CN101096650A

CN101096650A - One strain clausius bacillus mutant and fermentation production of alkaline pectic enzyme

Info

Publication number: CN101096650A
Application number: CNA200710100383XA
Authority: CN
Inventors: 李祖明; 陈艳; 李鸿玉; 白志辉
Original assignee: Beijing Union University
Current assignee: Beijing Union University
Priority date: 2007-06-11
Filing date: 2007-06-11
Publication date: 2008-01-02
Anticipated expiration: 2027-06-11
Also published as: CN101096650B

Abstract

The invention provides a Bacillus clausii N-10 mutant of a high-yield basic pectolase, which also provides a method for fermenting the basic pectolase with the bacterialin the biological engineering technique field. The invention adds proper maltose, phosphate, sodium carbonate and water with beet pulp as the carton source and inducer and the compressed cotton as the nitrogen source, which inoculates Bacillus clausii N-10 mutant, cultures about 72 hours at air and 40Deg. C condition and reaches the peak of producing enzyme. The method comprises the following steps: extracting the culture with Na2CO3/NaHCO3 buffer solution; filtering; getting the basic pectolase liquid; getting concentrated enzyme liquid by hyperconcentration or getting the basic pectolase powder by spray drying. The yield of the basic pectolase reaches 3300 unit /g dry substrate by measuring enzyme vitality with DNS method (the definition of the enzyme vitality unit is the enzyme quantity with which 1mu mol galacturonic acid is produced by degrading sodium polygalacturonate per minute at PH 10, 55Deg. C).

Description

One one strain clausius bacillus mutant and fermentation production of alkaline pectic enzyme thereof

Technical field

The invention belongs to technical field of bioengineering.

Technical background

At present alkaline pectase in the application of bioengineering field more and more widely, its Application Areas comprises: biological concise, the plant bast fiber degumming of cotton fibre, papermaking, processing pectin waste water, coffee ﹠ tea fermentation, washing composition etc.Chinese patent (publication number: disclose a bacillus pumilus (Bacillus pumilus) CCTCC No:M203042 CN 1480529A) and produced alkaline pectase, can be used for the cotton-spinning fabric refining processing through liquid submerged fermentation.Chinese patent (publication number: disclose a strain Alkaliphilic bacillus (Bacillus alcalophilus) CCTCCM96009 CN 1177003A) and can be directly used in China grass degumming.Chinese patent (publication number: disclose a bacillus subtilis (Bacillus Subtilis) CCTCC No:0213 CN 1113951A) and produced alkaline pectase through high density liquid state fermentation.We separate a strain alkaliphile from alkaline soil, has higher product alkaline pectase ability, through being accredited as gram Lloyd's genus bacillus (Bacillus clausii), we also studied the character (Zhang Baoguo that this strain fermentation is produced alkaline pectase, Bai Zhihui, Li Zuming, Guozheng ZHANG, Zhang Hongxun. gram Lloyd's genus bacillus S-4 bacterial strain solid state fermentation produces alkaline pectase. food and fermentation industries .2005,31 (3): 8-11).The present invention carries out ultraviolet mutagenesis with this original strain, obtains the mutant strain of high-production alkalescence pectinase, and this mutant strain bacterial strain also of no use at present carries out the report of fermentation production of alkaline pectic enzyme.

Summary of the invention

The purpose of this invention is to provide the mutant strain of strain alkaliphile gram Lloyd's genus bacillus, it has very high product alkaline pectase ability; And provide a kind of method of utilizing this strain fermentation to produce alkaline pectase.

This bacterial strain is to produce the alkaline pectase bacterial strain with separate the gram Lloyd's genus bacillus that obtains from alkaline soil, the alkaline pectase high productive mutant (Bacillus clausii N-10) that obtains through ultraviolet mutagenesis, its energy force rate original strain that produces alkaline pectase improves 50%, through the cultured continuously stable performance.Culture presevation is numbered CGMCC No.2073 at China Committee for Culture Collection of Microorganisms common micro-organisms center.

It is as follows to adopt this bacterial strain to carry out the step of production for solid state fermentation of alkali polygalacturonase:

1, culture presevation.Gram Lloyd's bacillus mutant N-10 is preserved in alkaline nutrient agar inclined-plane in 4 ℃ of refrigerators, and transfer once half a year.Substratum is formed: Tryptones 1.5%, and soy peptone 0.2%, yeast extract 0.3%, glucose 0.2%, NaCl 0.2%, K ₂HPO ₄0.12%, NaCO ₃0.3%, agar 1.5% was in 115 ℃ of sterilizations 15 minutes.

2, seed culture.Liquid nutrient medium is formed: pectin 0.5%, yeast extract 1%, MgSO ₄7H ₂O 0.05%, K ₂HPO ₄0.1%, NaCO ₃0.3%; In 115 ℃ of sterilizations 15 minutes.Inoculation, 35 ℃ of shake-flask culture 24 hours, both liquid seeds.

3, solid state fermentation.Producing the alkaline pectase solid medium is that the cotton dregs of interpolation 3% weight in containing pectin solid biomass (beet pulp, wheat bran, Receptaculum Helianthi, orange peel, rice bran, apple residue etc.) and the nutritive medium of 2.5 times of weight are made, nutritive medium is formed: maltose 1.2%, potassium primary phosphate 0.12%, yellow soda ash 1.2%.Behind beet pulp and the cotton dregs mixing and nutritive medium respectively at 115 ℃ of sterilizations 15 minutes, the weight ratio of nutritive medium and beet pulp was 2.5: 1, mixes, and inoculates liquid seeds, in 40 ℃ of static cultivation 72 hours.

4, the extraction of enzyme and purification.Material behind the solid state fermentation, the pH that adds 10 times of weight are the Na of 10 0.05mol/L ₂CO ₃/ NaHCO ₃Buffered soln soaked 30 minutes, carried out solid-liquid separation in supercentrifuge, and extracting solution is removed thalline with the micro-filtrate membrane filtration of aperture 0.1 μ m, and filtrate is that 10000 ultra-filtration membrane concentrates with molecular weight cut-off, collects concentrated solution, i.e. alkaline pectin enzyme solution, storage.

Below describe enforcement of the present invention in detail by specific embodiment, purpose is to help the reader to understand spirit of the present invention better, but not as to the qualification of the scope of the present invention.

Embodiment 1: different nitrogen sources is to producing the influence of enzyme

Add the nutritive medium of 2.5 times of weight in beet pulp, nutritive medium is formed: different nitrogen sources 1.2%, potassium primary phosphate 0.12%, yellow soda ash 1.0%.Beet pulp and nutritive medium mix by weight 1: 2.5 respectively at 115 ℃ of sterilizations 15 minutes, and the inoculation liquid seeds was in 40 ℃ of static cultivation 72 hours.The results are shown in Table 1.

Table 1: different nitrogen sources is to producing the influence of enzyme

Nitrogenous source	Relative enzyme % alive
Nitrogenous source	Relative enzyme % alive	Wheat bran (NH ₄) ₂SO ₄ NaNO ₃Tryptones soybean protein isolate soy peptone yeast soaks powder fish meal corn plumule powder	100 46 42 101 66 101 102 63 66

Sesame meal dregs of beans dish dregs of rice cotton dregs do not add nitrogenous source

86 84 67 113 60

Embodiment 2: different yellow soda ash additions are to producing the influence of enzyme

Add the nutritive medium of 2.5 times of weight in the mixture of beet pulp and cotton dregs in (5 parts of beet pulp+3% part cotton dregs), nutritive medium is formed: maltose 1.2%, potassium primary phosphate 0.12%, the different amounts of yellow soda ash.The mixture of beet pulp and cotton dregs and nutritive medium mix by weight 1: 2.5 respectively at 115 ℃ of sterilizations 15 minutes, and the inoculation liquid seeds was in 40 ℃ of static cultivation 72 hours.The results are shown in Table 2.When yellow soda ash 1.2%, zymogenic rate is the highest, reaches 3300 units/gram dry bottom things.

Table 2: various pectin biomass and the substratum different humidity of containing are to producing the enzyme influence

Carbonate content in the nutritive medium (%)	Enzyme productive rate (* 10 ³U/g dry bottom thing)
Carbonate content in the nutritive medium (%)	Enzyme productive rate (* 10 ³U/g dry bottom thing)	0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8	1.3 2.4 3.0 3.3 2.8 1.8 1.6 1.2

Annotate: adopt the DNS method to measure enzyme and live, 1 enzyme activity unit (u) is defined as: at 55 ℃, under pH 10 reaction conditionss, per minute degraded polygalacturonic acid sodium produces the amount of the required enzyme of 1 μ mol galacturonic acid.The result is the mean value of three parallel laboratory tests in the table, and relative deviation is less than 10%.

Claims

1. the alkaliphile of alkaline pectase is produced in a strain, it is characterized in that, this alkaliphile is an one strain clausius bacillus mutant, can utilize the beet pulp fermentation production of alkaline pectic enzyme, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, be numbered CGMCC No.2073.

2. method of utilizing the described alkaliphile of claim 1 to produce alkaline pectase, it is characterized in that, be carbon source with the beet pulp, adds nitrogenous source cotton dregs, phosphoric acid salt, yellow soda ash and water, the described alkaliphile of inoculation claim 1 adopts process for solid state fermentation to produce alkaline pectase.

3. the described method of claim 2 is characterized in that, the pH of the 10 times of weight of material adding behind the solid state fermentation is 10 Na ₂CO ₃/ NaHCO ₃Buffered soln soaked 30 minutes, carried out solid-liquid separation in supercentrifuge, and extracting solution is removed thalline with the micro-filtrate membrane filtration of aperture 0.1 μ m, and filtrate is that 10000 ultra-filtration membrane concentrates with molecular weight cut-off, collects concentrated solution, i.e. alkaline pectin enzyme solution, storage.