CN103555620A - Alkaline catalase high-yielding strain and fermentation-method production process thereof - Google Patents

Abstract

The invention relates to an alkaline catalase high-yielding strain and a fermentation-method production process thereof, belonging to the technical field of bioengineering. According to the alkaline catalase high-yielding strain disclosed by the invention, shown by assays, the alkaline catalase high-yielding strain belongs to Arthrobacter sp. and has the collection number of CGMCC (China General Microbiological Culture Collection Center) NO. 8181. The invention further discloses a method for producing alkaline catalase from the alkaline catalase high-yielding strain. According to the alkaline catalase high-yielding strain provided by the invention, the alkaline catalase can be prepared from the strain through shake-bottle liquid submerged fermentation, the activity of the catalase reaches 49706U/mL, the catalase can be applied to dyeing and finishing treatment processes for cotton textiles, the quality of products can be improved, the existing cotton textile dyeing and finishing treatment processes are improved effectively, and the production process is more environmental-friendly.

Description

A kind of hydrogen peroxidase through fermenting Producing Strain and this strain fermentation method production technique

Technical field

Producing Strain and by this strain fermentation method production hydrogen peroxidase through fermenting, particularly a plant height produces the bacterial strain of hydrogen peroxidase through fermenting, belongs to technical field of bioengineering.

Background technology

In numerous industries, textile industry is conventional industries and the mainstay industry of China.In textile industry, the research report playing a role in the process of weaving for hydrogen peroxide endonuclease capable is in the last few years also of common occurrence, and using catalase as a kind of material of removing residue hydrogen peroxide economic in yarn process, equally also in this operation of bleaching of fabric, play a role, by comparison, traditional water cleaning way is to have larger drawback: for example after using as SYNTHETIC OPTICAL WHITNER, to remove be not to be easy to and remaining reductive agent also will have some bad impacts for fiber to hydrogen peroxide, and after using catalase cleaning instead, not only water saving can also be increased work efficiency.The advantage of the biological cleaning technique of catalase is embodied in:

1) significantly reduce water consumption, energy consumption and wastewater flow rate;

2) efficient catalytic, reduces the production time, improves throughput, increases the output value;

3) enzyme agent consumption is few, reduces production costs;

4) better quality of fabric, intensity is higher, and pliability is better;

5) promote accuracy and the security of dyeing;

6) operation safe and easy;

7) product is oxygen G&W, is conducive to environmental protection.

Though use catalase to realize bleaching, there is good economy and environmental benefit in fabric bleaching technique, but just at present, the subject matter that realizes this technique is catalatic stability problem under high temperature and strong alkaline condition, comprise catalase storage stability problem and reaction stability problem, but large multi-product does not still reach the requirement of textile industry strong basicity (pH>10), in while document at home and abroad, Arthrobacter product catalase enzyme is alive very low, is far from reaching industrialized requirement.Hydrogen peroxide enzyme source is abundant, is almost present in all easy-to-keep biologicals, and along with the maturation of bacteria cell wall crushing technology, catalase application will be more extensive.

Summary of the invention

The object of this invention is to provide a kind of hydrogen peroxidase through fermenting Producing Strain and by this strain fermentation method production hydrogen peroxidase through fermenting, prepared hydrogen peroxidase through fermenting is for the dyeing and finishing treating processes of cotton textiles.

Technical scheme of the present invention: separation obtains a strain bacterium LHM7719 from the soil of paper mill, through identifying that it belongs to Arthrobacter arthrobacter sp.and on September 13rd, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCC NO.8181.

The method of hydrogen peroxidase through fermenting Producing Strain production hydrogen peroxidase through fermenting, according to following step, carry out:

Adopting CGMCC NO:8181 is starting strain, through seed culture and liquid submerged fermentation, makes hydrogen peroxidase through fermenting,

Seed culture medium is LB substratum: in g/L, and glucose 20, peptone 10, beef extract 5, sodium-chlor 5, pH 7.2; Culture condition: incubation time is 12～16 hours, temperature is 29～32 ℃, shaking speed is 160 revs/min.

Fermention medium is LB substratum: in g/L, and tapioca (flour) 15～20, bean cake powder 50～70, potassium primary phosphate 0.5, initial pH is 7.0; Fermentation condition: fermentation time is 35～45 hours, inoculum size is 1%～8%(v/v), liquid amount is 40ml/250 ml, and culture temperature is 30～40 ℃, and shaking speed is 200～250 revs/min.

Get 15ml fermented liquid, 12000 revs/min centrifugal 20 minutes, after then adding 15ml phosphoric acid buffer to mix, ultrasonic disruption is got, 12000 revs/min centrifugal 15 minutes, get supernatant liquor and obtain hydrogen peroxidase through fermenting.

The catalatic application of China's common micro-organisms culture presevation administrative center deposit number CGMCC NO:8181 fermentation gained: prepared hydrogen peroxidase through fermenting enzyme work reaches 49706U/mL, can be used for the dyeing and finishing treating processes of cotton textiles.

Beneficial effect of the present invention: the Producing Strain that has proposed a kind of hydrogen peroxidase through fermenting, with this bacterial strain, through shaking flask liquid submerged fermentation, can prepare hydrogen peroxidase through fermenting, enzyme work reaches 49706U/mL, the dyeing and finishing treating processes that can be used for cotton textiles, can improve product quality, effectively improve traditional cotton textile dyeing and finishing treatment process, make production process environmental protection.

Embodiment

A standard enzyme unit alive (lu) is defined as: at 37 ℃, decompose 1 umol H in 1 min ₂o ₂(optical extinction coefficient is 39.4L/mol/cm) required enzyme amount.H ₂o ₂rate of decomposition with ultra-violet and visible spectrophotometer S53 at 240 nm, under 30 ℃ of conditions, measure.Reaction cumulative volume is 3 mL, containing 0.1ml enzyme liquid and 2.9ml, contains 120 mmol/L H ₂o ₂potassium primary phosphate, dipotassium hydrogen phosphate damping fluid (pH 7.0) with 50 mmol/L.

Bacterial strain that the present invention adopts, has been preserved in Chinese common micro-organisms culture presevation administrative center, and deposit number is CGMCC NO:8181.

The screening method of China common micro-organisms culture presevation administrative center deposit number CGMCC NO:8181 is screening cultivating through flat board from the soil of paper mill, drips aerogenesis bubble situation and screens again, and catalase activity size and obtaining relatively.Paper mill soil is cultivated through twice LB substratum increment, be coated with dull and stereotyped, cultivate the rear 0.2ml3% of dropping hydrogen peroxide on bacterium colony of uniform size, selection aerogenesis steeps rapidly and time length bacterium colony of a specified duration carries out test tube fermentation culture, by comparing hydrogen peroxidase through fermenting decomposing H ₂o ₂the size of ability, finally determines that bacterial strain is LHM7719.Through identifying that it belongs to Arthrobacter arthrobacter sp.and on September 13rd, 2013, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (be called for short CGMCC, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number is CGMCC NO.8181.

LHM7719 bacterial strain some physiological-biochemical characteristics identifies that cell is shaft-like as shown in table 1, and gramstaining is positive, and is this bacterial strain 16S rDNA and identifies.16SrDNA sequence is passed through comparison and Arthrobacter sp. TM4_3(DQ279376 in rrna database) the most approaching, so determine that this bacterial strain is genus arthrobacter (Arthrobacter) microorganism.Final decision bacterial strain LHM7719 should belong to a kind of (the Arthrobacter sp.) of genus arthrobacter, called after Arthrobacter sp. LHM7719.

Embodiment 1

Scraping two ring bacterial classifications from inclined-plane, are placed in the 250ml triangular flask that 30ml seed culture medium is housed and carry out seed culture.Condition is: pH 7.2, and incubation time is 16 hours, and temperature is 30 ℃, and shaking speed is 160 revs/min.With the access of 2% inoculum size, be equipped with in the 250ml triangular flask of 40ml fermention medium and carry out fermentation culture again.Condition is: pH is 7.0, and fermentation time is 45 hours, and temperature is 30 ℃, and shaking speed is 250 revs/min, can obtain the fermented liquid of enzyme 49706U/mL alive.

Embodiment 2

Scraping two ring bacterial classifications from inclined-plane, are placed in the 250ml triangular flask that 30ml seed culture medium is housed and carry out seed culture.Condition is: pH 7.2, and incubation time is 16 hours, and temperature is 30 ℃, and shaking speed is 160 revs/min.With the access of 2% inoculum size, be equipped with in the 250ml triangular flask of 40ml fermention medium and carry out fermentation culture again.Condition is: pH is 7.0, and fermentation time is 36 hours, and temperature is 32 ℃, and shaking speed is 250 revs/min, can obtain the fermented liquid of enzyme 41000U/mL alive.

          

table 1 bacterial strain LHM7719 some physiological-biochemical characteristics

"+" positive wherein, "-" feminine gender.

Claims (2)

1. hydrogen peroxidase through fermenting Producing Strain, through identifying that it belongs to Arthrobacter Arthrobacter sp., deposit number is CGMCC NO.8181.

2. the method for hydrogen peroxidase through fermenting Producing Strain production hydrogen peroxidase through fermenting claimed in claim 1, according to following step, carry out:

Adopting CGMCC NO:8181 is starting strain, through seed culture and liquid submerged fermentation, makes hydrogen peroxidase through fermenting,

Seed culture medium is LB substratum: in g/L, and glucose 20, peptone 10, beef extract 5, sodium-chlor 5, pH 7.2; Culture condition: incubation time is 12～16 hours, temperature is 29～32 ℃, shaking speed is 160 revs/min;

Fermention medium is LB substratum: in g/L, and tapioca (flour) 15～20, bean cake powder 50～70, potassium primary phosphate 0.5, initial pH is 7.0; Fermentation condition: fermentation time is 35～45 hours, inoculum size is 1%～8%(v/v), liquid amount is 40ml/250 ml, and culture temperature is 30～40 ℃, and shaking speed is 200～250 revs/min;

Get 15ml fermented liquid, 12000 revs/min centrifugal 20 minutes, after then adding 15ml phosphoric acid buffer to mix, ultrasonic disruption is got, 12000 revs/min centrifugal 15 minutes, get supernatant liquor and obtain hydrogen peroxidase through fermenting.