 gram Daqu or wine unstrained spirits and be dissolved in 15～150ml stroke-physiological saline solution shaking table concussion 30～150 minutes, redilution, single bacterium colony moves and connects slant medium behind the spread plate, 25～35 ℃ of constant temperature culture 2～5 days, and single bacterium colony moves and connects the inclined-plane, after treating the slant pore maturation, 25～35 ℃, 150rpm/m, shake-flask culture 2～5 days, relatively enzyme is alive to carry out primary dcreening operation, and the bacterium that picking transparent circle and/or fluorescent ring are bigger shakes the multiple sieve of bottle; Selected have a bacterial strain that higher lipase enzyme is lived, and add and contain in the substratum of 1～10% heptane, 25～35 ℃, 150rpm/m, shake-flask culture 2～5 days is measured enzyme and is lived, and carries out multiple sieve ratio than the tolerance of microorganism to solvent environment; Screening obtains the bacterial strain of higher vigor, 25～35 ℃, 100～300rpm/m, shake-flask culture 2～5 days, nutrient solution is under 6000rpm/m after centrifugal 5 minutes, supernatant liquor and thalline respectively lyophilize to make after the zymin with organic synthesis fatty acid ester ability be index sieve again again, the thalline lyophilize makes full cellular enzymes preparation;
Culture condition is: inoculum size is 0.1～5 * 10
6Individual/mL, product enzyme culture medium condition is: soybean cake powder 3～10%, peptone 3～10%, glucose 0.2～5%, K
2HPO
40.1～3%, MgSO
4.7H
2O0.01～0.5%, CaCL
20.001～0.05%, pH6～9,50～300rpm, shake-flask culture 48～120h, enzyme work can reach 400u/g.
Primary dcreening operation is enzyme two kinds of methods alive relatively:
Tributyrin emulsion transparent circle method (is substrate with the short-chain aliphatic ester):
Contain the emulsion of 100g/L tributyrin with 3.0% P.V.A formulations prepared from solutions, add the agar powder of 25mL tributyrin emulsion and 1.5% in the 225mL phosphoric acid buffer (pH7.5), 0.1MPa, sterilization 20min.Hot solution 20mL above pouring in aseptic plate gets the aperture of diameter 0.5cm after solidifying, inject 30uL enzyme liquid, surveys the transparent circle size behind 34 ℃ of insulation 24h, the height of living with preliminary relatively esterase enzyme.
Sweet oil fluorescent ring method (is substrate with the long chain fatty acid ester):
P.V.A formulations prepared from solutions with 3.0% contains 250mL/L sweet oil emulsion, the agar powder that adds 25mL sweet oil emulsion and 1.5% in the 225mL phosphoric acid buffer (pH7.5), 0.1MPa sterilization 20min, be cooled to the rhodamine B of the filtration sterilization of adding 0.001% after 60 ℃, in aseptic plate, pour this solution 20mL into, get the aperture of diameter 0.5cm after solidifying, inject 30uL enzyme liquid, 34 ℃ of insulation 24h survey the fluorescent ring size under natural light, to compare the lipase activity height.
The lipase enzyme activity determination:
Get two of 100mL triangular flasks, add emulsion (3%PVA: the phosphoric acid buffer 5mL of 4mL and 0.02mol/L, pH7.5 sweet oil=3: 1) respectively, put preheating 5～10min in 40 ℃ of water-baths, add 1mL enzyme liquid therein in one bottle earlier, timing immediately, reaction adds 95% ethanol 15mL termination reaction behind the 15min immediately.The blank ethanol that adds earlier, all the other are the same.Phenolphthalein is made indicator, with the titration of 0.05mol/LNaOH solution to terminal, calculates the poor of the volume that consumed.Under 40 ℃, the condition of pH7.5, the enzyme amount that lipase hydrolysis fat per minute produces 1umol lipid acid is defined as 1 enzyme unit that lives.
The mensuration of fatty acid ester synthesis capability in the lipase organic phase:
Fermented supernatant fluid and/or thalline lyophilize, get lyophilize enzyme powder some (enzyme work is more than 300u), adding through the caproic acid of the 0.4mol/L of molecular sieve dehydration and ethanol in the 10mL heptane, in 150mL band screen mill mouth triangular flask, 150r/m, 38 ℃ of down reactions 1.5 days, the reaction solution of getting after the filtration carries out gas chromatographic analysis, with the ability of reflection lipase Acrawax.
The primary dcreening operation of microorganism
From the Daqu of five-Grain Liquor, Shahe, gladiolus spring, Yanghe River brewery and wine unstrained spirits, obtain 730 multi-strain bacterias altogether, comprise bacterium, yeast, actinomycetes and mould through plate isolation.Wherein, bacterium 17 strains, yeast 20 strains, actinomycetes 5 strains, other is mould.Find that through primary dcreening operation most moulds all produce certain lipase.Find that simultaneously there are inconsistent situation in fluorescent ring and transparent circle, show that lipase that these bacterium produce there are differences substrate-function, perhaps the enzyme that produces is the mix products of esterase and lipase.Get transparent circle and shake the multiple sieve of bottle greater than the bacterium of 0.9cm greater than 1.2cm and/or fluorescent ring.
Sieve the first time of microorganism again
Obtain the bigger bacterial strain of 56 strain transparent circle/fluorescent rings through primary dcreening operation, add after the culture media shaking vase contain 2% heptane cultivates, measure the size of its transparent circle and/or fluorescent ring two Artenkreis respectively and and survey enzyme with volumetry and live.To observe the adaptive faculty of microorganism strains, filter out organic solvent-resistant and have the bacterial strain of higher lipase activity solvent environment.The bacterial strain that screening obtains the higher vigor of 5 strains sieves again again.
Sieve again again
According to sieving for the first time the result again, select transparent circle wherein and/or fluorescent ring and the enzyme all higher strain fermentation of living and to measure the ability of its synthesizing ethyl hexanoate in organic solvents such as heptane after the thalline lyophilize after centrifugal, obtain two strains have higher ester synthesis of dynamic in multiple sieve again bacterial strain.Wherein not only to produce enzyme the highest for Rhizopuschinensis CCTCC M201021, and synthesis capability is also very strong.Simultaneously as seen, for different bacterial strains, there is no dependency between work of titration enzyme and the esterification yield.Therefore, when primary dcreening operation is parameter with lytic enzyme work, leak sieve, should select more dull and stereotyped sieve as far as possible for reducing.Higher because of Rhizopuschinensis CCTCC M201021 esterification yield, enzyme is lived also higher, and in organic synthesis, the transformation efficiency of ethyl hexanoate is also higher.
The preliminary evaluation of Rhizopus chinensis CCTCC M201021
The genus of identifying bacterial classification is other, understands its basic physio-biochemical characteristics, to effectively utilizing existing document, instructing further experiment that vital role is arranged.For this reason, method according to document has been carried out the evaluation of form and physio-biochemical characteristics to Rhizopus chinensisCCTCC M201021, and compares with reference culture zhizopchin (Rhizopus chinensis Saito AS3.1166), rhizopus arrhizus (Rhizopus arrhizus Fisher AS3.2893).
Morphological specificity:
Rhizopus chinensis CCTCC M201021 is inoculated in the PDA substratum, 28 ℃ of cultivations, at first the velvet-like mycelia of hairiness grows, then to around spread, grow that the flora height reaches about 0.5cm when vigorous, be hair-like.The mycelia initial stage is a white, becomes grey black to black after aging, and has a large amount of black spores to produce.Its colonial morphology is seen Fig. 1.
Physiological and biochemical property relatively
Slide glass is cultivated and be can be observed Rhizopus chinensis CCTCC M201021 mycelia under the opticmicroscope and be and crawl dendriticly, and barrier film is arranged, and the packing handle is upright, no branch, rhizoid is the brachydactylia shape, but extremely not obvious, sporocyst sphere or almost spherical, it is spherical that black, spore mostly are, and elliposoidal is also arranged, dark-coloured striped is arranged, but spore can an end the also multiterminal growth of sprouting, sexual spore is a zygospore, sees Fig. 2.
Approaching on reference standard bacterial strain zhizopchin and the Rhizopus chinensis CCTCC M201021 form, but poor growth is a little, and bacterium colony will loosen; The rhizopus arrhizus growth is more slower, becomes grey after mycelia is aging, and produces grey spore, no rhizoid.
Rhizopus chinensis CCTCC M201021 on morphological specificity more near zhizopchin, but on physiological and biochemical property, relatively also has significant difference with selected zhizopchin as standard, as Rhizopuschinensis CCTCC M201021 in the experimental temperature scope 41 ℃ the growth, do not grow for 45 ℃, and the standard zhizopchin is at 45 ℃ of well-growns still; Rhizopus chinensis CCTCC M201021 reaches at 8.1 o'clock at environment pH and does not grow, and standard zhizopchin well-grown still when pH8.5.This may be the different generations of bacterium source.Comprehensive above feature according to the classification of fungi system of Ainsworth, belongs to Zygomycotina, zygomycetes, mucorales, Mucoraceae, Rhizopus, the zhizopchin kind obtains because of this strain bacterium separates from the Yang He Da Qu, and is special with its called after Rhizopus chinensis CCTCC M201021.
Lipase produces the application of bacterium: the full cellular enzymes preparation that makes is used for the conversion of organic phase aromatic ester on the 2500L scale and synthesizes; Lipid acid is the saturated monoprotic acid of 3～18C, and the oleic acid of 18C, Fatty Alcohol(C12-C14 and C12-C18) are the primary alconol of 2～18C, the secondary alcohol of 4～5C, and under the concentration of substrate of 0.1～2.0mol/L, stirring reaction 24～120 hours, transformation efficiency reaches more than 96%, and half life of enzyme is more than 1000 hours.
3) beneficial effect
The Rhizopus chinensis CCTCC M201021 zymin that fermented liquid obtains through lyophilize is used for the conversion of ethyl hexanoate and synthesizes, and esterification yield is higher, reaches more than 96%, and enzyme is lived also higher, near 21U/ml.Compare with free enzyme or immobilized enzyme, full cellular enzymes provides " microenvironment " of catalyzed reaction in organic solvent, keeps the katalysis of efficient stable.
Description of drawings
Fig. 1: Rhizopus chinensis CCTCC M201021 mycelia form Photomicrograph
Fig. 2: Rhizopus chinensis CCTCC M201021 spore shape electromicroscopic photograph
Embodiment
Embodiment 1
Get bent sample of 0.5g or wine unstrained spirits, add 15mL stroke-physiological saline solution shaking table vibration 30min, again through suitably dilution, spread plate, put 28 ℃ of constant incubators and cultivated 2～3 days, single bacterium colony is drawn the inclined-plane, treats to connect after the slant pore maturation one and encircles in primary dcreening operation and shake bottle, 28～30 ℃, the rotary shaker fermentation of 150r/m 3 days.Fermented liquid is at the centrifugal 5min of 6000r/m, get supernatant liquor 30uL and annotate the dull and stereotyped aperture of primary dcreening operation, get the bigger bacterium of fluorescent ring and/or transparent circle and shake the multiple sieve of bottle, sieve again again, fermented liquid is through centrifugal, supernatant liquor is surveyed enzyme and is lived, and supernatant liquor and thalline make full cell zhizopchin lipase zymin respectively through lyophilize.
Claims (3)
1, a kind of lipase produces bacterium, it is characterized in that bacterial strain separates a strain zhizopchin that obtains and (has been preserved in Wuhan China typical culture collection center from Chinese Daqu, called after Rhizopus chinensis CCTCCM201021), its on morphological specificity more near zhizopchin, but on physiological and biochemical property, relatively go back some difference with selected zhizopchin (Rhizopus chinensis Saito AS3.1166) as standard, classification of fungi system according to Ainsworth, belong to Zygomycotina, zygomycetes, mucorales, Mucoraceae, Rhizopus, zhizopchin kind (Rhizopus chinensis).
2, the described lipase of a kind of claim 1 produces the screening method of bacterium, it is characterized in that adopting short-chain aliphatic ester and/or long chain fatty acid ester earlier is substrate, is the primary dcreening operation index with the ability of microbiological degradation short-chain aliphatic ester and/or long chain fatty acid ester; Serves as to sieve index for the first time again with microorganism to the tolerance performance of organic solvent; Again with the ability of microorganism Acrawax in organic phase for sieving index more again; Make up above three kinds of screening methods from bent sample of the traditional Daqu of China or wine unstrained spirits, through the dilution spread plate, single bacterium colony moves and connects slant medium, shake-flask culture, centrifugal, supernatant liquor is annotated the primary dcreening operation plate well, the bigger bacterial strain of picking transparent circle or fluorescent ring adds and contains in 1～10% heptane solvent environment shake-flask culture and measure the solvent environment lipase activity and carry out multiple sieve the first time, centrifugal with passing through behind the microorganism shake-flask culture that multiple sieve obtains for the first time, it is index sieve again again that the thalline lyophilize makes after the zymin with organic synthesis fatty acid ester ability, and the thalline lyophilize makes full cellular enzymes preparation;
Get bent sample of 0.5～5 gram Daqu or wine unstrained spirits and be dissolved in 15～150ml stroke-physiological saline solution shaking table concussion 30～150 minutes, redilution, single bacterium colony moves and connects slant medium behind the spread plate, 25～35 ℃ of constant temperature culture 2～5 days, and single bacterium colony moves and connects the inclined-plane, after treating the slant pore maturation, 25～35 ℃, 150rpm/m, shake-flask culture 2～5 days, relatively enzyme is alive to carry out primary dcreening operation, and the bacterium that picking transparent circle and/or fluorescent ring are bigger shakes the multiple sieve of bottle; Selected have a bacterial strain that higher lipase enzyme is lived, and add and contain in the substratum of 1～10% heptane, 25～35 ℃, 150rpm/m, shake-flask culture 2～5 days is measured enzyme and is lived, and carries out multiple sieve ratio than the tolerance of microorganism to solvent environment; Screening obtains the bacterial strain of higher vigor, 25～35 ℃, 100～300rpm/m, shake-flask culture 2～5 days, nutrient solution is under 6000rpm/m after centrifugal 5 minutes, supernatant liquor and thalline respectively lyophilize to make after the zymin with organic synthesis fatty acid ester ability be index sieve again again, the thalline lyophilize makes full cellular enzymes preparation;
Culture condition is: inoculum size is 0.1～5 * 10
6Individual/mL, product enzyme culture medium condition is: soybean cake powder 3～10%, peptone 3～10%, glucose 0.2～5%, K
2HPO
40.1～3%, MgSO
4.7H
2O0.01～0.5%, CaCL
20.001～0.05%, pH6～9,50～300rpm, shake-flask culture 48～120h, enzyme work can reach 400u/g.
3, the described lipase of a kind of claim 1 produces the application of bacterium, and it is synthetic to it is characterized in that the full cellular enzymes preparation that makes is used for the conversion of organic phase aromatic ester; Lipid acid is the saturated monoprotic acid of 3～18C, and the oleic acid of 18C, Fatty Alcohol(C12-C14 and C12-C18) are the primary alconol of 2～18C, the secondary alcohol of 4～5C, and under the concentration of substrate of 0.1～2.0mol/L, stirring reaction 24～120 hours, half life of enzyme 1000 hours.
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| CN100543130C (en) * | 2007-04-13 | 2009-09-23 | 新疆农业科学院微生物应用研究所 | A low-temperature neutral lipase and Geotrichum candidum producing the enzyme |
| CN1928116B (en) * | 2006-08-25 | 2010-07-21 | 江南大学 | A rapid assay method for lipase synthase activity in non-aqueous phase |
| CN101984053A (en) * | 2010-12-09 | 2011-03-09 | 江南大学 | Efficient preparation method of lipase by fermentation |
| CN101638644B (en) * | 2007-04-13 | 2011-05-11 | 新疆农业科学院微生物应用研究所 | A kind of preparation method of Geotrichum candidum producing low-temperature neutral lipase |
| CN101130757B (en) * | 2006-07-14 | 2011-05-11 | 新疆农业科学院微生物应用研究所 | Low-temperature lipase mycopremna, low-temperature lipase and method of preparing the same |
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| CN102604908A (en) * | 2009-11-11 | 2012-07-25 | 江南大学 | Lipase mutant with high catalytic activity |
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| CN101130757B (en) * | 2006-07-14 | 2011-05-11 | 新疆农业科学院微生物应用研究所 | Low-temperature lipase mycopremna, low-temperature lipase and method of preparing the same |
| CN1928116B (en) * | 2006-08-25 | 2010-07-21 | 江南大学 | A rapid assay method for lipase synthase activity in non-aqueous phase |
| CN101638644B (en) * | 2007-04-13 | 2011-05-11 | 新疆农业科学院微生物应用研究所 | A kind of preparation method of Geotrichum candidum producing low-temperature neutral lipase |
| CN100543130C (en) * | 2007-04-13 | 2009-09-23 | 新疆农业科学院微生物应用研究所 | A low-temperature neutral lipase and Geotrichum candidum producing the enzyme |
| CN101406204B (en) * | 2007-10-10 | 2012-09-05 | 王定立 | Plant powder pesticide and preparation method thereof |
| CN101693914B (en) * | 2009-09-24 | 2012-03-14 | 江南大学 | Process for preparing (R)-2-methylbutanoic acid and (R)-2-methylbutanoic acid ethyl ester via lipase resolution |
| CN102604908A (en) * | 2009-11-11 | 2012-07-25 | 江南大学 | Lipase mutant with high catalytic activity |
| CN102604908B (en) * | 2009-11-11 | 2013-01-23 | 江南大学 | Lipase mutant with high catalytic activity |
| CN102140426A (en) * | 2010-09-20 | 2011-08-03 | 江南大学 | High-yield lipase gene engineering bacterium and construction method therefor |
| CN102080092B (en) * | 2010-12-08 | 2012-02-08 | 江南大学 | Highly expressed lipase gene and its secreted expression vector and application |
| CN102080092A (en) * | 2010-12-08 | 2011-06-01 | 江南大学 | High expression lipase gene and secretory expression vector and application thereof |
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| CN102719387A (en) * | 2012-07-13 | 2012-10-10 | 光明乳业股份有限公司 | Screening method for high-producing strains of lipase |
| CN102719387B (en) * | 2012-07-13 | 2014-12-10 | 光明乳业股份有限公司 | Screening method for high-producing strains of lipase |
| US10731110B2 (en) | 2013-12-20 | 2020-08-04 | Novozymes A/S | Compositions and processes for treatment with lipases |
| CN108441428A (en) * | 2018-03-22 | 2018-08-24 | 江南大学 | One plant degradation alcohol soluble protein rhizopus chinensis and its application |
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