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Pectinase gene, pectinase, recombinant vector and application thereof in blueberry processing

A technology of recombinant vector and pectinase, applied in the direction of genetic engineering, application, plant gene improvement, etc., can solve the problems of limited products, unsuitable for large-scale application, etc., and achieve the effect of efficient degradation

Active Publication Date: 2022-07-15
青岛紫斐农业科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The application of pectinase in the extraction and purification of wine, blueberry and other fruit juices, and the treatment of pectin wastewater is less, and the products produced by the traditional enzyme preparation technology are limited, which is not suitable for large-scale application

Method used

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  • Pectinase gene, pectinase, recombinant vector and application thereof in blueberry processing
  • Pectinase gene, pectinase, recombinant vector and application thereof in blueberry processing
  • Pectinase gene, pectinase, recombinant vector and application thereof in blueberry processing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Screening of pectinase-producing strains

[0053] The screening medium was prepared with pectin as the sole carbon source: pectin 5g / L, NaNO 3 2g / L, NaCl 1g / L, MgSO 4 1g / L, K 2 HPO 4 ·3H 2 O 1g / L, KH 2 PO 4 0.5g / L, bromophenol blue 0.01g / L. Take 10 g of fresh blueberry peel samples and shake them in 50 mL of sterile normal saline for 30 min. After standing, draw 4 mL of it and inoculate it into 100 mL of screening medium, and culture with shaking at 30 °C for 24 h. Take 0.5 mL of bacterial suspension for appropriate gradient dilution, and then take 0.1 mL of bacterial suspension of different dilutions and spread it on the primary screening medium plate (the composition of the primary screening medium plate is the addition of 2% agar to the screening medium) After culturing at 30°C for 48 hours, single colonies that can generate yellow circles on the bromophenol blue plate were selected for streak separation and purification. The purified strains wer...

Embodiment 2

[0056] Example 2: Strain identification and pectinase gene cloning

[0057] The strain LM1 was cultured on YPD solid medium at 28°C for two days, and the colony characteristics and cell shape were observed. The results are shown in image 3 , Aureobasidium pullulans LM1 screened by the present invention is preserved in the China Center for Type Culture Collection, and the preservation time is March 09, 2022. The address of the preservation unit is Wuhan, China, and the preservation number is CCTCC NO: M2022182.

[0058] The product was obtained by PCR amplification, and the sequence of 26S rDNA was obtained after the product was sequenced. The primers used for PCR amplification of D1 / D2 26SrDNA sequences are:

[0059] Upstream primer NL-1: 5'-GCATATCAATAAGCGGAGGAAAAG-3' (SEQ ID NO. 3);

[0060] Downstream primer NL-4: 5'-GGTCCGTGTTTCAAGACGG-3 (SEQ ID NO. 4).

[0061] The sequencing results were analyzed by the Basic Local Alignment Search Tool (BLAST) in the GenBank databas...

Embodiment 3

[0067] Example 3: Heterologous expression and purification of pectinase ZF05 in Pichia pastoris

[0068] The pectinase gene ZF05 was linked with the pPIC9K Pichia vector to construct a recombinant vector, and electrotransformed according to the Pichia expression manual method. When a single colony was grown, it was screened and subjected to an induced expression experiment. The expression and purification of pectinase ZF05 were detected by polyacrylamide gel electrophoresis, and the results were as follows figure 1 As shown, the purified pectinase ZF05 showed a single band on the electrophoresis gel, and the position (about 49 kDa) matched the predicted molecular weight (49.30 kDa).

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Abstract

The invention discloses a pectinase gene, pectinase, a recombinant vector and application of the pectinase gene, the pectinase and the recombinant vector in blueberry processing. The amino acid sequence of the pectinase disclosed by the invention is as shown in SEQ ID NO. 2. The similarity between the amino acid sequence of the pectinase provided by the invention and the sequence of the existing pectinase with known functions is only 56%. The pectinase ZF05 disclosed by the invention has the characteristic of thermal stability, and can keep more than 80% of activity after being incubated at 60 DEG C for 2 hours. The pectinase disclosed by the invention is stable in property and high in yield, and can be used for degrading pectic polysaccharides.

Description

technical field [0001] The invention relates to the biological field, in particular to a pectinase gene, a pectinase, a recombinant vector and its application in blueberry processing. Background technique [0002] Aureobasidium pullulans ( Aureobasidium app.) is widely distributed in various ecological environments, including forest soil, seawater, animal and plant tissues, etc. The cell morphology of Aureobasidium pullulans mainly includes yeast-like cells, blastospores, chlamydospores and mycelium. It can be used to hydrolyze green algae in the ocean and various industrial wastes. Studies have shown that in addition to the production of pullulan polysaccharide, Aureobasidium pullulans can also produce a variety of enzymes, such as sucrase, amylase, pectinase and so on. [0003] Pectin is a general term for a class of plant cell wall polysaccharides rich in galacturonic acid, and its basic skeleton is a homopolymer linked by α-1,4 glycosidic bonds. Pectin is mainly com...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N15/56C12N15/60C12N15/81C12N1/19C12N1/14C12N9/18C12N9/24C12N9/88C12N1/02C08B37/06A23L2/02A23L2/84C12R1/645C12R1/84
CPCC12N9/18C12N9/2402C12N9/88C12N15/815C12N1/14C12N1/02C08B37/0045A23L2/02A23L2/84C12Y301/01011C12Y302/01015C12Y402/02002C12N2800/102
Inventor 张羽陈静辛凌锋
Owner 青岛紫斐农业科技发展有限公司
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