Method for producing chitinase through fermentation of thermobifida fusca

A chitinase and thermal cracking technology, applied in the field of bioengineering, can solve the problem of not many high-yield and high-temperature resistant chitinase, and achieve the effects of wide tolerance range, easy cultivation and short fermentation time

Inactive Publication Date: 2016-07-20
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many microorganisms have the ability to produce chitinase, but not many have the ability to produce high-temperature resistant chitinase

Method used

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  • Method for producing chitinase through fermentation of thermobifida fusca
  • Method for producing chitinase through fermentation of thermobifida fusca
  • Method for producing chitinase through fermentation of thermobifida fusca

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Experimental program
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Effect test

Embodiment 1

[0032] Embodiment 1: Preliminary experiment of brown thermobifidafusca (Thermobifidafusca) producing chitinase

[0033]The production of chitinase by Thermobista spp. was confirmed by the plate transparent circle method. The selection medium for producing the transparent circle is 5g of casein, 2g of water-soluble chitin, 7ml of 5% dipotassium hydrogen phosphate, 5ml of magnesium sulfate 1 / 10,000, 20g of agar and 1000ml of distilled water. Spot the test strains on a plate (diameter 9cm), spot 6 strains per plate, and observe whether a transparent circle is formed after culturing at 30°C for 5-7 days. Serratia marcescens known to produce chitinase was used as Control strain. The bacterial strains that produced transparent circles on the chitin plate were collected on the same plate and spot-connected, and the colonies and transparent circles were observed after 7 days. The results are shown in figure 1 .

Embodiment 2

[0034] Embodiment 2: the method for producing chitinase by fermentation of brown thermobifidafusca (Thermobifidafusca)

[0035] Put Thermophilia brown into a 50mL Erlenmeyer flask containing 10mL of enriched medium, cultivate it at 55°C and 220r / min for 24h, and transfer the first-grade seed liquid into the flask with 3% inoculum In a 250mL Erlenmeyer flask with 100mL of enriched medium, culture at 55°C and 220r / min for about 24h to prepare the secondary seed solution. The secondary seed solution was introduced into the fermentation medium with an inoculation amount of 2.5%, and cultured at 55° C. and 220 r / min for 52 hours to obtain a chitinase product.

[0036] Enriched medium components are in mass percent, including NaCl1.20%, (NH 4 ) 2 SO 4 2.49%, KH 2 PO 4 0.72%, Na 2 HPO 4 7.30%, EDTA0.02%, MgSO 4 ·7H 2 O0.08%, FeSO 4 ·7H 2 O0.008%, MnSO 4 .7H 2 O0.006%, CaCl 2 .2H 2 O0.005%, biotin 0.00008%, ammonium sulfate 0.00008%, yeast powder 0.016%, and adjust the ...

Embodiment 3

[0038] Embodiment 3: wavelength selection and standard curve drawing

[0039] Accurately weigh 0.258 g of glucosamine hydrochloride dried at 100°C to constant weight, add distilled water to dissolve and make the volume to 100 ml, and prepare a standard solution with a concentration of 12 μmol / mL. According to Table 1, add the solution to each 25ml graduated tube, develop the color in a boiling water bath for 5min, add distilled water to make the volume to 25ml after cooling. Firstly, with No. 0 tube (distilled water) as a reference, scan the absorbance wavelength curves of No. 0 tube and any other tube (No. 5 tube is selected in this experiment), and determine the measured absorption wavelength according to the maximum difference or maximum peak value of the absorption value. .

[0040] For the drawing of the standard curve, select tube No. 1 as a reference, scan the absorption wavelengths of tubes 2 to 7, and obtain the standard curve and linear correlation coefficient at di...

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Abstract

The invention discloses a method for producing chitinase through fermentation of thermobifida fusca.The method includes the steps of culturing thermobifida fusca in a fermentation culture medium at a temperature of 50-55 DEG C and a rotating speed of 200-240 r/min for 20-24 hours to prepare a first-grade seed solution, transferring the first-grade seed solution into a fermentation culture medium at an inoculum size of 2-3% to be cultured at a temperature of 50-55 DEG C and a rotating speed of 200-240 r/min for 20-24 hours to prepare a second-grade seed solution, and transferring the second-grade seed solution into a fermentation culture medium at an inoculum size of 2-3% to be cultured at a temperature of 50-55 DEG C and a rotating speed of 200-240 r/min for 50-54 hours to obtain a chitinase product.Thermobifida fusca is easy to culture, rapid in growth and short in fermentation time, and produced chitinase is high in activity, resistant to high temperature and large in pH tolerance range.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a method for fermenting and producing chitinase from Thermobista spp. Background technique [0002] Chitinase (Chitinase, EC3.2.1.14) is an enzyme widely present in animals, plants and microorganisms. Chitinase can catalyze the decomposition of chitin to produce chitooligosaccharides and chitobiose. The hydrolyzate oligosaccharides of chitinase have antibacterial effects, can regulate the immunity of organisms, and can also inhibit the growth of tumor cells. Chitinase also plays a role in the control of plant fungal diseases. Therefore, chitinase has unique application value in industries such as food, medicine, agriculture and cosmetics. [0003] Since microbial chitinases have a wider range of pH and temperature than animal and plant chitinases, and are relatively easier to prepare, the current method of obtaining chitinases at home and abroad is mainly microbial fermentation. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/42C12R1/01
CPCC12N9/2442C12Y302/01014
Inventor 邓禹张晓娟赵梅刘晓迪
Owner JIANGNAN UNIV
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