Chitinolyticbacter Meiyuanensis Isolation, Identification, And Fermentation For Chitinase Production

Chitin, an insoluble linearβ-1,4-linked polymer of N-acetylglucosamine （GlcNAc）, is frequently regarded as the second most abundant carbohydrate after cellulose in nature,and the second most abundant nitrogen organic after protein. N-acetylglucosamine（GlcNAc）, chitin oligosaccharide and its derivatives have a wide range of applications in industries of food, medical, agriculture, chemistry. As a group of enzymes capable of degrading N-acet- ylglucosamine monomers, chitinases hydrolyze chitin into chitin oligosaccharide, chitin disaccharide and N-acetylglucosamine.This dissertation reported a high chitinase-producing bacterium isolated from abandoned pond sediment. According to various characteristics, the bacterium was identified as a new species. The high chitinase-producing bacterium was deeply explored and researched, including strain isolation, optimization of chitinase-producing conditions, purification and characterization of chitinase, biosynthesis mechanism of chitinase, cloning and expresssion of chitinase gene, and its application in hydrolyzing chitin and shrimp by chitinase. The results were as follows:1)The 56 bacteria producing chitinase were isolated from 180 samples in four provinces through see-through circle way, including soil, insect, stricks and crustacean cadavers and so on. Among them, five bacteria have high chitinas-producing ability and one strain was selected and identified as a new species. According to the experiments of physiology and biochemistry, fatty acid composition, quinines, G+C content and 16S rRNA, it was named C. meiyuanensis SYBC-H1. The 16S rRNA has been submitted to GenBank （GQ981314）. This bacterium was preserved in the China General Microbiological Culture Collection Center （CGMCC 3438）and American Type Culture Collection（ATCC BAA-2140）.2)The submerged culture conditions and medium components of C. meiyuanensis SYBC-H1 were optimized. Effects of various carbon sources, nitrogen sources, metal ions and other factors were investigated firstly and ten important factors of chitinase production were screened. Furthermore some key factors for chitinase production were determined through Plackett-Burman design. The optimized medium （g/L） using central composite design were urea 3.1, inulin 3.55, chitin 3.80, Na₂SO₄ 0.32, K₂HPO₄·3H₂O 0.70, KH₂PO₄ 0.30, MgSO₄·7H₂O 0.5, FeSO₄·7H₂O 0.01. And then the optimum culture conditions were investigated through Central Composite Design,and the results optimum fermentration condition as rotate speed 233 r/min, initial pH 6.44 and temperature 26.21℃. The maximum enzyme activity was 5.16 U/mL while the tested enzyme activity was 5.20±0.32 U/mL. Meanwhile, the effects of cell age, inoculums size and surfactant on production of chitinase were studied.3)The crude chitinase was purified through three effective steps involving （NH₄）2SO₄ salt precipitation, DEAE-cellulose anion-exchange chromatography and Sephadex G-100 gel filtration. Pure electrophoresis grade chitinase was obtained. Enzymatic properties were studied,and its optimum temperature is 40℃, which aldso found that the enzyme activity decreases rapidly with rising temperature especially above 43℃. The chitinase activity with a wider range pH from 3.5 to 9.0 and the optimum pH of 6.5. Mercaptoethanol and EDTA effectively improved the stability of chitinase. Zn²⁺,Cu²⁺,Mn²⁺,Fe²⁺ and Fe（3+） have different inhibition degrees on chitinase , while Na⁺ and K⁺ stimulated it.4)The physiological characteristics of C. meiyuanensis SYBC-H1 during the chitinase production were investigated. It was found that secretion of chitinase and biomass had certain correlation. When the strain cultivated in the optimal medium, the oxidation level was relatively high, the contents of malondialdehyde, H2O2 and Vc were increased, and the activities of SOD and CAT were enhanced. These indicated that the strain was under oxidative stress during the chitinase production period.5)By observing the effect of carbon sources on enzyme activity, it was found that fructose as a kind of carbon source is far more effective than glucose. The cloning and expression experiment of chitinase gene enables us to clone and testify the ABC transportors system including ATPase, periplasmic sugar-binding protein and aminoglycoside transport protein. These offered theoretical base for further steps of molecule modification of this bacteria to design and construct bioengineering one.6)Two chitinase genes（CHI1,CHI2） were cloned and their full-length were 497 bp and 1151 bp. Three-dimensional structures were studied with Swiss-MODEL. E. coli expression vector pET28（a）-CHI2 was constructed and expressed into E. coli BL21 successfully. SDS-PAGE showed chitinase had significant expression but its activity was low, indicating that the expressed protein existed in the form of inclusion bodies.7)According to mass spectrometry （MS） and high performance liquid chromatography （HLPC） identification of hydrolysis products of chitin, there are mainly N acetyl glucosamine and few chitobiose. Chitinase degradation of chitin and dried small shrimps were investigated. Study showed that temperature, time, pH, beta mercaptoethanol, metal ions and oxygen supply affected the hydrolysis of chitin. Particle size of 100 hole of chitin powder was in favor of chitinase hydrolysis, but 30-70 chitin powder did not significantly affect. At the same time also we used Levenberg-Marquardt and general global optimization method to establish the chitinase hydrolysis chitin model, and the results showed that when the temperature was 37.5℃, pH 7.0 and the hydrolysis time 13.5 hours, the highest efficiency was reached, and sugar concentration was 90.4μmol / L.