Part:BBa_K2593004

Biology

LHyal gene encodes LHAase (Mw=58kD), which is a hyaluronidases from the hyaluronate 3-glycanohydrolases sub-family of leech origin. Hyaluronidase (HAase) is a large family of glycoside hydrolases that predominantly degrades hyaluronic acid (HA), which is a polysaccharide composed of disaccharides unit of N-acetyl glucosamine and glucuronic acid polymerization. Based on substrate specificity and hydrolysis products, HAases are commonly grouped into three families: first group is hyaluronate lyases (EC 4.2.2.1, Streptococcus hyaluronate lyase), it’s commonly use to produce LHAase but has some potential risk on product. Second group is hyaluronate 4-glycanohydrolases (EC 3.2.1.35, Bovine testicular hyaluronidase, BTH) Commercial BTH has been widely used in clinical medicine, and its hydrolysis mechanism has been studied extensively. The disadvantages of the enzymatic production of specific or narrow-spectrum HA oligosaccharides by BTH include the limited source material (bovine testes), its considerably high price and the broad range of degradation products. Third group is hyaluronate 3-glycanohydrolases(EC 3.2.1.36, Leech HAase).Compared with BTH and Streptococcus hyaluronate lyase, leech HAase has higher substrate specificity and a narrow-spectrum of enzymatic products16,17. In particular, leech HAase is unable to degrade chondroitin or chondroitin sulfate compared to other HAase sources due to its strong substrate specificity. Although mammalian HAase has been widely used as a drug diffusion agent, such HAase activity is susceptible to heparin inhibition. Leech HAase activity, in theory, is not affected by heparin, and has more medical value in clinic and other medical aspects. In addition, the use of recombinant leech HAase does not pose any risk of animal cross-infection. Therefore, high-level production of recombinant leech HAase would be of great significance for both clinical medical treatment (such as surgery, ophthalmology and internal medicine) and producing narrow-spectrum HA oligosaccharides at the industrial scale.

Usage

Fig 1 . the LHyal gene was validated by using PCR primer pMA-LHyal-F and pMA-LHyal-R,the expected size is 520 bp

In our project, this device worked to produce hyaluronic acid hydrolase (hyaluronidase) which is derived from leech genome. The SDS-PAGE method was used to prove the expression of the hydrolases, A clear band was shown with a molecular weight approximately 58 kDa (Figure 1), which is consistent with the value published in previous research.

Figure 1: SDS-PAGE analysis of LHAase in total extracellular crude protein fraction from recombinant B.subtilis harboring pMA0911-AmyX-H6LHAyal. A 12% SDS-PAGE gel was used. Lane 1: purified LHAase by Ni-NAT affinity column; Lane 2: the crude extracellular protein fraction .

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 157
  

Reference

Peng Jin, Guocheng Du, Zhen Kang. High-yield novel leech hyaluronidase to expedite the preparation of specific hyaluronan oligomers[J].Scientific Reports, 2014 : 1-2
Jinpeng, Kangzhen, Biosynthesis of hyaluronan oligosaccharides and construction of DNA editing and assembly tools[D]Jiangnan University: Jinpeng,2016.25-27.